Design, synthesis, and biological evaluation of peptidomimetic aldehydes as broad-spectrum inhibitors against enterovirus and sars-cov-2

Article abstract of DOI:10.1021/acs.jmedchem.0c02258

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3Cpro) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound 18p demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3Cpro in complex with 18p determined at a resolution of 1.2 ? revealed that 18p covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CLpro and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound 18p (IC50 = 0.034 μM, EC50 = 0.29 μM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088, compound 18p also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.

Full text of DOI:10.1021/acs.jmedchem.0c02258

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Article
Design, Synthesis, and Biological Evaluation of Peptidomimetic
Aldehydes as Broad-Spectrum Inhibitors against Enterovirus and
SARS-CoV‑2
Wenhao Dai,○ Dirk Jochmans,○ Hang Xie,○ Hang Yang,○ Jian Li, Haixia Su, Di Chang, Jiang Wang,
Jingjing Peng, Lili Zhu, Yong Nian, Rolf Hilgenfeld, Hualiang Jiang, Kaixian Chen, Leike Zhang,*
Yechun Xu,* Johan Neyts,* and Hong Liu*
Cite This: https://doi.org/10.1021/acs.jmedchem.0c02258
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ABSTRACT: A novel series of peptidomimetic aldehydes was
designed and synthesized to target 3C protease (3Cpro) of
enterovirus 71 (EV71). Most of the compounds exhibited high
antiviral activity, and among them, compound 18p demonstrated
potent enzyme inhibitory activity and broad-spectrum antiviral
activity on a panel of enteroviruses and rhinoviruses. The crystal
structure of EV71 3Cpro in complex with 18p determined at a
resolution of 1.2 Å revealed that 18p covalently linked to the
catalytic Cys147 with an aldehyde group. In addition, these
compounds also exhibited good inhibitory activity against the
3CLpro and the replication of severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), especially compound 18p (IC50
=
0.034 μM, EC50 = 0.29 μM). According to our previous work,
these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088, compound 18p also exhibited
good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.
As far as we know, none of the known human proteases
possessed a similar cleavage specificity, which makes 3Cpro
become a highly prospective target for developing broad-
spectrum drugs.3,4 Recent efforts in drug discovery also
furnished several inhibitors against the EV71 3Cpro (Figure 1).
Those peptidomimetic compounds with a warhead in P1′ and a
lactam ring in P1 could be briefly divided into reversible and
irreversible inhibitors according to their binding modes.
Compounds 1−6 are reversible inhibitors, among which
compounds 1, 2, 4, and 5 are covalent reversible inhibitors,
while 3 and 6 are noncovalent inhibitors. Those compounds
exhibited good anti-EV71 activity with EC50 values in the range
of 0.009−3.7 μM, while the broad-spectrum antiviral activity
and the drug-like properties were rarely evaluated.5 Rupintrivir
(AG7088)5a was reported to have the excellent antivirus activity
against EV71 with an EC50 value of 0.009 μM and has entered
INTRODUCTION
■
Enterovirus 71 (EV71) belongs to the enterovirus genus of the
picornaviridae family, and the genome of EV71 is composed of a
single-stranded positive-sense RNA. To the best of our
knowledge, EV71 is not only the primary pathogen of hand,
foot, and mouth disease (HFMD) but also closely associated
with neurological syndromes such as severe encephalitis and
aseptic meningitis, and these diseases caused by the EV71 have
become a worldwide health problem. Especially, young children
are more susceptible to be infected by the enterovirus.1
However, to date, there are no approved drugs to prevent or
treat the associated diseases.2 Considering the detriment of
HFMD and the central neurological syndromes in children, the
development of effective antiviral drugs is urgently needed.
The genome of enterovirus 71 encodes a polyprotein
precursor, which is cleaved by the 3C protease (3Cpro) and 2A
protease (2Apro) into structural proteins and nonstructural
proteins, of which the 3Cpro is responsible for most of the
cleavages. The 3Cpro of enteroviruses and rhinoviruses (RV) is
essential for viral replication, and it not only shared a high degree
of homology at an amino acid level but also contained a Cys-His-
Asp/Glu catalytic triad. In addition, the 3Cpro is an exceptional
cysteine protease with unique folding and catalytic mechanism,
and a Gln is almost required in the P1 position of the substrates.
clinical trials as a protease inhibitor targeting rhinovirus 3Cpro
,
Special Issue: COVID-19
Received: December 29, 2020
https://doi.org/10.1021/acs.jmedchem.0c02258
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Figure 1. Representatives of reported EV71 3C protease inhibitors.
but its activity against coronaviruses is weak. Compounds 15b
and 25c,d showed good anti-EV71 activity, and the selectivity of
compounds 35e and 45f toward other common mammalian
proteases was high, but their pharmaceutical properties were not
satisfied; no more research progresses have been reported. Our
compound (55g) exhibited broad-spectrum antiviral activity, but
its activity against EV71 was weak. As reported, the plasma
stability of compounds 65h and 75i was impressive, while their
pharmacokinetic properties needed to be further improved.
Although many inhibitors with different warheads had been
reported, there was still no effective drug on the market. With
those in mind, novel broad-spectrum antiviral inhibitors with
good pharmacokinetic properties and safety need to be
designed.
the EV71 3Cpro, which is the key point for maintaining the
antivirus activity. The (S)-γ-lactam ring at P1 position forms
hydrogen bonds with the crucial residues including Thr142 and
His161 in the S1 subsite, and the substituted phenyl group at the
P2 position occupies the S2 subsite well. The complex also
revealed that the isopropyl in the P3 moiety is solvent-exposed,
and the P4 moiety forms hydrogen bonds with Gly164, Asn145,
and Ser128.6h Unfortunately, the α,β-unsaturated ester is easily
hydrolyzed, the plasma stability of the AG7088 is poor, and its
half-life in rat plasma is less than 2 min; in addition, AG7088 was
inactive to the SARS-CoV 3CL protease (IC50 > 100 μM).7 We
want to overcome this shortcoming of AG7088 and obtain some
novel compounds.
In our previous work, peptidomimetic α-ketoamides (com-
pound 5) exhibited broad-spectrum antivirus activity.5g Former
reports showed that compound 1 also had good anti-EV71
activity (EC50 = 0.096 μM). After analyzing the crystal structure
of AG7088 with EV71 3Cpro and comparing the structure of
compound 1, compound 5, and AG7088, we found that they
shared similar important key pharmacophore fragments with a
warhead and (S)-γ-lactam ring. In this core structure, the
warheads could be covalently linked to Cys147, and the lactam
ring could interact with the crucial residues, and we envisioned
AG7088 showed better antivirus activity might benefit from the
additional interaction at the P3 and P4 position. When we
embarked on designing the novel compounds (Figure 3), the
key pharmacophore fragments in compounds 1, 5, and AG7088
were extracted, and then some common warheads were
investigated to get compounds 19a and 19b. Subsequently, we
introduced some heterocyclic moieties into the P3 position to
obtain the corresponding peptidomimetic aldehydes 18b−18p.
In a second approach, a valine moiety was introduced in the P3
position to keep the chain length similar to AG7088, and
different heterocycles were investigated in the P4 position to get
compounds 26a−26e.
RESULTS AND DISCUSSION
■
Compound Design. AG7088 has potent anti-EV71 activity
(EC50 = 0.009 μM), so we analyzed the crystal structure of EV71
3Cpro with AG7088 (Figure 2) for our compound’s design.6 The
results demonstrate that α,β-unsaturated ester of AG7088 forms
a covalent linkage with the Cys147 residue in the S1′ subsite of
Figure 2. X-ray structure of the surface representation of EV71 3Cpro
(PDB ID: 4GHT) complexed with the AG7088 (yellow).
B
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Figure 3. Design of novel EV71 3C protease inhibitors.
a
Scheme 1. Synthesis Procedure of Target Compounds
a
Reagents and conditions: (a) LiHMDS, THF, −78 °C; (b) NaBH4, CoCl2, 0 °C; (c) 4 M HCl, 12 h; (d) HATU, DIPEA, CH2Cl2, −20 °C, 12 h;
(e) 4 M HCl, 12 h; (f) HATU, DIPEA, CH2Cl2, −20 °C, 12 h; (g) NaBH4, CH3OH; (h) Dess−Martin periodinane, NaHCO3, CH2Cl2; (i)
Ph3PCH2COOR4, Et3N, DCM.
Chemistry. The synthetic routes and chemical structures of
the compounds (19a, 19b, and 18b−18p) are shown in Scheme
1. The starting material 8 was obtained from commercial
suppliers and used without further purification, and the key
C
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a
Scheme 2. Synthesis Procedure of Target Compounds
a
Reagents and conditions: (a) HATU, DIPEA, CH2Cl2, −20 °C, 12 h; (b) 4 M HCl, 12 h; (c) HATU, DIPEA, CH2Cl2, −20 °C, 12h; (d) NaBH4,
CH3OH; (e) Dess−Martin Periodinane, NaHCO3, CH2Cl2.
intermediate 115e was synthesized by following the literature.
The intermediate 13 was synthesized starting from the amino 11
and N-(t-butoxycarbonyl)-L-phenylalanine 12. After the t-
butoxycarbonyl group was removed from 13 by 4 M HCl in
dioxane, the intermediate 14 was obtained. A subsequent
coupling reaction between compound 14 and the corresponding
acids 15 resulted in esters 16a−16p. The peptidomimetic
aldehydes 1 and 18b−18p were approached via a two-step
route, in which the ester derivatives 16a−16p were first reduced
with NaBH4 to generate the primary alcohols 17a−17p, and
subsequently, 17a−17p were oxidized into aldehydes 1 and
18b−18p with Dess−Martin periodinane (DMP). Finally,
compounds 19a and 19b were obtained from 1 by the Wittig
reaction.
The synthetic procedure of compounds 26a−26e was shown
in Scheme 2. Compound 21 was obtained via a condensation
reaction between the same intermediate 14 with N-(t-
butoxycarbonyl)-L-valine 20, and then, the t-butoxycarbonyl
group of 21 was removed by 4 M HCl in dioxane to obtain 22.
Subsequently, compound 22 was coupled with the correspond-
ing acid 23 to afford esters 24a−24e. After completion of a
reduction of the esters 24a−24e, the desired products (25a-
25e) were reoxidized by DMP to obtain the final products 26a−
26e.
work. IC50 values of compound 1 were inconsistent with
previous reports, which might be caused by a difference in
enzyme concentration.5b These data indicated that the enzyme
inhibitory activity of peptidomimetic aldehyde (compound 1,
IC50 = 4.57 0.27 μM) was weaker than the compounds, in
which α,β-unsaturated ester was defined as the warhead
(compounds 19a and 19b), while compound 1 displayed better
anti-EV71 activity (EC50 = 0.10
0.01 μM) than α,β-
unsaturated methyl ester 19a (EC50 = 1.21 0.14 μM) and
α,β-unsaturated benzyl ester 19b (EC50 = 3.10 0.09 μM), and
the antiviral results showed that a small group might be more
suitable in P1′. Based on the antiviral activity, an aldehyde was
selected as a new warhead for further optimization. When
aldehyde on R2 was incorporated and the R1 moiety was
replaced with heterocyclic motifs, most of the target compounds
(18b−18l, 18n, and 18p) showed better 3Cpro inhibitory
activity (IC50 < 4.0 μM) than compound 1 (IC50 = 4.57 μM),
indicating that the introduction of the heterocyclic ring in P3
might be able to form additional interactions with the S4 subsite
to improve the inhibitory activity of 3Cpro. The inhibitory
activity of compound 11m was decreased, which might be due to
the fact that the nitrogen atom in this compound could not form
an additional interaction. As we know, the antiviral activity is a
result caused by multiple factors, which was related not only to
enzyme inhibitory activity but also the other properties such as
permeability. So the EC50 values deserved further discussion.
