Development of peptide epoxyketones as selective immunoproteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113556

A series of epoxyketone analogues with varying N-caps and P3-configurations were designed, synthesized and evaluated. We found that D-Ala in P3 was crucial for β5i selectivity over β5c. Notably, compounds 20j (β5i IC₅₀ = 26.0 nM, 25-fold selectivity) and 20l (β5i IC₅₀ = 25.1 nM, 24-fold selectivity) with the D-configuration at P3 were the most selective inhibitors. Although 20j and 20l showed only moderate anti-proliferative activity against RPMI-8226 and MM.1S cell lines, based on our experiments, it indicates that the inhibition of β5i alone is not sufficient to exert anticancer effects and may rely on the complementary inhibition of β1i, β5c and β5i. These data further increase our understanding of immunoproteasome inhibitors in hematologic malignancies.

Full text of DOI:10.1016/j.ejmech.2021.113556

European Journal of Medicinal Chemistry 221 (2021) 113556
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development of peptide epoxyketones as selective
immunoproteasome inhibitors
Xuemei Li , Duidui Hong 1, Mengmeng Zhang c 1, Lei Xu c d, Yubo Zhou c
Jia Li
a
a
,
1
b
,
,
,
, d, e, **
,
c
,
d,
e,
***
a, *
, Tao Liu
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, Zhejiang, 310058, PR China
Jiangsu Shengdia Industrial Co. Ltd., NO. 161 Shaoxing Road, Xiacheng District, Hangzhou, 310004, PR China
b
c
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,
2
01203, PR China
d
Zhongshan Institute of Drug Discovery, Institution for Drug Discovery Innovation, Chinese Academy of Science, Zhongshan, 528400, PR China
University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, PR China
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 January 2021
Received in revised form
A series of epoxyketone analogues with varying N-caps and P3-configurations were designed, synthe-
sized and evaluated. We found that D-Ala in P3 was crucial for
pounds 20j (
the D-configuration at P3 were the most selective inhibitors. Although 20j and 20l showed only mod-
b5i selectivity over b5c. Notably, com-
5i IC50 ¼ 26.0 nM, 25-fold selectivity) and 20l ( 5i IC50 ¼ 25.1 nM, 24-fold selectivity) with
b
b
6
May 2021
Accepted 14 May 2021
Available online 25 May 2021
erate anti-proliferative activity against RPMI-8226 and MM.1S cell lines, based on our experiments, it
indicates that the inhibition of
complementary inhibition of
b
5i alone is not sufficient to exert anticancer effects and may rely on the
1i, 5c and 5i. These data further increase our understanding of
b
b
b
Keywords:
Epoxyketones
Immunoproteasome inhibitor
Selectivity
immunoproteasome inhibitors in hematologic malignancies.
©
2021 Elsevier Masson SAS. All rights reserved.
Multiple myeloma
1. Introduction
containing three catalytic subunits, namely, b1 (caspase-like ac-
tivity),
b
2 (trypsin-like activity), and
b5 (chymotrypsin-like activ-
The ubiquitin proteasome system, discovered in the 1980s, is the
major intracellular pathway mediating protein turnover in
eukaryotic cells [1]. The system is responsible for the degradation of
approximately 80% of all cellular proteins, including misfolded and
regulatory proteins [2].
ity). The function of the
proteins can enter the proteolytic chamber [6].
a
-rings is to maintain a gate through which
Three types of proteasomes exist in vertebrate cells. The
constitutive proteasome (cCP) is present in all tissues, whilst the
immunoproteasome (iCP) is restricted to hematopoietic cells [7],
particularly lymphocytes and monocytes, where it mediates the
production of peptides that are subsequently presented by major
histocompatibility complex class I (MHC-I) molecules [8,9]. In
cortical thymic epithelial cells, the thymoproteasome (tCP) is
The proteasome is a supramolecular complex composed of a
catalytic core particle (CP) and regulatory particles (RPs) [3]. The
2
0S CP consists of four stacked homologous rings of seven subunits
each, two outer -rings and two inner -rings, forming a barrel-
shaped structure [4,5]. The -rings act as a proteolytic chamber,
a
b
b
expressed, which is essential for the optimal positive selection of
þ
CD8 T cells [10,11]. In regards to their proteolytic
b
-subunits of
5c, the iCP contains 1i,
1i, 2i and 5t.
Proteasome inhibition has emerged as an anticancer strategy.
each, the cCP bears subunits
b
1c,
b
2c and
b
b
*
Corresponding author.;
b
2i and 5i, and the tCP holds subunits
b
b
b
b
** Corresponding author. National Center for Drug Screening, State Key Laboratory
of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sci-
ences, Shanghai, 201203, PR China.
Three proteasome inhibitors, namely bortezomib [12] (1), carfil-
zomib [13] (2), and ixazomib [14] (3), have been approved by the
Food and Drug Administration (FDA) for the treatment of multiple
myeloma (MM) (Fig. 1A).
Despite their remarkable therapeutic efficacy in MM, the use of
conventional proteasome inhibitors is often limited because of
*** Corresponding author. National Center for Drug Screening, State Key Labora-
tory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, PR China.
E-mail addresses: ybzhou@simm.ac.cn (Y. Zhou), jli@simm.ac.cn (J. Li), Lt601@
zju.edu.cn (T. Liu).
1
Both authors contribute equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113556
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
their side effects due to their non-selective inhibition of protein
degradation. Thus, selectively targeting proteasome elements
would increase efficacy and reduce side effects [15]. Given the
relatively limited expression of the immunoproteasome in cells of
lymphoid origin, immunoproteasome-specific inhibitors can target
hematologic malignancies that originate from lymphoid cells, non-
cancer tissues [16]. Inhibiting the immunoproteasome may there-
fore maintain antimyeloma efficacy whilst reducing toxicity,
increasing the therapeutic index.
Compound 11a significantly inhibited b5i, but no compounds dis-
played improved selectivity over ONX-0914. Compound 11f showed
improved selectivity compared to Eꢀ83. Further docking studies
(Fig. 3) indicated the presence of a 4-(thiazol-2-yl)piperidine N-cap
in which the D-Ala may be too long, which encouraged us to further
modify this region. Based on 11a and 11f, 20a-20n with short N-
caps and the general ONX-0914 formula were designed and syn-
thesized (Fig. 2).
The immunoproteasome is a constituent of cells of hematopoi-
etic origin, encompassing 30%e90% of the total proteasome in MM
cells [17]. Increased immunoproteasome activity has been observed
3
. Results and discussion
3.1. Chemistry
in relapsed/refractory MM [18]. ONX-0914 (4,
IC50 ¼ 422 nM, Fig. 1B) was the first described immunoproteasome
inhibitor that is selective for 5i [19]. Studies using in vitro and
in vivo MM models showed that the immunoproteasome inhibitor
PR-924 (5,
b
5i IC50 ¼ 39 nM,
b5c
11a-11f were synthesized according to Scheme 1. In route a,1 was
b
reacted with 2-aminothiazole to produce 2, which was deprotected
with trifluoroacetic acid (TFA) to obtain 3. In route b, 4a or 4b were
first reacted with triphosgene to obtain isocyanate intermediates 5a
or 5b, followed by the addition of 3 to generate the intermediates 6a-
b
5i IC50 ¼ 22 nM, 5c IC50 ¼ 2900 nM, Fig. 1B) displayed
b
antitumor activity [20], highlighting the immuno-specific protea-
some as a potential treatment strategy.
