Discovery of juglone and its derivatives as potent SARS-CoV-2 main proteinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113789

SARS-CoV-2 as a positive-sense single-stranded RNA coronavirus caused the global outbreak of COVID-19. The main protease (M^pro) of the virus as the major enzyme processing viral polyproteins contributed to the replication and transcription of SARS-CoV-2 in host cells, and has been characterized as an attractive target in drug discovery. Herein, a set of 1,4-naphthoquinones with juglone skeleton were prepared and evaluated for the inhibitory efficacy against SARS-CoV-2 M^pro. More than half of the tested naphthoquinones could effectively inhibit the target enzyme with an inhibition rate of more than 90% at the concentration of 10 μM. In the structure-activity relationships (SARs) analysis, the characteristics of substituents and their position on juglone core scaffold were recognized as key ingredients for enzyme inhibitory activity. The most active compound, 2-acetyl-8-methoxy-1,4-naphthoquinone (15), which exhibited much higher potency in enzyme inhibitions than shikonin as the positive control, displayed an IC₅₀ value of 72.07 ± 4.84 nM towards M^pro-mediated hydrolysis of the fluorescently labeled peptide. It fit well into the active site cavity of the enzyme by forming hydrogen bonds with adjacent amino acid residues in molecular docking studies. The results from in vitro antiviral activity evaluation demonstrated that the most potent M^pro inhibitor could significantly suppress the replication of SARS-CoV-2 in Vero E6 cells within the low micromolar concentrations, with its EC₅₀ value of about 4.55 μM. It was non-toxic towards the host Vero E6 cells under tested concentrations. The present research work implied that juglone skeleton could be a primary template for the development of potent M^pro inhibitors.

Full text of DOI:10.1016/j.ejmech.2021.113789

European Journal of Medicinal Chemistry 225 (2021) 113789
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of juglone and its derivatives as potent SARS-CoV-2 main
proteinase inhibitors
*
*
Jiahua Cui , Jinping Jia
School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2021
Received in revised form
SARS-CoV-2 as a positive-sense single-stranded RNA coronavirus caused the global outbreak of COVID-
pro
19. The main protease (M ) of the virus as the major enzyme processing viral polyproteins contributed
to the replication and transcription of SARS-CoV-2 in host cells, and has been characterized as an
attractive target in drug discovery. Herein, a set of 1,4-naphthoquinones with juglone skeleton were
8
August 2021
Accepted 15 August 2021
Available online 18 August 2021
pro
prepared and evaluated for the inhibitory efficacy against SARS-CoV-2 M . More than half of the tested
naphthoquinones could effectively inhibit the target enzyme with an inhibition rate of more than 90% at
the concentration of 10 mM. In the structure-activity relationships (SARs) analysis, the characteristics of
Keywords:
Naphthoquinones
substituents and their position on juglone core scaffold were recognized as key ingredients for enzyme
inhibitory activity. The most active compound, 2-acetyl-8-methoxy-1,4-naphthoquinone (15), which
exhibited much higher potency in enzyme inhibitions than shikonin as the positive control, displayed an
Juglone
pro
M
inhibitors
pro
SARS-CoV-2
COVID-19
IC50 value of 72.07 ± 4.84 nM towards M -mediated hydrolysis of the fluorescently labeled peptide. It fit
well into the active site cavity of the enzyme by forming hydrogen bonds with adjacent amino acid
residues in molecular docking studies. The results from in vitro antiviral activity evaluation demonstrated
pro
that the most potent M inhibitor could significantly suppress the replication of SARS-CoV-2 in Vero E6
cells within the low micromolar concentrations, with its EC50 value of about 4.55
mM. It was non-toxic
towards the host Vero E6 cells under tested concentrations. The present research work implied that
pro
juglone skeleton could be a primary template for the development of potent M
inhibitors.
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
COVID-19 treatment and the side effects were still controversial
issues in academia [9e12]. The monoclonal antibody therapy
employing bamlanivimab and antibody combination (bamlanivi-
mab/etesevimab) has been authorized by FDA recently. However,
certain variants of SARS-CoV-2 (B.1.351 and B.1.1.248) might escape
from these neutralizing antibodies [13,14]. Therefore, it is urgent to
explore targeted antiviral chemotherapeutics against SARS-CoV-2.