The enzyme inhibitory activity of monocyclic moiety-
substituted derivatives (18b, 18c) was increased, while a lower
antiviral activity was observed when comparing to compound 1.
Those results indicated that monocyclic moieties might not be a
good choice on R1, and then bicyclic heterocycles were
investigated. The anti-EV71 activities of compounds with
heterocyclic acene moieties 18d−18h were decreased, and the
Structure−Activity Relationship of the Compounds.
All the synthesized compounds were tested for inhibitory
activity of EV71 3Cpro and antiviral activity of EV71, and results
are summarized in Table 1 and Table 2.
Modification on R1 and R2. Structural modifications on R1
and R2 were first conducted, and a number of derivatives have
been designed, synthesized, and biologically evaluated. The
results were summarized in Table 1, and two compounds
(compound 1 and AG7088) were used as references in this
D
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Table 1. Enzyme Inhibitory Activity and Anti-EV71 Activities
of Peptidomimetic Aldehydes with R1 and R2 Modifications
Table 2. Enzyme Inhibitory Activity and anti-EV71 Activities
of Peptidomimetic Aldehydes with R3 Modifications
a
a
a
Each value represented the average results from three independent
experiments.
by introducing a valine motif at the P3 position, and most of the
target compounds (26a−26e) showed good enzyme inhibitory
activities (ranging from 2.43 μM to 7.49 μM), while the
inhibitory activities of those compounds were weaker than
AG7088 (IC50 = 1.89 μM). We proposed the reason might be
that, after replacing the carbon atom in AG7088 with a nitrogen
atom, the conformation of those compounds (26a−26e)
changes a lot, which made those compounds unable to occupy
the binding pocket very well, and α,β-unsaturated ester showed
more potent inhibitory activity against 3Cpro than aldehyde. The
results of antiviral activities showed that the introduction of 5-
methyl-1,2-oxazole and benzoheterocycles moiety on R3 made
the anti-EV71 activity of the corresponding compounds (26a−
26d) comparable to compound 1. Especially when the quinoline
group was introduced into the R3 position, compound 26d
showed good activity (EC50 = 0.043 0.010 μM), while the
activities of these tripeptide compounds were decreased
compared with compound 18p and AG7088. In addition,
a
Each value represented the average results from three independent
experiments.
introduction of 7-bromoimidazo[1,2-a]pyridine (compound
18i) also reduced the anti-EV71 activity. Subsequently,
benzoheterocycles were introduced on R1 (18j−18p), and
compounds 18j, 18k, and 18m had similar activities compared
to compound 1. The introduction of methyl on quinoxaline
(18l) reduced the antiviral activity; when R1 was replaced with a
substituted quinoline (18n), the anti-EV71 activity was also
increased. It was noteworthy that compound 18o (EC50 = 0.07
0.01 μM) showed better anti-EV71 activity than compound 1,
albeit the cytotoxicity of these compounds was slightly increased
(CC50 = 16.4 μM). The 2-indole scaffold was incorporated, and
the activity of 18p against EV71 (EC50 = 0.030 0.002 μM) was
increased. The discrepancy of the activities between compounds
including 18h and 18j with 18p indicated that the NH moiety in
the 2-indole scaffold was vital for maintaining the anti-EV71
activity.
compounds 26d and 18p exhibited safety cytotoxicity (CC50
82.8 μM and CC50 > 100 μM, respectively).
=
Crystal Structure of EV71 3Cpro in Complex with 18p.
To understand the binding mode of these inhibitors with the
protease, the complex structure of EV71 3Cpro bound with 18p
was determined at a resolution of 1.2 Å. Compound 18p bound
into the substrate-binding site at the surface of the protease and
occupied S1, S2, and S4 subsites (Figure 4A). At the S1′ subsite,
the aldehyde group of 18p covalently linked to the catalytic
Cys147 of the protease (Figure 4B,C). In addition to this
covalent bond, the oxygen atom of the aldehyde group
established H-bonds with the side chain of catalytic His40
directly and the NH of the Gly145 main chain (part of the
oxyanion hole) through a water molecule (Figure 4C). The (S)-
γ-lactam ring of 18p perfectly engaged within the S1 subsite. The
oxygen atom of the (S)-γ-lactam ring formed H-bonds with the
side chains of His161 and Thr142, while the nitrogen atom of
Modification on the R3 Position. On the other hand, as
showed in Table 2, structural modifications had been continued
E
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of 18p and Ser128, mediated by a water molecule (Figure S1B,
C).
Activity on a Panel of Enteroviruses and Rhinoviruses.
Considering that the 3C proteases are conserved between
different viruses, four compounds (1, 18p, 19a, and 26b) were
further tested against a panel of relevant enteroviruses and
rhinoviruses, and results were summarized in Table 3. The
results demonstrated that the derivatives with aldehyde warhead
had potent broad-spectrum antiviral activities. Compound 1
showed better broad-spectrum antiviral activity than the α, β-
unsaturated ester 19a. Additionally, the dipeptide compound
18p was proved to have more efficacy than the tripeptide
compound 26b. Besides, compounds 18p also exhibited
excellent anti-EV68 activity (EC50 = 0.03
0.01 μM), and
compounds 18p and 26b also exhibited high anti-CoxA21
activity (EC50 = 0.43 0.11 μM and EC50 = 0.51 0.01 μM,
respectively), while its antiviral activities against CoxB3 and
HRV were weak, which might be caused by the difference in
substrate binding pockets (Figure S2). According to these
results, compound 18p has a lot of potential to be accessed in a
broad-spectrum antiviral drug.
Antiviral Activity on SARS-CoV-2. In late December 2019,
an emerged coronavirus called SARS-CoV-2 (severe acute
respiratory syndrome coronavirus 2), causes the pandemic
COVID-19 (coronavirus disease 2019). There are no specific
antiviral drugs approved by the FDA except Remdesivir, so the
development of more effective antiviral drugs is of great
significance.8 SARS-CoV-2 is an enveloped, positive-sense,
single-stranded RNA virus that belongs to the β-lineage of the
coronavirus. The genome is translated into two polyproteins,
and then the polyproteins were cleaved by 3C-like protease
(3CLpro, also named the main protease) and papain-like
proteinase (PLpro). The majority of this proteolytic processing
utilizes the 3CLpro, which plays a vital role in SARS-CoV-2′s
replication. Unlike enterovirus 3Cpro, the active form of the
SARS-CoV-2 3CLpro is a dimer, and it has a catalytic dyad
containing cysteine and histidine.9,10 As we know, the 3Cpro of
EV71 and 3CLpro of SARS-CoV-2 share some similarities; for
example, both the catalytic sites contain cysteine and histidine,
and a glutarnine (Gln) is almost always required in the P1
position of their substrates. In our previous work, we found the
active sites of 3Cpro and 3CLpro also are similar and usually
composed of four sites: S1′, S1, S2, and S4.5g,9d Recently, many
inhibitors targeting SARS-CoV-2 3CLpro have been reported,
and most of them shared similar key pharmacophore fragments
(warhead and lactam ring) and exhibited good inhibitor activity
against 3CLpro and replication of SARS-CoV-2 (Figure 5).9b,c,11
Among them, two peptide inhibitors developed by Pfizer (PF-
007304814 is a phosphate prodrug of PF-00835231)11b and
our group (compound 29)9d show potent antiviral activity and
good safety, and both have entered phase I clinical trials. Our
antienterovirus compounds show some similarity to compound
Figure 4. Crystal structure of the EV71 3Cpro in complex with 18p. (A)
The binding mode of 18p at the substrate-binding site of the EV71
3Cpro (PDB code: 7DNC). The EV71 3Cpro was shown as a molecular
surface, and 18p was shown by light orange sticks. (B) 2Fo-Fc density
maps contoured at 2.0 σ are shown for 18p and C147. (C) Interactions
of 18p with the surrounding residues revealed by the crystal structure.
Residues are shown as light blue sticks, and H-bonds are represented by
black dashed lines.
the (S)-γ-lactam ring formed a H-bond with the main chain of
Thr142. The benzyl ring of 18p occupied the S2 subsite by
forming hydrophobic interactions with His40, Glu71, and
Leu127. The indole group of 18p fitted into the S4 subsite and
donated a H-bond to the main chain of Gly164. In addition, the
amide bonds of 18p also participated in binding by establishing
H-bonds with Ile162 and Gly164 directly and with Ser128
through a water molecule (Figure 4C).
A structure overlay of the EV71 3Cpro-18p and the EV71
3Cpro-AG7088 complexes revealed that these two compounds
adopted similar binding modes (Figure S1A). The difference
mainly lied in the interactions with the S1′ and S4 subsites.
AG7088 covalently bound to the catalytic Cys147 with its α,β-
unsaturated ketone, while the aldehyde group of 18p was used to
covalently link to the catalytic Cys147. Simultaneously, the
oxygen atom of the aldehyde group participated in forming
multiple H-bonds with the surrounding residues including the
catalytic His40. At the S4 subsite, the indole ring of 18p flipped
up and established a shorter H-bond (2.9 Å) with Gly164
compared to the H-bond (3.1 Å) between AG7088 and Gly164.
In addition, a new H-bond was formed between the amide bond
a
Table 3. Activity of Inhibitors against a Panel of Enteroviruses and Rhinoviruses
compd
EV71 EC50 (μM)
EV68 EC50 (μM)
CoxA21 EC50 (μM)
CoxB3 EC50 (μM)
RV-A02-WT EC50 (μM)
RV-B14-WT EC50 (μM)
1
0.10 0.01
0.030 0.002
1.21 0.10
0.12 0.02
0.08 0.03
0.03 0.01
0.10 0.01
0.26 0.10
1.73 0.82
0.43 0.11
3.62 1.19
0.51 0.01
15.87
4.19
77.67
9.15
6.60
1.62
1.68
1.02
1.19
0.81
1.65
0.98
18p
19a
26b
a
The value of EV71, EV68, and CoxA21 represented the average results from three independent experiments.
F
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Figure 5. Representatives of reported SARS-CoV-2 3CL protease inhibitors
29, so some 3Cpro inhibitors were selected to be further tested
against 3CLpro as well as the replication of SARS-CoV-2.