6
b, which were hydrolyzed to the corresponding carboxylic de-
In this study, to explore the anti-MM activity of immunopro-
teasome inhibitors, a series of epoxyketone analogues bearing
varying N-cap and L- or D-configurations at P3 were designed and
synthesized. All compounds were evaluated for their potency
against human b5c and b5i. Moreover, inhibitory activity against
other subunits and anti-proliferative activity against MM cells were
evaluated for selected compounds.
rivatives 7a-7b. In route c, epoxyketone warheads 8a-8b (the syn-
thesis of which have been reported in the literature [23]) were
condensed with Boc-tryptophane or Boc-4-methoxyphenylalanine
to generate compounds 9a-9c, which were deprotected with TFA
to generate dipeptide epoxyketone intermediates 10a-10c. Finally,
dipeptide epoxyketone intermediates 10a-10c were condensed with
7
a-7b to obtain target compounds 11a-11f.
The synthetic routes for the target compounds 20a-20n are
2
. Design rationale
outlined in Scheme 2. The isocyanate intermediates 5a or 5b were
reacted with the corresponding amine to generate the in-
termediates 12a-12d and 12g-12n, which were hydrolyzed to
produce the carboxylic derivatives 13a-13d and 13g-13n, respec-
tively. Carbazole 14 was first reacted with 4-nitrophenyl carbon-
ochloridate to obtain compound 15, followed by treatment with 5a
or 5b to produce the intermediates 16a and 16b, which were hy-
drolyzed to produce 17a and 17b. The intermediates 13a-13d, 13g-
In our previous studies, we identified a potent proteasome in-
hibitor Eꢀ83 [21] that can indiscriminately inhibit
similar levels ( 5c IC50 ¼ 12 nM). ONX-0914 and
5i IC50 ¼ 21 nM,
PR-924 are selective 5i inhibitors. Based on the chemical tem-
plates of Eꢀ83 and ONX-0914/PR-924, we herein report the dis-
covery of a series of selective 5i inhibitors through fragment-
based drug design. We initially incorporated P1, P2 and P3 in PR-
24 and the N-cap fragments in Eꢀ83. It has been reported by
others that a cyclohexyl sidechain in P1 would enhance selectivity
for 5i [22]. Consequently, we introduced a cyclohexyl side chain
b5i and b5c to
b
b
b
b
13h and 17a-17b underwent condensation with O-methyl- -tyro-
L
9
sine methyl ester hydrochloride to afford the dipeptide in-
termediates 18a-18n, which were subsequently hydrolyzed to
provide the carboxylic intermediates 19a-19n. The epoxyketone
warheads 8a was condensated with 19a-19n to generate the target
compounds 20a-20n.
b
into P1 and further incorporated P2 in ONX-0914, whilst main-
taining a cyclohexyl side chain in P1 to design compounds 11a-11f.
Fig. 1. Proteasome inhibitors. (A) FDA-approved proteasome inhibitors bortezomib, carfilzomib, and ixazomib. (B) Immunoproteasome inhibitors ONX-0914, PR-924.
2

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
Fig. 2. Design strategy of the selective b5i inhibitors.
ꢁ
Scheme 1. Synthetic routes for the target compounds 11a-11f. Reagents and conditions: (a) 2-aminothiazole, HOBt, EDCI, DIPEA, DCM, rt, 3 h; (b) TFA, DCM, 0 C to rt, 3 h; (c)
ꢁ
Triphosgene, Saturated NaHCO
3
, DCM, 0 C, 15 min; (d) 3, DIPEA, DCM, rt, 1 h; (e) LiOH$H
2
2
O, Acetone, H O, 6 h; (f) Boc-tryptophane or Boc-4-methoxyphenylalanine, HOBt, EDCI,
ꢁ
DIPEA, DCM, rt, 3 h; (g) TFA, DCM, 0 C, 2 h; (h) 7a-7b, HOBt, EDCI, DIPEA, DCM, rt, 3 h.
3

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
2 2
Scheme 2. Synthetic routes for the target compounds 20a-20n. Reagents and conditions: (a) Corresponding amine, DIPEA, DCM, rt, 1 h; (b) LiOH$H O, Acetone, H O, 6 h; (c) 4-
ꢁ
ꢁ
nitrophenyl carbonochloridate, NaH, THF, N
methyl ester hydrochloride, HOBt, EDCI, DIPEA, DCM, rt, 3 h; (g) LiOH$H
2
, overnight; (d) 5a or 5b, DIPEA, MeCN, rt, overnight; (e) LiOH$H
2
O, Acetone, H
2
O, -5 Ce0 C, 30 min; (f) O-methyl-L-tyrosine
O, ꢀ5 ꢁC to 0 C, 30 min; (h) 8a, HOBt, EDCI, DIPEA, DCM, rt, overnight.
ꢁ
2
O, Acetone, H
2
4

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
3.2. Structure-activity relationship (SAR) studies
preference for
b
5i, indicating that a 4-methoxyphenyl motif may
be beneficial to the selectivity of b5i versus b5c.
Compounds 11a-11f is presented in Table 1. All compounds were
evaluated for their IC50 to inhibit b5i and b5c. No compounds dis-
To further elucidate the reasons why the potency of compounds
with the D-configuration in P3 was weaker than the L-configura-
tion, we performed docking simulations of compounds 11a, 11b,
Eꢀ83 with proteasome catalytic binding sites (PDB 5L5D, chimeric
played improved selectivity over ONX-0914, and all compounds
with a L-Ala at the R3 position were more potent than compounds
with a D-Ala (11a vs 11b; 11c vs 11d; 11e vs 11f), whereas com-
pounds with D-Ala were more selective than those with L-Ala (11b
vs 11a; 11d vs 11c; 11f vs 11e). When the R2 group was a 3-indolyl,
the phenyl group at R1 contributed to the more potent inhibition of
yeast proteasomes that incorporate key regions of human b5i and
the neighboring
11b (D-Ala in P3) with
cap of 11b and the bottom of the S3* pocket [24], also defined as the
5i S3 pocket formed at the neighboring 6 [25]., This severe steric
clash disappeared when an L-Ala was present in P3 (11a). With
-Leucine in P3, the 4-(thiazol-2-yl)
b
6 subunit [27]). As depicted in Fig. 3, modeling of
b
5i/b6 showed a steric clash between the N-
b
5i compared to the cyclohexyl group (11a vs 11c; 11b vs 11d).
Analogue 11a also exhibited higher 5i inhibitory activity with IC50
values of 21.1 ± 0.3 nm compared to the positive control ONX-0914
IC50 ¼ 30.5 ± 0.7 nm). We speculated that the phenyl group at R1
contributes to improved 5i activity. When R1 group was a cyclo-
b
b
b
regards to Eꢀ83 with an
L
(
piperidine N-cap was a large distance from the S3* pocket, sug-
gesting that such a 4-(thiazol-2-yl)piperidine N-cap in compounds
with a D-configuration may be too long (Fig. 3).
b
hexyl group, 4-methoxyphenyl substituted analogues were slightly
more potent and selective than 3-indolyl substituted compounds
11e vs 11c; 11f vs 11d), particularly 11f, that showed a 3.8-fold
Based on these findings, we shortened the N-cap whilst main-
taining P1 as a phenyl group, P2 as a 4-methoxyphenyl group
(
Table 1
Structures and biological data of compounds 11a-11f.
5

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
Fig. 3. (a) Complex structure of the human
b5i/
b6 chimeric proteasome with ONX-0914 (green) (PDB 5L5D). (b) Overlay of ONX-0914 (green), 11a (blue) and 11b (yellow). (c)
Predicted binding mode of Eꢀ83 into the
b5i/
b6.
(
analogue to ONX-0914) and synthesized analogues of ONX-0914
against
b
5i, with high
b5i-selectivity (20l, 20j), moderate
b5i-
featuring shorter N-cap moieties.
selectivity (20m), and weak
b5i-selectivity (11a). These were
We next synthesized ten analogues of ONX-0914 featuring a
pyrrolidine (20a and 20b), tetrahydroisoquinoline (20c and 20d),
carbazole (20e and 20f), 3-methoxypyrrolidine (20g and 20h) and
assayed with ONX-0914 as the positive control compound. These
data are shown in Table 3.