SARS-CoV-2 virus is a positive-sense single-stranded RNA virus
[15,16], and its genome is translated to two overlapping poly-
proteins upon entry into host cells. The two polypeptides are pro-
teolytically processed, mainly by a 33.8-kDa virus-specific main
Coronavirus disease 2019 (COVID-19) is a serious infectious
disease caused by a new coronavirus named severe acute respira-
tory syndrome coronavirus 2 (SARS-CoV-2) [1,2]. The rapid spread
of this pneumonia disease is an ongoing global threat that gener-
ates over 197 million diagnosed cases and more than 4.21 million
deaths over 233 countries and territories globally by 03 Aug 2021
[
3]. Until now, no clinically specific antiviral chemotherapeutics
were available to treat the disease. The approved chemotherapeutic
drugs against COVID-19 included favipiravir [4], lopinavir/ritonavir
pro
[
5], chloroquine/hydroxychloroquine (FDA revoked emergency use
authorization for chloroquine and hydroxychloroquine on June 15,
020) [6], and remdesivir [7,8]. All of these drugs had been devel-
protease (M ), to afford proteins with different structures and
pro
functions required for replication [17,18]. The M also referred to
2
as the 3C-like protease modified the polyproteins at no less than 11
oped for the treatment of other related viruses, such as SARS and
conserved amide linkages and played a pivotal role in the replica-
MERS coronavirus, Ebola, and HIV. Their degree of efficacy in
tion cycle of SARS-CoV-2 in host cells. Since closely related ho-
pro
mologues of M
have never been identified in host cells, the
protease is identified as a potential therapeutic target for the con-
*
Corresponding authors.
E-mail addresses: cpucjh@sjtu.edu.cn (J. Cui), jpjia@sjtu.edu.cn (J. Jia).
trol of virus replication [19,20].
https://doi.org/10.1016/j.ejmech.2021.113789
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

J. Cui and J. Jia
European Journal of Medicinal Chemistry 225 (2021) 113789
Natural products are a wellspring of lead compounds in drug
subsequent column chromatography afforded 1,4-naphthoquinone
5. Further Thiele-Winter acetoxylation of 1,4-naphthoquinone and
subsequent hydrolysis of naphthalene-1,2,4-triyl triacetate (6)
afforded 2-hydroxy-1,4-naphthoquione (7) in high yield [32]. 2-
Methyl-1,4-naphthoquinone (vitamin K3, 3) was obtained by the
oxidation of 2-methylnaphthalene (8) using chromic anhydride in
acidic conditions.
discovery [21], and several phytochemicals have been investigated
for their therapeutic potentials against SARS-CoV-2 [22,23]. Previ-
ous studies conducted by Jin et al. indicted that shikonin (Fig. 1, 1), a
natural naphthoquinone isolated from Lithospermum erythrorhizon
pro
Sieb. et Zucc., was a strong inhibitor of SARS-CoV-2 M
IC50 value of 15.75 ± 8.22
with its
m
M [18]. However, due to the Michael
addition of shikonin naphthazarin nucleus as electrophiles [24,25]
and bioreductive alkylation of its side chain with nucleophilic
biomolecules such as glutathione, proteins or DNA [26], shikonin
demonstrated significant cytotoxic effects at concentrations
The reactions used to synthesize juglone (2) and its derivatives
were outlined in Scheme 2. Juglone (2) was synthesized by the
oxidation of 1,5-naphthelendiol (9) using the Fremy's salts, Soxhlet
extraction, and further column chromatographic purification [33].
Juglone methyl ether (10) was obtained by the reductive methyl-
ation of juglone (1) with the presence of dimethyl sulfonate and
sodium dithionate in alkali conditions and further cerium (IV)
ammonium nitrate-mediated oxidative demethylation [34]. Pro-
pionyl juglone (11) and acetyl juglone (12) were prepared by the
acylation of the parent compound by corresponding acid anhydride
with sulfuric acid as the catalyst [35]. Acetyl juglone (12) was
reduced by sodium dithionate to yield the hydroquinone inter-
mediate. Migration of the acetyl group of hydroquinone in alkali
conditions and further methylation afforded 4,8-dimethoxy-1-
naphthol acetate (13) as the key intermediate [35]. Fries rear-
rangement of naphthol acetate (13) and subsequent CAN-mediated
oxidation produced 2-acetyl-8-methoxy-1,4-naphthoquinone (15).