The results of enzyme inhibitory activity and anti-SARS-CoV-
2 activity were summarized in Table 4. Most of the compounds
inhibitory activities were decreased (18m, 18n, and 26d). In
our previous work, the 2-indole moiety could form an additional
H-bond with Glu166 in the S4 subsite.9d When 2-quinoline and
the derivative (18m, 18n, and 18o) were introduced, the H-
bond could not be formed, so this might be the reason for the
decrease of inhibitory activity against 3CLpro. Then, the antiviral
activity against SARS-CoV-2 was evaluated, of which six
benzoheterocyclic dipeptide compounds exhibited excellent
inhibitory activity (EC50 < 0.5 μM), and the EC50 values of 18o
and 18p were 0.25 μM and 0.29 μM, respectively. In addition,
the selection index of 18p (SI = 2786) is better than 18o (SI =
1192). The results showed the inhibitory activities of the
tripeptide compounds (26a−26d) were greater than 1 μM, and
the reason might be that the tripeptide compounds showed poor
membrane permeability.11 Those results indicate the compound
18p also is a good starting point for further optimization as a
SARS-CoV-2 inhibitor.
Preliminary Pharmacokinetic (PK) Evaluation of
Compounds 18p and 26d. To explore the further
druggability of the novel peptide aldehydes, compounds 18p
and 26d were evaluated for their pharmacokinetic properties in
mice after intraperitoneal (20 mg/kg), subcutaneous (5 mg/kg),
and intravenous (5 mg/kg) administration. As shown in Table 5,
compound 18p given intraperitoneally and subcutaneously
displayed a much higher area under the curve (AUC) value than
that of 26d. Compound 18p also displayed a longer half-life
(T1/2) of 5.85 h when administrated intravenously. Those results
indicate that compound 18p has better PK properties than 26d
Table 4. Enzyme Inhibitory Activities and Anti-SARS-CoV-2
a
Activities of Peptidomimetic Aldehydes
compound
IC50 (μM)
EC50 (μM)
CC50 (μM)
18d
18e
18g
18j
0.078 0.016
0.059 0.005
0.097 0.017
0.080 0.002
0.065 0.011
0.140 0.012
0.240 0.042
0.120 0.003
0.034 0.004
0.067 0.014
0.068 0.012
0.067 0.014
0.18 0.029
1.35 0.16
0.76 0.26
8.21 0.68
0.49 0.001
0.44 0.04
0.43 0.05
0.35 0.03
0.25 0.04
0.29 0.06
>2
>1000
>1000
>1000
>1000
18l
602.6
18m
18n
18o
18p
26a
26b
26c
26d
627.2 16.3
823.1 32.2
298.7 4.9
808.7 20.4
>1000
>1000
>1000
604.1 5.9
4.22 0.25
5.62 1.26
4.12 0.52
a
Each value represented the average results from three independent
experiments
showed excellent inhibitory activity of 3CLpro (IC50 < 0.10 μM),
especially when the IC50 value of 18p was 0.034 μM. However,
when the quinoline group was incorporated, the enzyme
a
Table 5. Preliminary Pharmacokinetic (PK) Evaluation of Compounds 18p and 26d
T
T
C
AUC
AUC
CL
MRT
(h)
Vss
_obs
F
1/2
max
max
last
INF_obs
compd admin
(h)
(h)
0.25
0.58 0.29
(ng/mL)
(h ng/mL)
(h ng/mL)
(mL/min/kg)
(mL/kg)
(%)
18p
ip
sc
iv
ip
sc
iv
5.36 1.12
4.96 0.71
5.85 0.75
5.38 0.28
5.03 4.30
2.98 2.50
14572 3105
2762 689
15952 3468
3568 490
1732 161
4637 472
1321 222
1378 116
16080 3559
3579 489
1745 163
4651 472
1338 231
1390 114
1.69 0.60
1.34 0.21
1.42 0.12
1.11 0.01
1.82 0.89
0.98 0.06
230
206
48.0 4.7
60.2 4.7
4119 676
3536 298
26d
0.25
0.25
4542 457
1159 46
84.2
96
a
The value represented the average results from three independent experiments.
G
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purified by flash column chromatography (petroleum ether/ethyl
acetate = 4:1) to give product 9 (7.58 g, 55%) as a colorless oil. H
NMR (600 MHz, CDCl3): δ 5.11 (d, J = 7.5 Hz, 1H), 4.38 (s, 1H), 3.77
(s, 3H), 3.75 (s, 3H), 2.92−2.82 (m, 1H), 2.81−2.71 (m, 2H), 2.24−
2.08 (m, 2H), 1.44 (s, 9H).
to warrant further study. The reason why the bioavailability of
compound 18p is greater than 100% may be due to changes in
the clearance rate in different routes of administration, and the
clearance rate of 18p is fast by intravenous injection.12
1
Then, in a round-bottomed flask, compound 9 (6.0 g, 19.09 mmol)
was dissolved in anhydrous MeOH (100 mL) before CoCl2·6H2O
(2.72 g, 11.45 mmol) was added at 0 °C. Subsequently, NaBH4 (4.35 g,
114.78 mmol) was added porionwise, and the reaction mixture was
warmed to room temperature and stirred for 12 h. After the reactant was
consumed, the reaction was quenched by NH4Cl (30 mL). MeOH in
the mixture was evaporated, and the residual mixture was extracted with
ethyl acetate (50 mL × 3). The organic layers were washed by saturated
NH4Cl solution (100 mL × 3) and brine (100 mL × 3); then, the
organic phase was dried (MgSO4) and concentrated. The residue was
purified by flash column chromatography (petroleum ether/ethyl
CONCLUSION
■
In summary, a series of novel protease inhibitors with an
aldehyde warhead was designed, synthesized, and biologically
evaluated on EV71 by analyzing the crystal structure of EV71 3C
protease with AG7088. Most of the compounds have potent
inhibitory activity of 3Cpro and EV71. The SAR study indicated
that the introduction of aldehyde at P1′ position presented
better antiviral activities than the α, β-unsaturated ester.
Heteroaromatic scaffolds were introduced at the R1 group,
and most of the compounds displayed good antiviral activities,
especially when R1 is an indole moiety (18p). Compound 18p
1
acetate = 2:1) to give product 10 (2.18g, 40%) as a white solid. H
NMR (600 MHz, CDCl3): δ 6.64 (s, 1H), 5.56 (s, 1H), 4.29 (d, J = 9.1
Hz, 1H), 3.71 (s, 3H), 3.37−3.26 (m, 2H), 2.47−2.42 (m, 2H), 2.13−
2.08 (m, 1H), 1.84−1.81 (m, 2H), 1.41 (s, 9H).
not only has high inhibitory activity of 3Cpro and EV71 (3Cpro
:
IC50 = 2.36 μM, EV71: EC50 = 0.030 μM), but also against EV68
(EC50 = 0.03 μM), CoxA21 (EC50 = 0.43 μM) and RV-B14-WT
(EC50 = 0.81 μM). It also showed moderate activity against
CoxB3 (EC50 = 4.19 μM) and RV-A02-WT (EC50 = 1.62 μM)
and low toxicity (CC50 > 100 μM). The crystal structure of EV71
3Cpro in complex with 18p was also determined at a resolution of
1.2 Å. It showed that 18p fitted into the S1′, S1, S2, and S4 sites
perfectly and established multiple H-bonds with the surround-
ing residues including the catalytic His40. In addition, the
aldehyde group of 18p covalently bound to the catalytic Cys147,
which was essential for maintaining the potency of these newly
designed inhibitors, and the NH in the indole group formed a
hydrogen bond with the main chain of Gly164. Some
compounds were further evaluated against 3CLpro and SARS-
CoV-2, and the 18p also showed excellent inhibitory activity
(3CLpro: IC50 = 0.034 μM, SARS-CoV-2: EC50 = 0.29 μM). In
addition, this class of compounds also has no reasons for
concern regarding acute toxicity.9d Compared with AG7088,
compound 18p exhibited good PK properties and more potent
anticoronavirus activity, and it is an excellent starting point for
further optimization toward a broad-spectrum antiviral drug.
Compound 10 (1.0 g, 3.5 mmol) was dissolved in 10 mL of DCM;
then, the HCl (9 mL, 4 M in dioxane) was added. The reaction mixture
was stirred at 20 °C for 12 h, and the mixture was concentrated in vacuo
to get a white solid 11, which could be used for the following step
without purification.
Synthesis Procedure of Compound 13. To a solution of Boc-L-Phe-
OH 12 (1.1 g, 3.5 mmol) in DCM (40 mL) was added HATU (1.9 g,
4.9 mmol) sequentially at −20 °C, and then the residue concentrated
crude product 11 (0.77g 3.5 mmol) was added. After 30 min later,
DIPEA (1.7 mL, 10.5 mmol) was added dropwise. Then, the reaction
mixture was stirred at −20 °C for 12 h. The resulting mixture was
washed by saturated ammonium chloride solution (100 mL × 3),
saturated NaHCO3 solution (100 mL × 3), and brine (100 mL × 3).
The organic phase layer was dried over Na2SO4 and concentrated in
vacuo. The resulting mixture residue was purified by column
chromatography (DCM/CH3OH, 40:1 v/v) to afford the pure product
13 (1.26 g, 83%) as a light solid. 1H NMR (400 MHz, CDCl3): δ 7.61
(d, J = 7.0 Hz, 1H), 7.21 (m, 5H), 6.82 (s, 1H), 5.26 (s, 1H), 4.50 (d, J =
6.8 Hz, 2H), 3.69 (s, 3H), 3.36−3.25 (m, 2H), 3.12 (m, 1H), 2.99 (dd, J
= 13.4, 7.3 Hz, 1H), 2.34 (s, 2H), 2.19−2.10 (m, 1H), 1.81 (m, 2H),
1.35 (s, 9H).
General Synthesis Procedure of Compounds 16a−16p. To a dry
100 mL flask in which 13 (1.5 g, 3.5 mmol) was dissolved with dry
DCM was added 4 M HCl (9 mL, 35 mmol) slowly at 20 °C, and the
resulting mixture was stirred at an ambient temperature for 12 h. The
solvent was removed in vacuo, and the crude product 14 was directly
used in the next step without further purification. Then, trans-3-
phenylacrylic acid 15a (0.49 g, 1.5 mmol) was dissolved in a dry 100 mL
flask with CH2Cl2, HATU (0.68g, 1.8 mmol) was added sequentially at
−20 °C, and then compound 14 (0.55g 1.5 mmol) was added. DIPEA
(0.73 mL, 4.5 mmol) was added dropwise after 30 min. Then, the
reaction mixture was stirred at −20 °C for 12 h, followed by washing
with a saturated ammonium chloride solution (100 mL × 3), saturated
NaHCO3 solution (100 mL × 3), and brine (100 mL × 3). The organic
phase was dried over Na2SO4 and concentrated, and the residue was
purified by column chromatography (CH2Cl2/CH3OH, 30:1 v/v) to
afford the pure product 16a (0.55 g, 80%) as a light solid. 1H NMR (600
MHz, acetone-d6): δ 8.45 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H),
7.54−7.47 (m, 3H), 7.37−7.31 (m, 5H), 7.26 (t, J = 7.6 Hz, 2H), 7.21−
7.16 (m, 2H), 6.75 (d, J = 15.7 Hz, 1H), 5.01 (td, J = 8.4, 5.1 Hz, 1H),
4.53 (m, 1H), 3.69 (s, 3H), 3.33−3.24 (m, 3H), 3.04 (dd, J = 13.9, 8.5
Hz, 1H), 2.46 (dd, J = 9.8, 4.7 Hz, 1H), 2.31 (dd, J = 7.4, 4.8 Hz, 1H),
2.24−2.15 (m, 1H), 1.79 (m, 2H).