Inhibition profiling studies revealed in addition to potent
4
-methoxypiperidine (20i and 20j) in the N-cap and an L-Ala or a D-
Ala at the P3 position. As shown in Table 2, those with a shortened
N-cap exhibited moderate to potent 5i inhibitory activity.
inhibitory activity against
against 5c and 1i with IC50 values of 196.7 nM and 267.1 nM,
respectively. Apart from 5i and 5c, compounds 11a also
demonstrated moderate potency against 1i with a IC50 of
154.0 nM, whereas 20m, 20j, 20l minimally affected 1i, and IC50
values of all the compounds against other subunits demonstrated
>4 M.
b5i, ONX-0914 was moderately potent
b
b
b
b
b
When P3 was an L-Ala, compounds featuring a hydrophilic N-
cap (20g and 20i) showed an improved selectivity profile compared
to those bearing a hydrophobic N-cap (20a, 20c and 20e). Notably,
b
b
the chirality of the P3 position had a significant influence on
b
5i
m
selectivity and potency. When the N-cap was a small tetrahy-
dropyrrole group, 20b (featuring D-Ala in P3) showed increased
3.4. Anti-proliferative activity against cancer cell lines
To investigate whether targetting 5i selectively is efficacious
for the treatment of hematologic malignancies, 20l, 20j, 20m, 11a
were also evaluated in human multiple myeloma cells (RMPI-8226
and MM.1S) to assess their anti-proliferative activity in vitro. Assays
were validated using ONX-0914 as a reference compound. The re-
sults are summarized in Table 4.
selectivity and enhanced potency for
L-Ala in P3). Although Compound 20b (
slightly less active than the positive compound ONX-0914, it was
more selective than ONX-0914 for 5i over 5c (21-fold vs 7-fold).
b5i compared to 20a (bearing
b5i IC50 ¼ 62.9 nM) was
b
b
b
Compound 20d with a D-Ala in P3 also showed improved selec-
tivity than L-Ala in the P3 analogue 20c, showing a 12-fold pref-
erence for
b5i. Next, we expanded the N-cap and synthesized
carbazole analogues 20e and 20f. Although 20f exhibited high af-
finity, its selectivity was lower than that of compounds with a
smaller group in the N-cap (20b and 20d). This indicates that a
Unexpectedly, 20j (25-fold selectivity) and 20l (24-fold selec-
tivity), the most selective compounds over
b5c showed only
moderate cell growth inhibitory effects against RPMI-8226 and
MM.1S cells, whereas the growth inhibitory effects of the less se-
lective compound 11a (1-fold selectivity) was comparable to ONX-
bulky hydrophobic N-cap was not beneficial for
most prominent improvement in selectivity was noticed for 20h
and 20j which displayed 25-fold selectivity for 5i over 5c. And
0j ( 5i
5i IC50 ¼ 26.0 nM) was as potent as ONX-0914 (
IC50 ¼ 28.2 nM).
b5i selectivity. The
b
b
0914 against RPMI-8226 cells, with IC50 values of 0.11
0.15 M, respectively. And compound 20m which exhibited 8-fold
selectivity over 5c displayed higher anti-proliferative activity
mM and
2
b
b
m
b
We concluded that the selectivity of compounds with P3-D (20b,
0d, 20f, 20h and 20j) were higher than those with P3-L (20a, 20c,
0e, 20g and 20i) while remaining or even enhancing the binding
than 20l and 20j and lower activity than ONX-0914.
2
2
We next analyzed the possible causes leading to the apparent
discrepancy between activities of different subunits and anti-
proliferative activity against MM cell lines. As shown in Table 3,
affinity toward b5i.
To determine the optimal moiety, we selected 20h and 20j (P3-
D-Ala) for further optimization. As a result of investigating the
substitution pattern of the methoxyl group, a decrease in selectivity
and inhibitory activity was observed when the 4-methoxyl group
was replaced with the 3-methoxyl group at the piperidine (20m or
ONX-0914 strongly inhibited
against 5c and 1i. Although compound 20m showed comparable
inhibitory activities to ONX-0914 against 5c and 5i, its anti-
proliferative activities against two human MM cell lines were
weaker 3e5 fold than ONX-0914, which be explained by its poor
b5i and was moderately potent
b
b
b
b
2
0n versus 20j). Considering that 20h was a mixture (a mixture of
inhibitory activity against
proliferative activity showed similar inhibitory activity against
to ONX-0914. Only moderate inhibitory activities were observed
against 5c for compounds 20j and 20l, which demonstrated
micromolar inhibitory potency against 1i. This may explain why
20j and 20l showed weak cell growth inhibitory effects. Based on
these data, we conclude that the inhibition of 5i alone may be
insufficient to exert the anticancer effects and the co-inhibition of
1i, 5c and 5i may be required for its anti-leukemic effects.
These results were consistent with Niewerth et al. who assessed
b
1i. Compound 11a with strong anti-
compounds featuring R-3-methoxy-pyrrolidine N-cap and S-3-
methoxy-pyrrolidine N-cap), we further took them apart to
examined the activity and selectivity of each isomer. Compound
b1i
b
2
0k and 20l were similar in selectivity, but 20l with S-3-methoxy-
b
pyrrolidine showed improved potency than 20k featuring the R
isomer.
b
3.3. Inhibitory activity against
b
1c/b
1i/b
2c/b
2i
b
b
b
To establish the IC50 values more accurately and also to obtain
insights into inhibitory activities against 1c/ 1i/ 2c/ 2i, we
selected four potent inhibitors that were comparable to ONX-0914
the anti-leukemic activity of PR-924 against the MM cell line RPMI-
8226. They showed that PR-924 displayed cytotoxic activity in the
micromolar range [26]. And at higher concentrations of 1e10 mM,
b
b
b
b
6

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
Table 2
Structures and biological data of compounds 20a-20n.
Table 3
IC50 values of inhibitors against
Cpd IC50(nm)
1c
b
1c/
b1i/b
2c/b2i.
b
b1i
b
2c
b
2i
b
5c
b
5i
Ratio (b5c/b5i)
1
2
2
2
1a
0m
0l
4355.5 ± 696.5
>10000
>10000
>10000
>10000
154.0 ± 16.1
4273.0 ± 14.1
5770.0 ± 1074.8
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
25.6 ± 1.0
21.1 ± 0.3
29.9 ± 5.1
25.1 ± 3.6
26.0 ± 6.28
28.2 ± 9.2
1.2
8.2
24.1
24.9
7.0
244.1 ± 45.3
606.4 ± 212.3
647.1 ± 121.0
196.7 ± 77.0
0j
ONX-0914
267.1 ± 73.8
1603.5 ± 208.6
1697.00 ± 287.1
a
The IC50 values are shown as an average of three independent determinations.
7

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
Table 4
increase the understanding of the immunoproteasome inhibitors in
hematologic malignancies.
IC50 values of inhibitors against RPMI-8226 and MM.1S cells.
Cpd
Cytotoxicity (IC50
RPMI-8226
, m
M)a
MM.1S
5. Experimental section
1
2
2
2
1a
0m
0l
0.11 ± 0.02
0.68 ± 0.08
1.88 ± 0.38
1.92 ± 0.26
0.15 ± 0.01
0.11 ± 0.02
0.22 ± 0.24
1.37 ± 0.35
1.79 ± 0.39
0.06 ± 0.01
5
5
.1. Chemistry
0j
.1.1. General methods
All reagents and solvents were purchased from commercial
ONX-0914
a
The IC50 values are shown as an average of three independent determinations.
suppliers and used without further purification unless otherwise
indicated. Flash-column chromatography was performed using silica
1
13
gel (200e300 mesh). H and C NMR spectra were recorded on a
near complete inhibition of
observed, which indicated 5i inhibition alone is not sufficient to
elicit an anti-leukemic effect, with PR-924 paralleling a concomi-
tant inhibition of 1i, 5c and 5i at drug concentrations >1
b5c and b1i catalytic activities was
Bruker AV-400 (400 MHz) or AV-500 (500 MHz) spectrometer.
b
Chemical shifts (d) are given in ppm (d) relative to Chloroform-d or
DMSO‑d as internal standard. Melting points were determined us-
6
b
b
b
mM
ing a Buchi B-540 capillary melting point apparatus. LCꢀMS analysis
was performed on a Shimadzu LCMS-2020 mass spectrometer with
mobile phases as methanol and water containing 0.1% formic acid.
