A new class of α-ketoamide derivatives with potent anticancer and anti-SARS-CoV-2 activities

Article abstract of DOI:10.1016/j.ejmech.2021.113267

Inhibitors of the proteasome have been extensively studied for their applications in the treatment of human diseases such as hematologic malignancies, autoimmune disorders, and viral infections. Many of the proteasome inhibitors reported in the literature target the non-primed site of proteasome's substrate binding pocket. In this study, we designed, synthesized and characterized a series of novel α-keto phenylamide derivatives aimed at both the primed and non-primed sites of the proteasome. In these derivatives, different substituted phenyl groups at the head group targeting the primed site were incorporated in order to investigate their structure-activity relationship and optimize the potency of α-keto phenylamides. In addition, the biological effects of modifications at the cap moiety, P1, P2 and P3 side chain positions were explored. Many derivatives displayed highly potent biological activities in proteasome inhibition and anticancer activity against a panel of six cancer cell lines, which were further rationalized by molecular modeling analyses. Furthermore, a representative α-ketoamide derivative was tested and found to be active in inhibiting the cellular infection of SARS-CoV-2 which causes the COVID-19 pandemic. These results demonstrate that this new class of α-ketoamide derivatives are potent anticancer agents and provide experimental evidence of the anti-SARS-CoV-2 effect by one of them, thus suggesting a possible new lead to develop antiviral therapeutics for COVID-19.

Full text of DOI:10.1016/j.ejmech.2021.113267

European Journal of Medicinal Chemistry 215 (2021) 113267
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A new class of
a-ketoamide derivatives with potent anticancer and
anti-SARS-CoV-2 activities
Juan Wang a, Boqiang Liang b, Yiling Chen b, Jasper Fuk-Woo Chan c, Shuofeng Yuan c,
,
Hui Ye b, Linlin Nie b, Jiao Zhou b g, Yi Wu b, Meixian Wu d, Lina S. Huang d, Jing An d,
,
, d, g, *
Arieh Warshel e, Kwok-Yung Yuen c, Aaron Ciechanover b f, Ziwei Huang a
,
, g, **
Yan Xu b
a School of Life Sciences, Tsinghua University, Beijing, 100084, China
b Nobel Institute of Biomedicine, Zhuhai, 519000, China
c State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Pokfulam, Hong Kong, China
d Department of Medicine, Division of Infectious Diseases and Global Public Health, School of Medicine, University of California San Diego, La Jolla, CA,
92037, USA
e Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA
f Technion-Israel Institute of Technology, Haifa, 3109601, Israel
g Ciechanover Institute of Precision and Regenerative Medicine, School of Life and Health Sciences, Chinese University of Hong Kong, Shenzhen, 518172,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of the proteasome have been extensively studied for their applications in the treatment of
human diseases such as hematologic malignancies, autoimmune disorders, and viral infections. Many of
the proteasome inhibitors reported in the literature target the non-primed site of proteasome’s substrate
Received 28 September 2020
Received in revised form
30 January 2021
Accepted 30 January 2021
Available online 10 February 2021
binding pocket. In this study, we designed, synthesized and characterized a series of novel
a-keto
phenylamide derivatives aimed at both the primed and non-primed sites of the proteasome. In these
derivatives, different substituted phenyl groups at the head group targeting the primed site were
incorporated in order to investigate their structure-activity relationship and optimize the potency of a-
Keywords:
keto phenylamides. In addition, the biological effects of modifications at the cap moiety, P1, P2 and P3
side chain positions were explored. Many derivatives displayed highly potent biological activities in
proteasome inhibition and anticancer activity against a panel of six cancer cell lines, which were further
a
-ketoamides
Cancer
SARS-CoV-2
COVID-19
Proteasome
Drug discovery
rationalized by molecular modeling analyses. Furthermore, a representative
tested and found to be active in inhibiting the cellular infection of SARS-CoV-2 which causes the COVID-
19 pandemic. These results demonstrate that this new class of -ketoamide derivatives are potent
a-ketoamide derivative was
a
anticancer agents and provide experimental evidence of the anti-SARS-CoV-2 effect by one of them, thus
suggesting a possible new lead to develop antiviral therapeutics for COVID-19.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
signal transduction, antigen processing, protein quality control, etc
[1]. The proteasome, an ATP dependent multi-subunit complex, is a
The ubiquitinꢀproteasome system (UPS) plays essential roles in
central mediator of proteolysis in UPS. Diverse hybrid forms of
proteasome have been identified. In general, the hybrid proteasome
is composed of two parts: 20S core particle (CP) and regulatory
particle (RP) including PA700 (or the 19S cap), PA28 (also known as
11S), and Blm10/PA200 protein. One 20S core particle binds with
one or two regulatory particle to form singly (RP1eCP) or doubly
(RP2eCP) hybrid proteasome, respectively [2,3].
the turnover of numerous cellular proteins involved in cell cycle,
* Corresponding author. School of Life Sciences, Tsinghua University, Beijing
100084, China.
** Corresponding author. Chinese University of Hong Kong, Shenzhen, 518172,
China.
26S proteasome is the most common form of proteasome,
E-mail addresses: zwhny@yahoo.com (Z. Huang), yanxu@cuhk.edu.cn (Y. Xu).
https://doi.org/10.1016/j.ejmech.2021.113267
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
consisting of 20S core particle capped with two 19S regulatory
particles [4]. The 19S regulatory particle is responsible for the
ubiquitylated substrate recognition, deubiquitination, unfolding,
and translocation of the unfolded chain into the core particle. The
catalytic domain resides in the 20S core particle that is associated
with the degradation of damaged proteins and cell signaling pro-
teins. The 20S core particle consists of four stacked rings, with two
ixazomib [17] have been developed that mostly target the non-
primed substrate binding site of proteasome (Fig. 1A), few protea-
some inhibitors targeting both non-primed and primed sites or
only the primed site of the substrate binding pocket have been
reported. Among them, dipeptide
showing good selectivity for 1i subunit binds both non-primed
and primed sites [18]. -Lactone homobelactosin C primarily
binds to the primed site exhibiting high potency to 5 subunit
a,b epoxyketone UK-101 (Fig. 1B)
b
b
outer rings composed of seven
composed of seven subunits [5,6]. The catalytic subunits
and 1 reside in the inner ring that exhibits chymotrypsin-like
a
subunits and two inner rings
b
b
b
5,
b2
[19,20]. A recent study has shown that epoxyketone-based com-
pounds targeting P10 site have the efficacy against cell lines of de
novo or acquired resistance to carfilzomib [21]. Thus, discovery of
new compounds capable of binding the primed site remains an area
to be explored further.
b
b
(CT-L), trypsin-like (T-L) and caspase-like (C-L) proteolytic activ-
ity, respectively [7]. In vertebrates, three isoforms of the 20S CP
have been identified, including constitutive proteasome (cCP)
which harbors
harbors 1i (LMP2),
some (tCP) which harbors
teasome is involved in antigen processing that influences cytokine
production and T cell function [10]. Overexpression of immuno-
and constitutive proteasome is observed in hematologic malig-
nancies and autoimmune diseases. Different classes of proteasome
inhibitors have been developed for clinical intervention of hema-
tologic malignancies and autoimmune disorders [11e13].
b
1c,
b
b
2c and
2i (MECL-1) and
1i, 2i and b
b
5c, immunoproteasome (iCP) which
5i (LMP7), thymoprotea-
5t [8,9]. The immunopro-
It has been reported that a ketoamide derivative BSc2189
(Fig. 1A) binds to the active site in the 20S proteasome in a
reversible manner [22e24]. Its phenyl group at the head group
occupies the primed substrate binding channel of 20S proteasome
(Fig. 1A) [24]. Previous studies have utilized the primed site to
improve the specificity and potency of protease inhibitors [25e27].
In this study, we incorporated different substitutions into a-keto
phenylamide’s head group to examine their effects on the protea-
some inhibitory potency. Additional modifications were also made
b
b
b
b
Although many proteasome inhibitors including those clinically
approved drugs such as bortezomib [14,15], carfilzomib [16], and
on the cap moiety and the P1, P2 and P3 side chain positions of
keto phenylamide to investigate the influence of these positions on
a-
Fig. 1. Schematic illustration of (A) Bortezomib recognizing only the non-primed site and ketoamide derivatives BSc2189 capable of binding to both primed and non-primed sites of
the proteasome. (B) Chemical structures of ONX-0914, MG132 and UK101.
2

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
bioactivity. These modification studies should yield insight into the
2.2. Inhibition of
b5 catalytic subunit in a cell-based chymotrypsin-
structure-activity relationship (SAR) of
a-ketoamide-based pro-
like activity assay
teasome inhibitors which has not been reported before.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
is a novel betacoronavirus which causes the ongoing Coronavirus
Disease 2019 (COVID-19) pandemic [28]. Currently, antiviral treat-
ment options for COVID-19 are limited. Thus, the development of
effective drugs is an urgent challenge. Previous studies have shown
that proteasome inhibitors can inhibit coronavirus replication by
interfering with the early steps of the viral replication cycle [29,30].
For example, the proteasome inhibitor MG132 (Fig. 1B) and its
analogs have been reported to have inhibitory activity against se-
vere acute respiratory syndrome coronavirus (SARS-CoV) [31,32].
Mice infected with mouse hepatitis virus 1 and treated with MG132
had higher survival rate, less severe lung histopathological changes,
and reduced lung virus replication, lung STAT phosphorylation, and
expression of pneumoinflammatory cytokines. The anti-coronaviral
effect of MG132 was suggested to be due to its inhibition of viral
replication-dependent cysteine protease m-calpain [31]. Because of
the high homology between SARS-CoV-2 and SARS-CoV [33,34], we
hypothesized that proteasome inhibitors capable of blocking SARS-
CoV may also be effective against SARS-CoV-2. To test this hy-
pothesis, we examined the anti-SARS-CoV-2 activity of one of the
All compounds were evaluated with a cell-based proteasome
inhibition assay (Promega). Because the 5 catalytic subunit is the
primary target of these proteasome inhibitors [4], all -keto phe-
nylamide derivatives were screened for their inhibition of
chymotrypsin-like activity. The biological activity of these de-
rivatives is presented and discussed below in different groups ac-
cording to the structural modifications made at the N-cap position,
P1, P2 and P3 positions, and the head group.
b
a
2.2.1. Modifications at the N-cap position
Initially, we designed 18 and a hybrid compound 17 which
contains the peptide backbone of a b5i-selective inhibitor ONX-
0914 [11] and the head group of BSc2189 [24]. 17 and 18 exhibi-
ted decreased chymotrypsin-like activity compared with ONX-0914
and BSc2189. The subsequent modifications of 18 focused on the N-
cap position of the compound structure shown in Formula 1. A
variety of cap groups were attempted (Table 1), including hetero-
cycle (18), unbranched aliphatic alkyl groups (20), branched (21),
cycloalkyl (22), and aromatic rings (compound 23, 24, 25, 26). The
results indicated that compounds with substituted aromatic rings
at the cap position exhibited more potent activity. For example,
compounds 24, 25, 26 exhibited better potency against
chymotrypsin-like activity than 21 and 22. 20 with a decanoyl
group and 18 with a morpholinoacetyl group at the cap position
had modest activity. Of note, 5-(4-acetylphenyl)-furan-2-carbonyl
restored the activity of target compounds. Compound 26 retained
similar bioactivity to ONX-0914 in the cell-based CT-L inhibition
assay.
a
-ketoamide derivatives reported here, chosen based on its low
toxicity compared with others in Vero E6 cells which was used for
SARS-CoV-2 infection assay. This representative analog showed
potent inhibition of SARS-CoV-2 infection, thus demonstrating
experimentally that this and potentially some of the other
a-
ketoamide derivatives reported here may have the potential for the
development of anti-SARS-CoV-2 agents.
2.2.2. Modifications at P1, P2 and P3 positions
The sidechains on P1, P2 and P3 positions shown in Formula 2
were modified. Compound 17 with Phe at P1 position was slightly
more potent towards chymotrypsin-like subunit than compound 18
with Leu at P1 position (Table 2).
2. Results
2.1. Synthesis of
a
-ketoamide derivatives
Starting from a tripeptide scaffold with Cbz at the cap moiety,
Ala at P3 and Leu at P1, different aromatic side chains were incor-
porated at P2 position. The results show that compound 27 with
Trp at P2 is favorable to the biological activity. Compound 19 con-
taining P2-Tyr(OMe) and compound 28 containing P2-Phe dis-
played decreased inhibitory potency. Starting from a tripeptide
scaffold with Cbz in cap moiety, Leu at P3 and Leu at P1, different
side chains were incorporated at P2 position. The order of the
inhibitory potency of these compounds is: BSc2189 > 29 > 30. The
results indicate that Leu at P2 position is preferred over Trp,
The general synthetic route is shown in Schemes 1 and 2. In
general, amino acid methyl ester 1 was coupled with Boc protected
amino acid to afford 2a-b or Cbz protected amino acid to afford 9a-c
using HATU as the coupling reagent. The Boc group of 2a-b was
removed using trifluoroacetic acid (TFA) and the resulting com-
pound then coupled with commercially available carboxylic acid
derivatives except for carboxylic acid 16 which was prepared in our
lab (Scheme 2), affording compounds 4a-b. Compounds 4a-b or 9a-
c were hydrolyzed to give 5a-b or 10a-c. Acid 5a-b with different
cap moieties or acid 10a-c with Cbz cap moiety were coupled with
L-leucinol in the presence of isobutyl chloroformate and N-meth-
ylmorpholine to yield intermediate 6a or 11a-c. 6a or 11a was
oxidized into 7a or 12a using 2-iodoxybenzoic acid. Aldehyde 7a or
12a reacted with appropriate isonitrile by Passerini reaction
method to generate 8a or 13a, which was subsequently oxidized to
the desired target molecule with 2-iodoxybenzoic acid.
Tyr(OMe) or Phe for b5 inhibitory potency.
In addition, the potency of proteasome inhibition of compounds
bearing Ala or Leu at P3 position was determined. Compound with
Leu at P3 (29, 30) showed increased activity compared to Ala at P3
(27, 28). These results indicated that P3 position may favor bulky
side chains. Thus, a larger P3 side chain, cyclohexyl (31) was
introduced at this site. Compound 31 showed 3-fold increase in
potency relative to analog 28 with Ala at P3 site but ~3-fold reduced
activity compared with 30 with Leu at P3. Replacing Leu of 30 with
Ser (OMe) (32) at P3 site retained the potency.
The isonitrile derivatives were prepared according to a slightly
modified procedure reported in the literature [23]. Commercially
available aniline derivatives reacted with HCOOH to yield N-for-
mylated aniline derivatives which were converted into isonitrile
derivatives using POCl3 and DIEA in dry dichloromethane, exem-
plified by synthesis of 14 (Scheme 1C).
2.2.3. Modifications at the head group
The phenylamide group of BSc2189 could form
p-alkyl inter-
action with Ser129 in
b5’s primed site. In previous work by others,
The 5-aryl 2-furan carboxylic acid 16 was prepared according to
the reported procedure [35], as illustrated in Scheme 2. Recently, an
optimized reaction condition to prepare 2-arylated furan de-
rivatives was reported [36].
different electron donating groups such as methyl and electron
withdrawing groups such as cyano were incorporated into the para
position of phenylamide group [24], which found that compound
BSc4999 with 2, 4-dimethyl phenylamide was about 2 fold more
3