5,8-Dimethoxy-1,4-naphthoquinone (21) was prepared by the
methylation of 1,5-naphthalenediol (9), subsequent bromination
reactions, Cu(I)-catalyzed nucleophilic substitution [36], and
further CAN-mediated oxidation (Supplementary Information).
7-Methyl juglone (16) and its derivatives (22e25) were pre-
pared according to our reported procedures [37] resting on the
Stobbe condensation of 2,5-dimethoxy benzaldehyde with diethyl
succinate, intramolecular cyclization, reduction, acid-facilitated
debenzylation and further oxidation. Plumbagin (17) was synthe-
sized based on the reported procedure in which 1,5-
naphthalenediol served as the starting point [38]. The substituted
anthraquinone emodin (18), rhein (19) and aloe emodin (20) were
prepared by the D-A reactions between diene and corresponding
naphthoquinones [39,40].
ranging from 100 mg/mL to 10 ng/mL in vitro [27]. The toxicity of
shikonin prevented its further development as an antiviral drug
candidate. Therefore, rational structural modifications were
essential to overcome the defects in the structure of this hallmark
molecule.
The research results from mechanistic investigations implied
that the side chain and adjacent phenolic hydroxyl group on core
structure of shikonin tautomer (Fig. S1, Supplementary Informa-
tion) played pivotal roles in bioreductive alkylation and conjugate
addition with bionucleophiles [26], which gave rise to the cyto-
toxicity of shikonin. Accordingly, we decided to modify shikonin
skeleton through a scaffold simplification strategy to get juglone
derivatives with a more appropriate scaffold in terms of improving
cellular toxicity.
Juglone (2) is a naturally occurring 1,4-naphthoquione identified
in Juglandaceae species, which bears a simplified shikonin core
structure. It exhibited comparably low cytotoxicity against normal
peripheral blood mononuclear cells with its IC50 value of more than
5 mg/mL [28]. It had been prescribed as a remedy for the treatment
of a variety of skin diseases in the early 1900s [29]. The synthetic 2-
methyl-1,4-naphthoquione (menadione, 3) that served as a nutri-
tional supplement in animal feeding was also much less toxic [30].
The results from earlier clinical investigations demonstrated that
no toxic effects were observed in patients with hypopro-
thrombinemia receiving menadione doses from 1 to 200 mg [31].
All of the findings mentioned above supported our initial hypoth-
esis that scaffold simplification and modification of shikonin
naphthazarin nucleus would be reasonable approaches to reduce
Enzyme inhibition. Using a fluorescently labeled short peptide
containing Q-S scissile bond, the inhibitory activity of the prepared
pro
the cytotoxicity of shikonin as a natural SARS-CoV-2 M inhibitor.
pro
As a continuing investigation of biological activities of 1,4-
naphthoquinones with shikonin as a lead, and in order to
contribute to the drug discovery against COVID-19, the present
quinones against M of SARS-CoV-2 were evaluated according to
the reported procedure [17]. Firstly, we tested the enzymatic inhi-
bition rate of several naturally occurring naphthoquinones (juglone
2, 7-methyl juglone 16, lawsone 7, plumbagin 17 and shikonin 1),
9,10-anthraquinones (emodin 18, rhein 19 and aloe emodin 20) and
study afforded the discovery of juglone and its derivative as potent
pro
M
inhibitors of SARS-CoV-2, which are promising antiviral drug
candidates in future research and development.
the synthetic vitamin K3 (3) in the first library of compounds
against SARS-CoV-2 M at the concentration of 10 mM. The results
pro
from primary screening indicated that most of the natural quinones
were ineffective, with the inhibition rate of less than 10% at 10
Table S1, Supplementary Information). Vitamin K3 (3) with the
inhibition rate of 12.7% was also inactive. The natural naph-
thoquinone shikonin, which had been identified as one of the
2
. Results
mM
(
Chemistry. As shown in Scheme 1, the starting material for this
synthesis of 1,4-naphthoquinone (5) was 1-naphthylamine (4). The
oxidation of 1-naphthylamine (4) with hydrogen peroxide and
Fig. 1. The chemical structure of shikonin (1), juglone (2) and menadione (3).