EXPERIMENTAL SECTION
■
General Methods. The materials and solvents were purchased
from commercial sources and used without further purification. All
products were characterized by their NMR and MS spectra. 1H and 13
C
NMR spectra were recorded on a 400, 500, or 600 MHz instrument.
Compounds were purified by chromatography with silica gel (300−400
mesh). Analytical thin-layer chromatography (TLC) was HSGF 254
(0.15−0.2 mm thickness). Preparative thin-layer chromatography
(PTLC) was HSGF 254 (0.4−0.5 mm thickness). High-resolution
mass spectra (HRMS) were measured on a Micromass Ultra Q-TOF
spectrometer. HPLC analysis of all final compounds was performed on
Agilent-1100 HPLC with a binary pump and photodiode array detector
(DAD), using an Agilent Extend-C18 column (150 mm × 4.6 mm, 5
μm). All final compounds were analyzed using MeOH/H2O = 70:30
(v/v) (0.8 mL/min), and all of them had an at least 95% purity.
Synthesis Procedure of Compounds. Synthetic Procedure of
Compounds 9−11. The solution of lithium bis(trimethylsilyl)amide
(LHMDS) (94 mL, 1 M in THF) was added dropwise to a solution of
N-Boc-L-glutamic acid dimethyl ester (8) (12.0 g, 43.6 mmol) in THF
(100 mL) at −78 °C; then, the mixture was stirred at −78 °C for 1 h.
Subsequently, bromoacetonitrile (3.24 mL, 46.6 mmol) was added
dropwise to the mixture under the temperature of −78 °C, and the
reaction was kept at −78 °C for an additional 4 h. After the reactant was
consumed, the reaction was quenched by NH4Cl (40 mL). The
reaction mixture was warm up to room temperature and extracted with
ethyl acetate (50 mL × 3). The organic layers were concentrated and
General Synthesis Procedure of Compounds 17a−17p. In a dry
100 mL flask was dissolved 16a (0.92 g, 2.0 mmol) in dry THF, NaBH4
(0.6 g, 16 mmol) was added slowly at 0 °C, and then, the reaction
mixture was stirred at rt for 3 h. The completion of the reaction was
confirmed by TLC; then the reaction was quenched and concentrated
to get a crude residue. The residue was dissolved in DCM and washed
with saturated ammonium chloride solution (50 mL × 3), saturated
H
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NaHCO3 solution (50 mL × 3), and brine (50 mL × 3). The organic
phase was dried over Na2SO4 and concentrated, and the residue was
purified by column chromatography (DCM/CH3OH, 20:1 v/v) to
afford the pure product 17a (0.77 g, 90%) as a light solid. 1H NMR (500
MHz, methanol-d4): δ 7.97 (d, J = 8.8 Hz, 1H), 7.55−7.48 (m, 3H),
7.40−7.34 (m, 3H), 7.32−7.28 (m, 4H), 7.23 (td, J = 6.0, 3.2 Hz, 2H),
6.67 (d, J = 15.8 Hz, 1H), 4.74 (dd, J = 8.1, 6.7 Hz, 1H), 3.98−3.91 (m,
1H), 3.44 (dd, J = 11.0, 5.0 Hz, 1H), 3.34−3.22 (m, 4H), 3.17 (dd, J =
13.7, 6.6 Hz, 1H), 3.05 (dd, J = 13.7, 8.3 Hz, 1H), 2.46 (dd, J = 9.8, 2.5
Hz, 1H), 2.37−2.27 (m, 1H), 1.98−1.89 (m, 1H), 1.81−1.69 (m, 1H),
1.55 (m, 1H).
General Synthesis Procedure of Compounds 1 and 18b−18p. To
a solution of the 17a (0.87 g, 2.0 mmol) in CH2Cl2 was added DMP
(1.01 g, 2.4 mmol) slowly, and the reaction mixture was stirred at room
temperature for 5 h. The completion of the reaction was confirmed by
TLC, the reaction was quenched and concentrated, and the reaction
was filtered and washed with saturated NaHCO3 solution (50 mL × 3)
and brine (50 mL × 3). The organic phase was dried over Na2SO4 and
concentrated, and the residue was purified by column chromatography
(DCM/CH3OH, 20:1 v/v) to afford the pure product 1 (0.65 g, 76%)
as a light solid. 1H NMR (500 MHz, acetone-d6): δ 9.38 (s, 1H), 7.57
(dt, J = 10.6, 8.4 Hz, 4H), 7.42−7.36 (m, 3H), 7.34−7.25 (m, 4H), 7.22
(d, J = 6.7 Hz, 1H), 6.84 (s, 1H), 6.80−6.72 (m, 1H), 4.90 (dd, J = 7.9,
5.7 Hz, 1H), 4.54−4.19 (m, 1H), 4.06−3.80 (m, 1H), 3.29−3.16 (m,
3H), 3.11 (m, 1H), 2.40−2.17 (m, 2H), 1.97 (dd, J = 14.1, 3.8 Hz, 1H),
1.82−1.48 (m, 2H).
General Synthesis Procedure of Compounds 19a and 19b. To a
solution of the 1 (0.86 g, 2.0 mmol) in CH2Cl2 were added methyl-
(triphenylphosphoranylidene) acetate (0.80 g, 2.4 mmol) and Et3N
(0.56 mL, 4 mmol); then the reaction mixture was stirred at room
temperature for 12 h. The completion of the reaction was confirmed by
TLC; then the reaction was washed with saturated ammonium chloride
solution (50 mL × 3), saturated NaHCO3 solution (50 mL × 3), and
brine (50 mL × 3). The organic phase was dried over Na2SO4 and
concentrated, and the residue was purified by column chromatography
(DCM/CH3OH, 40:1 v/v) to afford the pure product 19a (0.78 g,
78%) as a light solid.
Synthesis Procedure of Compound 21. To a solution of Boc-L-Val-
OH 20 (0.76 g, 3.5 mmol) in DCM (40 mL) was added HATU (1.9 g,
4.9 mmol) sequentially at −20 °C, and then the residue concentrated
crude product 14 (1.28g 3.5 mmol) was added. After 30 min later,
DIPEA (1.7 mL, 10.5 mmol) was added dropwise. Then, the reaction
mixture was stirred at −20 °C for 12 h. The resulting mixture was
washed by saturated ammonium chloride solution (100 mL × 3),
saturated NaHCO3 solution (100 mL × 3), and brine (100 mL × 3).
The organic phase layer was dried over Na2SO4 and concentrated in
vacuo. The resulting mixture residue was purified by column
chromatography (DCM/CH3OH, 40:1 v/v) to afford the pure product
21 (1.58 g, 85%) as a light solid. 1H NMR (600 MHz, CDCl3): δ 7.67
(d, J = 7.7 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.24−7.14 (m, 6H), 7.01
(s, 1H), 5.11 (d, J = 9.1 Hz, 1H), 4.97−4.91 (m, 1H), 4.52 (t, J = 8.1 Hz,
1H), 3.84 (t, J = 8.1 Hz, 1H), 3.66 (s, 3H), 3.31 (m, 2H), 3.07 (d, J = 5.9
Hz, 2H), 2.39−2.27 (m, 1H), 2.18−2.06 (m, 2H), 1.99 (dd, J = 13.6,
6.8 Hz, 1H), 1.80−1.71 (m, 2H), 1.43 (s, 9H), 0.87 (d, J = 6.6 Hz, 3H),
0.82 (d, J = 6.6 Hz, 3H).
3.01 (dd, J = 13.8, 8.0 Hz, 1H), 2.49−2.40 (m, 1H), 2.23 (m, 3H),
1.92−1.77 (m, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H).
General Synthesis Procedure of Compounds 25a−25e. In a dry
100 mL flask was dissolved the 24d (0.88 g, 1.5 mmol) in dry THF,
NaBH4 (0.45 g, 12 mmol) was added slowly at 0 °C, and then the
reaction mixture was stirred at rt for 3 h. The completion of the reaction
was confirmed by TLC; then the reaction was quenched and
concentrated to get a crude residue. The residue was dissolved in
DCM and washed with saturated ammonium chloride solution (50 mL
× 3), saturated NaHCO3 solution (50 mL × 3), and brine (50 mL × 3).
The organic phase was dried over Na2SO4 and concentrated, and the
residue was purified by column chromatography (DCM/CH3OH, 20:1
v/v) to afford the pure product 25d (0.71 g, 85%) as a light solid. 1H
NMR (400 MHz, CDCl3): δ 8.60 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.5
Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.93 (t, J = 8.1 Hz, 2H), 7.84 (d, J =
7.7 Hz, 1H), 7.65 (t, J = 7.1 Hz, 1H), 7.62−7.52 (m, 2H), 7.19 (d, J =
7.3 Hz, 2H), 7.09 (t, J = 7.6 Hz, 2H), 7.02−6.87 (m, 2H), 4.91 (dd, J =
14.4, 8.4 Hz, 1H), 4.45 (dd, J = 8.0, 6.7 Hz, 1H), 4.10−3.98 (m, 1H),
3.57 (ddd, J = 33.4, 11.4, 4.6 Hz, 2H), 3.43−3.23 (m, 3H), 3.17 (dd, J =
13.7, 5.7 Hz, 1H), 3.02 (dd, J = 13.7, 8.4 Hz, 1H), 2.35 (m, 3H), 2.16−
2.00 (m, 1H), 1.78 (dd, J = 11.5, 9.3 Hz, 1H), 1.57 (m, 1H), 0.97 (d, J =
6.7 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H).
General Synthesis Procedure of Compounds 26a−26e. To a
solution of the 25d (0.56 g, 1.0 mmol) in CH2Cl2 was added DMP
(0.51 g, 1.2 mmol) slowly, and the reaction mixture was stirred at room
temperature. The completion of the reaction was confirmed by TLC;
then, the reaction was quenched and concentrated, and the reaction was
filtered and washed with saturated NaHCO3 solution (50 mL × 3) and
brine (50 mL × 3). The organic phase was dried over Na2SO4 and
concentrated, and the residue was purified by column chromatography
(DCM/CH3OH, 20:1 v/v) to afford the pure product 26d (0.42 g,
76%) as a light solid.