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Scheme 1. General synthetic procedure for a-ketoamide derivatives.
Reagents and conditions: (i) Boc-AA-OH or Cbz-AA-OH, HATU, HOBt, DIEA, THF, 0 ꢁC to room temperature, 1 h; (ii) TFA, CH2Cl2, 0 ꢁC, 3 h; (iii) carboxylic acid derivatives, HATU, HOBt,
DIEA, THF, 0 ꢁC to room temperature, 1 h; (iv) LiOH.H2O, THF, H2O, room temperature, 3 h; (v) L-Leucinol, isobutyl chloroformate, N-methylmorpholine, THF, ꢀ5 ꢁC; (vi) 2-
iodoxybenzoic acid, DMSO, room temperature, 4 h; (vii) pyridine, TFA, dry CH2Cl2, -15 ꢁCe0 ꢁC, 3 h; (viii) 2-iodoxybenzoic acid, DMSO, room temperature, 4e5 h; (ix) HCOOH,
reflux, 3 h; (x) POCl3, DIEA, dry CH2Cl2, 0 ꢁC to room temperature, 3 h.
4

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Scheme 2. General synthetic procedure for 5-(4-acetylphenyl)furan-2-carboxylic acid.
Reagents and conditions: (i) Pd(OAc)2, KOAc, DMAc, 130 ꢁC, 4 h; (ii) LiOH.H2O, THF, H2O, room temperature, 3 h.
Table 1
The cell-based CT-L inhibitory activity of cap moiety modified compounds.
Compound
Cap moiety
CT-La, IC50 (nM)
418.9 82.0
Compound
Cap moiety
CT-La, IC50 (nM)
311.0 19.7
18 (NI-04104)
24 (NI-04146)
19 (NI-01099)
20 (NI-04127)
1026 235.4
233.6 17.4
25 (NI-01114)
26 (NI-02133)
133.7 26.9
50.7 4.5
21 (NI-04154)
22 (NI-07014)
1753 392.1
1356 252.0
ONX-0914
BSc2189
58.5 4.3
34.5 1.7
23 (NI-02114)
716.5 139.2
a
The compounds were assayed in triplicate with the averaged IC50 SEM values shown.
potent than BSc2189 with an unsubstituted phenylamide whereas
most of other compounds showed decreased activity towards
chymotrypsin-like subunit compared with BSc2189.
Here, we focused on using primed site to improve the potency.
Phenyl group at the head position was deemed to be a suitable
starting point to introduce various modifications. Various groups at
the ortho and meta positions of phenylamide group were intro-
duced based on different tripeptide scaffolds (Table 3).
With 3-acetylphenyl substituent into the backbone of Cbz-Leu-
Phe-Leu, Cbz-Leu-Trp-Leu and Cbz-Leu-Leu-Leu, compounds 34, 35
and 38 showed 1.5e4 fold increase in inhibitory activity than
compounds 30, 29 and BSc2189 with unsubstituted phenyl. In
addition, the data indicated that the effect of the head group
modification on activity was also dependent of peptide backbone’s
interaction with the non-primed site when the substituent group at
the head position was the same. This was also demonstrated by 33
and 37. Compound 33 with 1-naphthyl at the head group showed
~3 fold increase in inhibitory potency over compound 19 of
unsubstituted phenyl whereas the same head group of 1-naphthyl
in 37 resulted in 2 fold decrease in inhibitory potency over 29 of
unsubstituted phenyl. Previous study showed that the compound
with 1-naphthyl at the head group exhibited low nanomolar
potency against the chymotryptic activity [37]. Incorporation of 3-
methylsulfonylphenyl (36), 3,5-dimethoxylphenyl (44, 45) at the
head group resulted in slightly improved activity compared with
the corresponding unsubstituted phenyl compounds.
In order to enhance the interaction with hydrophobic residue
Tyr168 of
b5, a phenyl was introduced in the ortho position of the
phenyl ring at the head group of the compound. However, this
resulted in up to 150-fold reduced inhibitory activity in compound
41 possibly caused by the phenyl ring introduced in the ortho po-
sition being too bulky and giving rise to steric hindrance to the
binding. The tripeptide with quinolinyl or isoquinolinyl at the head
group showed less potent activity compared to BSc2189 which was
synthesized as a control and tested together with other new
compounds in the assays.
The results above seemed to suggest that the head group posi-
tion is tolerant of introducing modification groups of suitable sizes
and independent of the electron density on the phenyl ring. Thus,
electron donating groups like 2, 3-dihydro-1H-indenyl (42) and 3-
ethylphenyl (43) were introduced at the head position. The results
showed that 42 and 43 had activity similar to BSc2189 with the IC50
values of 41.7 nM and 34.8 nM, respectively (Table 3).
The SAR results shown above suggested that a substituted
5

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Table 2
The cell-based CT-L inhibitory activity of P1, P2 and P3 modified compounds.
Compound
Cap moiety
P1
Ph
P2
P3
H
CT-La IC50 (nM)
364.6 42.2
17 (NI-01069)
(4-CH3O)-Ph
18 (NI-04104)
19 (NI-01099)
i-Pr
i-Pr
(4-CH3O)-Ph
(4-CH3O)-Ph
H
H
418.9 82.0
1026 235.4
27 (NI-07028)
28 (NI-06032)
29 (NI-07021)
30 (NI-04162)
31 (NI-18088)
32 (NI-18143)
BSc2189
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
1H-indol-3-yl
H
155.7 25.9
2008 219.7
66.5 11.0
230.8 14.5
603.3 26.7
243.4 32.8
34.5 1.7
Ph
H
1H-indol-3-yl
i-Pr
Ph
i-Pr
Ph
Cyclohexyl
CH3O
i-Pr
Ph
i-Pr
ONX-0914
58.5 4.3
a
The compounds were assayed in triplicate with the averaged IC50 SEM values shown.
aromatic ring at the cap moiety, Leu at the P1 position, Trp or Leu at
the P2 position, Leu at the P3 position, and 3-acetylphenyl or 3,5-
for the trypsin-like subunit (
b2). Recently others reported that dual
inhibition of 2 and 5 activity could achieve maximum protea-
b
b
dimethoxyphenyl at the head group are favored for
b5/CT-L
some inhibition and was most effective in proteasome inhibitor-
inhibitory activity. Based on this pharmacophore, we further
resistant multiple myeloma [38]. In light of this, 44 and 46 dis-
designed two hybrid molecules, 45 and 46 with all these features
playing activity in both b2 and b5 may merit further studies.
(Table 3). Both of them displayed potent inhibition of
tion, the 3-methoxyphenyl at the head position (47) showed strong
activity with an IC50 value of 28.8 nM against 5.
b5. In addi-
2.4. Anticancer activity in a panel of six cancer cell lines
b
Based on the results of cell-based proteasome inhibition assays
shown above in Tables 1e3, a group of 7 compounds were selected
for their more potent proteasome inhibitory activities than others
and for representing modifications at different sites (the head
group and cap moiety). These compounds were evaluated for
anticancer activity in various cancer cell lines including RPMI8226,
THP-1, Ramos (RA.1), SW480, HeLa, and MGC-803 using the MTS
cytotoxicity assay (Table 4). We also determined the IC50 values of
those compounds that exhibited ꢂ50% inhibition of RPMI8226 at 1
2.3. Inhibition of
like activity assays
b1 and b2 in cell-based caspase-like and trypsin-
In addition to
44 and 46 and control compound BSc2189 was determined against
1 and 2 in cell-based caspase-like and trypsin-like activity assay
(Fig. 2). All of these compounds were more active against the
subunit than the 1 subunit. 44 and 46 had a low nanomolar IC50
b5, the activity of representative compounds, 35,
b
b
b
2
b
6

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Table 3
The cell-based CT-L inhibitory activity of head group modified compounds.
Compound
Cap moiety
P2
P3
H
Head group
CT-La IC50 (nM)
385.6 36.9
33 (NI-01106)
(4-CH3O)-Ph
34 (NI-04192)
35 (NI-09016)
36 (NI-18030)
Ph
i-Pr
i-Pr
i-Pr
61.0 3.1
29.5 3.1
60.1 10.0
1H-indol-3-yl
1H-indol-3-yl
37 (NI-09062)
38 (NI-23067)
1H-indol-3-yl
i-Pr
i-Pr
i-Pr
165.1 7.6
22.1 1.2
39 (NI-18019)
40 (NI-18020)
i-Pr
i-Pr
i-Pr
i-Pr
99.7 24.7
64.1 16.6
41 (NI-09073)
i-Pr
i-Pr
4701 961
42 (NI-18073)
43 (NI-18060)
i-Pr
i-Pr
i-Pr
i-Pr
41.7 2.5
34.8 2.6
44 (NI-09083)
45 (NI-18050)
i-Pr
i-Pr
i-Pr
i-Pr
25.7 3.1
31.7 4.9
(continued on next page)
7

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Table 3 (continued)
Compound
Cap moiety
P2
P3
Head group
CT-La IC50 (nM)
25.3 3.8
46 (NI-15020)
i-Pr
i-Pr
47 (NI-18145)
BSc2189
i-Pr
i-Pr
i-Pr
i-Pr
28.8 3.4
34.5 1.7
58.5 4.3
ONX-0914
a
The compounds were assayed in triplicate with the averaged IC50 SEM values shown.
Fig. 2. The cell-based caspase-like (b1, shown on the upper panel) and trypsin-like (b2, lower panel) inhibitory activity of compounds BSc2189, 35, 44 and 46.
Table 4
Cytotoxicity of compounds against tumor cell lines (IC50 SEM, nM)a.
Compound
RPMI8226
THP-1
Ramos (RA 1)
SW480
Hela
MGC-803
25
26
35
43
44
46
47
BSc2198
455.0 42.1
474.8 53.3
90.99 11.1
40.41 3.23
37.57 2.46
38.11 1.19
43.4 1.04
67.76 5.79
567.9 49.48
475.4 53.17
78.12 1.09
71.12 2.02
70.67 6.11
94.41 1.45
97.86 2.79
88.07 3.85
464.0 9.53
428.1 7.28
38.35 2.46
45.73 3.46
26.92 0.40
26.13 0.18
38.33 3.04
53.6 2.21
2441 123.9
2002 213.2
94.25 4.77
82.86 4.67
72.55 7.54
79.26 1.87
74.50 3.13
106.3 14.93
8701 1195
3202 47.62
448.9 5.89
211.8 4.29
120.0 8.80
260.4 4.34
238.93 3.28
288.3 25.22
2017 123.4
1164 4.74
88.80 5.01
78.51 7.46
68.54 7.04
79.05 12.59
85.40 6.73
118.8 3.63
a
The IC50 was calculated with assay data measured at 7 concentrations while each value was determined from three independent experiments.
m
M (Supplemental Table S1). The results indicated good correlation
Compounds 25 and 26 were more effective on hematologic tu-
mor cell lines RPMI8226, Ramos and THP-1 than solid tumor cell
lines SW480, HeLa and MGC-803. On the other hand, 25 (THP-1
IC50: 567.9 nM, HeLa IC50: 8701 nM) and 26 (THP-1 IC50: 475.4 nM,
HeLa IC50: 3202 nM) showed less cytotoxicity than ONX-0914 (THP-
1 IC50: 202.4 nM, HeLa IC50: 590.5 nM) which may be related with
between proteasome inhibition potency and anticancer activity.
All tested compounds were highly effective on hematologic
tumor cell lines RPMI8226, Ramos and THP-1. The IC50 of control
compound BSc2189 was 288 nM in HeLa cells which was in
consistent with its previously published value of 254 nM [23]. 46
was the most potent compound in both cell-based proteasome
inhibition and cancer cell viability assays.
their higher b5i selectivity [39,40].
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J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
2.5. Antiviral activity against SARS-CoV-2
CoMFA using a selected set of 25 compounds with diverse chemical
structures and distribution of bioactivities. The data set was divided
into a training set (20 compounds) for model development and a
test set (5 compounds) for model validation. Selection of the test set
compounds was made based on structural and biological diversity.
These compounds are shown in Supplemental Table S3. The
calculation method is described in the method section.
After determining the potent anticancer activity of our new a-
ketoamide derivatives, in response to the most recent COVID-19
global pandemic caused by SARS-CoV-2, we decided to test the
hypothesis as described above that some of these compounds may
possess anti-SARS-CoV-2 activity. We first tested the cytotoxicity of
several representative derivatives in Vero E6 cells (Supplemental
Table S2). Compound 17 was found to be the least toxic and sub-
sequently tested in Caco-2 cells also. We selected compound 17 for
antiviral study using a plaque reduction assay to determine the 50%
antiviral effective dose (EC50) of the compound. In the plaque
reduction assay, Vero E6 cells were seeded at 4 ꢃ 105 cells/well in
12-well tissue culture plates on the day before carrying out the
assay. After 24 h of incubation, 50 plaque-forming units (PFU) of
SARS-CoV-2 were added to the cell monolayer with or without the
addition of the compound. The percentage of plaque inhibition
relative to the control (i.e. without the addition of compound) wells
were determined for each compound concentration. Remdesivir, a
drug used clinically for treating COVID-19 was used as a positive
control. Compound 17 displayed potent anti-SARS-CoV-2 activity in
3.2. Results of the CoMFA
The CoMFA model has good cross-validated correlation with a q2
value of 0.537 with 4 optimum components. The correlation coef-
ficient r2 is 0.959, and the predicted correlation coefficients r2pred is
0.814, which reveals that the CoMFA model has high predictive
capability. The plots of predicted versus actual activity for all the
data set are shown in Supplemental Figure S1. As to the steric and
electrostatic field contribution, the former accounts for 0.714 while
the latter 0.286, indicating that the steric factor contributes more to
the binding affinities.
3.3. CoMFA contour maps analysis
this plaque reduction assay with the EC50 of 1.28 mM (Fig. 3).
The CoMFA contour map is shown in Fig. 4. The cap position is
highlighted in green color, which indicates that bulky substituents
in this area may improve the activity. The bioactivity of 5-(4-
acetylphenyl) furan containing 26 is much higher than furan con-
taining 23. The yellow area at the P2 position indicates that the
activity can be improved by decreasing the size of the methoxyl
3. Comparative molecular field analysis (CoMFA)
3.1. Data set for analysis
The SAR of the synthesized compounds was further studied by
Fig. 3. Anti-SARS-CoV-2 activity of compound 17 in vitro. Upper panel: photos of plaque reduction assay using SARS-CoV-2-infected Vero E6 cells. Lower panel: compound 17
exhibits potent antiviral activity against SARS-CoV-2 with an EC50 of 1.28
presented as means standard deviations.
m
M as determined by plaque reduction assay, with Remdesivir as a positive control. The results are
9

