Strong inhibitory activity and action modes of synthetic maslinic acid derivative on highly pathogenic coronaviruses: Covid-19 drug candidate

Article abstract of DOI:10.3390/pathogens10050623

In late December 2019, a novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), escaped the animal–human interface and emerged as an ongoing global pandemic with severe flu-like illness, commonly known as coronavirus disea

Full text of DOI:10.3390/pathogens10050623

pathogens
Article
Strong Inhibitory Activity and Action Modes of Synthetic
Maslinic Acid Derivative on Highly Pathogenic Coronaviruses:
COVID-19 Drug Candidate
Raya Soltane 1,2, Amani Chrouda 3,4,5,*, Ahmed Mostafa 6,
*
, Ahmed A. Al-Karmalawy 7 , Karim Chouaïb 8,
Abdelwaheb dhahri 9, Rami Adel Pashameah 1, Ahlam Alasiri 1, Omnia Kutkat 6, Mahmoud Shehata 6
Hichem Ben Jannet 8, Jawhar Gharbi 10 and Mohamed A. Ali 6
,
1
Department of Basic Sciences, Adham University College, Umm Al-Qura University,
Adham 21971, Saudi Arabia; rasoltan@uqu.edu.sa (R.S.); rapasha@uqu.edu.sa (R.A.P.);
ajasiri@uqu.edu.sa (A.A.)
Faculty of Sciences, Tunis El Manar University, Tunis 1068, Tunisia
Department of Chemistry, College of Science Al-Zulfi, Majmaah University, Al-Majmaah 11952, Saudi Arabia
Laboratory of Interfaces and Advanced Materials, Faculty of Sciences, Monastir University,
Monastir 5000, Tunisia
Institute of Analytical Sciences, UMR CNRS-UCBL-ENS 5280, 5 Rue la Doua,
CEDEX 09, 69100 Villeurbanne, France
Center of Scientific Excellence for Influenza Viruses, National Research Centre, Dokki, 12622 Cairo, Egypt;
omniakutkat@gmail.com (O.K.); Shehata_mmm@hotmail.com (M.S.);
Mohamedahmedali2004@yahoo.com (M.A.A.)
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus University-Egypt,
34518 New Damietta, Egypt; ahmed.alkarmalawy2019@gmail.com
Laboratory of Heterocyclic Chemistry, Faculty of Science of Monastir, University of Monastir,
Natural Products and Reactivity (LR11ES39), Team: Medicinal Chemistry and Natural Products,
Avenue of Environment, Monastir 5019, Tunisia; karimchouaib1@hotmail.fr (K.C.);
hichem.bjannet@gmail.com (H.B.J.)
Polymer Materials Engineering, University of Lyon, UMR CNRS 5223, Lyon, 69100 Villeurbanne, France;
abdelwaheb.dhahri@gmail.com
Department of Biological Sciences, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia;
jagharbi@kfu.edu.sa
2
3
4
5
6
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
7
8
Citation: Soltane, R.; Chrouda, A.;
Mostafa, A.; Al-Karmalawy, A.A.;
Chouaïb, K.; dhahri, A.; Pashameah,
R.A.; Alasiri, A.; Kutkat, O.; Shehata,
M.; et al. Strong Inhibitory Activity
and Action Modes of Synthetic
Maslinic Acid Derivative on Highly
Pathogenic Coronaviruses:
9
10
COVID-19 Drug Candidate. Pathogens
2021, 10, 623. https://doi.org/
10.3390/pathogens10050623
*
Correspondence: amain.c@mu.edu.sa (A.C.); ahmed_elsayed@daad-alumni.de (A.M.)
Abstract: In late December 2019, a novel coronavirus, namely severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), escaped the animal–human interface and emerged as an ongoing global
pandemic with severe flu-like illness, commonly known as coronavirus disease 2019 (COVID-19).
In this study, a molecular docking study was carried out for seventeen (17) structural analogues
prepared from natural maslinic and oleanolic acids, screened against SARS-CoV-2 main protease. Fur-
thermore, we experimentally validated the virtual data by measuring the half-maximal cytotoxic and
inhibitory concentrations of each compound. Interestingly, the chlorinated isoxazole linked maslinic
acid (compound 17) showed promising antiviral activity at micromolar non-toxic concentrations.
Thoughtfully, we showed that compound 17 mainly impairs the viral replication of SARS-CoV-2.
Furthermore, a very promising SAR study for the examined compounds was concluded, which could
be used by medicinal chemists in the near future for the design and synthesis of potential anti-SARS-
CoV-2 candidates. Our results could be very promising for performing further additional in vitro
and in vivo studies on the tested compound (17) before further licensing for COVID-19 treatment.
Academic Editor: Alfonso Zecconi
Received: 23 April 2021
Accepted: 17 May 2021
Published: 19 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright:
© 2021 by the authors.
Keywords: maslinic acid; oleanolic acid; SARS-CoV-2; MERS-CoV; molecular docking; SAR
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Since its emergence, coronavirus disease 2019 (COVID-19) has triggered a scary and
contagious global pandemic of severe pneumonia-like disease, which causes respiratory
Pathogens 2021, 10, 623. https://doi.org/10.3390/pathogens10050623
https://www.mdpi.com/journal/pathogens

Pathogens 2021, 10, 623
2 of 22
illness and acute respiratory distress syndrome in human. Up to now, the race is still on to
discover specific and efficient anti-viral drugs against SARS-CoV-2 to control COVID-19
to minimize its awful spread and transmission [1]. To this point, researchers have tried to
make use of the existing anti-viral drugs that might combat the infection based on in silico
predictions, or based on previous knowledge of other coronaviruses such as SARS-CoV
and MERS-CoV. Therefore, a lot of FDA approved drugs have been tested or repositioned
to act as anti-SARS-CoV-2 [2,3]. In parallel, several trials to virtually recommend or
practically test the anti-SARS-CoV-2 activity of synthetic compounds, natural extracts and
immunomodulatory agents have been attempted [4–7].
Triterpenes are a class of natural molecules made up of 30 carbon atoms. Biosyntheti-
cally, the 200 different skeletons known to date and isolated from natural sources come from
the cyclization of (3S)-2,3-epoxy-2,3-dihydrosqualene and sometimes of squalene itself.
These molecules are in most cases hydroxylated at C-3 position due to the opening of the
epoxide during cyclization [8]. In the plant kingdom, terpenoids are secondary metabolites,
whose ecological role has been proven, especially in the process of communications and
defense. Triterpenes, of which more than 30,000 structures were previously isolated and
characterized, is derived from natural sources or enzymatic reactions. Additionally, they
comprise 47 sub-classes with enormous chemical diversity [8–11].
Triterpenes are among the natural substances with promising biological, therapeu-
tic and industrial value. In recent years, due to the broad spectrum of their biological
potentials, triterpenes have been the subject of an intensive number of research works,
mainly focused on their biopharmaceutical development. Indeed, triterpenes and their
structural analogues display very varied biological effects such as hemolytic, hypocholes-
terolimic, immunostimulant, anti-inflammatory, antimicrobial, antiparasitic, cytotoxic, and
anti-ulcer [12–18]. Moreover, triterpenes are known for their many important antiviral
activities such as anti-hepatitis B virus activity, anti-HIV1 and 2, AIDS, and (to remove)
anti-hepatitis C virus activity, and anti-Herpes simplex virus activity (Figure 1) [11,19].
Figure 1. Examples of bioactive triterpenes.
These compounds and their antiviral activity have seriously attracted the attention
of organic chemists, who have thought of chemically modifying them in different ways
in order to improve their activities. Some of these analogues are employed as medicinal
drugs to treat liver-related diseases [20].
Oleanolic acid (OA, 1) and maslinic acid (MA, 2) are triterpenoids isolated from several
plants; in particular, from Olea europaea L. that possesses a variety of interesting biological
activities. OA displays several biological potentials including antimicrobial, anticancer,
and anti-inflammatory effects [19
viruses such as HIV1 replication in acutely infected H9 lymphocyte cells [21
,
21
,
22]. It has been found to inhibit the entry of several
23]. In recent
–
decades, OA has undergone several chemical modifications in order to synthesize new
structural analogues with promising biological activities. These chemical modifications
mainly concerned the C-3 alcohol and C-17 carboxylic acid functions as well as the C12-C13
unsaturation. Various OA congeners have shown antibacterial, anti-acetylcholinesterase,