Methyl (S,E)-4-((S)-2-Cinnamamido-3-phenylpropanamido)-5-
((S)-2-oxopyrrolidin-3-yl)pent-2-enoate (19a). 1H NMR (500 MHz,
acetone-d6): δ 8.15 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.77−
7.61 (m, 1H), 7.55 (s, 1H), 7.52 (dd, J = 6.7, 2.6 Hz, 2H), 7.39−7.34
(m, 3H), 7.31 (d, J = 7.2 Hz, 2H), 7.26 (t, J = 7.6 Hz, 3H), 7.21−7.18
(m, 1H), 7.15 (s, 1H), 6.86 (dd, J = 15.7, 5.3 Hz, 1H), 6.80 (d, J = 15.8
Hz, 1H), 5.83 (dd, J = 15.7, 1.6 Hz, 1H), 4.96 (dd, J = 14.8, 7.6 Hz, 1H),
4.75−4.68 (m, 1H), 3.69 (s, 3H), 3.26−3.22 (m, 1H), 3.11 (dd, J =
13.6, 7.6 Hz, 1H), 2.30−2.23 (m, 1H), 2.05−1.97 (m, 2H), 1.80−1.73
(m, 1H), 1.61 (td, J = 9.7, 4.9 Hz, 1H); 13C NMR (125 MHz, acetone-
d6): δ 179.4, 171.4, 166.2, 165.4, 148.5, 140.0, 137.5, 135.2, 132.0,
131.9, 131.8, 129.4, 128.8, 128.7, 128.6, 128.3, 127.7, 126.6, 121.6,
120.1, 55.2, 50.8, 48.5, 40.0, 38.0, 35.2. HRMS (ESI) m/z: [M − H]−
calcd for C28H30N3O5, 488.2191; found, 488.2182. Purity: 98.6%.
Benzyl (S,E)-4-((S)-2-Cinnamamido-3-phenylpropanamido)-5-
((S)-2-oxopyrrolidin-3-yl)pent-2-enoate (19b). 1H NMR (600 MHz,
CDCl3): δ 7.90 (s, 1H), 7.55 (dd, J = 15.6, 2.7 Hz, 1H), 7.47−7.41 (m,
2H), 7.40−7.29 (m, 9H), 7.19 (d, J = 4.1 Hz, 4H), 7.10 (m, 1H), 6.92
(s, 1H), 6.72 (dd, J = 15.6, 5.4 Hz, 2H), 6.47 (d, J = 15.6 Hz, 1H), 5.76
(d, J = 15.7 Hz, 1H), 5.15 (s, 2H), 5.08 (d, J = 6.7 Hz, 1H), 4.49 (d, J =
5.3 Hz, 1H), 3.25 (m, 2H), 3.19−3.13 (m, 1H), 3.07 (dd, J = 13.5, 7.2
Hz, 1H), 2.27 (s, 2H), 1.95 (t, J = 10.8 Hz, 1H), 1.73−1.62 (m, 1H),
1.54−1.47 (m, 1H), 1.26 (d, J = 1.3 Hz, 1H). 13C NMR (150 MHz,
CDCl3): δ 179.7, 170.9, 165.6, 147.1, 141.2, 135.9, 135.5, 134.2, 129.4,
129.1, 128.4, 128.2, 128.1, 127.5, 126.6, 120.5, 119.9, 66.0, 54.1, 48.8,
48.7, 40.3, 38.7, 37.9, 34.4, 28.0. HRMS (ESI) m/z: [M − H]− calcd for
C34H34N3O5, 564.2504; found, 564.2508. Purity: 98.1%.
5-Methyl-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl)amino)-3-phenylpropan-2-yl)isoxazole-3-carboxa-
mide (18b). 1H NMR (600 MHz, acetone-d6): δ 11.14 (d, J = 8.4 Hz,
1H), 9.41 (s, 1H), 8.28−8.23 (m, 1H), 7.78 (dd, J = 12.5, 8.8 Hz, 1H),
7.58 (d, J = 1.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.37−7.33 (m, 2H),
7.23 (d, J = 7.7 Hz, 2H), 7.16 (dd, J = 6.3, 1.9 Hz, 4H), 5.44 (dd, J =
70.6, 8.0 Hz, 1H), 5.06 (dd, J = 7.1, 4.5 Hz, 1H), 4.51 (m, 1H), 4.13−
3.99 (m, 1H), 3.32 (d, J = 2.0 Hz, 1H), 3.20 (m, 2H), 2.44−2.34 (m,
1H), 2.33−2.25 (m, 1H), 1.79−1.55 (m, 2H).13C NMR (125 MHz,
acetone-d6): δ 199.6, 179.0, 171.0, 170.8, 158.3, 136.8, 129.0, 127.9,
126.2, 100.7, 57.0, 54.0, 39.6, 37.4, 29.3, 27.6, 10.8. HRMS (ESI) m/z:
General Synthesis Procedure of Compounds 24a−24e. To a dry
100 mL flask in which 21 (1.86 g, 3.5 mmol) was dissolved in dry DCM
was added 4 M HCl (9 mL, 35 mmol) slowly at 20 °C, and the resulting
mixture was stirred at an ambient temperature for 12 h. The solvent was
removed in vacuo, and the crude product 22 was directly used in next
step without further purification. Then compound 22 was coupled with
quinaldic acid 23d to get the esters 24d (0.96 g, 80%), and the synthesis
procedure is similar to 17a. 1H NMR (400 MHz, CDCl3): δ 8.60 (d, J =
9.5 Hz, 1H), 8.31 (d, J = 8.5 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.11 (d, J
= 8.5 Hz, 1H), 7.97−7.86 (m, 2H), 7.81−7.75 (m, 1H), 7.72 (d, J = 8.4
Hz, 1H), 7.67−7.59 (m, 1H), 7.27 (s, 1H), 7.16 (d, J = 7.3 Hz, 2H),
7.02 (t, J = 7.6 Hz, 2H), 6.81 (t, J = 7.4 Hz, 1H), 5.00 (td, J = 8.3, 5.1 Hz,
1H), 4.64 (ddd, J = 11.6, 8.4, 3.2 Hz, 1H), 4.39 (dd, J = 9.2, 8.2 Hz, 1H),
3.69 (s, 3H), 3.40 (t, J = 8.6 Hz, 2H), 3.18 (dd, J = 13.8, 5.0 Hz, 1H),
I
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[M − H]− calcd for C21H23N4O5, 411.1674; found, 411.1682. Purity:
95.0%.
125.8, 125.1, 120.4, 119.9, 110.4, 102.1, 57.1, 54.6, 39.5, 37.4. HRMS
(ESI) m/z: [M − H]− calcd for C25H25N4O4, 445.1881; found,
445.1873. Purity: 95.1%.
5-Fluoro-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl)amino)-3-phenylpropan-2-yl)picolinamide (18c). 1H
NMR (600 MHz, acetone-d6): δ 9.42 (s, 1H), 8.87 (d, J = 12.0 Hz,
1H), 8.75 (d, J = 6.0 Hz, 1H), 8.63 (d, J = 2.7 Hz, 1H), 8.24 (d, J = 8.1
Hz, 1H), 7.98−7.94 (m, 1H), 7.37 (d, J = 7.3 Hz, 2H), 7.30 (dd, J =
10.4, 4.7 Hz, 2H), 7.22 (m, 1H), 5.01 (m, 1H), 4.32−4.27 (m, 1H),
3.38−3.34 (m, 1H), 3.31−3.27 (m, 1H), 3.19 (dd, J = 13.9, 8.9 Hz,
1H), 2.51−2.40 (m, 1H), 2.39−2.33 (m, 1H), 1.97−1.88 (m, 1H), 1.82
(m, 1H), 1.39 (s, 1H), 1.31 (d, J = 2.5 Hz, 2H). 13C NMR (150 MHz,
acetone-d6): δ 200.1, 179.3, 171.7, 163.9, 160.0, 158.3, 144.7, 140.3,
137.7, 129.4, 128.3, 126.6, 121.7, 58.0, 55.3, 40.1, 38.2, 37.6, 30.8.
HRMS (ESI) m/z: [M − H]− calcd for C22H22FN4O4, 425.1631;
found, 425.1631. Purity: 95.7%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl) propan-2-
yl)amino)-3-phenylpropan-2-yl)benzo[d][1,3]dioxole-5-carboxa-
mide. (18d). 1H NMR (500 MHz, CDCl3): δ 9.25 (s, 1H), 8.47 (d, J =
6.3 Hz, 1H), 7.32−7.20 (m, 8H), 6.89 (s, 1H), 6.78−6.73 (m, 1H), 5.97
(s, 2H), 5.16−4.92 (m, 1H), 4.44−4.24 (m, 1H), 3.31−3.21 (m, 3H),
2.33 (m, 2H), 2.03−1.45 (m, 3H). 13C NMR (125 MHz, CDCl3): δ
199.9, 180.0, 172.4, 166.5, 150.5, 147.8, 136.6, 129.5, 128.5, 127.9,
126.9, 122.1, 107.9, 107.7, 101.7, 57.7, 54.6, 40.6, 38.8, 38.0, 29.7, 28.4.
HRMS (ESI) m/z: [M − H]− calcd for C24H24N3O6, 450.1671; found,
450.1663. Purity: 96.7%.
6-Bromo-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl)amino)-3-phenylpropan-2-yl)imidazo[1,2-a]pyridine-
1
2-carboxamide (18i). H NMR (600 MHz, acetone-d6): δ 9.40 (s,
1H), 8.79 (s, 1H), 8.53 (d, J = 6.2 Hz, 1H), 8.27 (d, J = 4.9 Hz, 1H),
8.04 (d, J = 7.3 Hz, 1H), 7.58−7.52 (m, 1H), 7.47−7.40 (m, 1H), 7.32
(t, J = 6.4 Hz, 2H), 7.28−7.22 (m, 2H), 7.18 (t, J = 7.2 Hz, 1H), 6.91 (s,
1H), 5.03−4.93 (m, 1H), 4.44 (m, 1H), 3.32 (t, J = 6.9 Hz, 1H), 3.27−
3.23 (m, 1H), 2.45−2.26 (m, 2H), 1.99−1.94 (m, 1H), 1.80−1.75 (m,
1H), 1.37 (s, 1H), 1.29 (d, J = 3.3 Hz, 2H).13C NMR (150 MHz,
acetone-d6): δ 200.0, 178.9, 171.4, 161.5, 142.8, 137.3, 129.5, 129.3,
128.3, 128.2, 127.6, 126.6, 118.7, 114.8, 107.2, 98.7, 57.6, 54.0, 39.9,
38.4, 37.8, 30.8. HRMS (ESI) m/z: [M + H] + calcd for C24H25BrN5O4,
526.1084; found, 526.1093. Purity: 98.8%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)benzofuran-2-carboxamide (18j).
1H NMR (500 MHz, acetone-d6): δ 9.43 (s, 1H), 8.64 (d, J = 6.8
Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.56−7.50
(m, 1H), 7.49−7.41 (m, 2H), 7.37 (dd, J = 9.2, 2.3 Hz, 2H), 7.32−7.22
(m, 3H), 7.19 (d, J = 7.5 Hz, 1H), 7.12 (s, 1H), 5.10−5.01 (m, 1H),
4.58−4.32 (m, 1H), 3.38 (m, 1H), 3.25 (dd, J = 9.0, 2.5 Hz, 3H), 2.53−
2.27 (m, 2H), 2.04 (m, 1H), 1.86−1.62 (m, 2H). 13C NMR (125 MHz,
acetone-d6): δ 199.6, 178.8, 171.1, 157.8, 154.3, 148.5, 137.0, 129.0,
127.9, 127.1, 126.4, 126.1, 123.2, 122.2, 111.3, 109.6, 97.9, 57.1, 54.0,
39.6, 37.4. HRMS (ESI) m/z: [M − H]− calcd for C25H24N3O5,
446.1721; found, 446.172. Purity: 95.6%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)benzofuran-5-carboxamide (18e).