2

J. Cui and J. Jia
European Journal of Medicinal Chemistry 225 (2021) 113789
ꢀ
ꢀ
ꢀ ꢀ
3 3 3
ONa, CH OH, 5 C; then conc. HCl; d) CrO , CH COOH, 40 C for
Scheme 1. Reagents and conditions: a) CH
3
COOH, H
2
4 2
SO , H O
2
, 80 C, 3 h; b) (CH
3
CO)
2
O, H SO
2 4
, 10 C, 8 h; c) CH
3
ꢀ
30 min, then 65 C for 20 min.
ꢀ
ꢀ
ꢀ
Scheme 2. Reagents and conditions: e) (CH
3
CO)
2
O, H
2
O
SO
2
, 40e60 C, 1 h; f) (CH
3
)
2
SO
O/H
OH-DMF, reflux, 48 h.
4
, NaOH, Na
2
S
2
O
4
, Et
2
O/H
I, K
2
O, 5 C, overnight; g) CAN, DCM-ACN (3:1), 5 C; h) (CH
3
CH
2 2
CO) O,
ꢀ
ꢀ
ꢀ
ꢀ
Cat. Conc. H
2
SO
4
, 5 C, 4 h; i) (CH
3
CO)
2
O, Cat. Conc. H
2
4
, 5 C, 4 h; j) Na
2
S
2
O
4
, Et
2
2
O, r.t., 2 h; then CH
3
2
CO
3
, DMF, 10 C, overnight; k) BF
3
eEt
2
O, 60 C, 0.5 h; l) CAN, DCM-
ꢀ
ꢀ
CAN (3:1), 5 C, 0.5 h; m) NBS, ACN, ꢁ10 C, overnight; then CH
3
ONa, CuI, CH
3
strong Mpro inhibitors in previous studies (IC50 ¼ 15.75 ± 8.22
m
M)
0.1
m
M entered the IC50 value screening (Table S3).
[
17], was employed as the positive control. It demonstrated mod-
As shown in Table S3, within the tested synthetic 1,4-
pro
erate inhibitory effects towards the target enzyme at the concen-
tration of 10 M. In the first library of naphthoquinones, juglone (2)
naphthoquinones as strong M
1,4-naphthoquinone (15) was characterized as the most potent
inhibitors, 2-acetyl-8-methoxy-
m
and 7-methyl juglone (16) exhibited the strongest inhibition with
the completely loss of the hydrolytic efficacy of M . The two
natural naphthoquinones were employed as the lead compounds
for further structural modifications.
In the second library, the derivatives of juglone (2) and 7-methyl
juglone (16) were produced by the addition of a few groups on their
naphthoquinone scaffold and modifications on the phenolic hy-
droxyl group on the B-ring. The enzyme inhibition rate of com-
pounds in the second library was displayed in Table S2. The results
implied that almost all of the derivatives in the second library
maintained the high inhibitory potency of juglone under concen-
inhibitor against the target enzyme with its IC50 value of
pro
72.07 ± 4.84 nM, which was comparable to the recently reported
pro
IC50 value of a short peptide as SARS-CoV-2 M
inhibitor
(IC50 ¼ 53 ± 5 nM) [17]. The 1,4-naphthoquinone (5) and propionyl
juglone (11) have also been identified as potent inhibitors with IC50
value of 110.13 ± 7.04 and 129.77 ± 0.45 nM, respectively. 7-Methyl
juglone ethyl acetate (23) and its benzyl ether (25) exhibited much
higher IC50 values than propionyl juglone did.
Structure-activity relationship studies. In the first library of
compounds (Table S1), the natural naphthoquinone juglone (2)
exhibited potent inhibitory effects against the target SARS-CoV-2
pro
trations of both 10
mM and 1
mM. At the concentration of 0.1
mM, a
M
with the inhibition rate of 99% at the concentration of
few analogues exhibited much higher potency as compared with
the parent compounds (2 and 16). Then, the compounds with an
enzymatic inhibition rate of more than 25% at the concentration of
10
m
M. By contrast, lawsone (7) as its analogue did not exhibit any
pro
detectable inhibition against M
The sharp decrease in the activity should be ascribed to the
under the same concentration.
3

J. Cui and J. Jia
European Journal of Medicinal Chemistry 225 (2021) 113789
transposition of the B-ring hydroxyl group to the quinone ring.
the methyl group attached to the phenolic hydroxyl group of
juglone.