Pathogens 2021, 10, 623
3 of 22
anticancer, anti-inflammatory, and anti-influenza virus activities and were found to be
more active than OA [11,15,17,18,21,23–27].
Maslinic acid (MA) has shown a broad spectrum of biological activity. Indeed, it has
been found to be inhibit glycogen phosphorylases [28] and protein tyrosine phosphatase
1B [29]. It also exhibited anti-inflammatory [20], antitumor [30], and anti-HIV-1 activi-
ties [31]. Several other interesting biological activities were also reported [15,17,18]. This
triterpene acid has attracted the attention of several chemists, who have taken advantage of
its various chemical functions to synthesize new series of structural analogues in order to
improve some of their activities and also to understand the structure–activity relationship;
we cite the example of esters, 3,5-isoxazoles and 1,5- and 1,4-disubstituted triazoles [15,17].
Compounds bearing isoxazole moiety are of high importance due to their broad spec-
trum of biological properties including antiviral [32], antidiabetic [33], neuroprotective [34],
anticancer and anti-inflammatory activities (Figure 2) [17,35].
Figure 2. Previously reported bioactive isoxazoles.
1,2,3-Triazoles, constituting another important class of heterocycles, widely used in
medicinal chemistry, act as pharmacophores and linkers between two or more substances
of interest in molecular hybridization approaches [36]. Several of these triazoles have a
wide spectrum of biological effects such as anti-HIV [37], antiviral [38], cytotoxic, anti-
inflammatory [16,18], and antibacterial [39] activities (Figure 3).
Figure 3. Previously reported bioactive triazoles.
In our previous works, Chouaïb et al. have described the isolation of OA and MA
from pomace olive (Olea europaea L.) which have been used as a starting material to prepare
new series of congeners constituting a library of novel pentacyclic triterpenoid derivatives
bearing esters, 3,5-disubstituted isoxazoles, 1,5- and 1,4-disubstituted triazoles and were

Pathogens 2021, 10, 623
4 of 22
synthesized as antimicrobial, anti-inflammatory and anticancer agents [15
–18]. In order to
broaden the range of our biological tests on these derivatives taking into account the antivi-
ral effect of oleanolic and maslinic acids as well as of isoxazoles and triazoles, molecular
docking studies of OA, MA and most previously synthesized derivatives has been carried
out targeting SARS-CoV-2 main protease which allowed one to select seventeen com-
pounds to be evaluated for their antiviral activity against hCoV-19/Egypt/NRC-03/2020
(Accession Number on GSAID: EPI_ISL_430820) “NRC-03-nhCoV” virus [40].
Molecular docking is an important method of computational studies which are very
promising tools in the new process of drug discovery and development as well [39].
Concerning the aforementioned crucial rule of main protease (Mpro) for SARS-CoV-2
maturation, and in continuation to our previous work targeting SARS-CoV-2 [2,3,41–44],
we decided to target SARS-CoV-2 Mpro using molecular docking studies and we further
validated the antiviral activity of the 17 compounds representing OA, MA and their
derivatives. Moreover, we further evaluated the impact of promising anti-SARS-CoV-2
compounds on different stages of the viral replication cycle.
2. Results and Discussion
Respiratory viruses represent a major public health concern, as they are highly mu-
tated, resulting in the emergence of new strains with high pathogenicity. Currently, the
whole world is suffering from a new evolving severe acute respiratory syndrome coron-
avirus 2 (SARS-CoV-2), which originally spread from the Hubei region, concisely in Wuhan
City in early December 2019, and subsequently spread globally. By the end of January,
and due to the increasing rate of SARS-CoV-2-infected confirmed cases associated with
relatively high mortality rate, the WHO raised awareness and declared an emergency,
claiming a potentially pandemic lethal infectious disease named COVID-19.
Initially, SARS-CoV-2 was considered as a zoonotic disease, based on a high number
of infected people exposed to a wet animal market in the Wuhan Chinese city; however,
transmission from person to person was confirmed, making the situation even more harsh.
This condition resulted in many put quarantines in place, cancelling flights and suspending
travel, with the consequence of huge economic burden.
The perceived threat of this pandemic has led many researchers and academic insti-
tutions all over the world to dissect COVID-19 from different perspectives, including but
not limited to the route of virus transmission, diagnosis, virus inhibition treatments, and
vaccine preparation. Apparently, no treatment or vaccine until now has been discovered;
nevertheless, the reposition of the currently approved antiviral drugs are in use. Special
consideration for more susceptible people such as elderly people, patients with chronic
diseases, and health care takers should be applied. This study is considered as a serious
attempt to synthesize a unique series of oleanolic acid (
as anti-SARS-CoV-2 candidates.
1) and maslinic acid (2) derivatives
2.1. Chemistry
2.1.1. Green Chemistry Extraction of Compounds 1 and 2
In this study, the ultrasound-assisted extraction of triterpenoids
1 and 2 from po-
mace olive of Olea europaea L. was optimized compared to previous methods used. The
triterpenoid yield was 3.6 and 9.2 mg/g dry weight (DW), respectively, under optimal
conditions. As shown in Table 1, the present study is the first report on the simultaneous
extraction of triterpenoids
1 and 2 from pomace olive (Olea europaea L.) cultivar: Chemlali
with a large amount under green chemistry conditions (ultrasonic-assisted extractions, then
centrifugation, and purification), compared with those of previous works. This developed
process could be useful in the preparation of a triterpenoid-rich ingredient that holds great
promise for application in the therapeutic industry.

Pathogens 2021, 10, 623
5 of 22
Table 1. Methods and extraction yield of oleanolic acid (1) and maslinic acid (2) from different pomace olive cultivars.
Yield of Extraction (mg/g DW)
Cultivar
Extraction Methods
References
Oleanolic Acid 1 Maslinic Acid 2
Picual
Hojiblanca
Arbequina
Non-indicated
Manzanilla
Hojiblanca
Cacereña
Kalamata
Picual
Kalamon
0.500
0.500
0.400
0.015
0.274
0.565
0.185
0.841
1.003
0.838
3.400
1.200
1.300
1.500
0.034
0.824
0.904
0.295
1.318
2.440
2.100
8.500
[41]
[42]
Solid–liquid extraction (maceration)
Solid–liquid extraction (centrifugation)
Ultrasonic-assisted extraction
[43]
[44]
[15]
Chemlali
Solid–liquid, then ultrasonic-assisted extractions
Ultrasonic-assisted extractions, then
centrifugation isolation
Chemlali
3.6
9.2
This work
2.1.2. Synthesis
To prepare the propargylated ester
3, derived from oleanolic acid
1, we started by the
protection of the hydroxyl group at C-3 via its oxidation in order to direct the propargy-
lation towards the carboxylic acid function in C-17 (Scheme 1). Then, we subjected the
oxidized intermediate to the propargylation in dry N,N-Dimethylformamide (DMF) at
room temperature in the presence of NaH and propargyl bromide for 2 h, yielding the
desired propargylated intermediate which was reduced with sodium borohydride under
microwave irradiation, to obtain the propargyl-(3β)-3-hydroxyolean-12-en-28-oate (3).
◦
Scheme 1. Propargylation of oleanolic acid (
1
). Reagents and conditions: (a) Jones oxidation (CrO3, H2SO4/acetone), 0 C
(b) propargyl bromide, NaH, dry DMF, rt (c) NaBH4, CH3OH/THF, Microwave (250 W, 3 min).
Similarly, maslinic acid 2 was subjected to the same reaction of propargylation, with-
out oxidation beforehand, yielding a mixture of differently propargylated compounds
which were separated over silica gel column chromatography to afford compounds 4–7
(Table 2) [16,17].