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
groups with proteasome may also contribute to high binding af-
finity of the compound towards its target.
4.2. Docking of compounds to m-calpain and SARS-CoV-2 main
protease
To examine the possible mechanism for the observed anti-SARS-
CoV-2 effect of compound 17, we performed molecular docking
calculation of this compound with m-calpain because previous
studies by others showed that a proteasome inhibitor MG132
blocked SARS-CoV virus replication by targeting m-calpain [31].
The binding modes of proteasome inhibitors MG132 and 17 as
predicted by our docking calculations were shown in Fig. 6A and B.
Fig. 4. The CoMFA contour map of the training set.
Both the warhead
a-ketoamide group of 17 and the warhead
aldehyde group of MG132 formed a covalent bond with Cys105 of
m-calpain while the backbone’s amide of both compounds formed
hydrogen bond with Gly261 of m-calpain. The N-terminal mor-
pholine cap moiety of 17 had weak van der Waals interaction with
the protein while the N-terminal benzyl ring cap moiety of MG132
had stronger hydrophobic interaction with Trp214. The backbone’s
carbonyl group of 17 formed hydrogen bond with Gly198 while the
backbone’s carbonyl group of MG132 formed hydrogen bond with
Trp106. Moreover, the methoxyl group at P2 position of 17 formed
extra hydrogen bond with Arg337 of m-calpain. The carbonyl oxy-
gen and amide at the warhead of 17 formed hydrogen bond with
Gln99 and Gly261, respectively. The phenyl group at the warhead of
17 extended into the primed site of m-calpain. Although the reac-
tive groups of BSc2189 and MG132 were not the same, the overall
binding modes of these two compounds were basically consistent
within the binding site of the receptor (Fig. 6C).
group of the phenyl core. The green area around the isopropyl
group shows that it plays an important role in the contribution to
bioactivities. The isopropyl group at P2 position has higher bioac-
tivity than an aromatic group such as a phenyl or indole ring. The
blue area at the warhead region suggests that a positive charge on
the phenyl core may improve the activity.
4. Molecular docking and dynamics simulations
4.1. Docking of compounds to 20S proteasome
Molecular docking simulation was performed to predict and
visualize the possible binding modes of compound 46 to 20S pro-
teasome. To evaluate the stability of receptor-ligand complexes, the
docking pose closest to the experimentally determined binding
mode of control BSc2189 (PDB: 4NO8) [23] were used to carry out
molecular dynamics (MD) simulation to generate stable confor-
mations. In the proteasome binding mode of compound 46 (Fig. 5),
the amide group of the peptide backbone of the compound formed
Recent studies show that
a-ketoamide derivatives such as
boceprevir, narlaprevir [42], and 13b [43] act as potent SARS-CoV-2
main protease inhibitors. Therefore, we also examined the potential
docking of compound 17 to the active site of SARS-CoV-2 main
protease (Fig. 6D). Docking models indicated that
and the amide oxygen of -ketoamide head group formed two
hydrogen bonds with His41 and Gly143 on SARS-CoV-2 main pro-
tease, which might suggest a possible advantage of -ketoamide
a-keto oxygen
hydrogen bonds with proteasome0s residues Gly47, Ala49, and Thr21
.
a
Additional hydrogen bonds were observed between the acetyl
group at the cap position of the compound and hydroxyl group of
proteasome0s residue Tyr305. The furan group at the cap position
and phenyl group at the head position of the compound formed H-
a
compared with other head group such as aldehyde and trans-a, b-
unsaturated ethyl ester. The compound0s benzyl group at P1 posi-
tion occupied S1 pocket formed by Glu166 and Phe140 on the main
protease. The amide group of the compound formed a hydrogen
p
bond with proteasome’s residues Pro326 and Ser130. These results
suggest that the interactions of the compound0s cap and head
Fig. 5. The predicted complex structure of compound 46 (salmon stick) bound to human 20S proteasome (PDB ID: 5LEY) [41] generated from molecular dynamics simulation.
Hydrogen bonds and H- bonds are shown by red and green dashed lines.
p
10

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Fig. 6. Predicted m-calpain (PDB ID: 3BOW) [44] bound structures of MG132 (A) and 17 (B); (C) Superposition of the structures of MG132 (green stick) and 17 (cyan stick) bound to
m-calpain (wheat cartoon). (D) Predicted structure of 17 (cyan stick) bound to SARS-CoV-2 main protease (PDB ID: 6Y2F) [43].
bond with His41. The side chain of Tyr(OMe) at the P2 position of
the compound didn0t properly insert into the hydrophobic S2
pocket but extended into another more flexible site, forming a
hydrogen bond with Gln192. The N-terminal morpholine cap moiety
of 17 did not occupy the small hydrophobic S4 pocket and showed a
kinked conformation.
substituents targeting the primed site may affect the reaction rate
between
-keto moiety and catalytic residue Thr1 or the lowest
energy conformation of the inhibitory compound important for its
potency. How the substituents targeting the primed site residues
affect the potency and selectivity of the inhibitor needs to be
further clarified.
a
Our data also show that compounds with the same moiety at the
head position but attached to different peptide sequences display
varying fold increase in inhibitory activity compared with com-
pounds with an unsubstituted moiety at the head position. For
example, 3-acetyl group at the head position gives rise to ~4 fold
increase in inhibitory activity of the compound with a peptide
backbone Cbz-Leu-Phe-Leu, 2 fold increase for a peptide backbone
Cbz-Leu-Trp-Leu, and 1.5 fold increase for a peptide backbone Cbz-
Leu-Leu-Leu. Taken together, these results and those published
previously by others demonstrate that the primed site, a region on
the proteasome previously less explored compared to the non-
primed site, modulates the activity of proteasome inhibitors and
that incorporation of suitable substituent groups targeted at this
site leads to better inhibitory activity. Besides the primed site, our
studies also reveal new insights into the role of the non-primed site
and effect on bioactivity of chemical modifications targeted at this
site. The overall summary of SAR obtained from our studies is
illustrated in Fig. 7. In particular, biphenyl-4-carbonyl and 5-(4-
acetylphenyl)furan-2-carbonyl at the cap position, and 3-
methoxyphenyl, 3,5-dimethoxylphenyl, 3-acetylphenyl and 3-
methylsulfonylphenyl at the head position are beneficial to com-
pound’s inhibition of proteasome. Modifications at the side chains
5. Discussion
Many previous studies have demonstrated that targeting the
primed site can be an effective strategy for developing potent or
selective inhibitors of proteases of viruses such as human immu-
nodeficiency virus and hepatitis C virus [25e27]. Recently, several
groups have reported a-keto phenylamide and epoxyketone based
proteasome inhibitors targeting the primed site to increase potency
or overcome resistance [21,45]. It was shown that the targeting of
the primed site by 3-phenoxy phenyl could increase b5 inhibition of
a compound. Here we studied the impact on bioactivity of different
groups at the head position that interact with the primed site
(Fig. 1A). Electron withdrawing groups, acetyl (34, 35, 38) and
methylsulfonyl (36) incorporated into the meta-position of the
phenyl ring at the head position led to 1.5e4 fold increase in
inhibitory activity. Incorporation of an electron donating group
methoxyl (44, 45, 47) also led to increase in inhibitory activity
while doing so with an ethyl or cyclopentyl moiety resulted in
similar inhibitory activity. These results indicate that the activity of
ketoamide derivatives may not depend on the strength of the
electron donating substituents at the head position. We reason that
11

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Fig. 7. Summary of the SAR of
a
-keto phenylamide derivatives.
at P1, P2 and P3 positions also affect proteasome inhibitor’s activity.
It is worth noting that one of our -ketoamide derivatives re-
pandemic, thus suggesting that the novel
reported here, besides their value for developing anticancer drugs,
may offer new lead in finding effective anti-SARS-CoV-2
therapeutics.
a-ketoamide derivatives
a
ported here, 17 was chosen as a representative compound to be
tested in SARS-CoV-2 cellular infection assay and found to be active
in inhibiting the viral infection. It has been previously reported that
proteasome inhibitors such as MG132 can inhibit the replication of
SARS-CoV likely via targeting and inhibiting m-calpain activity
necessary for the viral replication cycle. Our molecular docking
studies show that 17 binds m-calpain in a similar manner as
MG132, which is consistent with the notion that proteasome in-
hibitors may intervene with the replication of SARS-CoV-2, which is
highly homologous with SARS-CoV, by interfering with host
cellular processes mediated by proteins like m-calpain that are
a
7. Experimental section
7.1. Chemistry
Commercially available reagents were used directly without
further purification. The high resolution MS (HRMS) of target
compounds were analyzed by using Waters Xevo G2 QTof. NMR
spectra were obtained on the Bruker Ascend™ 400. The chemical
essential for the virus life cycle. Recently, some
a
-ketoamide de-
shifts (d) are reported in parts per million (ppm) using suitable
rivatives are reported to target SARS-CoV-2 main protease [42,43],
which raises the possibility of an additional or alternative mecha-
nism of 17 action on SARS-CoV-2. It awaits further studies to
delineate the detailed mechanism of 17 and whether this com-
deuterated NMR solvents in reference to tetramethyl silane (TMS)
at 0 ppm. Multiplicities are defined as follows: s (singlet), brs
(broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of
doublets), and m (multiplets). The reaction was monitored by TLC
(silica gel GF254). The compounds were purified by silica chro-
matography (silica gel 100e200 mesh or 200e300 mesh) or Prep-
HPLC. HPLC Conditions, Column: XBridge Prep OBD™ C18
pound or other
a-ketoamide derivatives might exert anti-SARS-
CoV-2 effect by targeting a host and/or a viral protein. Given the
urgency and severity of the COVID-19 pandemic and need for
effective prevention and treatment measures, the observed anti-
19 mm ꢃ 150 mm ꢃ 5
mm; detection: 220 nm; flow rate: 15 mL/
min; run time: 30 min; mobile phase A: water (0.01% TFA); mobile
phase B: acetonitrile (0.01% TFA).
viral effect of our new
a
-ketoamide derivative against SARS-CoV-2
suggests that this may be a new lead for developing therapeutics
against coronaviruses. If successfully developed, such therapeutics
will add to the clinical arsenal for combinational therapy for the
COVID-19 pandemic.
The experimental details of compounds 35 and 46 are described
below. The synthetic procedure of compounds 17e26, 45, 47 is
similar to that of compound 46, as shown in Scheme 1A. The syn-
thetic procedure of compounds 19, 27e34, 36e44 is similar to that
of compound 35, as shown in Scheme 1B.
6. Conclusion
We have designed, synthesized, and characterized the biological
7.1.1. Procedure for the preparation of (S)-2-((S)-2-(5-(4-
activities and SAR of a new class of
a
-keto phenylamide derivatives.
acetylphenyl)furan-2-carboxamido)-4-methylpentanamido)-4-
methylpentanoic acid (5a)
These novel compounds target both non-primed and primed sites
of the proteasome and contain a variety of chemical modifications
at the cap moiety, P1, P2 and P3 side chain positions, and the head
group. Cell-based proteasome inhibition and anticancer activity
assays have led to the identification of many compounds from this
series that possess highly potent biological activity at the nano-
molar concentrations. Among them, compounds 35, 44, 46 and 47
display most potent activities and may merit further studies of their
potential as new anticancer agents. Another interesting and
possibly important finding of our studies is that a representative
compound 17 is found to be active in inhibiting the cellular infec-
tion of SARS-CoV-2 that causes the ongoing COVID-19 global
To the mixture of H-Leu-OMe$HCl (12.7 g, 69.9 mmol, 1.0 equiv),
Boc-Leu-OH (17.8 g, 76.9 mmol, 1.1 equiv) in THF (150 mL) was
added HATU (29.22 g, 76.9 mmol, 1.1 equiv), HOBt (10.4 g,
76.9 mmol,1.1 equiv). The reaction mixture was cooled to 0 ꢁC, DIEA
(3.5 equiv) was added. The temperature was increased to room
temperature naturally and stirred for 1 h. After addition brine, the
reaction mixture was extracted with ethyl acetate, the organic
phase was washed with brine, dried over anhydrous Na2SO4,
filtered, concentrated under vacuum. The crude product was pu-
rified by silica gel column chromatography (30% EtOAc/petroleum
ether) to afford Boc-Leu-Leu-OMe 2a (20.8 g, 83% yield). 1H NMR
12