HPLC analysis was performed using an Agilent 1260 Series system
with a COSMOSIL 5C18-MS-II (4.6 mm ꢂ 250 mm) column and
detected at 220 nm wavelength. The details can be found in sup-
plementary data. All tested compounds are >95% pure.
[
26].
3.5. Molecular docking analysis
Docking is an effective method to propose the binding in-
teractions between a ligand and the receptor. In this study, we have
chosen the most potent and selective compound 20l against 5i/
b
b6
for molecular modeling using Discovery Studio (version 2.1). As
depicted in Fig. 4, The proposed binding pose of compound 20l in
complex with b5i/b6 has four important H-bonds. Two of these H-
5
1
.1.2. General procedure A for the synthesis of intermediates 6a-6b,
2a-12d & 12g-12n
Alanine methyl ester hydrochloride 4a or 4b (1.0 equiv) was
dissolved in DCM (500 mg/25 mL) and saturated NaHCO
bonds are located between the carbonyl group of epoxyketone
warhead and Ser 21 and Gly 23 amino acid residues. The third H-
bond of compound 20l is observed between the NH of the amide
group of phenylalanine in P1 and the carbonyl group of Ser 21
amino acid. And the last H-bond was formed between the carbonyl
group of N-cap and the hydroxyl group of Ser 27. These H-bonds
demonstrate that the structure of the ligand is stabilized by the
three key amino acids.
3
(V
DCM:Vsaturated NaHCO3 ¼ 1:1) was added. The mixture was then
ꢁ
cooled to 0 C, and triphosgene was added. The mixture was stirred
at 0 C for 15min, after which the organic layer was separated and
ꢁ
washed with brine (1 ꢂ ), dried over Na
2 4
SO . The organic layer was
ꢁ
then cooled to 0 C and corresponding amine (0.8 equiv) and DIPEA
(
5 equiv) were added. The reaction mixture was allowed stirred at
room temperature for 3 h. Then the mixture was washed with brine
SO . The organic layer was evaporated in
(1 ꢂ ) and dried over Na
2
4
4
. Conclusions
vacuo and the crude product was purified by flash-column chro-
matography on silica gel.
In summary, this study describes a series of epoxyketone in-
hibitors with varying N-caps and P3 configurations. SARs indicated
that the D-Ala in the P3 residue was crucial to increasing the
5
.1.2.1. Methyl (4-(thiazol-2-ylcarbamoyl)piperidine-1-carbonyl)-L-
alaninate (6a). Compound 6a was obtained by the general pro-
cedure A from commercial intermediate 3 and 5a. Yield: 83%;
White solid; H NMR (500 MHz, DMSO‑d
selectivity for
b5i over
b5c whilst maintaining or enhancing
b5i-
potency. Analogues 20j (
b
5i IC50 ¼ 26.0 nM, 25-fold) and 20l (
b
5i
1
6
)
d
12.07 (s, 1H), 7.46 (d,
IC50 ¼ 25.1 nM, 24-fold) with D-Ala at the P3 site were the most
J ¼ 3.0 Hz, 1H), 7.19 (d, J ¼ 2.5 Hz, 1H), 6.74 (d, J ¼ 5.8 Hz, 1H),
selective inhibitors, which showed higher selectivity than ONX-
4
.18e4.11 (m, 1H), 4.03 (m, 2H), 3.61 (s, 3H), 2.69 (m, 3H), 1.77 (d,
0
914. Docking studies on compound 20l revealed key interactions
J ¼ 12.4 Hz, 2H), 1.57e1.43 (m, 2H), 1.28 (d, J ¼ 7.0 Hz, 3H); ESI-MS:
with the catalytic binding sites. Although 20j and 20l showed only
moderate anti-proliferative activity against RPMI-8226 and MM.1S
þ
m/z ¼ 341 [MþH] .
cell lines, based on our experiments, the inhibition of
insufficient to exert anticancer effects, which likely rely on com-
plementary inhibition of 1i, 5c and 5i. Our results will further
b5i alone was
5.1.2.2. Methyl (4-(thiazol-2-ylcarbamoyl)piperidine-1-carbonyl)-D-
alaninate (6b). Compound 6bwas obtained by the general procedure
b
b
b
Fig. 4. Predicted binding mode of 20l into the b5i/b6.
8

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
1
A fromcommercialintermediate 3 and 5b. Yield:85%;White solid; H
NMR (500 MHz, Chloroform-d)
7.38 (d, J ¼ 3.5 Hz, 1H), 6.97 (d,
5.1.2.11. Methyl ((R)-3-methoxypyrrolidine-1-carbonyl)-D-alaninate
d
(12k). Compound 12k was obtained by the general procedure A
from (R)-3-methoxypyrrolidine and 5b. Yield: 75%; Colorless oil; H
1
J ¼ 3.5 Hz,1H), 4.44 (q, J ¼ 7.0 Hz,1H), 4.06e3.96 (m, 2H), 3.73 (s, 3H),
2
.90 (m, 2H), 2.65e2.56 (m, 1H), 1.92 (m, 2H), 1.83e1.74 (m, 2H), 1.38
NMR (500 MHz, Chloroform-d)
d
4.79 (d, J ¼ 6.0 Hz, 1H), 4.52 (p,
þ
(
d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 341 [MþH] .
J ¼ 7.0 Hz, 1H), 3.99e3.95 (m, 1H), 3.74 (s, 3H), 3.48e3.42 (m, 4H),
3
.32 (s, 3H), 2.09e2.03 (m, 1H), 2.01e1.93 (m,1H),1.40 (d, J ¼ 7.0 Hz,
þ
5.1.2.3. Methyl
(pyrrolidine-1-carbonyl)-L-alaninate
(12a).
3H); ESI-MS: m/z ¼ 231 [M þ H] .
Compound 12a was obtained by the general procedure A from
commercial pyrrolidine and 5a. Yield: 81%; Yellowish-white solid;
5.1.2.12. Methyl ((S)-3-methoxypyrrolidine-1-carbonyl)-D-alaninate
1
H NMR (500 MHz, Chloroform-d)
d
4.74 (brs, 1H), 4.57e4.49 (m,
(12l). Compound 12l was obtained by the general procedure A from
1
1
H), 3.74 (s, 3H), 3.42e3.28 (m, 4H), 1.95e1.85 (m, 4H), 1.40 (d,
(S)-3-methoxypyrrolidine and 5b. Yield: 78%; Colorless oil; H NMR
þ
J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 201 [M þ H] .
(500 MHz, Chloroform-d)
d
4.78 (d, J ¼ 6.0 Hz, 1H), 4.56e4.47 (m,
1
H), 3.99e3.95 (m, 1H), 3.74 (s, 3H), 3.51 (d, J ¼ 11.0 Hz, 1H),
5.1.2.4. Methyl
(pyrrolidine-1-carbonyl)-D-alaninate
(12b).
3.46e3.39 (m, 3H), 3.32 (s, 3H), 2.12e2.04 (m, 1H), 2.03e1.96 (m,
þ
Compound 12b was obtained by the general procedure A from
commercial pyrrolidine and 5b. Yield: 79%; white solid;
1H), 1.40 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 231 [M þ H] .
ꢁ
1
mp ¼ 91.2e92.0 C; H NMR (500 MHz, Chloroform-d)
d
4.74 (s,1H),
5.1.2.13. Methyl ((R)-3-methoxypiperidine-1-carbonyl)-D-alaninate
4
4
.56e4.48 (m, 1H), 3.73 (s, 3H), 3.40e3.29 (m, 4H), 1.93e1.85 (m,
H), 1.39 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 201 [M þ H] .