Pathogens 2021, 10, 623
6 of 22
Table 2. Oleanolic acid (
1), maslinic acid (2) and their derivatives 3–17 screened for their anti- SARS-CoV-2 activity in
addition to N3 (the co-crystallized native inhibitor of SARS-CoV-2).
Code
Nomenclature
Chemical Structures
Yield (%)
1
2
3
Oleanolic acid
-
Maslinic acid
-
Propargyl-(3β)-3-
hydroxyolean-12-en-28-oate
99
Propargyl-(2α,3β)-2,3-
dihydroxyolean-12-en-28-
oate
4
5
25
23
Propargyl-(2α)-2-
(propargyloxy)-(3β)-3-
hydroxy-olean-12-en-28-oate
Propargyl-(3α)-3-
(propargyloxy)-(2β)-2-
hydroxy-olean-12-en-28-oate
6
7
20
31
Propargyl-(2α,3β)-2,3-
bis(propargyloxy)-olean-12-
en-28-oate

Pathogens 2021, 10, 623
7 of 22
Table 2. Cont.
Chemical Structures
Code
Nomenclature
Yield (%)
(1-(3-methylphenyl)-1H-1,2,3-
triazol-5-yl)methyl-(3β)-3-
hydroxyolean-12-en-28-oate
8
90
(1-(3-methylphenyl)-1H-1,2,3-
triazol-5-yl)methyl-(2α,3β)-
2,3-dihydroxyolean-12-en-28-
oate
9
92
98
(1-(3-methylphenyl)-1H-1,2,3-
triazol-4-yl)methyl-(3β)-3-
hydroxyolean-12-en-28-oate
10
(1-Phenyl-1H-1,2,3-triazol-4-
yl)methyl-(2α,3β)-2,3-
dihydroxyolean-12-en-28-
oate
11
12
13
96
94
96
(1-(4-Chlorophenyl)-1H-1,2,3-
triazol-4-yl)methyl-(2α,3β)-2-
((1-(4-chlorophenyl)-1H-1,2,3-
triazol-4-yl)methoxy)-3-
hydroxyolean-12-en-28-oate
(1-(4-Methoxyphenyl)-1H-
1,2,3-triazol-4-yl)methyl-
(2α,3β)-2-hydroxy-3-((1-(4-
methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)-olean-
12-en-28-oate
(1-(4-Methoxyphenyl)-1H-
1,2,3-triazol-4-yl)methyl-
(2α,3β)-2,3-bis((1-(4-
methoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)-olean-
12-en-28-oate
14
92

Pathogens 2021, 10, 623
8 of 22
Table 2. Cont.
Chemical Structures
Code
Nomenclature
Yield (%)
(3-(4-methoxyphenyl)
isoxazol-5-yl) methyl-(3β)-3-
hydroxyolean-12-en-28-oate
15
98
(3-(4-methoxyphenyl)
isoxazol-5-yl) methyl-(2α,3β)-
2,3-dihydroxyolean-12-en-28-
oate
16
17
87
96
(3-(4-chlorophenyl)
isoxazol-5-yl) methyl-(2α,3β)-
2,3-dihydroxyolean-12-en-28-
oate
N3 (co-crystallized native
inhibitor of SARS-CoV-2)
18
-
A regiospecific, facile and practical multicomponent synthesis of 1,5-triazolyl deriva-
tives ( and ) by Ru(II)-catalyzed, and mono-, bis- and tri-1,4-triazolyl derivatives (10–14
8
9
)
by Cu(I)-catalyzed was carried out (Scheme 2).
Scheme 2. Synthesis of the 1,4- and 1,5-triazolyl (8–14) derivatives.

Pathogens 2021, 10, 623
9 of 22
According to the same method as mentioned above, the dipolarophiles
3
and
4
were
found to be readily cyclized to the 3,5-disubstituted isoxazoles (15–17) (Scheme 3). In fact,
copper-catalyzed microwave-assisted multicomponent 1,3-dipolar cycloaddition between
pentacyclic triterpenoid alkyne derivatives
3 and 4, and the appropriate aldehyde, re-
giospecifically afforded 3,5-disubstituted isoxazoles (15–17) in quantitative yields (Table 2).
Scheme 3. Synthesis of the 3,5-disubstituted isoxazol (15–17).
All the reactions were assisted by microwave irradiation avoiding toxic reagents and
solvents. The products were obtained from the reaction mixture by simple purification in
almost quantitative yields (Table 2).
2.2. Anti-SARS-CoV-2 Activity
2.2.1. Molecular Docking Studies
By studying the binding pocket of the SARS-CoV-2Mpro, it was found that the co-
crystallized native inhibitor (N3) fitted inside its binding pocket of the main protease
receptor asymmetrically. N3 is a designed inhibitor for SARS-CoV-2 Mpro that was built
from amino acids based on the Mpro pocket amino acids and cannot used medicinally
(Table 2).
Molecular docking of oleanolic acid (1), maslinic acid (2), and other oleanolic acid
and maslinic acid derivatives (3–17) (Table 2), and N3 inhibitor (18) into the main protease
binding site was performed. Almost all the tested compounds (1–17) were stabilized inside
the Mpro binding site by variable scores and binding interactions with the amino acids of the
receptor pocket. It is worth mentioning that, especially for both compounds 17 and
3, the
obtained binding scores and modes were greatly similar to that of the docked N3 inhibitor
(
(
18), as depicted in Table 3. Additionally, the 2 D binding modes of the tested compounds
1–17) with the amino acids of the SARS-CoV-2 Mpro pocket are presented in Supplementary
Table S2. All of the tested compounds (1–17) achieved promising binding scores (from
6.55 to 10.20 kcal/mol), compared to the docked co-crystallized N3 inhibitor (18) with
a binding score of 9.70 kcal/mol. The RMSD_refine values of the selected poses were
within the acceptable range (from 0.9 to 2.25).
−
−
−

Pathogens 2021, 10, 623
10 of 22
Table 3. 3D representations showing the binding interactions and positioning between the two promising tested compounds
(17 and 3) and the N3-binding pocket in comparison to the docked N3 inhibitor (18).
Code
3D Binding Interactions
3D Pocket Positioning
17
3
N3 (18)
The docked N3 inhibitor (18) was stabilized inside the SARS-CoV-2 Mpro pocket
through a hydrogen bond formation with Thr26 amino acid at 3.08 A◦. Additionally, the
most promising cytotoxic compound (17) fitted inside the binding site of the SARS-CoV-2
Mpro by three hydrogen bond formations, two of them with ◦Thr24 at 2.71 and 3.19 A◦,
respectively, and the third one was formed with Thr26 at 3.28 A . Moreover, compound (3)
formed three hydrogen bonds with the previously mentioned two amino acids as well. It
formed two hydrogen bonds with Thr24 at 2.80 and 3.18 A◦, respectively, and the third