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
(400 MHz, CDCl3)
d
6.50e6.41 (m, 1H), 4.97e4.82 (m, 1H),
light yellow solid.
4.67e4.58 (m, 1H), 4.19e4.03 (m, 1H), 3.74 (s, 3H), 1.73e1.64 (m,
4H), 1.63e1.48 (m, 2H), 1.46 (s, 9H), 0.99e0.90 (m, 12H).
To a solution of 2b (3.7 g, 9.73 mmol, 1 equiv) in CH2Cl2 (10 mL)
was added TFA (11.08 g, 97.3 mmol, 10 equiv) at 0 ꢁC. The solution
was stirred for 4 h at 0 ꢁC. Then the solution was concentrated. The
product was precipitated from ethyl ether. The precipitate was
collected to give NH2-Ala-Tyr(OMe)eOMe TFA salt 3b as off white
Boc-Leu-Leu-OMe (20.8 g, 58.14 mmol, 1.0 equiv) was dissolved
in CH2Cl2 (60 mL). The reaction solution was cooled to 0 ꢁC and TFA
(53 g, 465.1 mmol, 8.0 equiv) was added in portions. After the
addition was completed, the reaction solution was stirred at 0 ꢁC for
3 h. The reaction solution was evaporated in vacuum. The crude
product was precipitated from ethyl ether and filtered to give 3a
TFA salt (19.4 g, 94% yield) as off white solid. The crude product was
used directly in the next step without further purification. 1H NMR
solid. 1H NMR (400 MHz, DMSO‑d6)
d
9.09 (d, J ¼ 7.4 Hz, 1H), 8.37 (s,
3H), 7.17 (d, J ¼ 8.5 Hz, 2H), 6.89 (d, J ¼ 8.5 Hz, 2H), 4.48e4.39 (m,
1H), 3.91e3.81 (m, 1H), 3.72 (s, 3H), 3.61 (s, 3H), 3.06e2.85 (m, 2H),
1.37 (t, J ¼ 10.6 Hz, 3H).
To a solution of 2-morpholinoacetic acid (0.9 g, 6.21 mmol, 1.0
equiv) in THF was added HATU (2.6 g, 6.83 mmol, 1.1 equiv) and
HOBt (0.9 g, 6.83 mmol, 1.1 equiv) subsequently. The reaction so-
lution was cooled to 0 ꢁC. Then 3b (2.6 g, 6.21 mmol, 1.0 equiv) was
added. DIEA (3.52 g, 27.32 mmol, 4.0 equiv) was added dropwise.
The reaction solution was stirred at the same temperature for 1 h.
TLC showed the reaction was completed. The mixture reaction was
partitioned between ethyl acetate and water. The aqueous layer
was separated and extracted again with ethyl acetate. The com-
bined organic layer was washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated under vacuum. The crude
product was purified by silica gel column eluted with CH3OH:
CH2Cl2 (30:1) to yield (S)-methyl 3-(4-methoxyphenyl)-2-((S)-2-
(2-morpholinoacetamido)propanamido)propanoate 4b (1.9 g, 75%
yield) as light yellow oil.
(400 MHz, DMSO‑d6)
4.38e4.29 (m, 1H), 3.84e3.75 (m, 1H), 3.63 (s, 3H), 1.76e1.46 (m,
6H), 0.97e0.81 (m, 12H).
d
8.84 (d, J ¼ 7.5 Hz, 1H), 8.19 (s, 3H),
To a mixture of 3a TFA salt (8.6 g, 24.2 mmol, 1.0 equiv), com-
pound 16 (6.2 g, 26.62 mmol, 1.1 equiv) in THF was added HATU
(10.1 g, 26.62 mmol, 1.1 equiv), HOBt (3.6 g, 26.62 mmol, 1.1 equiv).
The reaction solution was cooled to 0 ꢁC and DIEA (12.5 g,
96.8 mmol, 4.0 equiv) was added. The temperature was increased to
room temperature naturally and stirred for 1 h. The mixture reac-
tion was partitioned between ethyl acetate and water. The aqueous
layer was separated and extracted again with ethyl acetate. The
combined organic layer was washed with brine, dried over anhy-
drous sodium sulfate, filtered, concentrated under vacuum. The
crude product was purified by silica gel column chromatography to
afford 4a (6.4 g, 56% yield) as light yellow powder.
To a solution of 4b (1.9 g, 4.67 mmol, 1.0 equiv) in THF (15 mL)
and H2O (5 mL), LiOH.H2O (0.2 g, 4.67 mmol, 1.0 equiv) was added
at 0 ꢁC. The reaction was stirred for 3 h at 0 ꢁC. TLC showed the
reaction was completed. Ethyl acetate was added, the organic layer
was separated. The aqueous layer was adjusted pH to 5e6 with 1 N
HCl. The crude product was purified by reverse phase silica gel
eluted with 20% acetonitrile/H2O (1:4) and lyophilized to get
compound 5b (1.5 g, 83% yield) as white solid. 1H NMR (400 MHz,
Compound 4a (7.2 g, 15.3 mmol, 1.0 equiv) was dissolved in THF
(80 mL), H2O (20 mL). The reaction solution was cooled to 0 ꢁC.
LiOH.H2O (1.9 g, 45.9 mmol, 3.0 equiv) was added. The reaction
solution was stirred for 2 h at room temperature. TLC showed the
reaction was completed. THF was removed under reduced pressure.
The residue was diluted with water, then adjusted pH to 3 with 1 N
HCl. The resulting mixture was extracted with ethyl acetate. The
organic layers were combined, washed with brine, dried and
concentrated under vacuum to give compound 5a (6.3 g, 90% yield)
as light yellow powder. The product was used directly in the next
DMSO‑d6)
d
7.84 (d, J ¼ 7.8 Hz, 1H), 7.44 (d, J ¼ 7.0 Hz, 1H), 7.03 (d,
J ¼ 8.6 Hz, 2H), 6.72 (d, J ¼ 8.6 Hz, 2H), 4.27e4.18 (m, 1H), 4.03e3.95
(m, 1H), 3.68 (s, 3H), 3.56e3.53 (m, 4H), 3.03e2.95 (m, 4H),
2.42e2.36 (m, 4H), 1.19 (d, J ¼ 7.0 Hz, 3H).
step. 1H NMR (400 MHz, DMSO‑d6)
d 12.48 (s, 1H), 8.57 (d,
J ¼ 8.5 Hz, 1H), 8.26 (d, J ¼ 7.9 Hz, 1H), 8.12e8.01 (m, 4H), 7.33e7.29
(m, 2H), 4.64e4.53 (m, 1H), 4.29e4.13 (m, 1H), 2.62 (s, 3H),
1.78e1.44 (m, 6H), 0.98e0.77 (m, 12H).
7.1.3. Procedure for the preparation of Cbz-Leu-Trp-OH (10a), Cbz-
Leu-Leu-OH (10b), Cbz-Leu-Phe-OH (10c)
To the solution of Cbz-Leu-OH (3.3 g, 12.4 mmol, 1.0 equiv) and
H-Trp-OMe.HCl (3.16 g, 12.4 mmol, 1.0 equiv) in DMF (40 mL) was
added HATU(5.2 g, 13.6 mmol, 1.1 equiv), HOBt (1.84 g, 13.6 mmol,
1.1 equiv). The reaction mixture was cooled to 0 ꢁC, DIEA (5.6 g,
43.4 mmol, 3.5 equiv) was added. The temperature was increased to
room temperature naturally and stirred for 1 h. After addition brine,
the resulting mixture was extracted with ethyl acetate. The com-
bined organic phase was washed with brine, dried over anhydrous
Na2SO4, filtered, concentrated under vacuum. The crude product
was purified by silica gel column chromatography (30% EtOAc/pe-
troleum ether / 50% EtOAc/petroleum ether) to afford Cbz-Leu-
Trp-OMe (5.3 g, yield 93%) as white solid. 1H NMR (400 MHz,
7.1.2. Procedure for the preparation of (S)-2-((S)-3-(4-
methoxyphenyl)-2-(2-morpholinoacetamido)propanamido)-4-
methylpentanoic acid (5b)
To a solution of Boc-Tyr(OMe)eOH (3.6 g, 12.19 mmol, 1.0 equiv)
in dry methanol was added SOCl2 (2.9 g, 24.38 mmol, 2.0 equiv)
dropwise at 0 ꢁC. The resulting solution was warmed to room
temperature and stirred for 2 h. The reaction solution was
concentrated under vacuum. The product was precipitated by the
addition of ethyl ether and the solid was collected by filtration to
give NH2-Tyr(OMe)eOH HCl salt 1b (2.72 g, 91% yield) as off white
solid. 1H NMR (400 MHz, DMSO‑d6)
d
8.81 (s, 3H), 7.17 (d, J ¼ 8.5 Hz,
2H), 6.89 (d, J ¼ 8.5 Hz, 2H), 4.20e4.13 (m, 1H), 3.74 (s, 3H), 3.66 (s,
DMSO‑d6)
d
8.06 (s, 1H), 7.50 (d, J ¼ 7.8 Hz, 1H), 7.37e7.26 (m, 6H),
3H), 3.23e3.14 (m, 1H), 3.12e3.03 (m, 1H).
7.19e7.12 (m, 1H), 7.11e7.04 (m, 1H), 6.94 (s, 1H), 6.59 (d, J ¼ 7.3 Hz,
1H), 5.20e5.11 (m, 1H), 5.09e4.96 (m, 2H), 4.93e4.84 (m, 1H),
4.29e4.16 (m, 1H), 3.65 (s, 3H), 3.29 (d, J ¼ 5.2 Hz, 2H), 1.68e1.52
(m, 2H), 1.50e1.38 (m, 1H), 0.92e076 (m, 6H).
To a solution of Boc-L-analine (2.1 g,11.11 mmol,1.0 equiv) in THF
was added HATU (4.64 g, 12.22 mmol 1.1 equiv) and HOBt (1.65 g,
12.22 mmol, 1.1 equiv). The resulting solution was cooled to 0 ꢁC.
Compound 1b HCl salt (2.72 g, 11.11 mmol, 1.0 equiv) and DIEA
(5.02 g, 38.9 mmol, 3.5 equiv) was added. The reaction solution was
stirred at the same temperature for 1 h. Water was added and the
mixture was extracted with ethyl acetate. The organic layer was
separated and washed with brine, dried over anhydrous sodium
sulfate, filtered, and concentrated under vacuum. The crude prod-
uct was purified by silica gel column eluted with 1% CH3OH in
CH2Cl2 to yield Boc-Ala-Tyr(OMe)eOMe 2b (3.7 g, 87% yield) as
Cbz-Leu-Trp-OMe (5.3 g, 11.4 mmol, 1.0 equiv) was dissolved in
THF (60 mL), H2O (15 mL). The reaction solution was cooled to 0 ꢁC.
LiOH.H2O (1.4 g, 34.2 mmol, 3.0 equiv) was added. The reaction
solution was stirred for 2 h at room temperature. TLC showed the
reaction was completed. THF was removed under reduced pressure.
The residue was diluted with water, then adjusted to pH 3 with 1 N
HCl. The resulting mixture was extracted with ethyl acetate. The
organic layers were combined, washed with brine, dried and
13