1H NMR (500 MHz, acetone-d6): δ 9.43 (s, 1H), 8.59 (d, J = 6.8
Hz, 1H), 8.18 (dd, J = 15.6, 1.8 Hz, 1H), 8.08−7.99 (m, 1H), 7.92−
7.82 (m, 2H), 7.53 (t, J = 8.1 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.28 (t, J
= 7.5 Hz, 2H), 7.19 (t, J = 7.3 Hz, 1H), 7.13 (s, 1H), 6.97−6.90 (m,
1H), 5.07 (m, 1H), 4.37 (m, 1H), 3.37−3.33 (m, 1H), 3.29−3.20 (m,
3H), 2.52−2.24 (m, 2H), 2.05−1.98 (m, 1H), 1.87−1.62 (m, 2H). 13C
NMR (125 MHz, acetone-d6): δ 200.1, 179.3, 172.3, 166.9, 156.5,
146.7, 137.9, 129.5, 128.3, 127.4, 126.5, 124.1, 121.1, 110.8, 107.1, 57.6,
55.3, 40.0, 37.9, 37.8. HRMS (ESI) m/z: [M − H]− calcd for
C25H24N3O5, 446.1721; found, 446.1715. Purity: 95.2%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)quinoxaline-2-carboxamide (18k).
1H NMR (500 MHz, acetone-d6): δ 9.47 (s, 1H), 8.69 (d, J = 8.0 Hz,
1H), 8.15 (m, 2H), 7.95 (m, 2H), 7.80 (d, J = 8.0 Hz, 1H), 7.34 (d, J =
7.3 Hz, 2H), 7.25 (t, J = 7.3 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 6.83 (s,
1H), 5.03−4.95 (m, 1H), 3.99 (dd, J = 7.3, 4.3 Hz, 1H), 3.50 (m, 2H),
3.31−3.20 (m, 3H), 2.39−2.28 (m, 2H), 1.95−1.87 (m, 1H), 1.78−
1.68 (m, 1H), 1.62−1.51 (m, 1H). 13C NMR (151 MHz, acetone-d6): δ
199.5, 179.0, 170.7, 162.3, 143.1, 139.7, 136.7, 131.3, 130.6, 129.2,
129.1, 128.9, 127.9, 1277, 126.2, 97.9, 57.4, 53.9, 53.6, 39.5, 37.6.
HRMS (ESI) m/z: [M − H]− calcd for C25H24N5O4, 458.1834; found,
458.1823. Purity: 98.3%.
3-Methyl-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl)amino)-3-phenylpropan-2-yl)quinoxaline-2-carboxa-
mide (18l). 1H NMR (500 MHz, CDCl3): δ 9.30 (s, 1H), 8.63 (d, J =
8.5 Hz, 1H), 8.33 (d, J = 6.2 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 8.02 (d, J
= 8.5 Hz, 1H), 7.80 (m, 1H), 7.75 (dd, J = 11.0, 4.0 Hz, 1H), 7.39−7.27
(m, 5H), 7.25 (d, J = 7.1 Hz, 1H), 6.56 (s, 1H), 5.12 (q, J = 7.1 Hz, 1H),
4.32 (m, 1H), 3.31 (dd, J = 12.8, 6.2 Hz, 3H), 3.02 (s, 3H), 2.40−2.30
(m, 2H), 1.99−1.93 (m, 1H), 1.88−1.75 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 199.8, 179.9, 171.8, 164.5, 153.9, 142.9, 139.1, 136.4,
131.7, 129.7, 129.6, 129.4, 128.6, 128.4, 127.1, 57.7, 54.5, 40.5, 39.1,
37.9, 29.7, 28.4, 24.5. HRMS (ESI) m/z: [M + H]+ calcd for
C26H28N5O4, 474.2136; found, 474.215. Purity: 98.8%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-
1
6-carboxamide (18f). H NMR (500 MHz, CDCl3): δ 9.22 (s, 1H),
8.36 (d, J = 6.3 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.23 (s, 5H), 7.02 (d, J
= 8.1 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.55 (s, 1H), 5.07 (m, 1H),
4.27−4.21 (m, 6H), 3.31−3.24 (m, 2H), 3.20 (dd, J = 14.4, 6.7 Hz,
2H), 2.35−2.28 (m, 2H), 1.90 (dd, J = 10.2, 6.4 Hz, 1H), 1.80 (m, 2H).
13C NMR (125 MHz, CDCl3): δ 200.0, 180.0, 172.3, 166.4, 146.7,
143.3, 136.5, 129.5, 128.5, 127.0, 120.7, 117.2, 116.7, 64.5, 64.2, 57.7,
54.5, 40.6, 38.8, 38.0, 29.5, 28.5. HRMS (ESI) m/z: [M − H]− calcd for
C25H26N3O6, 464.1827; found, 464.1824. Purity: 97.5%.
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-
5-carboxamide (18g). 1H NMR (600 MHz, acetone-d6): δ 11.14 (d, J
= 8.8 Hz, 1H), 9.41 (s, 1H), 8.28−8.23 (m, 1H), 7.78 (m, 1H), 7.58 (d,
J = 1.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.37−7.33 (m, 2H), 7.23 (d, J
= 7.7 Hz, 2H), 7.16 (dd, J = 6.3, 1.9 Hz, 4H), 5.44 m, 1H), 5.06 (dd, J =
7.1, 4.5 Hz, 1H), 4.51 (m, 1H), 4.13−3.99 (m, 1H), 3.32 (d, J = 2.0 Hz,
1H), 3.20 (m, 2H), 2.44−2.34 (m, 1H), 2.33−2.25 (m, 1H), 1.79−1.55
(m, 2H). 13C NMR (125 MHz, acetone-d6): δ 199.8, 178.9, 171.4,
163.7, 143.5, 142.2, 136.8, 129.2, 127.9, 126.3, 122.8, 121.6, 120.3, 97.9,
64.5, 63.3, 57.1, 54.5, 53.4, 39.6, 37.4, 27.7. HRMS (ESI) m/z: [M −
H]− calcd for C25H26N3O6, 464.1827; found, 464.183. Purity: 97.6%.
N-((R)-1-Oxo-1-(((R)-1-oxo-3-((R)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)-1H-indole-5-carboxamide (18h).
1H NMR (500 MHz, acetone-d6): δ 10.53 (s, 1H), 9.39 (s, 1H),
8.19−8.16 (m, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.66 (dd, J = 8.5, 1.7 Hz,
1H), 7.43 (d, J = 8.5 Hz, 1H), 7.40−7.38 (m, 1H), 7.37−7.33 (m, 2H),
7.26 (m, 2H), 7.19−7.15 (m, 1H), 6.96 (s, 1H), 6.54 (m, 1H), 5.01 (dt,
J = 8.2, 4.0 Hz, 1H), 4.31−4.26 (m, 1H), 3.31 (dd, J = 8.2, 5.6 Hz, 1H),
3.26−3.18 (m, 3H), 2.45−2.36 (m, 1H), 2.32−2.21 (m, 1H), 1.98−
1.95 (m, 1H), 1.81−1.68 (m, 2H). 13C NMR (125 MHz, acetone-d6):
δ 199.7, 178.6, 171.9, 167.3, 137.6, 137.5, 129.0, 127.8, 127.2, 126.0,
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)quinoline-2-carboxamide (18m).
1H NMR (500 MHz, CDCl3): δ 9.22 (s, 1H), 8.84 (d, J = 8.5 Hz,
1H), 8.28 (d, J = 8.5 Hz, 1H), 8.22 (dd, J = 10.1, 6.0 Hz, 1H), 8.11 (m,
2H), 7.86 (d, J = 8.1 Hz, 1H), 7.77 (dd, J = 11.2, 4.1 Hz, 1H), 7.62 (m,
1H), 7.36−7.27 (m, 4H), 7.24 (t, J = 7.2 Hz, 1H), 5.08 (m, 1H), 4.33−
4.28 (m, 1H), 3.35−3.29 (m, 2H), 3.25 (t, J = 6.8 Hz, 2H), 2.38−2.30
(m, 2H), 1.93−1.84 (m, 2H), 1.79−1.71 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 199.8, 179.8, 171.7, 164.4, 149.1, 146.6, 137.4, 136.5,
129.6, 129.4, 128.6, 127.1, 118.7, 57.7, 54.7, 40.5, 39.0, 37.8, 29.6, 28.6.
HRMS (ESI) m/z: [M − H]− calcd for C26H25N4O4, 457.1881; found,
457.1888. Purity: 95.2%.
7-Bromo-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl) amino)-3-phenylpropan-2-yl)quinoline-2-carboxa-
1
mide (18n). H NMR (600 MHz, acetone-d6): δ 11.14 (d, J = 8.4
Hz, 1H), 9.41 (s, 1H), 8.28−8.23 (m, 1H), 7.78 (dd, J = 12.5, 8.8 Hz,
1H), 7.58 (d, J = 1.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.37−7.33 (m,
2H), 7.23 (d, J = 7.7 Hz, 2H), 7.16 (dd, J = 6.3, 1.9 Hz, 4H), 5.44 (m,
J
https://doi.org/10.1021/acs.jmedchem.0c02258
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1H), 5.06 (dd, J = 7.1, 4.5 Hz, 1H), 4.51 (m, 1H), 4.13−3.99 (m, 1H),
3.32 (d, J = 2.0 Hz, 1H), 3.20 (m, 2H), 2.44−2.34 (m, 1H), 2.33−2.25
(m, 1H), 1.79−1.55 (m, 2H). 13C NMR (150 MHz, acetone-d6): δ
200.0, 178.9, 171.3, 163.3, 150.7, 147.0, 138.0, 137.2, 131.6, 131.3,
129.8, 129.7, 129.6, 128.4, 128.1, 126.7, 123.8, 119.1, 57.8, 54.6, 54.1,
39.9, 38.3, 37.9. HRMS (ESI) m/z: [M − H]− calcd for C26H24BrN4O4,
535.0986; found, 535.0992. Purity: 95.0%.
6-Chloro-N-((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)-
propan-2-yl)ami no)-3-phenylpropan-2-yl)-2H-chromene-3-car-
boxamide (18o). 1H NMR (600 MHz, CDCl3): δ 9.24 (s, 1H), 8.55
(d, J = 6.0 Hz, 1H), 7.29−7.26 (m, 2H), 7.22 (m, 5H), 7.10 (m, 1H),
6.99 (d, J = 2.6 Hz, 1H), 6.95 (s, 1H), 6.72 (d, J = 8.6 Hz, 1H), 6.58 (s,
1H), 5.01 (dd, J = 14.9, 6.9 Hz, 1H), 4.91−4.85 (m, 2H), 4.25−4.20
(m, 1H), 3.32−3.26 (m, 2H), 3.18 (m, 2H), 2.34−2.29 (m, 2H), 1.94−
1.87 (m, 1H), 1.82−1.77 (m, 1H). 13C NMR (150 MHz, CDCl3): δ
201.6, 181.9, 174.3, 166.5, 155.1, 138.2, 132.7, 131.4, 130.5, 129.6,
129.1, 129.0, 128.9, 128.3, 124.2, 119.2, 66.8, 59.9, 56.1, 42.6, 40.6,
40.1, 31.4, 30.5. HRMS (ESI) m/z: [M − H]− calcd for C26H25ClN3O5,
494.1488; found, 494.1481. Purity: 96.2%.