Pathogens 2021, 10, 623
11 of 22
one with Thr26 at 3.37 A◦. Additionally, it is worth mentioning that the achieved binding
scores for compounds 17 and
3
were
−
8.00 and
−
7.55 kcal/mol, respectively, compared to
the docked co-crystallized N3 inhibitor (18) (−9.70 kcal/mol) (Table 3).
By considering the docking findings of the selected tested compounds (1–17) against
the binding pocket of the SARS-CoV-2 main protease compared to its co-crystallized
inhibitor (N3) as a reference standard, and its great matching with the cytotoxicity studies
applied, we can build a very promising and recommended idea about their mechanism
of action.
2.2.2. Compound 17 Showed Promising Anti-SARS-CoV-2 Activity at
Non-Toxic Concentrations
To identify the proper concentrations to define the antiviral activity of the selected
compounds, half maximal cytotoxic concentration “CC50” was calculated for each indi-
vidual drug (Figure 4). The antiviral screening revealed that one compound “17” out of
the tested panel compounds exhibits a promising in vitro activity against SARS-CoV-2
“NRC-03-nhCoV” (IC50 = 4.12
and Table 4).
µ
M) with a satisfactory selectivity index ( 7.5) (Figure 4
≥
Table 4. Selectivity index of tested compounds 1–17.
Compound
CC50
189.9
97.30
97.40
157.2
90.62
208.8
97.42
170
IC50
476.1
99.87
42.01
236.3
85.21
135
Selectivity Index (CC50/IC50)
1
2
0.39
0.97
2.32
0.67
1.06
1.55
0.90
0.98
1.40
3.68
1.62
4.42
2.24
2.96
0.79
1.48
34.36
3
4
5
6
7
108
8
173.9
131.9
75.31
134.7
116.1
158.9
136.7
523
9
185.3
277.2
218.1
513.8
356.4
405.4
415.3
324.2
141.6
10
11
12
13
14
15
16
17
218.6
4.12

Pathogens 2021, 10, 623
12 of 22
(a)
Figure 4. Cont.

Pathogens 2021, 10, 623
13 of 22
(b)
Figure 4. Anti-SARS-CoV-2 activities of tested compounds (1–17). (
a) Half-maximal cytotoxic concentrations (CC50) on Vero
E6 cells, and ( ) half-maximal inhibitory concentrations (IC50) against NRC-03-nhCoV in Vero E6. Inhibitory concentration
b
50% (IC50) values were calculated using nonlinear regression analysis of GraphPad Prism software (version 5.01) by plotting
log inhibitor versus normalized response (variable slope).

Pathogens 2021, 10, 623
14 of 22
2.2.3. Compound 17 Showed Promising Anti-MERS-CoV Activity and
Non-Toxic Concentrations
To assess the antiviral activity of compound 17 against other highly pathogenic coron-
aviruses, we assayed its activity against MERS-CoV virus “NRCE-HKU270”. Interestingly,
compound 17 showed promising antiviral activity against NRCE-HKU270 at micromolar
concentrations (Figure 5).
Figure 5. Half-maximal inhibitory concentrations (IC50) against Middle East respiratory syndrome-
related coronavirus isolate NRCE-HKU270 (accession number: KJ477103.2; NRCE-HKU270 is ob-
tained from CSEIVs) [45] in Vero E6 cells using crystal violet assay. Inhibitory concentration 50% (IC50
)
values were calculated using nonlinear regression analysis of GraphPad Prism software (version
5.01) by plotting log inhibitor versus normalized response (variable slope).
2.2.4. Structure–Activity Relationship (SAR) Study
By studying the relations between the structures of the selected tested compounds
(1–17) and their cytotoxic IC50 values (Figure 6) and using the molecular docking results
as a way to relate their activities to their binding affinities as SARS-CoV-2 main protease
inhibitors, we may conclude that;
(a) The common shared steroidal nucleus of the selected tested compounds (1–17) ap-
peared to be very promising for their antiviral activities. Molecular docking revealed
the great stabilization of the tested compounds inside the branched large pocket of
SARS-CoV-2 main protease as a proposed mechanism of action.
(b) The presence of OH groups at positions 2 and 3 of the steroidal nucleus seems to be
very crucial for the antiviral activity. Most compounds (17
,
3, and 10) containing both
2- and 3-OH groups achieved high to moderate activities (4.12–99.87
µM). Docking
studies referred to the involvement of both OH groups in hydrogen bond formations
with Thr24 and/or Thr26 amino acids of the binding pocket.
(c) Compound (17) containing the isoxazole side chain with a p-chlorophenyl substitution
(an electron withdrawing lipophilic group) achieved a very high cytotoxic activity
(IC50 = 4.12
side chain with a p-methoxyphenyl substitution (an electron donating hydrophilic
group) achieved low cytotoxic activities (IC50 = 218.8 and 523 M, respectively).
µM). However, both compounds (16 and 15) containing the isoxazole
µ
Therefore, the lipophilic p-chloro group is very important for the antiviral activity.
Again, compound (16) containing both 2- and 3-OH groups showed a higher cytotoxic
activity compared to compound (15) with only 3-OH group as mentioned before.

Pathogens 2021, 10, 623
15 of 22
(d) The presence of the acetyl methylene group at positions 2 and/or 3 in compounds
(5, 7, and 6) gave better cytotoxic activities compared to the heteroaromatic ring
substitutions at the same positions in compounds (12, 14, and 13).
(e) The cytotoxic activity of compound (17) containing the isoxazole side chain with a
p-chlorophenyl substitution (IC50 = 4.12
µM) was clearly higher than the respective
same compound (10) with a phenyl triazole side chain instead (IC50 = 75.31 µM).
Figure 6. Structure–activity relationship (SAR) of the selected tested compounds (1–17) based on both their cytotoxic and
docking studies against SARS-CoV-2.
Collectively, these studies greatly suggested the very promising affinities of the se-
lected tested compounds (1–17) against SARS-CoV-2, especially compounds 17 and 3.
Accordingly, we recommend such compounds for more advanced in vitro and in vivo
studies to obtain an effective therapeutic against the pandemic SARS-CoV-2. Moreover,
the aforementioned studied compounds could be used alone or in combinations with each
other’s against SARS-CoV-2.
2.2.5. Mechanism of Actions
The virucidal effect of the antiviral compound 17 against SARS-CoV-2 virus has
been studied, and interference with viral adsorption and replication was demonstrated.
Besides the virucidal effect of compound 17, it could efficiently impair viral replication and
adsorption (Figure 7).

Pathogens 2021, 10, 623
16 of 22
Figure 7. Mode of antiviral action of compound 17 against SARS-CoV-2 in Vero E6 cells as measured
by plaque reduction assay. Inhibitory percent values were calculated and plotted against inhibitor
concentration using GraphPad Prism software (version 5.01).
3. Materials and Methods
3.1. Chemistry
3.1.1. Green Chemistry Condition for Extraction and Purification of OA (1) and MA (2)
from the Pomace Olive of Olea europaea L.
Using cultivar Chemlali pomace olive resulting from olive fruit pressing as a natural
substance, the most suitable conditions to carry out the extraction and purification of
compounds
1
and
2
were explored. The optimal conditions were determined to be the
◦
ultrasonic power of 250 W, extraction temperature of 60 C, ratio of liquid to solid of
30 mL/g, and 60 min extraction time, using aqueous ethanol (concentration of 70%) as
the extracting solvent. After the evaporation of the ethanol under reduced pressure, the
extract was precipitated at +4 ◦C for 48 h. After filtration, the extract was centrifuged at
5000 rpm/5 min, yielding a mixture of compounds
(13.5% (w/w)).The crude mixture was recrystallized from the mixture EtOH/AcOEt (8:2,
v/v) to obtain pure maslinic acid (9.2 mg/g DW). The filtrate was concentrated and
1 and 2 according to TLC analysis
2
recrystallized from ethanol to obtain pure oleanolic acid 1 (3.6 mg/g DW).
3.1.2. Synthesis
Multicomponent Synthesis of 1,5-Disubstituted Triazoles in Water Catalyzed by
Cp*RuCl (PPh3)2
To a solution of alkyne 3 or 4 (0.1 g) and NaN3 (1.1 mmol) in water, Cp*RuCl(PPh3)2
(5 mol%) was added at room temperature. To this mixture, alkyl halide (1.0 mmol) was
added, and the reaction mixture was subjected to microwave irradiations at 250 W until
total conversion of the starting alkyne (TLC). The crude mixture was extracted with EtOAc
(3
×
30 mL). The organic layer was dried over Na2SO4. After the removal of solvent under
reduced pressure, the resulting residue was purified by recrystallization from ethanol, to
afford the desired products 8 and 9 in 90% and 92% yields, respectively [46,47].
Cu(I))-Cuatalyzed Multicomponent Huisgen 1,3-Dipolar Cycloaddition Reaction
To a suspension of the corresponding terminal alkyne (100 mg) and NaN3 (1.1 mmol)
in water (3 mL), the corresponding alkyl halide (1.0 mmol) was added, followed by the
addition of the CuI catalyst (0.1 equiv) (Scheme 1). The reaction mixture was subjected to
microwave irradiations at 250 W, which was completed within 4–6 min (TLC). The crude
mixture was extracted with EtOAc (3
over sodium sulfate, concentrated under reduced pressure and purified through recrystal-
×
30 mL) and the combined organic layer was dried
lization from ethanol, to give pure 1,4-triazolyl derivatives 10–14 in 90–98% yields [48].
Multicomponent Synthesis of 3,5-Disubstituted Isoxazoles in Water Catalyzed by CuI
At room temperature and stirring, to a mixture of alkyl derivatives
3 or 4 (0.1 g),
NH2OH.HCl (1.2 equiv) and 0.1 equiv copper(I) iodide (CuI) in water, the appropriate