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
concentrated under vacuum to give compound 10a (4.8 g, 94%
(m, 1H), 7.23e7.15 (m, 3H), 6.94e6.85 (m, 1H), 6.36e6.19 (m, 1H),
5.40e5.30 (m, 1H), 5.15e4.92 (m, 2H), 4.62 (q, J ¼ 7.4 Hz, 1H),
4.20e4.09 (m, 1H), 4.02e3.90 (m, 1H), 3.52e3.32 (m, 2H),
3.18e2.99 (m, 2H), 2.41 (s, 2H), 1.65e1.39 (m, 4H), 1.38e1.17 (m,
2H), 0.91e0.83 (m, 12H).
yield) as light yellow powder. The product was used in the next step
directly. 1H NMR (400 MHz, DMSO‑d6)
d 10.86 (s, 1H), 8.05 (d,
J ¼ 7.6 Hz, 1H), 7.53 (d, J ¼ 7.8 Hz, 1H), 7.44e7.22 (m, 7H), 7.17e7.12
(m, 1H), 7.10e7.02 (m, 1H), 7.01e6.93 (m, 1H), 5.15e4.86 (m, 2H),
4.55e4.43 (m, 1H), 4.14e3.99 (m, 1H), 3.23e2.99 (m, 2H), 1.67e1.33
(m, 3H), 0.92e0.74 (m, 6H).
7.1.5. General procedure for the preparation of 7a and 12a
To a solution of 6a or 11a (1 equiv) in DMSO, IBX (1.5 equiv) was
added. The resulting solution was stirred at room temperature for
4 h. The reaction mixture was partitioned between ethyl acetate
(30 mL) and saturated NaHCO3 (30 mL). The organic layer was
separated and the aqueous layer was extracted with ethyl acetate
(2 ꢃ 30 mL). The combined organic layer was washed with satu-
rated NaHCO3 and brine, dried over anhydrous sodium sulfate,
filtered, concentrated under vacuum. The crude product was pu-
rified by silica gel column to yield 7a or 12a.
Cbz-Leu-Leu-OH (10b) The synthetic procedure is similar to
compound 10a using H-Leu-OMe$HCl as starting material. 1H NMR
(400 MHz, CDCl3)
d 7.38e7.27 (m, 5H), 6.80e6.72 (m, 1H),
5.66e5.54 (m, 1H), 5.10 (s, 2H), 4.65e4.54 (m, 1H), 4.33e4.08 (m,
1H), 1.76e1.45 (m, 6H), 0.98e0.84 (m, 12H).
Cbz-Leu-Phe-OH (10c) The synthetic procedure is similar to
compound 10a using H-Phe-OMe$HCl as starting material. 1H NMR
(400 MHz, CDCl3)
d 8.66 (s, 1H), 7.45e7.29 (m, 5H), 7.25e7.19 (m,
3H), 7.16e7.11 (m, 2H), 7.07e6.96 (m, 1H), 5.73e5.58 (m, 1H),
5.22e5.03 (m, 2H), 4.92e4.77 (m, 1H), 4.39e4.08 (m, 1H),
3.26e3.13 (dd, J ¼ 13.9, 5.4 Hz, 1H), 2.99 (dd, J ¼ 13.9, 6.6 Hz, 1H),
1.73e1.35 (m, 3H), 0.95e0.80 (m, 6H).
7.1.5.1. (S)-2-((5-(4-acetylphenyl)furan-2-yl)amino)-4-methyl-N-
((S)-4-methyl-1-(((S)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopentan-2-yl)pentanamide (7a). White solid. Yield 72%.1H NMR
7.1.4. General procedure for the preparation of 6a and 11a-c
(400 MHz, DMSO‑d6)
d
9.38 (s, 1H), 8.60 (d, J ¼ 8.2 Hz, 1H), 8.27 (d,
To a solution of 5a or 10a-c (1.0 equiv) in THF (10 mL) was added
NMM (3.0 equiv). The resulting solution was cooled to ꢀ5 ꢁC. Iso-
butyl chloroformate (1.1 equiv) was added. The resulting solution
was stirred at ꢀ5 ꢁC for 30 min. Then L-leucinol (1.6 equiv) in THF
(10 mL) was added dropwise. The reaction mixture was stirred
at ꢀ5 to 0 ꢁC for 2 h. Water was added and the mixture was
extracted with ethyl acetate. The organic layer was separated and
the aqueous layer was extracted with ethyl acetate again. The
combined organic layer was washed with brine, dried over anhy-
drous sodium sulfate, filtered, concentrated under vacuum. The
crude product was purified by silica gel column to yield 6a or 11a-c.
J ¼ 7.3 Hz, 1H), 8.21e8.13 (m, 1H), 8.12e7.97 (m, 4H), 7.36e7.24 (m,
2H), 4.68e4.46 (m, 1H), 4.43e4.27 (m, 1H), 4.18e4.07 (m, 1H), 2.62
(s, 3H), 1.75e1.35 (m, 9H), 0.97e0.72 (m, 18H).
7.1.5.2. ((S)-1-(((S)-3-(1H-indol-2-yl)-1-(((S)-4-methyl-1-
oxopentan-2-yl)amino)-1-oxopropan-2-yl)amino)-4-methyl-1-
oxopentan-2-yl) carbamic acid benzyl ester (12a). Off white solid.
Yield 68%. 1H NMR (400 MHz, DMSO‑d6)
d 10.86 (s, 1H), 9.14 (s, 1H),
8.34 (d, J ¼ 7.3 Hz,1H), 8.10 (d, J ¼ 8.0 Hz,1H), 7.58 (d, J ¼ 7.8 Hz,1H),
7.44 (d, J ¼ 8.2 Hz, 1H), 7.40e7.25 (m, 6H), 7.16 (d, J ¼ 2.0 Hz, 1H),
7.09e6.93 (m, 2H), 5.08e4.95 (m, 2H), 4.68e4.54 (m,1H), 4.11e3.99
(m, 2H), 3.22e3.07 (m, 1H), 3.06e2.86 (m, 1H), 1.65e1.25 (m, 6H),
0.92e0.70 (m, 12H).
7.1.4.1. 5-(4-acetylphenyl)-N-((S)-1-(((S)-1-(((S)-1-hydroxy-4-
methylpentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)furan-2-carboxamide (6a). Light yellow
7.1.6. Procedure for the preparation of 1-(3-isocyanophenyl)
ethanone (14)
solid. Yield 41%. 1H NMR (400 MHz, CDCl3)
d
8.01 (d, J ¼ 8.5 Hz, 2H),
7.81 (d, J ¼ 8.4 Hz, 2H), 7.05e6.85 (m, 3H), 6.47 (d, J ¼ 8.5 Hz, 1H),
4.64e4.52 (m, 1H), 4.43e4.31 (m, 1H), 4.09e3.99 (m, 1H),
3.70e3.62 (m, 1H), 3.54e3.44 (m, 1H), 2.62 (s, 3H), 1.86e1.51 (m,
9H), 1.03e0.85 (m, 18H).
The mixture of 3-aminoacetophenone (6.0 g, 44.4 mmol, 1.0
equiv) and HCOOH (6.1 g, 133.2 mmol, 2.5 equiv) was stirred for
3 h at 60 ꢁC. After completion of the reaction, CH2Cl2 was added.
The organic layer was washed with cold water, saturated aqueous
NaHCO3 and brine, then dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. The crude N-(3-
acetylphenyl)formamide was used in the next step without further
purification.
7.1.4.2. ((S)-1-(((S)-1-(((S)-1-hydroxy-4-methylpentan-2-yl)amino)-
3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl) carbamic acid benzyl ester (11a). White solid. Yield 34%. 1H
NMR (400 MHz, DMSO‑d6)
d
10.81 (s, 1H), 8.01 (d, J ¼ 8.2 Hz, 1H),
N-(3-acetylphenyl)formamide (44.4 mmol, 1.0 equiv) and DIEA
(17.2 g, 133.2 mmol, 3.0 equiv) are dissolved in 100 mL dry CH2Cl2.
The reaction solution was cooled to 0 ꢁC. POCl3 (8.2 g, 53.3 mmol,1.1
equiv) was added dropwise at 0 ꢁC. The temperature of reaction
was increased to room temperature naturally and stirred for 3 h
CH2Cl2 was added. The organic layer was washed with cold water,
saturated aqueous NaHCO3 and brine. The organic layer was
separated, dried over Na2SO4, filtered and concentrated under
reduced pressure at room temperature. The crude product was
purified by silica gel column chromatography to afford 1-(3-
isocyanophenyl)ethanone 14 as light yellow solid (5.9 g, 92%
7.61e7.48 (m, 2H), 7.43 (d, J ¼ 8.2 Hz, 1H), 7.40e7.23 (m, 6H),
7.16e7.09 (m, 1H), 7.08e7.02 (m, 1H), 7.01e6.93 (m, 1H), 5.03 (q,
J ¼ 12.6 Hz, 2H), 4.62e4.46 (m, 2H), 4.08e3.98 (m, 1H), 3.84e3.71
(m, 1H), 3.28e3.18 (m, 1H), 3.17e3.03 (m, 2H), 2.98e2.84 (m, 1H),
1.63e1.47 (m, 2H), 1.44e1.16 (m, 4H), 0.88e0.77 (m, 12H).
7.1.4.3. ((S)-1-(((S)-1-(((S)-1-hydroxy-4-methylpentan-2-yl)amino)-
4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)
carbamic acid benzyl ester (11b). White solid. Yield 52%. 1H NMR
(400 MHz, CDCl3)
d 7.39e7.29 (m, 5H), 6.85e6.75 (m, 1H), 6.66 (d,
J ¼ 8.3 Hz, 1H), 5.57e5.46 (m, 1H), 5.16e5.04 (m, 2H), 4.45e4.36 (m,
1H), 4.25e4.12 (m, 1H), 4.07e3.95 (m, 1H), 3.64 (dd, J ¼ 11.2, 3.5 Hz,
1H), 3.52 (dd, J ¼ 11.2, 5.6 Hz, 1H), 2.97 (s, 1H), 1.78e1.46 (m, 7H),
1.45e1.27 (m, 2H), 0.94e0.82 (m, 18H).
yield).1H NMR (400 MHz, CDCl3)
d 8.01e7.93 (m, 2H), 7.61e7.51 (m,
2H), 2.63 (s, 3H). Due to the strong smell of isonitrile, all operations
were conducted in a well-ventilated hood.
7.1.7. Procedure for the preparation of 5-(4-acetylphenyl)furan-2-
carboxylic acid (16)
To a solution of 4-bromoacetophenone (15.0 g, 75.3 mmol, 1.0
equiv), methyl 2-furoate (126.1 g, 301.2 mmol, 4.0 equiv), KOAc
(22.2 g, 225.9 mmol, 3.0 equiv) in dimethylacetamide (150 mL) was
7.1.4.4. ((S)-1-(((S)-1-(((S)-1-hydroxy-4-methylpentan-2-yl)amino)-
1-oxo-3-phenylpropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)
carbamic acid benzyl ester (11c). White solid. Yield 43%. 1H NMR
(400 MHz, CDCl3) d 7.39e7.30 (m, 5H), 7.29e7.26 (m, 1H), 7.25e7.24
14