Compared with juglone, plambagin (17) with an additional methyl
pro
group on A-ring was also inactive towards M . Similarly, the
The annulation of the quinone moiety with another phenyl
group led to a drastic decrease in the enzymatic inhibition potency,
because emodin (18), rhein (19) and aloe emodin (20) as derivatives
of 7-methyl juglone showed the inhibition rate of less than 10%. The
increased steric hindrance of the annulated phenyl ring caused
detrimental effects on binding 7-methyl juglone with the target
enzyme.
The methylation of 7-methyl juglone led to a drop in the activity
at low concentrations, and 7-methyl juglone methyl ether (22) did
not show any inhibitory effects towards the target enzyme at the
synthetic vitamin K3 (3) with a methyl group on the quinone ring
displayed the enzymatic inhibition rate of only 12.7% as compared
with the negative control. The elimination of the A-ring methyl
group of vitamin K3 led to a significant increase in the efficacy in
pro
diminution of SARS-CoV-2
M
activity, because 1,4-
naphthoquinone (5) as a strong inhibitor exhibited an IC50 value
of only 110.13 ± 7.04 nM. The results demonstrated that both hy-
droxyl group and methyl substituent on the quinone ring had
detrimental effects on the enzyme inhibitory potency of juglone
(
see Fig. 2).
-Methyl juglone (16) with a methyl group on B-ring of juglone
was as potent as compound 2 at 10 M in the primary screening. At
much lower concentrations, the enzyme inhibitory potency of 7-
methyl juglone (inhibition rates of 99.6% and 21.7% at 1.0 M and
.1 M, respectively) was slightly more potent than juglone. The
concentration of 0.1 mM. Protection of the phenolic hydroxyl group
7
with a methoxy methyl ether moiety also had deleterious effects
pro
m
since the M inhibition rate of compound 24 was lower than 5% at
concentrations of less than 1.0
benzylation of the phenolic hydroxyl group of 7-methyl juglone
caused an increase in potency at the concentration of 0.1 M. The
mM. By contrast, both acylation and
m
0
m
m
results implied that the localization of a methyl group on C(2) of A-
ring had deleterious effects while the exchange of the hydrogen
atom on C(7) with a methyl group led to a slight increase in the
activity. Furthermore, juglone and 7-methyl juglone were nearly
two-fold more potent than shikonin as the control in inhibiting the
benzylated compound 25 exhibited an IC50 value of
160.68 ± 17.83 nM towards the target enzyme (Table S3), which was
less than three-quarters of the value for the acylated derivative 23
(IC50 ¼ 220.90 ± 14.03 nM).
Molecular docking. In order to gain an insight into the binding
target enzyme at the concentration of 10
m
M. It was envisioned that
interaction of investigated naphthoquinones with SARS-CoV-2
pro
the methyl substituent at C(7) on B-ring, instead of the corre-
sponding six-carbon side chain of shikonin, could enhance the
inhibitory potency.
M
enzyme, we performed molecular docking studies based on
pro
the crystal structures of M in complex with the peptide-like in-
hibitor N3 (PDB ID: 6LU7). As shown in Fig. 3, both juglone (a),
The modifications of juglone on the chelated phenolic hydroxyl
group have been shown to improve the inhibitory activity. Within
the synthetic analogues, acetyl juglone and propionyl juglone
showed higher potency than the parent compound juglone at the
propionyl
juglone
(b)
and
2-acetyl-8-methoxy-1,4-
naphthoquinone (c) tightly fit the active site cavity of the
enzyme. Juglone was bound to the target enzyme with the calcu-
lated binding energy of ꢁ8.6771 kJ/mol. The docking results indi-
cated that the C(1) carbonyl group formed a hydrogen bond with
concentration of 1 mM. Methylation of the chelated phenolic hy-
143
droxyl group of juglone was not preferred because the corre-
sponding methylated product (10) was less potent in enzymatic
inhibition assay. The introduction of another methoxy group at C(8)
on B-ring of juglone methyl ether (10) caused a further reduction in
activity since the enzymatic inhibition rate of compound 21 was
Gly
amino acid residue. Another hydrogen bonding interaction
166
between the phenolic hydroxyl group and Glu
observed.
The molecular docking study of propionyl juglone with the
crystal structure of M (Fiure 3, b) showed that this ligand fit well
was also
pro
pro
less than 80% at concentrations of 10
mM and 1.0
mM. Introduction
into the substrate binding site of M
enzyme. Propionyl juglone
of another acetyl substitution on C(2) adjacent to the C(1) carbonyl
moiety led to increased activity, and compound 15 was identified as
the most potent inhibitors among the prepared juglone derivatives.