Pathogens 2021, 10, 623
17 of 22
aldehyde (1.1 equiv) was added, and the mixture was subjected to microwave irradiations
at 250 W for 5–10 min (Scheme 2). The crude mixture was diluted with water and then
extracted with EtOAc (3
×
30 mL). The organic layer was dried over Na2SO4. After the
removal of solvent in vacuo, the resulting residue was purified by recrystallization from
ethanol, to obtain the desired products 15–17 in 98, 87 and 96% yield, respectively [49].
The spectral data of all the synthesized compounds are in agreement with the literature
data [16–18,50].
3.2. Biological
3.2.1. Molecular Docking Studies
Molecular docking studies using MOE 2019.012 suite [51] were performed to propose
the expected mechanism of action for the 17 tested compounds as SARS-CoV-2 Mpro
inhibitors through evaluating their binding scores and interaction modes at the SARS-CoV-
2 Mpro receptor active site. The co-crystallized native inhibitor (N3) was extracted from the
protein complex and inserted in our calculations as a reference standard.
3.2.2. Preparation of the Selected Tested Compounds
Each member of the previously mentioned selected compounds (1–17) was sketched
using the ChemDraw program, transferred to MOE, and then subjected to the preparation
process for the docking step as described earlier [52]. Then, all the prepared compounds
(1–17) were imported in one database together with the co-crystallized native inhibitor (N3,
18) and saved as an MDB file to be used during the docking procedure.
3.2.3. Preparation of the Active Site of the Applied SARS-CoV-2 Mpro Receptor
The Protein Data Bank website was used to extract the X-ray structure of the SARS-
CoV-2 Mpro receptor (PDB ID: 6LU7) [53]. Then, all default steps for its preparation were
run as described earlier in detail [54,55].
3.2.4. Validation of the Applied MOE Program
Before applying the docking process, we first performed a validation process for
the prepared target SARS-CoV-2 Mpro receptor by redocking its co-crystallized native
inhibitor (N3) inside its active site and comparing the resulted RMSD values between the
co-crystallized (native) and docked N3 molecules. The obtained low RMSD value (1.20)
indicated a promised valid performance (Supplementary Table S1) [56].
3.2.5. Docking of the Prepared Database to the Binding Pocket of SARS-CoV-2
Mpro Receptor
The previously discussed database was selected and inserted at the site of ligand at
the beginning of the general docking process and the default methodology was performed
as described before [57]. Finally, we selected the best poses according to their binding
scores, RMSD values, and binding modes for further considerations.
3.2.6. In Vitro Antiviral Bioassay
Cytotoxicity (CC50) Determination
To assess the half maximal cytotoxic concentration (CC50), stock solutions of the test
compounds were prepared in 10% DMSO in ddH2O and diluted further to the working
solutions with DMEM. The cytotoxic activity of the extracts was tested in VERO-E6 cells by
using crystal violet assay as previously described [58
,59] with minor modifications. Briefly,
the cells were seeded in 96-well plates (100 L/well at a density of 3
µ
×
105 cells/mL)
and incubated for 24 h at 37 ◦C in a humidified 5% CO2 incubator. After 24 h, cells were
treated with various concentrations of the tested compounds in triplicates. Seventy two
hours later, the supernatant was discarded, and cell monolayers were fixed with 10%
formaldehyde for 1 h at room temperature (RT). The fixed monolayers were then dried and
stained with 50 µL of 0.1% crystal violet for 20 min on a bench rocker at RT. The monolayers

Pathogens 2021, 10, 623
18 of 22
were then washed, dried and the crystal violet dye in each well was then dissolved with
200
µL of methanol for 20 min on a bench rocker at RT. The absorbance of crystal violet
solutions was measured at
λmax 570 nm as a reference wavelength using a multi-well
plate reader. Cytotoxic concentration 50% (CC50) values were calculated using nonlinear
regression analysis of GraphPad Prism software (version 5.01) by plotting log inhibitor
versus normalized response (variable slope).
Inhibitory Concentration 50 (IC50) Determination
The IC50 values for the tested compounds were determined as previously described [2,3]
,
with minor modifications. Briefly, in 96-well tissue culture plates, 2.4
were distributed in each well and incubated overnight at a humidified 37◦C incubator
under 5% CO2 condition. The cell monolayers were then washed once with 1 PBS. An
×
104 Vero-E6 cells
×
aliquot of the SARS-CoV-2 “NRC-03-nhCoV” virus [40] containing 100 TCID50 was incu-
bated with serial diluted compounds and kept at 37 ◦C for 1 h. The Vero-E6 cells were
treated with virus/compound mix and co-incubated at 37 ◦C in a total volume of 200
µL
per well. Untreated cells infected with virus represented the virus control; however, cells
that were not treated and not infected were the cell control. Following incubation at 37 ◦C
in 5% CO2 incubator for 72 h, the cells were fixed with 100
for 20 min and stained with 0.5% crystal violet in distilled water for 15 min at RT. The
crystal violet dye was then dissolved using 100 L of absolute methanol per well and the
µL of 10% paraformaldehyde
µ
optical density of the color was measured at 570 nm using the Anthos Zenyth 200 rt plate
reader (Anthos Labtec Instruments, Heerhugowaard, the Netherlands). The IC50 of the
compound was that required to reduce the virus-induced cytopathic effect (CPE) by 50%,
relative to the virus control.
Mechanism of Action(s) for Compound 17
To investigate whether the tested compound with high selectivity index and an-
tiviral activity against NRC-03-nhCoV affect (a) viral adsorption, (b) viral replication or
(c) viricidal effect, a plaque infectivity reduction assay was performed according to the
following tprotocols:
(a) Viral adsorption mechanism
The viral adsorption mechanism was assayed according to a protocol by Zhang et al. [60
]
with minor modifications. Vero-E6 cells were cultivated in a 6-well plate (105 cells/mL) for
24 h at 37 ◦C. Each tested drug was appl◦ied in 500
µL of medium without supplements and
co-incubated with the cells for 2 h at 4 C. The non-absorbed drug in the applied inocula
was removed by washing cells three times with 1x PBS. The NRC-03-nhCoV was diluted to a
countable range “10−4” and co-incubated with the pretreated cells. About 1 h post-incubation,
the inocula were removed and 3 mL of agarose overlay (1:1 mixture of warmed 2
×
plaque
media and a stock solution of heated 2% agarose) were added. The plates were kept at room
◦
temperature to solidify and then incubated at 37 C to allow the formation of viral plaques. The
plaques were fixed using 10% formaldehyde, stained using crystal violet (1% crystal violet in
20% ethanol and distilled H2O), washed with rinse water and dried to calculate the percentage
reduction in plaque formation compared to control wells, which comprised untreated Vero-E6
cells directly infected with NRC-03-nhCoV.
(b) Viral replication mechanism
The impact of the tested drug on viral replication was determined as previously
described [50]. Vero-E6 cells were cultivated in a 6-well plate (105 cell/mL) for 24 h at
◦
37 C. The NRC-03-nhCoV o◦f the countable range was inoculated directly to the cells
and incubated for 1 h at 37 C. The inocula containing the non-adsorbed virus were
removed by washing infected cell monolayers three times with 1
×
PBS. The analyzed
compound was then applied in varying concentrations to infected cells for another 1 h
◦
contact time at 37 C. After removing the inocula containing the tested drug, agarose
overlay were added to the cells. The plates were kept at room temperature to solidify and