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
added Pd(OAc)2 (0.253 g, 1.13 mmol, 0.015 equiv) under nitrogen
atmosphere. The resulting mixture was stirred at 130 ꢁC for 4 h
under nitrogen atmosphere. Then saturated NH4Cl was added and
extracted with CH2Cl2. The organic layer was washed with 0.1 N HCl
(2 ꢃ ), brine, and concentrated under reduced pressure. The crude
product was applied onto a silica gel column with ethyl acetate/
petroleum ether (1:10) to give methyl 5-(4-acetylphenyl)-furan-2-
carboxylate 15 (10.1 g, 55% yield) as light yellow solid. 1H NMR
(m, 1H), 3.33e3.05 (m, 1H), 2.64 (s, 3H), 1.60e1.51 (m, 2H),
1.50e1.16 (m, 4H), 1.06e0.65 (m, 12H). 13C NMR (101 MHz,
DMSO‑d6)
d 197.52, 196.78, 172.13, 171.91, 159.70, 155.89, 137.99,
137.33, 137.02, 135.99, 135.86, 129.25, 128.36, 127.78, 127.68, 127.31,
124.90, 123.37, 120.84, 119.82, 118.32, 118.16, 111.23, 109.93, 65.40,
53.16, 52.56, 52.13, 40.67, 38.32, 27.69, 26.79, 24.54, 24.12, 23.13,
23.03, 21.42, 21.18. HRMS (ESI): m/z (M
40H48N5O7,710.8384; found, 710.3558.
þ
H)þ calcd for
C
(400 MHz, CDCl3)
d
8.02 (d, J ¼ 8.3 Hz, 2H), 7.87 (d, J ¼ 8.3 Hz, 2H),
7.29e7.27 (m, 1H), 6.92e6.85 (m, 1H), 3.94 (s, 3H), 2.63 (s, 3H).
Methyl 5-(4-acetylphenyl)-furan-2-carboxylate 15 (10.1 g,
41.4 mmol, 1.0 equiv) was dissolved in THF (100 mL), H2O (25 mL).
LiOH.H2O (5.1 g, 124.2 mmol, 3.0 equiv) was added. The reaction
solution was stirred for 2 h at room temperature. CH2Cl2 and water
was added. The water layer was separated, then adjusted pH to 3
with 2 N HCl. The resulting mixture was extracted with ethyl ace-
tate. The organic layers were combined, washed with brine, dried
and concentrated under vacuum to give compound 16 (8.5 g, 89%
yield) as off white solid.
7.1. 8. 3. (S)-3-((S)-3-(4-methoxyphenyl)-2-((S)-2-(2-
morpholinoacetamido)propanamido) propanamido)-2-oxo-N,4-
diphenylbutanamide (17). White solid. Yield 10%. 1H NMR
(400 MHz, CDCl3)
d 8.67 (s, 1H), 7.72e7.60 (m, 2H), 7.45e7.34 (m,
2H), 7.25e7.16 (m, 4H), 7.11e6.99 (m, 3H), 6.84e6.72 (m, 2H),
6.69e6.50 (m, 1H), 5.67e5.48 (m, 1H), 4.65e4.47 (m, 1H),
4.40e4.27 (m, 1H), 3.92e3.58 (m, 7H), 3.38e3.22 (m, 1H),
3.19e2.83 (m, 5H), 2.80e2.33 (m, 4H), 1.35e1.24 (m, 3H). HRMS
(ESI): m/z (M þ H)þ calcd for C35H42N5O7, 644.7373; found,
644.3080.
7.1.8. General procedure for the preparation of 35 and 46
To a solution of 7a or 12a (1.0 equiv) and 14 (1.5 equiv) in dry
CH2Cl2 (2 mL), trifluoroacetic acid (2.0 equiv) was added dropwise
at ꢀ5 ꢁC. The reaction mixture was allowed to stir for 2 h at 0 ꢁC.
Then pyridine (4.0 equiv) was added at 0 ꢁC. The resulting solution
was stirred at 0 ꢁC for 1 h CH2Cl2 was added, the pH value of the
reaction was adjusted to 5e6 with 1 N HCl in ice bath. The aqueous
layer was separated, the organic layer was washed with water
(30 mL) and brine (30 mL). The combined organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The crude product was applied onto a silica gel
column with ethyl acetate/petroleum ether (1 : 5) to get product as
white solid.
To a solution of compound 8a or 13a (1.0 equiv) in DMSO was
added IBX (2.0 equiv). The resulting solution was stirred at room
temperature for 5 h. Saturated NaHCO3 solution was added and
extracted with ethyl acetate (3 ꢃ 30 mL). The combined organic
layer was washed with saturated NaHCO3 solution, water, brine,
dried over anhydrous sodium sulfate, filtered, concentrated under
vacuum. The crude product was precipitated from ethyl acetate to
give final product 35 or 46.
7.1. 8. 4. (S)-3-((S)-3-(4-methoxyphenyl)-2-((S)-2-(2-
morpholinoacetamido)propanamido) propanamido)-5-methyl-2-
oxo-N-phenylhexanamide (18). White solid. Yield 60%. 1H NMR
(400 MHz, CDCl3)
d 8.67 (s, 1H), 7.71e7.47 (m, 3H), 7.41e7.32 (m,
2H), 7.24e7.06 (m, 3H), 6.88e6.67 (m, 3H), 6.66e6.49 (m, 1H),
5.41e5.28 (m, 1H), 4.71e4.56 (m, 1H), 4.48e4.24 (m, 1H), 3.82e3.69
(m, 7H), 3.19e2.92 (m, 4H), 2.72e2.41 (s, 4H), 1.75e1.67 (m, 1H),
1.63e1.54 (m, 1H), 1.50e1.42 (m, 1H), 1.39e1.32 (m, 3H), 1.01e0.95
(m, 3H), 0.94e0.89 (m, 3H). HRMS (ESI): m/z (M þ H)þ calcd for
C
32H44 N5O7, 610.7211; found, 610.3252.
7.1.8.5. ((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)carbamic acid benzyl ester (19).
White solid. Yield 54%. 1H NMR (400 MHz, CDCl3)
d 8.62 (s, 1H),
7.68e7.58 (m, 2H), 7.41e7.32 (m, 5H), 7.23e7.15 (m, 1H), 7.11 (d,
J ¼ 8.5 Hz, 2H), 6.86e6.74 (m, 2H), 6.63e6.41 (m, 2H), 5.39e5.28
(m, 1H), 5.18e4.94 (m, 3H), 4.70e4.55 (m, 1H), 4.24e4.10 (m, 1H),
3.77e3.65 (m, 3H), 3.15e2.89 (m, 2H), 1.77e1.66 (m, 1H), 1.61e1.52
(m, 1H), 1.49e1.43 (s, 1H), 1.38e1.30 (m, 3H), 1.04e0.86 (m, 6H).
HRMS (ESI): m/z (M þ Na)þ calcd for C34H40N4NaO7, 639.6938;
found, 639.2794.
7.1.8.1. 5-(4-acetylphenyl)-N-((S)-1-(((S)-1-(((S)-1-((3-acetylphenyl)
amino)-5-methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)furan-2-
carboxamide (46). White solid. Yield 35%. 1H NMR (400 MHz,
7.1.8.6. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)decanamide (20). White solid. Yield 56%.
CDCl3)
d
8.93 (s, 1H), 8.19 (s, 1H), 8.08e7.93 (m, 3H), 7.86e7.72 (m,
1H NMR (400 MHz, CDCl3)
d
8.63 (s, 1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 7.37
3H), 7.48 (t, J ¼ 7.9 Hz,1H), 6.94 (d, J ¼ 7.9 Hz,1H), 6.89 (d, J ¼ 3.5 Hz,
1H), 6.85 (d, J ¼ 6.9 Hz, 1H), 6.70 (d, J ¼ 7.1 Hz, 1H), 5.49e5.38 (m,
1H), 4.73e4.62 (m, 1H), 4.55e4.46 (m, 1H), 2.71e2.52 (m, 6H),
1.86e1.67 (m, 6H), 1.64e1.47 (m, 6H), 1.10e0.72 (m, 18H). 13C NMR
(t, J ¼ 7.9 Hz, 2H), 7.21e7.15 (m, 1H), 7.14e7.08 (m, 2H), 6.79 (d,
J ¼ 8.6 Hz, 2H), 6.58 (dd, J ¼ 21.0, 7.7 Hz, 2H), 5.96e5.79 (m, 1H),
5.39e5.28 (m, 1H), 4.70e4.53 (m, 1H), 4.52e4.35 (m, 1H), 3.72 (s,
3H), 3.15e2.90 (m, 2H), 2.13 (t, J ¼ 7.4 Hz, 2H), 1.77e1.65 (m, 2H),
1.59e1.52 (m, 2H), 1.49e1.41 (m, 1H), 1.37e1.32 (m, 3H), 1.31e1.22
(m, 12H), 1.01e0.96 (m, 3H), 0.93e0.84 (m, 6H). HRMS (ESI): m/z
(M þ H)þ calcd for C36H53N4O6, 637.8292; found, 637.3986.
(101 MHz, CDCl3)
d 197.78, 197.30, 196.95, 172.20, 172.10, 158.28,
157.45, 154.73, 147.4, 137.99, 137.19, 136.80, 133.54, 129.62, 129.08,
125.30, 124.59, 119.75, 117.34, 109.54, 53.05, 51.73, 41.30, 40.76,
40.24, 26.85, 26.73, 25.44, 25.01, 24.81, 23.30, 22.99, 22.78, 22.40,
22.34, 21.59. HRMS (ESI): m/z (M þ Na)þ calcd for C40H50N4NaO8,
737.8368; found,737.3535.
7.1.8.7. ((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)carbamic acid tert-butyl ester (21).
7.1.8.2. ((S)-1-(((S)-1-(((S)-1-((3-acetylphenyl)amino)-5-methyl-1,2-
dioxohexan-3-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)carbamic acid benzyl ester (35).
White solid. Yield 60%. 1H NMR (400 MHz, CDCl3)
d 8.69e8.62 (m,
1H), 7.66e7.61 (m, 1H), 7.40 (t, J ¼ 7.9 Hz, 2H), 7.21 (t, J ¼ 7.4 Hz,1H),
7.14 (d, J ¼ 8.6 Hz, 2H), 6.87e6.77 (m, 2H), 6.61e6.54 (m, 1H),
5.40e5.32 (m, 1H), 4.87e4.74 (m, 1H), 4.69e4.58 (m, 1H), 4.15e3.99
(m, 1H), 3.83e3.71 (m, 3H), 3.20e3.12 (m, 1H), 3.00e2.87 (m, 1H),
1.75e1.52 (m, 3H), 1.51e1.29 (m, 12H), 1.02e0.96 (m, 3H), 0.91e0.88
White solid. Yield 21%. 1H NMR (400 MHz, CDCl3)
d 8.84 (s, 1H), 8.34
(s, 1H), 8.16 (s, 1H), 7.96e7.84 (m, 1H), 7.81e7.69 (m, 1H), 7.64e7.43
(m, 2H), 7.42e7.27 (m, 4H), 7.15e6.94 (m, 3H), 7.84e6.61 (m, 1H),
5.35e5.21 (m, 1H), 5.11e4.93 (m, 2H), 4.83e4.72 (m, 1H), 4.22e4.04
(m, 3H). 13C NMR (101 MHz, CDCl3)
d 196.16, 172.47, 170.77, 158.81,
15

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
156.80, 155.76, 136.38, 130.48, 129.33, 128.26, 125.51, 119.99, 114.24,
80.66, 55.31, 54.30, 52.87, 50.97, 40.33, 36.89, 28.36, 25.16, 23.27,
21.54, 18.20. HRMS (ESI): m/z (M þ Na)þ calcd for C31H42N4NaO7,
605.6775; found, 605.2941.
7.1.8.13. ((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-methyl-1,2-dioxo-
1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)carbamic acid benzyl ester (27). White solid. Yield
20%.1H NMR (400 MHz, CDCl3)
d 8.63e8.44 (m, 1H), 8.00e7.83 (m,
1H), 7.71e7.56 (m, 2H), 7.50e7.30 (m, 6H), 7.29e7.26 (m, 1H),
7.25e7.24 (m, 1H), 7.23e7.01 (m, 3H), 6.76e6.56 (m, 1H), 6.49e6.35
(m, 1H), 5.36e5.23 (m, 1H), 5.11e4.92 (m, 2H), 4.84e4.66 (m, 1H),
4.26e4.07 (m, 1H), 3.43e3.26 (m, 1H), 3.23e3.06 (m, 1H), 1.73e1.56
(m, 2H), 1.54e1.42 (m, 1H), 1.39e1.27 (m, 3H), 0.93 (d, J ¼ 6.5 Hz,
3H), 0.89e0.80 (m, 3H). HRMS (ESI): m/z (M þ H)þ calcd for
7.1.8.8. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)adamantane-1-carboxamide
(22).
White solid. Yield 25%. 1H NMR (400 MHz, CDCl3)
d
8.66 (s, 1H), 7.62
(d, J ¼ 8.1 Hz, 2H), 7.37 (t, J ¼ 7.8 Hz, 2H), 7.18 (t, J ¼ 7.4 Hz, 1H), 7.10
(d, J ¼ 8.3 Hz, 2H), 6.84e6.71 (m, 3H), 6.47e6.37 (m, 1H), 5.99e5.85
(m, 1H), 5.49e5.35 (m, 1H), 4.71e4.61 (m, 1H), 4.39e4.26 (m, 1H),
3.72 (s, 3H), 3.33e3.19 (m, 1H), 2.93e2.78 (m, 1H), 2.23e2.06 (m,
2H), 2.05e1.96 (m, 3H), 1.78e1.55 (m, 12H), 1.54e1.46 (m, 1H),
1.45e1.34 (m, 3H), 1.08e0.97 (m, 3H), 0.94e0.82 (m, 3H). HRMS
(ESI): m/z (M þ H)þ calcd for C37H49N4O6, 645.8082; found,
645.3650.
C
35H40N5O6, 626.7220; found, 626.2984.
7.1.8.14. ((S)-1-(((S)-1-(((S)-5-methyl-1,2-dioxo-1-(phenylamino)
hexan-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)carbamic acid benzyl ester (28). White solid. Yield
3%. 1H NMR (400 MHz, CDCl3)
d 8.62 (s, 1H), 7.67e7.56 (m, 1H),
7.44e7.30 (m, 5H), 7.30e7.26 (m, 4H), 7.22e7.14 (m, 3H), 6.62e6.39
(m, 2H), 5.41e5.29 (m, 1H), 5.12e4.96 (m, 2H), 4.77e4.60 (m, 1H),
4.22e4.07 (m, 1H), 3.22e2.97 (m, 2H), 1.76e1.52 (m, 3H), 1.37e1.27
(m, 3H), 1.06e0.84 (m, 6H). HRMS (ESI): m/z (M þ H)þ calcd for
7.1.8.9. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)furan-2-carboxamide (23). White solid.
C
33H39N4O6, 587.6860; found, 587.2872.
Yield 43%.1H NMR (400 MHz, CDCl3)
d
8.66 (s,1H), 7.63 (d, J ¼ 7.9 Hz,
2H), 7.53e7.46 (m, 1H), 7.37 (t, J ¼ 7.8 Hz, 2H), 7.25e7.03 (m, 4H),
6.84e6.59 (m, 5H), 6.58e6.47 (m, 1H), 5.40e5.32 (m, 1H),
4.68e4.52 (m, 2H), 3.74e3.61 (m, 3H), 3.18e2.89 (m, 2H), 1.81e1.38
(m, 6H), 1.05e0.78 (m, 6H). HRMS (ESI): m/z (M þ H)þ calcd for
7.1.8.15. ((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-methyl-1,2-dioxo-
1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamic acid benzyl
ester (29).
d 10.89e10.77
White solid. Yield 48%.1H NMR (400 MHz, DMSO‑d6)
C
31H37N4O7, 577.6481; found, 577.2662.
(m, 1H), 10.67e10.58 (m, 1H), 8.50e8.31 (m, 1H), 8.28e7.95 (m, 1H),
7.85e7.76 (m, 2H), 7.62e7.49 (m, 1H), 7.48e7.21 (m, 8H), 7.19e7.08
(m, 2H), 7.07e7.00 (m, 1H), 6.99e6.90 (m, 1H), 5.22e5.12 (m, 1H),
5.10e4.92 (m, 2H), 4.75e4.53 (m, 1H), 4.09e3.92 (m, 1H), 3.18e3.03
(m, 1H), 2.96e2.84 (m, 1H), 1.79e1.16 (m, 6H), 0.98e0.60 (m, 12H).
HRMS (ESI): m/z (M þ H)þ calcd for C38H46N5O6, 668.8017; found,
668.3454.
7.1.8.10. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)-6-phenylpicolinamide (24). White solid.
Yield 15%. 1H NMR (400 MHz, CDCl3)
d 8.68 (s, 1H), 8.42 (d,
J ¼ 6.6 Hz, 1H), 8.13e7.84 (m, 5H), 7.72e7.46 (m, 5H), 7.42e7.31 (m,
2H), 7.22e7.10 (m, 1H), 7.08e6.98 (m, 1H), 6.89e6.72 (m, 2H),
6.63e6.45 (m, 2H), 5.45e5.26 (m, 1H), 4.85e4.46 (m, 2H),
3.81e3.42 (m, 3H), 3.19e2.91 (m, 2H), 1.80e1.43 (m, 6H), 1.07e0.78
(d, J ¼ 96.3 Hz, 6H). HRMS (ESI): m/z (M þ H)þ calcd for C38H42N5O6,
664.7700; found, 664.3145.
7.1.8.16. ((S)-4-methyl-1-(((S)-1-(((S)-5-methyl-1,2-dioxo-1-(phe-
nylamino)hexan-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopentan-2-yl)carbamic acid benzyl ester (30). White solid. Yield
48%. 1H NMR (400 MHz, CDCl3)
d 8.64 (s, 1H), 7.67e7.53 (m, 2H),
7.43e7.28 (m, 7H), 7.26e7.23 (m, 1H), 7.22e7.11 (m, 4H), 6.80e6.26
(m, 2H), 5.44e5.29 (m, 1H), 5.11e4.97 (m, 2H), 4.82e4.61 (m, 1H),
4.19e3.94 (m, 1H), 3.25e2.92 (m, 2H), 1.80e1.49 (m, 4H), 1.48e1.30
(m, 2H), 1.02e0.75 (m, 12H). HRMS (ESI): m/z (M þ Na)þ calcd for
7.1.8.11. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)-[1,10-biphenyl]-4-carboxamide
(25).
8.84 (s, 1H),
White solid. Yield 32%.1H NMR (400 MHz, CDCl3)
d
C36H44N4NaO6, 651.7475; found, 651.3157.
7.89e7.75 (m, 2H), 7.74e7.30 (m, 7H), 7.26e7.20 (m, 2H), 7.19e7.01
(m, 3H), 7.01e6.87 (m, 1H), 5.42e5.22 (m, 1H), 4.75e4.51 (m, 2H),
3.73 (s, 3H), 3.19e2.93 (m, 2H), 1.41e1.75 (m, 6H), 0.75e1.07 (m,
6H). HRMS (ESI): m/z (M þ H)þ calcd for C39H43N4O6,663.7819;
found, 663.3195.
7.1.8.17. ((2S)-3-cyclohexyl-1-(((2S)-1-((5-methyl-1,2-dioxo-1-(phe-
nylamino)hexan-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)carbamic acid benzyl ester (31). White solid. Yield
39%. 1H NMR (400 MHz, CDCl3)
d
8.61 (s, 1H), 7.62 (d, J ¼ 7.8 Hz, 2H),
7.43e7.29 (m, 6H), 7.25e7.22 (m, 2H), 7.21e7.13 (m, 3H), 6.59e6.43
(m, 2H), 5.40e5.29 (m, 1H), 5.12e4.94 (m, 2H), 4.72e4.60 (m, 1H),
4.21e4.07 (m, 1H), 3.21e2.95 (m, 2H), 1.77e1.51 (m, 8H), 1.49e1.35
(m, 2H), 1.34e1.08 (m, 4H), 1.03e0.80 (m, 7H). 13C NMR (101 MHz,
7.1.8.12. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-5-methyl-1,2-
dioxo-1-(phenylamino)hexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-1-oxopropan-2-yl)5-(4-acetylphenyl)furan-2-carboxamide
(26). White solid. Yield 61%. 1H NMR (400 MHz, CDCl3)
d
8.63 (s,
CDCl3) d 195.95, 172.05, 170.47, 156.51, 156.28, 136.31, 136.17, 135.91,
1H), 8.03 (d, J ¼ 8.5 Hz, 2H), 7.81 (d, J ¼ 8.5 Hz, 2H), 7.63 (d,
J ¼ 7.7 Hz, 2H), 7.44e7.33 (m, 2H), 7.25e7.16 (m, 2H), 7.14e7.06 (m,
2H), 6.90 (d, J ¼ 3.6 Hz, 1H), 6.83e6.77 (m, 1H), 6.75e6.65 (m, 3H),
6.56e6.43 (m,1H), 5.41e5.31 (m, 1H), 4.72e4.57 (m, 2H), 3.67e3.53
(m, 3H), 3.14e2.94 (m, 2H), 2.64 (s, 3H), 1.78e1.69 (m, 1H),
1.66e1.55 (m, 2H), 1.53e1.46 (m, 3H), 1.03e0.97 (m, 3H), 0.96e0.89
129.27, 129.23, 128.68, 128.62, 128.37, 128.09, 127.04, 125.44, 119.86,
67.29, 54.13, 53.24, 52.87, 40.11, 39.58, 37.71, 33.98, 33.64, 32.39,
26.30, 26.12, 25.94, 25.15, 23.17, 21.43. HRMS (ESI): m/z (M þ H)þ
calcd for C39H49N4O6, 669.8296; found, 669.3641.
7.1.8.18. ((S)-3-methoxy-1-(((S)-1-(((S)-5-methyl-1,2-dioxo-1-(phe-
nylamino)hexan-3-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl) carbamic acid benzyl ester (32). White solid. Yield
(m, 3H). 13C NMR (101 MHz, DMSO)
d 197.22, 196.95, 171.78, 171.27,
159.38, 157.72, 157.28, 153.50, 147.64, 137.49, 136.15, 133.24, 130.18,
129.42, 128.86, 128.73, 124.57, 124.34, 120.41, 116.26, 113.35, 109.97,
54.75, 53.75, 52.02, 48.22, 38.32, 36.48, 26.74, 24.51, 23.05, 21.14,
17.48. HRMS (ESI): m/z (M þ H)þ calcd for C39H43N4O8, 695.7807;
found, 695.3109.
5%. 1H NMR (400 MHz, CDCl3)
d 8.68e8.57 (m, 1H), 7.62 (d,
J ¼ 8.3 Hz, 2H), 7.48e7.30 (m, 6H), 7.25e7.09 (m, 4H), 6.97e6.61 (m,
2H), 6.57e6.37 (m, 1H), 5.66e5.48 (m, 1H), 5.45e5.29 (m, 1H),
5.17e4.95 (m, 2H), 4.83e4.61 (m, 1H), 4.34e4.18 (m, 1H), 3.84e3.62
16