39.5, 37.3, 30.5, 18.4, 17.4. HRMS (ESI) m/z: [M − H]− calcd for
C30H33N4O6, 545.2406; found, 545.2407. Purity: 98.7%.
N-((S)-3-Methyl-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
pyrrolidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)-
butan-2-yl)quinoline-2-carboxamide (26d). 1H NMR (500 MHz,
acetone-d6): δ 9.40 (s, 1H), 8.74 (d, J = 8.5 Hz, 1H), 8.52 (d, J = 8.5 Hz,
1H), 8.27 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 8.0
Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.88−7.82 (m, 1H), 7.72 (dd, J =
11.2, 4.0 Hz, 1H), 7.29 (d, J = 7.4 Hz, 2H), 7.19 (t, J = 7.6 Hz, 2H), 7.09
(d, J = 7.5 Hz, 1H), 4.85 (dd, J = 11.2, 5.0 Hz, 1H), 4.62−4.55 (m, 1H),
4.40−4.30 (m, 1H), 3.33−3.20 (m, 3H), 3.06−3.00 (m, 1H), 2.50−
2.41 (m, 1H), 2.38−2.27 (m, 2H), 2.06−2.00 (m, 1H), 1.80 (dt, J =
12.9, 9.6 Hz, 2H), 1.30 (d, J = 5.1 Hz, 1H), 0.99 (dd, J = 19.0, 6.8 Hz,
6H). 13C NMR (125 MHz, acetone-d6): δ 199.5, 178.7, 171.2, 170.3,
163.7, 149.2, 145.9, 137.4, 137.0, 129.9, 129.1, 128.8, 127.7, 127.6,
127.5, 125.9, 118.2, 58.0, 56.8, 54.3, 39.5, 37.3, 37.2, 30.8, 18.5, 17.0.
HRMS (ESI) m/z: [M − H]− calcd for C31H35N5O5, 556.2565; found,
556.2562. Purity: 95.4%.
N-((S)-3-Methyl-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
pyrrolidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)-
butan-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide (26e).
1H NMR (500 MHz, acetone-d6): δ 9.35 (s, 1H), 8.32 (d, J = 7.1 Hz,
N-((S)-1-Oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
yl)amino)-3-phenylpropan-2-yl)-1H-indole-2-carboxamide (18p).
1H NMR (600 MHz, acetone-d6): δ 10.52 (s, 1H), 9.40 (s, 1H), 8.16
(d, J = 13.6 Hz, 1H), 7.73−7.62 (m, 2H), 7.45−7.40 (m, 1H), 7.39 (d, J
= 2.4 Hz, 1H), 7.35 (t, J = 7.6 Hz, 2H), 7.28−7.21 (m, 2H), 7.18 (t, J =
6.6 Hz, 1H), 6.94−6.78 (m, 1H), 6.54 (s, 1H), 5.45−4.98 (m, 1H),
4.97−4.39 (m, 1H), 4.34−3.82 (m, 1H), 3.26−3.16 (m, 3H), 2.45−
2.22 (m, 2H), 2.00−1.86 (m, 1H), 1.81−1.43 (m, 2H).. 13C NMR (125
MHz, acetone-d6): δ 200.0, 179.6, 172.1, 161.6, 137.6, 137.0, 131.0,
129.5, 129.4, 128.3, 128.2, 127.7, 126.5, 123.9, 121.7, 120.0, 112.3,
103.5, 57.6, 54.9, 40.1, 37.9, 37.8. HRMS (ESI) m/z: [M − H]− calcd
for C25H25N4O4, 445.1881; found, 445.1881. Purity: 97.0%.
1H), 7.88 (d, J = 8.1 Hz, 1H), 7.61 (s, 1H), 7.46 (dd, J = 6.5, 2.1 Hz,
2H), 7.26 (d, J = 7.1 Hz, 2H), 7.20 (t, J = 7.4 Hz, 2H), 7.14 (s, 1H), 6.89
(d, J = 9.0 Hz, 1H), 4.81 (m 1H), 4.45 (dd, J = 15.3, 7.4 Hz, 1H), 4.36−
4.27 (m, 5H), 3.30−3.23 (m, 2H), 3.18−3.03 (m, 2H), 2.44−2.28 (m,
2H), 2.21 (d, J = 7.0 Hz, 1H), 1.98−1.92 (m, 1H), 1.77 (dd, J = 9.0, 3.1
Hz, 2H), 1.31 (s, 1H), 0.95 (t, J = 7.3 Hz, 6H). 13C NMR (125 MHz,
acetone-d6): δ 199.5, 171.2, 170.8, 165.9, 146.2, 142.8, 136.9, 128.9,
127.7, 127.0, 125.9, 120.4, 116.3, 64.1, 63.7, 59.0, 54.1, 37.3, 37.2, 29.2,
18.5, 17.6. HRMS (ESI) m/z: [M − H]− calcd for C30H35N4O7,
563.2511; found, 563.2501. Purity: 95.1%.
5-Methyl-N-((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-
((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-
yl)amino)butan-2-yl)isoxazole-3-carboxamide (26a). 1H NMR (500
MHz, acetone-d6): δ 9.35 (s, 1H), 8.30 (d, J = 6.8 Hz, 1H), 7.81−7.69
(m, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.31−7.22 (m, 4H), 7.17 (t, J = 7.1
Hz, 1H), 6.94 (s, 1H), 6.52 (d, J = 0.7 Hz, 1H), 4.79 (m, 1H), 4.54−
4.43 (m, 1H), 4.27 (m, 1H), 3.32−3.18 (m, 3H), 3.04 (dd, J = 13.8, 8.1
Hz, 1H), 2.44−2.28 (m, 2H), 2.22 (dd, J = 13.4, 6.7 Hz, 1H), 1.93 (d, J
= 5.6 Hz, 1H), 1.81−1.72 (m, 2H), 1.31 (s, 2H), 0.93 (dd, J = 10.3, 6.8
Hz, 6H). 13C NMR (125 MHz, acetone-d6): δ 199.4, 178.5, 171.1,
171.0, 169.8, 158.2, 136.9, 128.9, 127.7, 126.0, 100.7, 57.7, 56.9, 54.2,
39.4, 37.23,37.2, 29.1, 27.7, 18.3, 16.9, 10.7. HRMS (ESI) m/z: [M −
H]− calcd for C26H33N5O6, 510.2358; found, 510.2352. Purity: 96.7%.
N-((S)-3-Methyl-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
pyrrolidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)-
Materials and Methods. Protein Expression and Purification.
The full-length gene encoding the EV71 3Cpro and the SARS-CoV-2
3CLpro with an N-terminal 6 × His-SUMO2 fusion tag was cloned into
the pET-15b vector. The resulting plasmids were transformed into
BL21 (DE3) cells for protein expression. The expressed proteins were
purified by a Ni-NTA column (GE) and transformed into the cleavage
buffer (25 mM Tris, pH 7.5, 300 mM NaCl, 2 mM DTT) containing
SUMO Specific Peptidase 2 (SENP2) for removing the 6 × His-
SUMO2 fusion tag. The resulting protein samples were further purified
by Q-Sepharose (GE Healthcare) and Superdex200 (GE Healthcare).
The eluted EV71 3Cpro and SARS-CoV-2 3CLpro were stored in a
solution containing 25 mM Tris (pH 8.0), 500 mM NaCl, and 2 mM
DTT, and in a solution containing 10 mM Tris (pH 7.5), respectively.
Protein Crystallization and Structure Determination. The purified
EV71 3Cpro was concentrated to 10 mg/mL and incubated 1 h with 2
mM 18p before crystallization condition screening. Crystallization was
performed at 20 °C using a hanging drop vapor-diffusion method, by
mixing equal volumes (1:1 μL) of the protein and crystallization
solution. Crystals were finally yielded in a solution containing 0.1 M
MES monohydrate (pH 6.0), 20% 2-propanol, and 20% polyethylene
glycol monomethyl ether 2000. Then, crystals were flash-frozen in
liquid nitrogen in the presence of the reservoir solution supplemented
with 20% glycerol. X-ray diffraction data were collected at beamline
BL18U1 at the Shanghai Synchrotron Radiation Facility.13 The data
were processed with HKL3000 software packages.14 The complex
structure was solved by molecular replacement using the program
PHASER15 with a search model of PDB code 4GHT. The model was
built using Coot16 and refined with a simulated-annealing protocol
implemented in the program PHENIX.17 The refined structure was
deposited to the Protein Data Bank with an accession code, 7DNC.
Inhibition Assays of the EV71 3Cpro and the SARS-CoV-2 3CLpro. A
fluorescence resonance energy transfer (FRET) protease assay was
applied to measure the inhibitory activity of compounds against the
EV71 3Cpro. The fluorogenic substrate Dacyl-KTSAVLQSGFRKME-
Edans was synthesized by GenScript (Nanjing, China). The assay was
performed in a total volume of 120 μL. The recombinant EV71 3Cpro at
a final concentration of 5 μM was mixed with serial dilutions of each
compound in 80 μL of assay buffer (25 mM Tris, pH 8.0, 150 mM
1
butan-2-yl)-1H-indole-2-carboxamide (26b). H NMR (500 MHz,
acetone-d6): δ 11.20 (s, 1H), 9.46 (s, 1H), 8.22 (d, J = 7.4 Hz, 1H),
7.96−7.88 (m, 1H), 7.75 (dd, J = 11.9, 4.0 Hz, 1H), 7.62 (m, 2H),
7.33−7.29 (m, 1H), 7.26−7.20 (m, 3H), 7.19−7.06 (m, 4H), 7.03 (dd,
J = 16.8, 9.5 Hz, 1H), 4.88−4.80 (m, 1H), 4.54−4.32 (m, 2H), 3.38
(dd, J = 19.0, 10.1 Hz, 1H), 3.32−3.24 (m, 2H), 3.00 (m, 1H), 2.43 (m,
1H), 2.32 (m, 2H), 2.23 (tt, J = 9.0, 4.5 Hz, 1H), 1.87−1.81 (m, 1H),
1.31 (d, J = 1.7 Hz, 1H), 0.96 (dd, J = 10.7, 6.9 Hz, 6H). 13C NMR (125
MHz, acetone-d6): δ 199.8, 179.2, 171.5, 170.2, 162.5, 137.2, 130.2,
128.6, 127.7, 125.8, 123.4, 121.1, 119.6, 112.2, 103.2, 60.0, 56.7, 54.2,
39.5, 37.3, 36.5, 30.3, 29.5, 29.1, 27.0, 18.1, 17.5. HRMS (ESI) m/z: [M
− H]− calcd for C30H34N5O5, 544.2565; found, 544.2568. Purity:
97.6%.