Pathogens 2021, 10, 623
19 of 22
then incubated at 37 ◦C to allow the formation of viral plaques. The plaques were fixed
using 10% formaldehyde, stained using 1% crystal violet washed with rinse water and
dried to calculate the percentage reduction in plaque formation compared to control wells,
which comprised untreated Vero-E6 cells directly infected with NRC-03-nhCoV.
(c) Virucidal mechanism
The virucidal mechanism was assayed following a previously described protocol [61].
Briefly, Vero-E6 cells (105 cells/mL) were cultivated for 24 h at 37 ◦C in a 6-well plate. In
an eppendorf, 200 µL of stock NRC-03-nhCoV were mixed with the tested compound at
different concentrations. The mixture was kept for 1 h at room temperature against the
control untreated virus. After 1 h of incubation, the mixture was ten-fold serial diluted
four times using serum-free medium to obtain a countable range of the virus. An aliquot
of 100 µL of each dilution were added to the Vero-E6 cell monolayer. After 1 h contact
time, the agarose overlay was added to the cell monolayer. The plates were kept at room
◦
temperature to solidify and then incubated at 37 C to allow the formation of viral plaques.
The plaques were fixed using 10% formaldehyde, stained using 1% crystal violet washed
with rinse water and dried to calculate the percentage reduction in plaque formation
compared to control wells, which comprised untreated Vero-E6 cells directly infected with
NRC-03-nhCoV.
4. Conclusions
Oleanolic acid (1) and maslinic acid (2), isolated from pomace olive oil, and a series of
their synthesized structural analogues (3–17) were first evaluated using molecular docking
for their proposed expected inhibitory activities against SARS-CoV-2 main protease, and
then were screened for the same activity. Most of the tested compounds showed promising
affinities towards SARS-CoV-2 main protease, especially for the most active member (17).
Accordingly, we recommend such compounds for more advanced in vitro and in vivo
studies to obtain an effective therapeutic against the pandemic SARS-CoV-2. Moreover,
the aforementioned studied compounds could be used alone or in combinations with each
other against SARS-CoV-2.
5. Patents
This work presented in this manuscript is submitted as a patent application to Saudi
Authority for Intellectual Property under number 121420640.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/pathogens10050623/s1.
Author Contributions: Conceptualization, R.S., A.M., K.C., H.B.J., and M.A.A.; methodology, A.C.,
A.M., A.A.A.-K., K.C., A.A., A.d., O.K., R.A.P., and M.S.; formal analysis, R.S., A.M., J.G., H.B.J.,
and M.A.A.; investigation, A.M., A.C., R.S., M.S., and J.G., H.B.J., and M.A.A.; resources, R.S., A.M.,
H.B.J., and M.A.A.; data curation, R.S. and A.M.; writing—original draft preparation, A.M., K.C., J.G.,
H.B.J., and M.A.A.; writing—review and editing, A.M., K.C., J.G., H.B.J., and M.A.A.; supervision,
A.M., H.B.J., and M.A.A.; funding acquisition, R.S., H.B.J., A.M. and M.A.A. All authors have read
and agreed to the published version of the manuscript.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors gratefully acknowledge the Deanship of Scientific Research at Umm
Al-Qura University for supporting this work by Grant Code “20-SCI-4-13-0002”.
Conflicts of Interest: The authors declare no conflict of interest.