(12 m). Compound 12m was obtained by the general procedure A
þ
1
from (R)-3-methoxypiperidine and 5b. Yield: 75%; Colorless oil; H
NMR (500 MHz, Chloroform-d)
d
5.04 (d, J ¼ 6.0 Hz, 1H), 4.48 (p,
5.1.2.5. Methyl (1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-L-alani-
J ¼ 7.0 Hz, 1H), 3.81 (dd, J ¼ 13.0, 3.5 Hz, 1H), 3.74 (s, 3H), 3.52e3.46
(m, 1H), 3.37 (s, 3H), 3.28e3.23 (m, 1H), 3.13e3.05 (m, 1H), 2.99 (dd,
J ¼ 13.0, 8.0 Hz, 1H), 1.99e1.93 (m, 1H), 1.79e1.73 (m, 1H), 1.53e1.44
nate (12c). Compound 12c was obtained by the general procedure
A from commercial 1,2,3,4-tetrahydroisoquinoline and 5a. Yield:
0%; Colorless oil; 1H NMR (400 MHz, Chloroform-d)
d
7.22e7.10
(m, 2H), 1.39 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 245 [M þ H] .
þ
8
(
m, 4H), 5.09 (s, 1H), 4.56 (m, 3H), 3.75 (s, 3H), 3.62 (m, 2H), 2.88 (t,
þ
J ¼ 5.6 Hz, 2H), 1.43 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 263 [M þ H]
5.1.2.14. Methyl ((S)-3-methoxypiperidine-1-carbonyl)-D-alaninate
(
12n). Compound 12n was obtained by the general procedure A
1
5.1.2.6. Methyl (1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-D-alani-
from (S)-3-methoxypiperidine and 5b. Yield: 73%; Colorless oil; H
NMR (500 MHz, Chloroform-d) 5.09 (s, 1H), 4.51e4.41 (m, 1H),
nate (12d). Compound 12d was obtained by the general procedure
A from commercial 1,2,3,4-tetrahydroisoquinoline and 5b. Yield:
d
3.75e3.71 (m, 4H), 3.50e3.43 (m, 1H), 3.37 (s, 3H), 3.28e3.21 (m,
1H), 3.16e3.06 (m, 2H), 1.96e1.90 (m, 1H), 1.76 (m, 1H), 1.57e1.46
8
2%; Colorless oil; 1H NMR (400 MHz, Chloroform-d)
d 7.23e7.08
þ
(
(
m, 4H), 5.08 (d, J ¼ 7.0 Hz, 1H), 4.62e4.51 (m, 3H), 3.75 (s, 3H), 3.63
(m, 2H), 1.39 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 245 [M þ H] .
m, 2H), 2.88 (t, J ¼ 5.6 Hz, 2H), 1.43 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/
þ
z ¼ 263 [M þ H] .
5.1.3. Methyl (9H-carbazole-9-carbonyl)-L-alaninate (16a)
To a solution of carbazole 14 (669 mg, 4.0 mmol) in anhydrous
ꢁ
5.1.2.7. Methyl(3-methoxypyrrolidine-1-carbonyl)-
L
-alaninate (12g).
THF (20 mL) was added NaH (192 mg, 4.8 mmol) at 0 C. The
Compound 12g was obtained by the general procedure A from 3-
methoxypyrrolidine and 5a. Yield: 82%; Colorless oil; H NMR
mixture was stirred under argon for 30 min at the same tempera-
ture. 4-nitrophenyl carbonochloridate (947 mg, 4.7 mmol) was
added to the mixture and the mixture was stirred under argon at
1
(
(
1
500 MHz, Chloroform-d)
m, 1H), 3.72 (m, 3H), 3.47e3.38 (m, 4H), 3.30 (m, 3H), 2.04 (m, 1H),
.99e1.93 (m, 1H), 1.38 (m, 3H); ESI-MS: m/z ¼ 231 [M þ H] .
d
4.79 (s, 1H), 4.50 (q, J ¼ 7.0 Hz, 1H), 3.95
ꢁ
0 C for 1 h. After that, the reaction solution was allowed to warm to
þ
room temperature and stirred overnight. The volatiles were
removed under reduced pressure. The crude product was purified
by flash-column chromatography to provided 15. Yellow soild
5.1.2.8. Methyl
(3-methoxypyrrolidine-1-carbonyl)-D-alaninate
ꢁ
1
(
12h). Compound 12h was obtained by the general procedure A
(797 mg, 60%); mp ¼ 181.9e182.0 C. H NMR (500 MHz, DMSO‑d
6
)
1
from 3-methoxypyrrolidine and 5b. Yield: 85%; Colorless oil;
NMR (500 MHz, Chloroform-d)
4.77 (s, 2H), 4.51 (d, J ¼ 6.0 Hz,
H), 4.00e3.93 (m, 2H), 3.73 (s, 6H), 3.48e3.38 (m, 8H), 3.32 (s, 3H,
H
d
8.42 (d, J ¼ 9.0 Hz, 2H), 8.29 (d, J ¼ 8.5 Hz, 2H), 8.25 (d, J ¼ 7.5 Hz,
d
2H), 7.85 (d, J ¼ 9.0 Hz, 2H), 7.60e7.54 (m, 2H), 7.48 (m, 2H). ESI-MS:
þ
2
m/z ¼ 333 [M þ H] .
isomer), 3.31 (s, 3H, isomer), 2.09e2.03 (m, 2H), 1.98 (m, 2H), 1.39
To a solution of 15 (332 mg, 1.0 mmol) in THF (10 mL) was added
5a (1116 mg, 8.0 mmol) followed by DIPEA (1.36 mL, 8.0 mmol). The
mixture was stirred at room temperature overnight. The solvent
was evaporated in vacuum, and the residue was diluted with EtOAc
þ
(
d, J ¼ 7.2 Hz, 6H); ESI-MS: m/z ¼ 231 [M þ H] .
5.1.2.9. Methyl (4-methoxypiperidine-1-carbonyl)-L-alaninate (12i).
Compound 12i was obtained by the general procedure A from 4-
(10 mL). The organic layer was washed with saturated NaHCO
(10 mL ꢂ 2), brine (10 mL ꢂ 1), and dried over Na SO . Purification
by flash-column chromatography. White soild (130 mg, 44%);
NMR (500 MHz, Chloroform-d)
3
methoxypiperidine and 5a. Yield: 79%; Colorless oil; 1H NMR
2
4
1
(
400 MHz, Chloroform-d)
d
5.05 (brs, 1H), 4.52e4.43 (m, 1H), 3.74
H
(
s, 3H), 3.70e3.59 (m, 2H), 3.41e3.36 (m, 1H), 3.34 (s, 3H), 3.14 (m,
d
8.12 (d, J ¼ 8.5 Hz, 2H), 8.04 (d,
2
H), 1.92e1.81 (m, 2H), 1.62e1.51 (m, 2H), 1.39 (d, J ¼ 7.2 Hz, 3H);
J ¼ 7.5 Hz, 2H), 7.50 (ddd, J ¼ 8.5, 7.5, 1.0 Hz, 2H), 7.39e7.33 (m, 2H),
þ
ESI-MS: m/z ¼ 245 [M þ H] .
6.42 (d, J ¼ 7.0 Hz, 1H), 4.88 (m, 1H), 3.86 (s, 3H), 1.64 (d, J ¼ 7.0 Hz,
þ
3
H). ESI-MS: m/z ¼ 297 [M þ H] .