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
(m, 1H), 3.56e3.35 (m, 1H), 3.33e3.24 (m, 2H), 3.23e2.94 (m, 2H),
1.77e1.64 (m, 1H), 1.61e1.48 (m, 1H), 1.47e1.36 (s, 1H), 1.08e0.69
(m, 6H). HRMS (ESI): m/z (M þ Na)þ calcd for C34H40N4NaO7,
639.6938; found, 639.2814.
7.1.8.24. ((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-5-methyl-1,2-dioxo-
1-(quinolin-5-ylamino)hexan-3-yl)amino)-1-oxopentan-2-yl)
amino)-1-oxopentan-2-yl)carbamic acid benzyl ester (39).
White solid. Yield 27%. 1H NMR (400 MHz, CDCl3)
d 9.18 (s, 1H),
9.04e8.91 (m,1H), 8.47e8.29 (m, 1H), 8.25e8.08 (m, 2H), 7.87e7.72
(m, 1H), 7.65e7.50 (m, 1H), 7.39e7.31 (m, 3H), 7.30e7.27 (m, 1H),
7.20e6.88 (m, 1H), 6.48e6.29 (m, 1H), 5.39e5.28 (m, 1H), 5.17e5.06
(m, 2H), 4.57e4.42 (m, 1H), 4.22e4.06 (m, 1H), 1.83e1.47 (m, 9H),
7.1.8.19. (S)-3-((S)-3-(4-methoxyphenyl)-2-((S)-2-(2-
phenylacetamido)propanamido)propanamido)-5-methyl-N-(naph-
thalen-1-yl)-2-oxohexanamide (33). White solid. Yield 35%. 1H NMR
1.13e0.65 (m, 18H). HRMS (ESI): m/z (M
36H48N5O6, 646.7962; found, 646.3607.
þ
H)þ calcd for
(400 MHz, CDCl3)
d
9.24 (d, J ¼ 6.2 Hz, 1H), 8.19 (t, J ¼ 8.2 Hz, 1H),
C
7.95e7.81 (m, 2H), 7.77e7.70 (m, 1H), 7.63e7.45 (m, 3H), 7.40e7.28
(m, 5H), 7.17e7.08 (m, 2H), 6.81 (t, J ¼ 8.8 Hz, 2H), 6.70e6.46 (m,
2H), 5.45e5.35 (m, 1H), 5.16e4.95 (m, 3H), 4.74e4.60 (m, 1H),
4.23e4.07 (m, 1H), 3.70 (d, J ¼ 25.8 Hz, 3H), 3.16e2.92 (m, 2H),
1.82e1.69 (m, 1H), 1.66e1.44 (m, 2H), 1.33 (t, J ¼ 7.8 Hz, 3H),
7.1.8.25. ((S)-1-(((S)-1-(((S)-1-(isoquinolin-5-ylamino)-5-methyl-
1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamic acid benzyl
ester (40).
White solid. Yield 16%. 1H NMR (400 MHz, CDCl3)
d
9.33 (s, 1H), 9.23
1.05e0.88 (m, 6H). HRMS (ESI): m/z (M
38H42N4NaO7, 689.7524; found, 689.2951.
þ
Na)þ calcd for
(s, 1H), 8.67e8.55 (m, 1H), 8.52e8.41 (m, 1H), 7.96e7.87 (m, 1H),
7.80e7.63 (m, 2H), 7.40e7.27 (m, 4H), 7.20e7.02 (m, 1H), 6.97e6.84
(m, 1H), 6.44e6.30 (m, 1H), 5.43e5.32 (m, 1H), 5.15e5.04 (m, 2H),
4.55e4.43 (m, 1H), 4.20e4.07 (m, 1H), 1.88e1.58 (m, 9H), 1.13e0.58
(m, 18H). HRMS (ESI): m/z (M þ H)þ calcd for C36H48N5O6,
646.7962; found, 646.3597.
C
7.1.8.20. ((S)-1-(((S)-1-(((S)-1-((3-acetylphenyl)amino)-5-methyl-
1,2-dioxohexan-3-yl) amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamic acid benzyl ester (34).
White solid. Yield 65%. 1H NMR (400 MHz, CDCl3)
d 8.77 (s, 1H), 8.18
(s, 1H), 7.96e7.87 (m, 1H), 7.82e7.73 (m, 1H), 7.53e7.44 (m, 1H),
7.41e7.30 (m, 4H), 7.28e7.26 (m, 1H), 7.25e7.22 (m, 2H), 7.21e7.14
(m, 2H), 6.62e6.51 (m, 1H), 5.39e5.28 (m, 1H), 5.12e4.96 (m, 2H),
4.73e4.62 (m, 1H), 4.16e4.06 (m, 1H), 3.17e2.99 (m, 2H), 2.63 (s,
3H), 1.74e1.53 (m, 4H), 1.50e1.36 (m, 2H), 1.05e0.78 (m, 12H).
HRMS (ESI): m/z (M þ H)þ calcd for C38H47N4O7, 671.8024; found,
671.3445.
7.1.8.26. ((S)-1-(((S)-1-(((S)-1-([1,10-biphenyl]-2-ylamino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)carbamic acid benzyl ester (41).
White solid. Yield 32%. 1H NMR (400 MHz, CDCl3)
d 8.89 (s, 1H),
8.52e8.43 (m, 1H), 7.55e7.27 (m, 11H), 7.26e7.18 (m, 1H),
6.96e6.60 (m, 1H), 6.41e6.21 (m, 1H), 5.46e5.35 (m, 1H), 5.14e5.05
(m, 2H), 4.54e4.36 (m, 1H), 4.21e4.05 (m, 1H), 1.77e1.63 (m, 6H),
1.52e1.41 (m, 3H), 1.06e0.76 (m, 18H). HRMS (ESI): m/z (M þ Na)þ
calcd for C39H50N4NaO6, 693.8273; found, 693.3616.
7.1.8.21. ((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-methyl-1-((3-
(methylsulfonyl)phenyl)amino)-1,2-dioxohexan-3-yl)amino)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamic acid
benzyl ester (36). White solid. Yield 11%. 1H NMR (400 MHz,
7.1.8.27. ((S)-1-(((S)-1-(((S)-1-((2,3-dihydro-1H-inden-4-yl)amino)-
5-methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)carbamic acid benzyl ester (42).
DMSO‑d6)
d 11.02 (s, 1H), 10.80 (s, 1H), 8.51e8.44 (m, 2H), 8.07 (d,
J ¼ 8.1 Hz, 1H), 7.99 (d, J ¼ 8.1 Hz, 1H), 7.74e7.60 (m, 2H), 7.50 (d,
J ¼ 7.8 Hz, 1H), 7.40e7.27 (m, 6H), 7.17e7.10 (m, 1H), 7.08e7.00 (m,
1H), 6.99e6.89 (m, 1H), 5.17e5.08 (m, 1H), 5.06e4.94 (m, 2H),
4.69e4.58 (m, 1H), 4.06e3.96 (m, 1H), 3.18 (s, 3H), 3.14e3.05 (m,
1H), 2.95e2.86 (m, 1H), 1.76e1.45 (m, 4H), 1.36e1.18 (m, 2H),
White solid. Yield 6%. 1H NMR (400 MHz, CDCl3)
d
8.52 (d, J ¼ 6.1 Hz,
1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.40e7.28 (m, 4H), 7.22e7.13 (m, 1H),
7.09e7.03 (m, 1H), 6.95e6.70 (m, 1H), 6.38e6.27 (m, 1H), 5.45e5.33
(m, 1H), 5.15e5.06 (m, 2H), 4.53e4.40 (m, 1H), 4.22e4.09 (m, 1H),
3.01e2.77 (m, 4H), 2.19e2.06 (m, 2H), 1.87e1.48 (m, 9H), 1.11e0.68
(m, 18H). HRMS (ESI): m/z (M þ Na)þ calcd for C36H50N4NaO6,
657.7952; found, 657.3691.
0.96e0.68 (m, 12H). HRMS (ESI): m/z (M
39H48N5O8S, 746.8921; found, 746.3225.
þ
H)þ calcd for
C
7.1.8.22. ((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-methyl-1-(naph-
thalen-1-ylamino)-1,2-dioxohexan-3-yl)amino)-1-oxopropan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl) carbamic acid benzyl ester (37).
7.1.8.28. ((S)-1-(((S)-1-(((S)-1-((3-ethylphenyl)amino)-5-methyl-
1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)carbamic acid benzyl
ester (43).
White solid. Yield 32%. 1H NMR (400 MHz, CDCl3)
d 9.17 (d,
White solid. Yield 12%. 1H NMR (400 MHz, CDCl3)
d
8.68 (s, 1H),
J ¼ 14.8 Hz, 1H), 8.26e8.15 (m, 1H), 7.97e7.82 (m, 3H), 7.78e7.72
(m, 1H), 7.68e7.46 (m, 5H), 7.44e7.29 (m, 5H), 7.18e7.03 (m, 3H),
6.78e6.43 (m, 1H), 5.44e5.31 (m, 1H), 5.09e4.94 (m, 2H),
4.92e4.69 (m,1H), 4.21e4.04 (m,1H), 3.45e3.28 (m,1H), 3.24e3.09
(m, 1H), 1.76e1.49 (m, 9H), 0.99e0.94 (m, 3H), 0.93e0.84 (m, 9H).
HRMS (ESI): m/z (M þ H)þ calcd for C42H48N5O6, 718.8604; found,
718.3611.
7.55e7.41 (m, 2H), 7.40e7.36 (m, 1H), 7.35e7.31 (m, 2H), 7.31e7.24
(m, 1H), 7.04 (d, J ¼ 7.5 Hz, 1H), 6.94e6.85 (m, 1H), 6.66e6.51 (m,
1H), 5.46e5.28 (m, 2H), 5.13 (s, 2H), 4.58e4.46 (m, 1H), 4.30e4.16
(m, 1H), 2.67 (q, J ¼ 7.5 Hz, 2H), 1.85e1.47 (m, 9H), 1.25 (t, J ¼ 7.6 Hz,
3H), 1.11e0.85 (m, 18H). 13C NMR (101 MHz, CDCl3)
d 196.79, 172.49,
171.76, 156.86, 156.46, 145.73, 136.29, 136.16, 129.23, 128.71, 128.42,
128.19, 125.19, 119.51, 117.40, 67.36, 53.79, 53.02, 51.70, 41.34, 40.63,
40.21, 28.98, 25.40, 24.85, 23.36, 23.04, 22.88, 22.25, 22.08, 21.51,
15.62. HRMS (ESI): m/z (M þ H)þ calcd for C35H51N4O6, 623.8026;
found, 623.3800.
7.1.8.23. ((S)-1-(((S)-1-(((S)-1-((3-acetylphenyl)amino)-5-methyl-
1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl) carbamic acid benzyl ester (38).
White solid. Yield 21%. 1H NMR (400 MHz, DMSO‑d6)
d
10.81 (s, 1H),
7.1.8.29. ((S)-1-(((S)-1-(((S)-1-((3,5-dimethoxyphenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)carbamic acid benzyl ester (44).
8.50e8.38 (m, 2H), 8.06e7.95 (m, 1H), 7.91 (d, J ¼ 8.3 Hz, 1H), 7.75
(d, J ¼ 7.8 Hz, 1H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.44e7.24 (m, 6H),
5.09e4.93 (m, 3H), 4.43e4.33 (m, 1H), 4.09e3.98 (m, 1H), 2.57 (s,
3H), 1.78e1.66 (m, 1H), 1.64e1.47 (m, 4H), 1.46e1.29 (m, 4H),
1.01e0.61 (m,18H). HRMS (ESI): m/z (M þ H)þ calcd for C35H49N4O7,
637.7862; found, 637.3611.
White solid. Yield 65%. 1H NMR (400 MHz, CDCl3)
d 8.59 (s, 1H),
7.41e7.29 (m, 5H), 6.85 (d, J ¼ 2.2 Hz, 2H), 6.77e6.70 (m, 1H),
6.45e6.33 (m, 1H), 6.31e6.27 (m, 1H), 5.46e5.32 (m, 1H), 5.16e5.07
(m, 2H), 4.51e4.39 (m, 1H), 4.23e4.09 (m, 1H), 3.80 (s, 6H),
17