N-((S)-3-Methyl-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxo-
pyrrolidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)-
1
butan-2-yl)benzofuran-2-carboxamide (26c). H NMR (500 MHz,
acetone-d6): δ 9.37 (s, 1H), 8.36 (d, J = 7.1 Hz, 1H), 8.02 (d, J = 8.1 Hz,
1H), 7.77 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 6.2 Hz, 2H), 7.48 (dd, J = 8.4,
7.5 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.30−7.26 (m, 2H), 7.21−7.03
(m, 4H), 4.86 (m, 1H), 4.63−4.55 (m, 1H), 4.51−4.30 (m, 1H), 4.07
(d, J = 2.6 Hz, 1H), 3.30−3.16 (m, 3H), 3.05 (dd, J = 13.9, 8.2 Hz, 1H),
2.43−2.18 (m, 3H), 2.05−1.97 (m, 1H), 1.84−1.75 (m, 1H), 1.60 (m,
1H), 0.98 (dd, J = 6.6, 2.2 Hz, 6H). 13C NMR (125 MHz, acetone-d6): δ
199.5, 179.5, 178.8, 171.2, 170.2, 158.0, 154.3, 148.5, 136.9, 128.9,
127.7, 126.5, 125.9, 123.2, 122.2, 111.3, 109.8, 58.0, 56.8, 54.3, 53.6,
K
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NaCl, and 10% glycerol) and incubated for 10 min. The reaction was
initiated by adding 40 μL of a fluorogenic substrate at a final
concentration of 25 μM. The reaction solution was then incubated at 30
°C for 3 h. After that, the fluorescence signal at 340 nm (excitation)/
490 nm (emission) was measured immediately with a Bio-Tek
Synergy4 plate reader.
The antiviral activity of selected compounds against EV68, CoxA21,
CoxB3, RV-A02, and RV-B14 was tested as described previously.20
To assess the cytotoxicity of the test compounds, Vero E6 cells
preseeded in a 96-well dish (2 × 104 cells/well) were treated with
different concentrations of the indicated compounds, and 24 h later, the
relative numbers of surviving cells were measured with cell counting kit-
8 (GK10001, GLPBIO) according to the manufacturer’s instructions.
Pharmacokinetic Profiles in CD-1 Mice. Male CD-1 mice (n = 3 per
group) were treated with a solution of compounds 18p and 26d
(DMSO/EtOH/PEG300/NaCl (5:5:40:50, v/v/v/v)) at doses of 20
mg/kg, 5 mg/kg, and 5 mg/kg via intraperitoneal (ip), subcutaneous
(sc), and intravenous (iv), respectively. Blood samples were collected at
0.05, 0.25, 0.75, 2, 4, 8, and 24 h after administration. Serum samples
were obtained through common procedures, and the concentrations of
the compound in the supernatant were analyzed by LC-MS/MS.
All procedures relating to animal handling, care, and treatment were
performed according to the guidelines approved by the Institutional
Animal Care and Use Committee of the contract research organizations
performing the study.
The inhibition assay of the SARS-CoV-2 3CLpro has been described
previously.18 In brief, the recombinant SARS-CoV-2 3CLpro at a
concentration of 30 nM was mixed with serial dilutions of each
compound in 80 μL of assay buffer (50 mM Tris−HCl, pH 7.3, 1 mM
EDTA) and incubated for 10 min. The reaction was initiated by adding
40 μL of a fluorogenic substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-
NH2) at a final concentration of 20 μM. After that, the fluorescence
signal at 320 nm (excitation)/405 nm (emission) was measured
immediately every 35 s for 3.5 min with a Bio-Tek Synergy4 plate
reader. The velocities of reactions with compounds added at various
concentrations compared to the reaction added with DMSO were
calculated and used to generate inhibition profiles. For each compound,
at least three independent experiments were performed for the
determination of IC50 values. The IC50 values were expressed as the
mean SD and determined via nonlinear regression analysis using
GraphPad Prism software 8.0 (GraphPad Software, Inc., San Diego,
CA, USA).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02258.
■
sı
Cells. RD cells were maintained in MEM Rega-3 medium
supplemented with 2% FBS, 2 mM L-glutamine, and 0.075%
NaHCO3, all supplied by Gibco, Life Technologies. Cells were
maintained at 37 °C in a humidified atmosphere of 5% CO2.
The Vero E6 cell line was obtained from American Type Culture
Collection (ATCC, no. 1586) and maintained in Dulbecco’s modified
Eagle medium (DMEM; Gibco Invitrogen) supplemented with 10%
fetal bovine serum (FBS; Gibco Invitrogen), 1% antibiotic/antimycotic
(Gibco Invitrogen), at 37 °C in a humidified 5% CO2 incubator.
Viruses. Enterovirus 71 strain BrCr (EV71 BrCr), kindly provided by
Prof. Dr. F. van Kuppeveld (Universiteit Utrecht, The Netherlands),
was grown on RD cells. When a full cytopathic effect (CPE) was
observed, the virus was harvested from the supernatants after
centrifugation (10 min, 3000 rpm) and stored at −80 °C. The viral
titer was determined by end point titration.
An overlay of EV71 3Cpro-18p and AG7088 complex
structures; the binding pocket of 3Cpro of CoXB3, RV-
A02, and EV71; the anti-SARS-CoV-2 activities of
compounds; the 1H NMR and 13C NMR spectra of target
compounds; the purity of target compounds; and the
crystallography data collection and refinement statistics
(PDF)
Molecular formula stings (CSV)
Accession Codes
The atomic coordinates and structure factors have been
deposited into the Protein Data Bank with accession code
7DNC (EV71 3Cpro-18p). The authors will release the atomic
coordinates and experimental data upon article publication.
A clinical isolate of SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/
WIV04/2019) was propagated in Vero E6 cells, and a viral titer was
determined by TCID50.8a All infection experiments were performed at
biosafety level-3 (BSL-3).
AUTHOR INFORMATION
Corresponding Authors
■
Antiviral Assay. The anti-EV71 activity of selected compounds was
tested in RD cells and seeded in a 96-well plate (2.5 × 104 cells/well).
Cells were allowed to adhere overnight, after which cells were infected
with EV71; a serial dilution of selected compounds was added and
incubated for 4 days, i.e., until complete CPE was observed in the
untreated and infected virus control conditions. CPE was subsequently
quantified using an MTS-reduction assay (MTS = 3f-(4,5-dimethylth-
iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt). For this, a MTS/phenazine methosulfate (PMS)
stock solution (2 mg/mL MTS (Promega, Leiden, The Netherlands)
and 46 μg/mL PMS (Sigma-Aldrich, Bornem, Belgium) in PBS at pH
6−6.5) was diluted 1:20 in MEM (Life Technologies, Gent, Belgium
cat. no. 21090-022). The medium was aspirated from wells, and 75 μL
of MTS/PMS solution was added. After 1−2 h of incubation at 37 °C,
the absorbance was measured at 498 nm. The % inhibition for each well
is then calculated by normalization of the absorbance to the condition
of untreated-infected cells (=0% inhibition) and the condition of
untreated−uninfected cells (=100% inhibition). From the obtained
dose−response curve, the EC50 is calculated by curve fitting using
Dotmatics software.
Leike Zhang − State Key Laboratory of Virology, Wuhan
Institute of Virology, Center for Biosafety Mega-Science,
Chinese Academy of Sciences, Wuhan, Hubei 430071, China;
orcid.org/0000-0002-2593-2571; Email: zhangleike@
wh.iov.cn
Yechun Xu − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
School of Pharmaceutical Science and Technology, Hangzhou
Institute for Advanced Study, Beijing 100049, China;
orcid.org/0000-0002-1581-6155; Email: ycxu@
simm.ac.cn
Johan Neyts − KU Leuven, Department of Microbiology and
Immunology, Rega Institute for Medical Research, Laboratory
of Virology and Chemotherapy, Leuven B-3000, Belgium;
Email: johan.neyts@kuleuven.be
Hong Liu − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; College of Pharmacy, Nanjing University of
Chinese Medicine, Nanjing 210023, China; University of
Chinese Academy of Sciences, School of Pharmaceutical Science
and Technology, Hangzhou Institute for Advanced Study,
To assess the antiviral activity of compounds against SARS-CoV-2,
preseeded Vero E6 cells (5 × 104 cells/well) were treated with different
concentrations of the indicated compounds for 1 h and then were
infected with SARS-CoV-2 at an MOI of 0.01. At 24 h pi, the cell
supernatant was collected, and a viral RNA copy number in the cell
supernatant was measured using real-time PCR, as described
previously.19
L
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Beijing 100049, China; orcid.org/0000-0003-3685-6268;
Email: hliu@simm.ac.cn
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
Beijing 100049, China
Authors
Complete contact information is available at:
Wenhao Dai − State Key Laboratory of Drug Research, CAS
Key Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
Beijing 100049, China
Dirk Jochmans − KU Leuven, Department of Microbiology and
Immunology, Rega Institute for Medical Research, Laboratory
of Virology and Chemotherapy, Leuven B-3000, Belgium
Hang Xie − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
Hang Yang − State Key Laboratory of Virology, Wuhan Institute
of Virology, Center for Biosafety Mega-Science, Chinese
Academy of Sciences, Wuhan, Hubei 430071, China
Jian Li − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; College of Pharmacy, Nanjing University of
Chinese Medicine, Nanjing 210023, China
Haixia Su − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
Beijing 100049, China
https://pubs.acs.org/10.1021/acs.jmedchem.0c02258
Author Contributions
○W.D., D.J., H.X., and H.Y. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Natural Science Foundation of
China (21632008, 21672231, 21877122, 21877118, and
81620108027), the Strategic Priority Research Program of the
Chinese Academy of Sciences (XDA12040107 and
XDA12040201), and the Youth Innovation Promotion
Association CAS (grants 2018367 to L.-K.Z.) for financial
support
ABBREVIATIONS USED
■
EV71, enterovirus 71; HFMD, hand, foot, and mouth disease;
SARS-CoV-2, severe acute respiratory syndrome coronavirus 2;
COVID-19, coronavirus disease 2019; 3Cpro, 3C protease;
3CLpro, 3C-like protease; HRV, human rhinoviruses; EV68,
enterovirus 68; CoxA21, coxsackievirus A21; DMP, Dess−
Martin periodinane; SAR, structure−activity relationship; PDB,
protein data bank; HATU, O-(7-; 2-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; DIPEA,
N,N-diisopropyl-ethyl-amine; TLC, thin-layer chromatography;
HRMS, high-resolution mass spectra
Di Chang − Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
Jiang Wang − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
School of Pharmaceutical Science and Technology, Hangzhou
Institute for Advanced Study, Beijing 100049, China
Jingjing Peng − State Key Laboratory of Drug Research, CAS
Key Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; University of Chinese Academy of Sciences,
Beijing 100049, China
Lili Zhu − Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
Yong Nian − State Key Laboratory of Drug Research, CAS Key
Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; College of Pharmacy, Nanjing University of
Chinese Medicine, Nanjing 210023, China
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