5.1.2.10. Methyl
(4-methoxypiperidine-1-carbonyl)-D-alaninate
(
4
(
3
2
12j). Compound 12j was obtained by the general procedure A from
-methoxypiperidine and 5b. Yield: 82%; Colorless oil; H NMR
5.1.4. Methyl (9H-carbazole-9-carbonyl)-D-alaninate (16b)
1
To a solution of 15 (332 mg, 1.0 mmol) in THF (10 mL) was added
5b (1116 mg, 8.0 mmol) followed by DIPEA (1.36 mL, 8.0 mmol). The
mixture was stirred at room temperature overnight. The solvent
was evaporated in vacuum, and the residue was diluted with EtOAc
500 MHz, Chloroform-d)
.73 (s, 3H), 3.69e3.59 (m, 2H), 3.38 (m, 1H), 3.34 (s, 3H), 3.13 (m,
d
5.01 (d, J ¼ 6.5 Hz, 1H), 4.48 (m, 1H),
H), 1.85 (m, 2H), 1.60e1.51 (m, 2H), 1.38 (d, J ¼ 7.0 Hz, 3H); ESI-MS:
þ
m/z ¼ 245 [M þ H] .
(10 mL). The organic layer was washed with saturated NaHCO
3
9

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
(
10 mL ꢂ 2), brine (10 mL ꢂ 1), and dried over Na
by flash-column chromatography. White soild (150 mg, 51%);
NMR (500 MHz, Chloroform-d)
8.12 (d, J ¼ 8.5 Hz, 2H), 8.03 (d,
J ¼ 7.5 Hz, 2H), 7.53e7.47 (m, 2H), 7.39e7.33 (m, 2H), 6.42 (d,
2
SO
4
. Purification
oxopropan-2-yl)carbamate (9a). Compound 9a was obtained by the
general procedure D from Phe-epoxyketone and (tert-butox-
1
H
ꢁ
1
d
ycarbonyl)-
L
-tryptophan. Yield: 60%; yellow soild; mp:83e85 C; H
NMR (500 MHz, DMSO‑d
6
)
d
10.79 (s, 1H), 8.28 (d, J ¼ 7.5 Hz, 1H),
13
J ¼ 7.0 Hz, 1H), 4.88 (m, 1H), 1.64 (d, J ¼ 7.0 Hz, 3H). C NMR
7.54 (d, J ¼ 8.0 Hz, 1H), 7.26 (m, 6H), 7.05 (t, J ¼ 7.5 Hz, 1H), 7.01 (s,
1H), 6.97 (t, J ¼ 7.5 Hz, 1H), 6.69 (d, J ¼ 8.5 Hz, 1H), 4.64e4.59 (m,
1H), 4.22e4.15 (m,1H), 3.16 (d, J ¼ 5.0 Hz,1H), 2.95 (m, 3H), 2.80 (m,
(
126 MHz, CDCl3)
d
173.59, 151.97, 138.28, 127.10, 125.24, 122.49,
þ
1
20.17, 113.77, 52.82, 49.53, 18.93. ESI-MS: m/z ¼ 297 [M þ H] .
1
H), 2.73 (m, 1H), 1.36 (s, 3H), 1.29 (s, 9H); ESI-MS: m/z ¼ 492
þ
5
.1.5. General procedure B for the synthesis of carboxylic derivatives
[MþH] .
ꢁ
Compounds (1.0 equiv) were dissolved in acetone and at ꢀ10 C
added 0.3 N LiOH (1.2 equiv) dropwise. The solution was stirred
5.1.8.3. Tert-butyl ((S)-1-(((S)-3-cyclohexyl-1-((R)-2-methyloxiran-
2-yl)-1-oxopropan-2-yl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-
yl)carbamate (9b). Compound 9b was obtained by the general
ꢁ
at ꢀ0 C for 30 min. The mixture was then acidified to pH ¼ 2e3
with 3 N HCl and extracted with EtOAc (3 ꢂ ) or N-butanol (3 ꢂ ) (if
necessary). The organic layer was dried over Na
2
SO
4
, evaporated in
procedure
ycarbonyl)-
D
from cyclohexyl-epoxyketone and (tert-butox-
-tryptophan. Yield: 61%; yellow soild;
10.81 (s, 1H), 8.15
vacuo to yield the crude product without further purification.
L
ꢁ
1
6
mp:151e152 C; H NMR (500 MHz, DMSO‑d ) d
5
.1.5.1. Carboxylic derivatives 7a-7b, 13a-13d, 13g-13n, 17a-17b and
(d, J ¼ 7.5 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.31 (d, J ¼ 8.0 Hz, 1H), 7.10
(s, 1H), 7.05 (t, J ¼ 7.5 Hz, 1H), 6.97 (t, J ¼ 7.5 Hz, 1H), 6.71 (d,
J ¼ 8.5 Hz, 1H), 4.45 (m, 1H), 4.22 (m, 1H), 3.15 (d, J ¼ 5.0 Hz, 1H),
3.05e2.94 (m, 2H), 2.84 (dd, J ¼ 14.5, 9.5 Hz, 1H), 1.75e1.54 (m, 5H),
19a-19n. Compound 7a-7b, 13a-13d, 13g-13n and 19a-19n were
obtained by the general procedure B. The crude product were used
in the next step without further purification.
1
.40 (m, 4H), 1.30 (s, 9H), 1.22e1.08 (m, 5H), 0.99e0.90 (m, 1H), 0.82
5
.1.6. N-(thiazol-2-yl)piperidine-4-carboxamide-trifluoroacetate
(m, 1H); ESI-MS: m/z ¼ 498 [MþH]þ.
(
3)
Compound 2 (5.0 g, 16.1 mmol) was dissolved in DCM and
cooled to 0 C, added dropwise trifluoroacetic acid (16 mL). The
mixture was allowed to warm to room temperature and stirred for
h. After completion of the reaction, the solvent was evaporated
5.1.8.4. Tert-butyl ((S)-1-(((S)-3-cyclohexyl-1-((R)-2-methyloxiran-
2-yl)-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-1-oxopropan-
2-yl)carbamate (9c). Compound 9c was obtained by the general
procedure D from cyclohexyl-epoxyketone and (S)-2-((tert-butox-
ycarbonyl)amino)-3-(4-Methoxyphenyl)propanoic acid. Yield:
ꢁ
3
under reduced pressure. Then the THF was added to the crude
ꢁ
1
ꢁ
1
product, precipitating 3.6 g white solid (70%); mp:188-189 C; H
NMR (500 MHz, DMSO‑d 12.26 (s, 1H), 8.62 (s, 1H), 8.37 (s, 1H),
63%; white soild; mp: 153e155 C; H NMR (500 MHz, Chloroform-
d)
6
)
d
d
7.12 (d, J ¼ 8.0 Hz, 2H), 6.83 (d, J ¼ 8.5 Hz, 2H), 6.12 (d, J ¼ 7.5 Hz,
7
2
1
.48 (d, J ¼ 3.5 Hz, 1H), 7.23 (d, J ¼ 3.5 Hz, 1H), 3.33 (d, J ¼ 13.0 Hz,
1H), 4.95 (d, J ¼ 5.0 Hz, 1H), 4.60e4.55 (m, 1H), 4.28 (m, 1H), 3.79 (s,
3H), 3.25 (d, J ¼ 2.5 Hz, 1H), 3.03 (dd, J ¼ 14.0, 6.5 Hz, 1H), 2.93 (dd,
J ¼ 14.0, 7.0 Hz, 1H), 2.88 (d, J ¼ 5.0 Hz, 1H), 1.81 (d, J ¼ 12.5 Hz, 1H),
H), 2.99e2.88 (m, 2H), 2.81e3.75 (m, 1H), 2.02e1.94 (m, 2H),
þ
.87e1.74 (m, 2H).; ESI-MS: m/z ¼ 212 [MþH] .
1
.73e1.52 (m, 6H), 1.49 (s, 3H), 1.42 (s, 9H), 1.22e1.09 (m, 5H), 0.89
þ
5
(
.1.7. General procedure C for the removal of t-butyloxycarbonyl
Boc) protecting group on dipeptide epoxyketone fragment 10a, 10b
and 10c
(m, 2H); ESI-MS: m/z ¼ 489 [MþH] .