The strong electron withdrawing effects possibly contributed to the
tight binding affinity of compound 15 with the target enzyme. The
results implied that there might be some positive or negative in-
teractions between the acetyl substituent on the quinone ring and
was bound to the target enzyme with the calculated binding energy
of ꢁ17.3199 kJ/mol, which was the lowest value for all of the pre-
dicted 30 binding models in MOE molecular docking. The oxygen
atom of the C(4) carbonyl group underwent simultaneous H-
143
bonding interactions with the backbone NH of Gly
and the hy-
144
droxyl group of Ser
.
2-Acetyl-8-methoxy-1,4-naphthoquinone (15) as the most
Fig. 2. SARs analysis of juglone and its derivatives as SARS-CoV-2 Mpro inhibitors.
4

5

J. Cui and J. Jia
European Journal of Medicinal Chemistry 225 (2021) 113789
Fig. 3. The predicted binding modes of juglone (a), propionyl juglone (b) and 2-acetyl-8-methoxy-1,4-naphthoquinone (c) in the active site cavity of Mpro
.
potent inhibitor against the target enzyme in the study was also
the
reported
procedures
[18].
2-Acetyl-8-methoxy-1,4-
bound to the substrate binding site of Mpro (Fig. 3, c). The C(4)
naphthoquinone (15) exhibited antiviral activity at concentrations
of more than 1
(EC50) of 4.55
41
carbonyl group was oriented towards the imidazole moiety of His
m
M, with the half-maximal effective concentrations
mM. The result indicated that the quinone (15)
with the formation of a hydrogen bonding interaction. The oxygen
atom on the acetyl substitution also hydrogen bonded with the
possibly penetrate cellular membranes and inhibit the target viral
143
pro
backbone of NH of Gly . The methoxy group of compound 15 was
placed towards Glu1 , and there was an H-bonding interaction
between the oxygen atom in the methoxy group and NH in the
amide backbone of Glu1 . The tight binding interaction between 2-
acetyl-8-methoxy-1,4-naphthoquinone (15) and the target enzyme
M
enzyme. The results from cytotoxicity evaluations implied that
the compound was much less toxic than juglone towards normal
HFF-1 cells. At the concentration of less than 20 M, it didn't affect
66
m
66
the growth of host Vero E6 cells (Fig. 5, b, cell viability of more than
90%). Balb/C mice that received the preparation of the target
compound (Fig. S2, 100 mg/kg, p.o., on every the other day, 10 times
should explain its potent inhibitory activity against the enzymatic
pro
activity of M
.
Cytotoxicity of Juglone and its derivatives. The ideal antivirus
agents were those ones that acted by inhibiting viral replication,
but without cytotoxicity towards host normal cells [41]. Therefore,
pro
juglone and its derivatives as SARS-CoV-2 M
inhibitors were
initially tested for their cytotoxic activity against human normal
fibroblast HFF-1 cells using the standard MTT assay. As presented in
Table S4, the naturally occurring juglone (2), 7-methyl juglone (16),
and shikonin (1) exhibited potent growth inhibition towards the
proliferation of HFF-1 cells with their IC50 values of less than 5 mM.
The methylation and acylation of the phenolic hydroxyl group of
juglone led to a minor decrease in cytotoxicity. Propionyl juglone
(
11) as a potent Mpro inhibitor was also toxic towards normal HFF-
1
cells. It possibly underwent hydrolysis catalyzed by cytoplasmic
enzymes to afford juglone (2) as a cytotoxic metabolite (Fig. 4). By
contrast, the absence of the B-ring hydroxyl group of juglone
caused
a
significant decrease in toxicity, because 1,4-
naphthoquinone (5) exhibited a much higher IC50 value towards
the normal HFF-1 cells.
The cytotoxicity of 7-methyl juglone (16) tended to be attenu-
ated by the benzylation of the hydroxyl group on B-ring, and the
IC50 value of compound 25 was 7-fold higher than that of the parent
compound 16. Lawsone (7) and vitamin K3 (3) with a substituent
on the quinone ring displayed almost no cytotoxic effects on HFF-
1
cells (IC50 > 50 mM). The electron donating effects and the steric
hindrance of the group adjacent to the quinoidal carbonyl group
prevented Michael addition of the quinone ring with nucleophilic
biomolecules. 2-Acetyl-8-methoxy-1,4-naphthoquinone (15) was
also much less toxic towards normal HFF-1 cells with its IC50 value
of 41.2 mM. The presence of the acetyl moiety on A-ring prohibited
the generation of ROS species and nucleophilic conjugate additions
of quinone moiety with nucleophiles. Due to its strong inhibitory
pro
potency towards SARS-CoV-2 M
and low cytotoxic profile, it
entered further in vitro antiviral activity evaluations.