Pathogens 2021, 10, 623
20 of 22
References
1.
Kumar, S.; Singh, R.; Kumari, N.; Karmakar, S.; Behera, M.; Siddiqui, A.J.; Rajput, V.D.; Minkina, T.; Bauddh, K.; Kumar, N.
Current understanding of the influence of environmental factors on sars-cov-2 transmission, persistence, and infectivity. Environ.
Sci. Pollut. Res. Int. 2021, 28, 6267–6288. [CrossRef] [PubMed]
2.
Mostafa, A.; Kandeil, A.; Elshaier, Y.A.M.M.; Kutkat, O.; Moatasim, Y.; Rashad, A.A.; Shehata, M.; Gomaa, M.R.; Mahrous, N.;
Mahmoud, S.H.; et al. Fda-approved drugs with potent in vitro antiviral activity against severe acute respiratory syndrome
coronavirus 2. Pharmaceuticals 2020, 13, 443. [CrossRef] [PubMed]
3.
4.
5.
Alnajjar, R.; Mostafa, A.; Kandeil, A.; Al-Karmalawy, A.A. 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, e05641. [CrossRef]
Mahmoud, D.B.; Shitu, Z.; Mostafa, A. Drug repurposing of nitazoxanide: Can it be an effective therapy for covid-19? J. Genet.
Eng. Biotechnol. 2020, 18, 35. [CrossRef]
Surti, M.; Patel, M.; Adnan, M.; Moin, A.; Ashraf, S.A.; Siddiqui, A.J.; Snoussi, M.; Deshpande, S.; Reddy, M.N. Ilimaquinone
(marine sponge metabolite) as a novel inhibitor of sars-cov-2 key target proteins in comparison with suggested covid-19 drugs:
Designing, docking and molecular dynamics simulation study. RSC Adv. 2020, 10, 37707–37720. [CrossRef]
Alzahrani, F.A.; Saadeldin, I.M.; Ahmad, A.; Kumar, D.; Azhar, E.I.; Siddiqui, A.J.; Kurdi, B.; Sajini, A.; Alrefaei, A.F.; Jahan, S.
The potential use of mesenchymal stem cells and their derived exosomes as immunomodulatory agents for covid-19 patients.
Stem Cells Int. 2020, 2020, 8835986. [CrossRef] [PubMed]
6.
7.
Fayed, M.A.A.; El-Behairy, M.F.; Abdallah, I.A.; Abdel-Bar, H.M.; Elimam, H.; Mostafa, A.; Moatasim, Y.; Abouzid, K.A.M.;
Elshaier, Y.A.M.M. Structure- and ligand-based in silico studies towards the repurposing of marine bioactive compounds to target
sars-cov-2. Arab. J. Chem. 2021, 14, 103092. [CrossRef]
8.
9.
Xu, R.; Fazio, G.C.; Matsuda, S.P.T. On the origins of triterpenoid skeletal diversity. Phytochemistry 2004, 65, 261–291. [CrossRef]
[PubMed]
Bruneton, J. Pharmacognosie, Phytochimie, Plantes Médicinales; Technique et Documentation Lavoisier: Paris, France, 1999.
10. Muffler, K.; Leipold, D.; Scheller, M.-C.; Haas, C.; Steingroewer, J.; Bley, T.; Neuhaus, H.E.; Mirata, M.A.; Schrader, J.; Ulber, R.
Biotransformation of triterpenes. Process Biochem. 2011, 46, 1–15. [CrossRef]
11. Hisham Shady, N.; Youssif, K.A.; Sayed, A.M.; Belbahri, L.; Oszako, T.; Hassan, H.M.; Abdelmohsen, U.R. Sterols and triterpenes:
Antiviral potential supported by in-silico analysis. Plants 2021, 10, 41. [CrossRef]
12. Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.; Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Pharmacological
activities of natural triterpenoids and their therapeutic implications. Nat. Prod. Rep. 2006, 23, 394–411. [CrossRef] [PubMed]
13. Sparg, S.G.; Light, M.E.; van Staden, J. Biological activities and distribution of plant saponins. J. Ethnopharmacol. 2004, 94, 219–243.
[CrossRef] [PubMed]
14. Cheng, K.; Liu, J.; Liu, X.; Li, H.; Sun, H.; Xie, J. Synthesis of glucoconjugates of oleanolic acid as inhibitors of glycogen
phosphorylase. Carbohydr. Res. 2009, 344, 841–850. [CrossRef] [PubMed]
15. Chouaïb, K.; Hichri, F.; Nguir, A.; Daami-Remadi, M.; Elie, N.; Touboul, D.; Ben Jannet, H.; Hamza, M.A. Semi-synthesis of new
antimicrobial esters from the natural oleanolic and maslinic acids. Food Chem. 2015, 183, 8–17. [CrossRef] [PubMed]
16. Chouaïb, K.; Delemasure, S.; Dutartre, P.; Jannet, H.B. Microwave-assisted synthesis, anti-inflammatory and anti-proliferative
activities of new maslinic acid derivatives bearing 1,5- and 1,4-disubstituted triazoles. J. Enzym. Inhib. Med. Chem. 2016, 31,
130–147. [CrossRef] [PubMed]
17. Chouaïb, K.; Romdhane, A.; Delemasure, S.; Dutartre, P.; Elie, N.; Touboul, D.; Ben jannet, H.; Ali Hamza, M.h. Regiospecific
synthesis, anti-inflammatory and anticancer evaluation of novel 3,5-disubstituted isoxazoles from the natural maslinic and
oleanolic acids. Ind. Crop. Prod. 2016, 85, 287–299. [CrossRef]
18. Chouaïb, K.; Romdhane, A.; Delemasure, S.; Dutartre, P.; Elie, N.; Touboul, D.; Ben Jannet, H. Regiospecific synthesis by copper-
and ruthenium-catalyzed azide–alkyne 1,3-dipolar cycloaddition, anticancer and anti-inflammatory activities of oleanolic acid
triazole derivatives. Arab. J. Chem. 2019, 12, 3732–3742. [CrossRef]
19. Chen, P.; Zeng, H.; Wang, Y.; Fan, X.; Xu, C.; Deng, R.; Zhou, X.; Bi, H.; Huang, M. Low dose of oleanolic acid protects against
lithocholic acid-induced cholestasis in mice: Potential involvement of nuclear factor-e2-related factor 2-mediated upregulation of
multidrug resistance-associated proteins. Drug Metab. Dispos. Biol. Fate Chem. 2014, 42, 844–852. [CrossRef]
20. Sheng, H.; Sun, H. Synthesis, biology and clinical significance of pentacyclic triterpenes: A multi-target approach to prevention
and treatment of metabolic and vascular diseases. Nat. Prod. Rep. 2011, 28, 543–593. [CrossRef]
21. Chen, D.-F.; Zhang, S.-X.; Wang, H.-K.; Zhang, S.-Y.; Sun, Q.-Z.; Cosentino, L.M.; Lee, K.-H. Novel anti-hiv lancilactone c and
related triterpenes from kadsura lancilimba. J. Nat. Prod. 1999, 62, 94–97. [CrossRef]
22. Kashiwada, Y.; Wang, H.K.; Nagao, T.; Kitanaka, S.; Yasuda, I.; Fujioka, T.; Yamagishi, T.; Cosentino, L.M.; Kozuka, M.; Okabe, H.;
et al. Anti-aids agents. 30. Anti-hiv activity of oleanolic acid, pomolic acid, and structurally related triterpenoids. J. Nat. Prod.
1998, 61, 1090–1095. [CrossRef]
23. Zhu, Y.M.; Shen, J.K.; Wang, H.K.; Cosentino, L.M.; Lee, K.H. Synthesis and anti-hiv activity of oleanolic acid derivatives. Bioorg.
Med. Chem. Lett. 2001, 11, 3115–3118. [CrossRef]
24. Shanmugam, M.K.; Dai, X.; Kumar, A.P.; Tan, B.K.; Sethi, G.; Bishayee, A. Oleanolic acid and its synthetic derivatives for the
prevention and therapy of cancer: Preclinical and clinical evidence. Cancer Lett. 2014, 346, 206–216. [CrossRef] [PubMed]