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
1.81e1.62 (m, 2H), 1.55e1.44 (m, 2H), 1.04e0.79 (m, 18H). HRMS
SARS-CoV-2 were added to the cell monolayer with or without the
addition of drug compounds and the plates were further incubated
for 1 h at 37 ꢁC in 5% CO2 before removal of unbound viral particles
by aspiration of the media and washing once with DMEM. Mono-
layers were then overlaid with media containing 1% low melting
agarose (Cambrex Corporation, New Jersey, USA) in DMEM and
appropriate concentrations of individual compound, inverted and
incubated as above for another 72 h. The wells were then fixed with
10% formaldehyde (BDH, Merck, Darmstadt, Germany) overnight.
After removal of the agarose plugs, the monolayers were stained
with 0.7% crystal violet (BDH, Merck) and the plaques counted. The
percentage of plaque inhibition relative to the control (i.e. without
the addition of compound) wells were determined for each drug
compound concentration. The EC50 was calculated using Sigma plot
(SPSS) in an Excel add-in ED50V10. The plaque reduction assay
experiments were performed in triplicate and repeated twice for
confirmation.
(ESI): m/z (M
found,677.35193.
þ
Na)þ calcd for C35H50N4NaO8, 677.78327;
7.1.8.30. N-((S)-1-(((S)-1-(((S)-1-((3,5-dimethoxyphenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)5-(4-acetylphenyl)-furan-2-
carboxamide (45). White solid. Yield 6%. 1H NMR (400 MHz, CDCl3)
d
8.59 (s, 1H), 8.02 (d, J ¼ 8.4 Hz, 2H), 7.80 (d, J ¼ 8.4 Hz, 2H),
7.25e7.23 (m, 1H), 6.89 (d, J ¼ 3.6 Hz, 1H), 6.86e6.83 (m, 2H),
6.82e6.77 (m, 1H), 6.68 (d, J ¼ 7.3 Hz, 1H), 6.57e6.51 (m, 1H),
6.31e6.27 (m,1H), 5.46e5.38 (m,1H), 4.70e4.62 (m,1H), 4.52e4.43
(m, 1H), 3.79 (s, 6H), 2.64 (s, 3H), 1.82e1.64 (m, 7H), 1.58e1.48 (m,
2H), 1.10e0.81 (m, 18H). HRMS (ESI): m/z (M þ H)þ calcd for
C
40H53N4O9, 733.8702; found, 733.3821.
7.1.8.31. N-((S)-1-(((S)-1-(((S)-1-((3-methoxyphenyl)amino)-5-
methyl-1,2-dioxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)5-(4-acetylphenyl)-furan-2-
carboxamide (47). White solid. Yield 11%. 1H NMR (400 MHz, CDCl3)
7.2.4. Cell viability assay
Before performing the above described plaque reduction assay,
the CellTiterGlo luminescent assay (Promega Corporation, Madison,
WI, USA) was conducted to detect the cytotoxicity of the selected
compound as we described previously [49]. Briefly, Vero E6 cells
and/or Caco-2 cells (2 ꢃ 104 cells/well) were incubated with the
serially diluted compound for 48 h, followed by addition of sub-
strate and measurement of luminance 10 min according to manu-
facturer’s protocol. The 50% cytotoxic concentrations (CC50) of the
drug were calculated by GraphPad Prism software.
d
8.62 (s, 1H), 8.02 (d, J ¼ 8.4 Hz, 2H), 7.81 (d, J ¼ 8.5 Hz, 2H),
7.37e7.32 (m, 1H), 7.29e7.26 (m, 1H), 7.25e7.23 (m, 1H), 7.15e7.07
(m, 1H), 6.94e6.85 (m, 2H), 6.76e6.71 (m, 1H), 6.70e6.65 (m, 1H),
6.64e6.55 (m, 1H), 5.49e5.38 (m, 1H), 4.71e4.62 (m, 1H),
4.52e4.42 (m, 1H), 3.82 (s, 3H), 2.64 (s, 3H), 1.86e1.67 (m, 6H),
1.65e1.47 (m, 3H), 1.10e0.73 (m, 18H). HRMS (ESI): m/z (M þ Na)þ
calcd for C39H50N4NaO8, 725.8261; found, 725.3524.
7.2. Biological assays
7.3. Molecular modeling
7.2.1. Proteasome-Glo™ chymotrypsin-like, trypsin-like and
caspase-like cell-based assays
7.3.1. CoMFA model
The cell based proteasome inhibition were assayed following
The CoMFA model is built by SYBYL_X 2.0 suite software (Tripos
Inc., USA). The set of compounds (25 in total) are minimized with
the Tripos force field and Gasteiger-Hückel charge. The conforma-
tion of compound BSc2198 is used as the template, the other 24
compounds are aligned by fitting the three chiral carbon atoms.
CoMFA descriptors calculation and partial least-squares (PLS)
model fitting are conducted in the QSAR module of SYBYL suite.
SAMPLS with leave-one-out (LOO) are carried out to calculate the
cross-validation value q2. The conventional correlation coefficient
r2 is then calculated with no validation using the optimum number
of component. In order to improve reduce ‘noise’, a minimum filter
value of 2.00 kcal/mol is used.
the promega protocol.1250H226 cells were plated in 45
96-well plate. Cells were incubated at 37 ꢁC, 5% CO2 overnight. And
then incubated with 5 L/well various inhibitor concentrations for
105 min with two repetitions of each. 50 L/well of Proteasome-
mL/well in a
m
m
Glo™ cell-based reagent containing luminogenic substrate Suc-
LLVY-aminoluciferin for chymotrypsin-like, Z-LRR-aminoluciferin
for trypsin-like, and Z-nLPnLD-aminoluciferin for caspase-like ac-
tivity was added. Incubate at room temperature for 10 min. The
luminescence was measured with a plate-reader. The IC50 values
were calculated by GraphPad Prism software.
7.2.2. Anticancer cell assay
RPMI8226, Ramos, THP-1 were grown in RPMI 1640 supple-
mented with 10% fetal bovine serum at 37 ꢁC in 5% CO2. MGC-803,
HeLa, SW480 were grown in DMEM supplemented with 10% fetal
bovine serum at 37 ꢁC in 5% CO2.
7.3.2. Molecular docking
The covalent docking studies are performed with GOLD version
2020.1. The crystal of human 20S proteasome complex with opro-
zomib (PDB: 5LEY) [41] is used for the docking studies. The initial
structure of protein and ligands for docking are prepared using the
software SYBYL_X 2.0. A radius of 10 Å from the centroid of the co-
crystallized ligand is regarded as the docking site. The GA runs are
50, the scoring function is ChemScore, and the other parameters are
A standard MTS assay was used to measure cancer cell growth. A
100 m
L RPMI8226 (1*104/well), THP-1(5*104/well), Ramos (5*104/
well), MGC-803 (5*103/well), HeLa (5*103/well), SW480 cells
(5*103/well) were seeded into 96-well plates. After treated with
tested compounds for 72 h, an amount of 20
m
L of a solution of MTS
keep the default. The carbonyl group of the a-Ketoamide is the link
was added to each well and incubated for another 3 h at 37 ꢁC.
Optical density was determined at 490 nm by microplate reader.
The assays were done in triplicate with triplicate wells per exper-
iment. The IC50 values were calculated by GraphPad Prism software.
atom that binds to the hydroxyl oxygen of Thr1.
The crystal structure of m-calpain (PDB: 3BOW) [44] was used
for 17 and MG132 docking studies. The initial structure of protein
and ligands for docking were prepared using the software SYBYL_X
2.0. The residue Ser105 of m-calpain was mutated to Cys105 before
docking. The hydrogen atom attached to the sulfur atom of Cys105
was removed. The carbonyl group (C]O) in the aldehyde group of
7.2.3. Plaque reduction assay
Plaque reduction assay was performed to plot the 50% antiviral
effective dose (EC50) as we described previously with slight modi-
fications [46e48]. Briefly, Vero E6 cells were seeded at 4 ꢃ 105 cells/
well in 12-well tissue culture plates on the day before carrying out
the assay. After 24 h of incubation, 50 plaque-forming units (PFU) of
MG132 or in the a-Ketoamide of our proteasome inhibitors was
replaced by carbon-sulfur (CeS) and carbon-oxygen (CeO). The
sulfur atoms of Cys105 and MG132 or our proteasome inhibitors
were set as the protein link atom and ligand link atom, respectively.
18

J. Wang, B. Liang, Y. Chen et al.
European Journal of Medicinal Chemistry 215 (2021) 113267
Baumeister, R. Baumeister W Fau - Huber, R. Huber, Crystal structure of the
20S proteasome from the archaeon T. acidophilum at 3.4 A resolution, Science
268 (1995) 533e539.
The binding site was defined as 12 Å around the sulfur atom of
Cys105. GoldScore scoring function was used and the number of GA
runs was 50. The side chains of Cys105 was set to be flexible with
‘crystal mode’, and the other parameters were set as defaults.
The crystal structure of SARS-CoV-2 main protease (PDB: 6Y2F)
[43]was used for compound 17 docking studies. The binding site
was defined as 10 Å around the sulfur atom of Cys145. ChemScore
scoring function was used and the number of GA runs was 50 and
the other parameters were set as defaults.
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The simulation systems are solvated by a standard MOLARIS surface
constrained all-atom solvent (SCAAS) model centered around the
ligand. This involved a simulation sphere of 18 Å with a 2 Å surface
constraint region surrounded by a 2 Å Langevin dipole surface. The
MD simulations are carried out with the ENZYMIX force field. All
complexes are relaxed for 0.5 ns, with 1 fs time step. The simula-
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J.W. performed chemical syntheses and wrote the manuscript;
B.L., L.N. participated in chemical syntheses; Y.C., H.Y. performed
the proteasome inhibition and cancer cell toxicity experiments;
J.F.W.C., S.Y. performed anti-SARS-CoV-2 assays; J.Z., Y.W., M.W.,
L.S.H., J.A. and A.W. performed the molecular modeling studies and
data analysis; K.Y.Y., A.C., Z.H, and Y. X. oversaw the project and
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Funding sources
[21] M.J. Lee, D. Bhattarai, J. Yoo, Z. Miller, J.E. Park, S. Lee, W. Lee, J.J. Driscoll,
K.B. Kim, Development of novel epoxyketone-based proteasome inhibitors as
a strategy to overcome cancer resistance to carfilzomib and bortezomib,
J. Med. Chem. 62 (2019) 4444e4455.
This work was supported by the grant from Guangdong prov-
ince (grant no. 00201503).
[22] H.A. Braun, S. Umbreen, M. Groll, U. Kuckelkorn, I. Mlynarczuk, M.E. Wigand,
I. Drung, P.M. Kloetzel, B. Schmidt, Tripeptide mimetics inhibit the 20 S pro-
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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.
[23] M.L. Stein, H. Cui, P. Beck, C. Dubiella, C. Voss, A. Krüger, B. Schmidt, M. Groll,
Systematic comparison of peptidic proteasome inhibitors highlights the
a-
ketoamide electrophile as an auspicious reversible lead motif, Angew. Chem.
Int. Ed. 53 (2014) 1679e1683.
[24] C. Voss, C. Scholz, S. Knorr, P. Beck, M.L. Stein, A. Zall, U. Kuckelkorn,
P.M. Kloetzel, M. Groll, K. Hamacher, B. Schmidt, alpha-Keto phenylamides as
P1’-extended proteasome inhibitors, ChemMedChem 9 (2014) 2557e2564.
[25] P. Ingallinella, D. Fattori, S. Altamura, C. Steinkühler, U. Koch, D. Cicero,
R. Bazzo, R. Cortese, E. Bianchi, A. Pessi, Prime site binding inhibitors of a
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113267.
serine Protease: NS3/4A of hepatitis
C virus, Biochemistry 41 (2002)
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M.G. Grütter, Exploring the S4 and S1 prime subsite specificities in caspase-3
with aza-peptide epoxide inhibitors,, Biochemistry 45 (2006) 9059e9067.
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E.A. Nalivaika, R. Swanstrom, N.K. Yilmaz, C.A. Schiffer, A. Ali, HIV-1 protease
inhibitors incorporating stereochemically defined P20 ligands to optimize
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