Antiviral activity. The antiviral activity of compound 15 to
inhibit SARS-CoV-2 replication in vitro was conducted according to
Fig. 5. In vitro inhibitory activity of compound 15 against SARS-CoV-2 in Vero E6. (a),
the host Vero E6 cells were incubated with different concentrations of the target
compound, and infected by SARS-CoV-2 in vitro with the MOI value of 0.05. The
reproduced virus in cell culture was quantified by qRT-PCR assay. (b), the cell viability
of host Vero E6 cells was determined by the standard MTT assay upon co-incubation of
the cells with a series of concentrations of the indicated compound for 24 h.
Fig. 4. The hydrolysis of propionyl juglone (11) and acetyl juglone (12).
6

J. Cui and J. Jia
European Journal of Medicinal Chemistry 225 (2021) 113789
in 20 days) did not show any obvious toxicity symptoms like
reduced activity, hypothermia, or body shivering. Meanwhile,
compound 15 induced no animal deaths and only caused a minor
body weight loss as compared with control animals after a total
treatment of 10 times in 20 days. As a potent Mpro inhibitor with
antivirus activity, the juglone derivative 15 deserved further in vivo
antiviral activity evaluation in future studies.
activity of remdesivir against SARS-CoV-2, might be used to test the
treatment efficacy of other antiviral drug candidates in future. The
in vitro research results confirmed by animal experiments would
provide the rational basis for further research and development.
Author contributions
J. Cui and J. Jia conceived the idea. J. Cui performed the experi-
ment and analyzed the data. J. Jia supervised the project. J. Cui and J.
Jia wrote the paper.
2
.1. Discussion and future perspectives
Herein, we have described the discovery of juglone and its de-
pro
rivatives as potent M
inhibitors against SARS-CoV-2. Earlier
Funding
chemical investigations disclosed the presence of juglone as a
bioactive ingredient in Exocarpium Juglandis Immaturum, a tradi-
tional Chinese medicine used to treat psoriasis, ichthyosis, sores,
and furuncles in the Orient [42]. It has also historically been used in
European folk medicines as a remedy for parasites, ringworm, and
other fungal infectious diseases [43]. The research results from
previous investigations demonstrated that the natural naph-
thoquinone juglone was active against the animal Vesicular Sto-
matitis Virus [44] and it could potently reactivate latent HIV-1 in
This program was financially supported by Medical and Engi-
neering Interdisciplinary Research Fund of Shanghai Jiao Tong
University (Grant No. 20X190020002).
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4
þ
the bcl-2-transduced primary CD T cell model [45].
The exact mechanism by which juglone acts against virus in-
fections, however, still remains unclear. In our studies, this naph-
Acknowledgement
thoquinone was characterized as a potent inhibitor against SARS-
pro
CoV-2 M
by a high-throughput screening assay. It completely
M. 3C-like
We want to express our gratitude to Prof. Hong Liu and Prof.
Yechun Xu in Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, for their valuable suggestions and also kind
help in the biological evaluation. We also want to express our great
appreciation to Prof. Gang Chen in School of Chemistry and
Chemical Engineering, Shanghai Jiao Tong University, for the pro-
fessional proof editing of our manuscript.
inactivated the main protease at the concentration of 1
m
pro
proteases (M in coronavirus), which belong to the cysteine pro-
tease family with a chymotrypsin-like fold, have been widely
characterized in positive-sense single-stranded RNA viruses. In
addition, 3C-like proteases shared several general similarities in
substrate specificity and also inhibitor effectiveness [46]. Therefore,
the structural features of juglone as a non-peptide inhibitor might
act as a valuable scaffold for further anti-coronavirus drug design.
Additionally, the results of our study also provided one explanation
of the antiviral molecular mechanism of juglone.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113789.
Since the cleavage of viral proteins by specific proteases was
crucial at post-entry stage in virus replication cycles, the SARS-CoV-
pro
2
M
was an attractive target for selective chemotherapeutic
pro
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