5.1.8.5. Methyl(S)-3-(4-methoxyphenyl)-2-((S)-2-(pyrrolidine-1-
carboxamido)propanamido)propanoate (18a). Compound 18a was
obtained by the general procedure D from 13a and methyl (S)-2-
The dipeptide epoxyketone fragment was dissolved in DCM and
ꢁ
cooled to 0 C, added dropwise trifluoroacetic acid. The mixture
ꢁ
was stirred for 3 h at 0 C, the solvent was evaporated under
amino-3-(4-methoxyphenyl)propanoate hydrochloride. Yield:
1
reduced pressure to get the light yellow oil and the crude product
83%; Colorless oil; H NMR (500 MHz, Chloroform-d)
d
7.04 (d,
were used in the next step without further purification.
J ¼ 8.5 Hz, 2H), 6.96 (brs, 1H), 6.78 (d, J ¼ 8.5 Hz, 2H), 4.78 (m, 2H,
CH þ NH), 4.46e4.39 (m, 1H), 3.76 (s, 3H), 3.71 (s, 3H), 3.31 (m, 4H),
3.09 (dd, J ¼ 14.0, 6.0 Hz, 1H), 3.00 (dd, J ¼ 14.0, 6.5 Hz, 1H), 1.91 (m,
5
9
.1.8. General procedure D for the synthesis of the intermediates 2,
a-9c, 18a-18n, 11a-11f and 20a-20n
þ
4H), 1.33 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 378 [M þ H] .
The crude acid (1.0 equiv) was dissolved in DCM and cooled to
C. The solution was then treated with HOBt (1.2 equiv), EDCI (1.8
ꢁ
0
5.1.8.6. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-(pyrrolidine-1-
carboxamido)propanamido)propanoate (18b). Compound 18b was
obtained by the general procedure D from 13b and methyl (S)-2-
equiv), corresponding amine or amino acid (1.2 equiv) and DIPEA
3.0 equiv). The mixture was allowed to warm to room temperature
and stirred for 3 h. After completion of the reaction, the mixture
was washed with 1 N HCl (2 ꢂ ), saturated NaHCO (2 ꢂ ), and brine
SO . The organic layer was evaporated in
(
amino-3-(4-methoxyphenyl)propanoate hydrochloride. Yield:
1
3
85%; Colorless oil; H NMR (500 MHz, Chloroform-d)
d
7.04 (d,
(
2 ꢂ ), and dried over Na
2
4
J ¼ 8.5 Hz, 2H), 6.98 (d, J ¼ 7.5 Hz, 1H), 6.80 (d, J ¼ 8.5 Hz, 2H), 4.77
vacuo and the crude product was purified by flash-column chro-
(m, 2H, CH þ NH), 4.47e4.39 (m, 1H), 3.77 (s, 3H), 3.70 (s, 3H), 3.32
matography on silica gel.
(m, 4H), 3.09 (dd, J ¼ 14.0, 6.0 Hz, 1H), 3.01 (dd, J ¼ 14.0, 6.5 Hz, 1H),
þ
1
.90 (m, 4H), 1.30 (d, J ¼ 7.0 Hz, 3H). ESI-MS: m/z ¼ 378 [M þ H] .
5.1.8.1. Tert-butyl 4-(thiazol-2-ylcarbamoyl)piperidine-1-carboxylate
(
1
2). Compound 2 was obtained by the general procedure D from
5.1.8.7. Methyl(S)-3-(4-methoxyphenyl)-2-((S)-2-(1,2,3,4-
tetrahydroisoquinoline-2-carboxamido)propanamido)propanoate
(18c). Compound 18c was obtained by the general procedure D
from 13c and methyl (S)-2-amino-3-(4-methoxyphenyl)prop-
ꢁ
1
and 2-aminothiazole. Yield:89%; white solid; mp:192-194 C; H
12.04 (brs, 1H), 7.41 (d, J ¼ 3.5 Hz,
NMR (500 MHz, Chloroform-d)
1
d
H), 7.03 (d, J ¼ 3.5 Hz, 1H), 4.18 (s, 2H), 2.91e2.76 (m, 2H), 2.63 (tt,
1
J ¼ 11.0, 4.0 Hz, 1H), 1.91 (d, J ¼ 11.5 Hz, 2H), 1.86e1.76 (m, 2H), 1.47
anoate hydrochloride. Yield: 81%; Colorless oil; H NMR (400 MHz,
þ
(
s, 9H); ESI-MS: m/z ¼ 312 [MþH] .
Chloroform-d)
d
7.23e7.09 (m, 4H), 7.01 (d, J ¼ 8.6 Hz, 2H), 6.79 (d,
J ¼ 7.4 Hz, 1H), 6.74 (d, J ¼ 8.6 Hz, 2H), 5.03 (d, J ¼ 7.0 Hz, 1H), 4.79
(m, 1H), 4.53 (s, 2H), 4.45 (m, 1H), 3.72 (s, 3H), 3.69 (s, 3H),
3.66e3.52 (m, 2H), 3.09 (dd, J ¼ 14.0, 5.6 Hz, 1H), 3.00 (dd, J ¼ 14.0,
5
.1.8.2. Tert-butyl ((S)-3-(1H-indol-3-yl)-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
10

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
6
.4 Hz, 1H), 2.87 (t, J ¼ 6.0 Hz, 2H), 1.36 (d, J ¼ 7.0 Hz, 3H); ESI-MS:
2H), 5.09 (d, J ¼ 6.5 Hz, 1H), 4.78 (m, 1H), 4.39 (m, 1H), 3.77 (s, 3H),
3.70 (s, 3H), 3.66e3.58 (m, 2H), 3.37 (m, 1H), 3.34 (s, 3H), 3.15e3.05
þ
m/z ¼ 440 [M þ H] .
(
m, 3H), 3.00 (dd, J ¼ 14.0, 6.5 Hz, 1H), 1.83 (m, 2H), 1.54 (m, 2H),
þ
5
.1.8.8. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-(1,2,3,4-
tetrahydroisoquinoline-2-carboxamido)propanamido)propanoate
18d). Compound 18d was obtained by the general procedure D
1.28 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 422 [M þ H] .
(
5.1.8.14. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-((R)-3-
methoxypyrrolidine-1-carboxamido)propanamido)propanoate (18k).
Compound 18k was obtained by the general procedure D from 13k
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
from 13d and methyl (S)-2-amino-3-(4-methoxyphenyl)prop-
anoate hydrochloride. Yield: 83%; Colorless oil; H NMR (400 MHz,
1
Chloroform-d)
J ¼ 7.8 Hz, 1H), 6.77 (d, J ¼ 8.6 Hz, 2H), 5.15 (d, J ¼ 7.0 Hz, 1H), 4.80
m, 1H), 4.53 (s, 2H), 4.46 (m, 1H), 3.72 (s, 3H), 3.69 (s, 3H),
d
7.20e7.09 (m, 4H), 7.03 (d, J ¼ 8.6 Hz, 2H), 6.93 (d,
1
chloride. Yield: 83%; Colorless oil; H NMR (500 MHz, Chloroform-
(
d)
d
7.52 (brs, 1H), 7.07 (d, J ¼ 8.5 Hz, 2H), 6.81 (d, J ¼ 8.5 Hz, 2H),
3
6
.66e3.52 (m, 2H), 3.10 (dd, J ¼ 14.0, 5.6 Hz, 1H), 3.02 (dd, J ¼ 14.0,
5.55 (brs, 1H), 4.78e4.72 (m, 1H), 4.53e4.45 (m, 1H), 4.00e3.96 (m,
1H), 3.77 (s, 3H), 3.71 (s, 3H), 3.52e3.42 (m, 4H), 3.32 (s, 3H), 3.11
(dd, J ¼ 14.0, 5.5 Hz, 1H), 3.04 (dd, J ¼ 14.0, 7.0 Hz, 1H), 2.12e2.05
.4 Hz, 1H), 2.86 (t, J ¼ 6.0 Hz, 2H), 1.32 (d, J ¼ 7.0 Hz, 3H); ESI-MS:
þ
m/z ¼ 440 [M þ H] .