Pathogens 2021, 10, 623
21 of 22
25. Hichri, F.; Jannet, H.B.; Cheriaa, J.; Jegham, S.; Mighri, Z. Antibacterial activities of a few prepared derivatives of oleanolic acid
and of other natural triterpenic compounds. Comptes Rendus Chim. 2003, 6, 473–483. [CrossRef]
26. Tsuji, M.; Sriwilaijaroen, N.; Inoue, H.; Miki, K.; Kinoshita, K.; Koyama, K.; Furuhata, K.; Suzuki, Y.; Takahashi, K. Synthesis and
anti-influenza virus evaluation of triterpene-sialic acid conjugates. Bioorg. Med. Chem. 2018, 26, 17–24. [CrossRef] [PubMed]
27. Blanco-Cabra, N.; Vega-Granados, K.; Moya-Andérico, L.; Vukomanovic, M.; Parra, A.; Álvarez de Cienfuegos, L.; Torrents, E.
Novel oleanolic and maslinic acid derivatives as a promising treatment against bacterial biofilm in nosocomial infections: An
in vitro and in vivo study. ACS Infect. Dis. 2019, 5, 1581–1589. [CrossRef] [PubMed]
28. Wen, X.; Zhang, P.; Liu, J.; Zhang, L.; Wu, X.; Ni, P.; Sun, H. Pentacyclic triterpenes. Part 2: Synthesis and biological evaluation of
maslinic acid derivatives as glycogen phosphorylase inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 722–726. [CrossRef]
29. Qiu, W.-W.; Shen, Q.; Yang, F.; Wang, B.; Zou, H.; Li, J.-Y.; Li, J.; Tang, J. Synthesis and biological evaluation of heterocyclic
ring-substituted maslinic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1b. Bioorg. Med. Chem. Lett. 2009
19, 6618–6622. [CrossRef]
,
30. Taniguchi, S.; Imayoshi, Y.; Kobayashi, E.; Takamatsu, Y.; Ito, H.; Hatano, T.; Sakagami, H.; Tokuda, H.; Nishino, H.; Sugita, D.;
et al. Production of bioactive triterpenes by eriobotrya japonica calli. Phytochemistry 2002, 59, 315–323. [CrossRef]
31. Parra, A.; Rivas, F.; Lopez, P.E.; Garcia-Granados, A.; Martinez, A.; Albericio, F.; Marquez, N.; Muñoz, E. Solution- and solid-phase
synthesis and anti-hiv activity of maslinic acid derivatives containing amino acids and peptides. Bioorg. Med. Chem. 2009, 17,
1139–1145. [CrossRef]
32. Agrawal, N.; Mishra, P. The synthetic and therapeutic expedition of isoxazole and its analogs. Med. Chem. Res. 2018, 27, 1309–1344.
[CrossRef] [PubMed]
33. Kónya, B.; Docsa, T.; Gergely, P.; Somsák, L. Synthesis of heterocyclic n-(
glycogen phosphorylase. Carbohydr. Res. 2012, 351, 56–63. [CrossRef]
β-d-glucopyranosyl)carboxamides for inhibition of
34. Koufaki, M.; Tsatsaroni, A.; Alexi, X.; Guerrand, H.; Zerva, S.; Alexis, M.N. Isoxazole substituted chromans against oxidative
stress-induced neuronal damage. Bioorg. Med. Chem. 2011, 19, 4841–4850. [CrossRef] [PubMed]
35. Filali, I.; Romdhane, A.; Znati, M.; Jannet, H.B.; Bouajila, J. Synthesis of new harmine isoxazoles and evaluation of their potential
anti-alzheimer, anti-inflammatory, and anticancer activities. Med. Chem. 2016, 12, 184–190. [CrossRef] [PubMed]
36. Agalave, S.G.; Maujan, S.R.; Pore, V.S. Click chemistry: 1,2,3-triazoles as pharmacophores. Chem. Asian J. 2011, 6, 2696–2718.
[CrossRef] [PubMed]
37. Gonzaga, D.T.G.; Souza, T.M.L.; Andrade, V.M.M.; Ferreira, V.F.; de C da Silva, F. Identification of 1-aryl-1h-1,2,3-triazoles as
potential new antiretroviral agents. Med. Chem. 2018, 14, 242–248. [CrossRef]
38. El-Sebaey, S.A. Recent advances in 1,2,4-triazole scaffolds as antiviral agents. ChemistrySelect 2020, 5, 11654–11680. [CrossRef]
39. Wang, X.L.; Wan, K.; Zhou, C.H. Synthesis of novel sulfanilamide-derived 1,2,3-triazoles and their evaluation for antibacterial
and antifungal activities. Eur. J. Med. Chem. 2010, 45, 4631–4639. [CrossRef] [PubMed]
40. Kandeil, A.; Mostafa, A.; El-Shesheny, R.; Shehata, M.; Roshdy, W.H.; Ahmed, S.S.; Gomaa, M.; Taweel, A.E.; Kayed, A.E.;
Mahmoud, S.H.; et al. Coding-complete genome sequences of two sars-cov-2 isolates from egypt. Microbiol. Resour. Announc.
2020, 9. [CrossRef] [PubMed]
41. Guinda, A.; Rada, M.; Delgado, T.; Gutiérrez-Adánez, P.; Castellano, J.M. Pentacyclic triterpenoids from olive fruit and leaf. J.
Agric. Food Chem. 2010, 58, 9685–9691. [CrossRef] [PubMed]
42. Gil, M.; Haïdour, A.; Ramos, J.L. Identification of two triterpenoids in solid wastes from olive cake. J. Agric. Food Chem. 1997, 45,
4490–4494. [CrossRef]
43. Romero, C.; García, A.; Medina, E.; Ruíz-Méndez, M.V.; Castro, A.d.; Brenes, M. Triterpenic acids in table olives. Food Chem. 2010
118, 670–674. [CrossRef]
,
44. Goulas, V.; Manganaris, G.A. Towards an efficient protocol for the determination of triterpenic acids in olive fruit: A comparative
study of drying and extraction methods. Phytochem. Anal. 2012, 23, 444–449. [CrossRef]
45. Chan, R.W.Y.; Hemida, M.G.; Kayali, G.; Chu, D.K.W.; Poon, L.L.M.; Alnaeem, A.; Ali, M.A.; Tao, K.P.; Ng, H.Y.; Chan, M.C.W.;
et al. Tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory
tract: An in-vitro and ex-vivo study. Lancet Respir. Med. 2014, 2, 813–822. [CrossRef]
46. Oakdale, J.S.; Fokin, V.V.; Umezaki, S.; Fukuyama, T. Preparation of 1,5-disubstituted 1,2,3-triazoles via ruthenium-catalyzed
azide alkyne cycloaddition. Org. Synth 2013, 90, 96–104. [PubMed]
47. Takasu, K.; Azuma, T.; Takemoto, Y. Synthesis of trifunctional thioureas bearing 1,5-disubstituted triazole tether by ru-catalyzed
huisgen cycloaddition. Tetrahedron Lett. 2010, 51, 2737–2740. [CrossRef]
48. Nador, F.; Volpe, M.A.; Alonso, F.; Feldhoff, A.; Kirschning, A.; Radivoy, G. Copper nanoparticles supported on silica coated
maghemite as versatile, magnetically recoverable and reusable catalyst for alkyne coupling and cycloaddition reactions. Appl.
Catal. A Gen. 2013, 455, 39–45. [CrossRef]
49. Hansen, T.V.; Wu, P.; Fokin, V.V. One-pot copper(i)-catalyzed synthesis of 3,5-disubstituted isoxazoles. J. Org. Chem. 2005, 70,
7761–7764. [CrossRef]
50. Kuo, Y.C.; Lin, L.C.; Tsai, W.J.; Chou, C.J.; Kung, S.H.; Ho, Y.H. Samarangenin b from limonium sinense suppresses herpes
simplex virus type 1 replication in vero cells by regulation of viral macromolecular synthesis. Antimicrob. Agents Chemother. 2002
,
46, 2854–2864. [CrossRef]

Pathogens 2021, 10, 623
22 of 22
51. Vilar, S.; Cozza, G.; Moro, S. Medicinal chemistry and the molecular operating environment (moe): Application of qsar and
molecular docking to drug discovery. Curr. Top. Med. Chem. 2008, 8, 1555–1572. [CrossRef]
52. Al-Karmalawy, A.A.; Khattab, M. Molecular modelling of mebendazole polymorphs as a potential colchicine binding site inhibitor.
New J. Chem. 2020, 44, 13990–13996. [CrossRef]
53. Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; et al. Structure of mpro from sars-cov-2
and discovery of its inhibitors. Nature 2020, 582, 289–293. [CrossRef]
54. Ghanem, A.; Emara, H.A.; Muawia, S.; Abd El Maksoud, A.I.; Al-Karmalawy, A.A.; Elshal, M.F. Tanshinone iia synergistically
enhances the antitumor activity of doxorubicin by interfering with the pi3k/akt/mtor pathway and inhibition of topoisomerase
ii: In vitro and molecular docking studies. New J. Chem. 2020, 44, 17374–17381. [CrossRef]
55. Khattab, M.; Al-Karmalawy, A. Revisiting activity of some nocodazole analogues as a potential anticancer drugs using molecular
docking and dft calculations. Front. Chem. 2021, 9, 92. [CrossRef] [PubMed]
56. McConkey, B.J.; Sobolev, V.; Edelman, M. The performance of current methods in ligand–protein docking. Curr. Sci. 2002, 83,
845–856.
57. Eliaa, S.G.; Al-Karmalawy, A.A.; Saleh, R.M.; Elshal, M.F. Empagliflozin and doxorubicin synergistically inhibit the survival of
triple-negative breast cancer cells via interfering with the mtor pathway and inhibition of calmodulin: In vitro and molecular
docking studies. ACS Pharmacol. Transl. Sci. 2020, 3, 1330–1338. [CrossRef]
58. Al-Rabia, M.W.; Alhakamy, N.A.; Ahmed, O.A.A.; Eljaaly, K.; Aloafi, A.L.; Mostafa, A.; Asfour, H.Z.; Aldarmahi, A.A.; Darwish,
K.M.; Ibrahim, T.S.; et al. Repurposing of sitagliptin- melittin optimized nanoformula against sars-cov-2; antiviral screening and
molecular docking studies. Pharmaceutics 2021, 13, 307. [CrossRef]
59. Feoktistova, M.; Geserick, P.; Leverkus, M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc.
2016, 2016. [CrossRef]
60. Zhang, J.; Zhan, B.; Yao, X.; Gao, Y.; Shong, J. Antiviral activity of tannin from the pericarp of punica granatum l. Against genital
herpes virus in vitro. China J. Chin. Mater. Med. 1995, 20, 556–558, 576, inside backcover.
61. Schuhmacher, A.; Reichling, J.; Schnitzler, P. Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus
type 1 and type 2 in vitro. Phytomed. Int. J. Phytother. Phytopharm. 2003, 10, 504–510. [CrossRef]