(
m, 1H), 2.01e1.92 (m, 1H), 1.32 (d, J ¼ 7.0 Hz, 3H). ESI-MS: m/
þ
5
.1.8.9. Methyl(S)-2-((S)-2-(9H-carbazole-9-carboxamido)prop-
z ¼ 408 [M þ H] .
anamido)-3-(4-methoxyphenyl)propanoate (18e). Compound 18e
was obtained by the general procedure D from 17a and methyl (S)-
5.1.8.15. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-((S)-3-
methoxypyrrolidine-1-carboxamido)propanamido)propanoate (18l).
Compound 18l was obtained by the general procedure D from 13l
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
2
8
-amino-3-(4-methoxyphenyl)propanoate hydrochloride. Yield:
2%; White solid; 1H NMR (500 MHz, Chloroform-d)
d
8.06 (d,
J ¼ 8.5 Hz, 2H), 8.04 (d, J ¼ 7.5 Hz, 2H), 7.52e7.46 (m, 2H), 7.39e7.33
1
(
(
(
m, 2H), 7.01 (d, J ¼ 8.5 Hz, 2H), 6.67 (d, J ¼ 8.5 Hz, 2H), 6.49e6.43
chloride. Yield: 79%; Colorless oil; H NMR (500 MHz, Chloroform-
m, 2H, NH), 4.89 (m,1H), 4.71 (m,1H), 3.77 (s, 3H), 3.59 (s, 3H), 3.15
d)
d
7.04 (d, J ¼ 8.5 Hz, 2H), 6.80 (d, J ¼ 8.5 Hz, 2H), 4.91 (brs, 1H),
dd, J ¼ 14.0, 5.5 Hz, 1H), 3.03 (dd, J ¼ 14.0, 6.5 Hz, 1H), 1.57 (d,
4.81e4.74 (m, 1H), 4.47e3.38 (s, 1H), 3.96 (m, 1H), 3.77 (s, 3H), 3.71
(s, 3H), 3.50 (m, 1H), 3.45e3.38 (m, 3H), 3.32 (s, 3H), 3.09 (dd,
J ¼ 14.0, 5.5 Hz, 1H), 3.02 (dd, J ¼ 14.0, 6.5 Hz, 1H), 2.11e2.04 (m,
1H), 1.98 (m, 1H), 1.31 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 408 [M þ
þ
J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 474 [M þ H] .
5
.1.8.10. Methyl(S)-2-((R)-2-(9H-carbazole-9-carboxamido)prop-
þ
anamido)-3-(4-methoxyphenyl)propanoate (18f). Compound 18f
was obtained by the general procedure D from 17b and methyl (S)-
H] .
2
8
-amino-3-(4-methoxyphenyl)propanoate hydrochloride. Yield:
5.1.8.16. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-((R)-3-
methoxypiperidine-1-carboxamido)propanamido)propanoate (18 m).
Compound 18m was obtained by the general procedure D from
13m and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate
0%; White solid; 1H NMR (500 MHz, Chloroform-d)
d
8.05 (d,
J ¼ 8.5 Hz, 2H), 8.02 (d, J ¼ 7.5 Hz, 2H), 7.48 (ddd, J ¼ 8.5, 7.5, 1.0 Hz,
2
2
H), 7.37e7.31 (m, 2H), 7.02 (d, J ¼ 8.5 Hz, 2H), 6.77 (d, J ¼ 8.5 Hz,
H), 6.54 (d, J ¼ 7.0 Hz, 1H), 6.49 (d, J ¼ 8.0 Hz, 1H), 3.72 (s, 3H), 3.71
1
hydrochloride. Yield: 80%; Colorless oil; H NMR (500 MHz, Chlo-
(
1
s, 3H), 3.16 (dd, J ¼ 14.0, 6.0 Hz, 1H), 3.09 (dd, J ¼ 14.1, 6.0 Hz, 1H),
roform-d)
J ¼ 13.5, 6.5 Hz, 1H), 4.42 (dd, J ¼ 13.0, 6.0 Hz, 1H), 3.77 (s, 3H),
.73e3.67 (m, 4H), 3.45e3.39 (m, 1H), 3.36 (s, 3H), 3.28e3.23 (m,
d
7.04 (d, J ¼ 8.5 Hz, 2H), 6.81 (d, J ¼ 8.5 Hz, 2H), 4.78 (dd,
þ
.53 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 474 [M þ H] .
3
5
.1.8.11. Methyl(2S)-3-(4-methoxyphenyl)-2-((2S)-2-(3-
1H), 3.22e3.14 (m, 2H), 3.10 (dd, J ¼ 14.0, 5.5 Hz, 1H), 3.02 (dd,
J ¼ 14.0, 6.5 Hz, 1H), 1.93e1.86 (m, 1H), 1.76 (m, 1H), 1.59e1.51 (m,
1H), 1.49e1.40 (m, 1H), 1.31 (d, J ¼ 7.0 Hz, 3H); ESI-MS: m/z ¼ 422
methoxypyrrolidine-1-carboxamido)propanamido)propanoate (18g).
Compound 18g was obtained by the general procedure D from 13g
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
þ
[M þ H] .
1
chloride. Yield: 83%; Colorless oil; H NMR (500 MHz, Chloroform-
d)
d
7.03 (d, J ¼ 8.5 Hz, 2H), 6.97 (m,1H), 6.82e6.77 (m, 2H), 4.76 (m,
5.1.8.17. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-((S)-3- methox-
2
H), 4.41 (m, 1H), 3.97 (m, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.50e3.36
ypiperidine-1-carboxamido)propanamido)propanoate
(18n).
(
1
m, 4H), 3.33 (m, 3H), 3.08 (dd, J ¼ 14.0, 5.5 Hz, 1H), 3.04e2.95 (m,
Compound 18n was obtained by the general procedure D from 13n
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
chloride. Yield: 88%; Colorless oil (153 mg, 86%); H NMR (500 MHz,
H), 2.08 (m, 1H), 1.97 (m, 1H), 1.32 (m, 3H); ESI-MS: m/z ¼ 408
þ
1
[M þ H] .
Chloroform-d)
d
7.02 (d, J ¼ 8.5 Hz, 2H), 6.81 (d, J ¼ 8.5 Hz, 3H), 5.09
5
.1.8.12. Methyl(2S)-3-(4-methoxyphenyl)-2-((2R)-2-(3-
(brs, 1H), 4.79 (m, 1H), 4.82e4.75 (m, 1H), 3.77 (s, 3H), 3.74e3.68
(m, 4H), 3.45e3.39 (m, 1H), 3.35 (s, 3H), 3.27e3.21 (m, 1H),
3.19e3.12 (m, 1H), 3.11e2.99 (m, 3H), 1.91 (m, 1H), 1.80e1.71 (m,
1H), 1.58e1.49 (m, 1H), 1.47e1.41 (m, 1H), 1.30 (d, J ¼ 7.0 Hz, 3H);
methoxypyrrolidine-1-carboxamido)propanamido)propanoate (18h).
Compound 18h was obtained by the general procedure D from 13h
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
chloride. Yield: 86%; Colorless oil; H NMR (500 MHz, Chloroform-
d)
2
3
3
1
þ
ESI-MS: m/z ¼ 422 [M þ H] .
d
7.03 (d, J ¼ 8.5 Hz, 2H), 6.90 (d, J ¼ 7.5 Hz, 1H), 6.83e6.78 (m,
H), 4.78 (m, 2H), 4.45e4.38 (m, 1H), 3.96 (m, 1H), 3.77 (s, 3H),
.72e3.68 (m, 3H), 3.51e3.36 (m, 4H), 3.31 (d, 3H), 3.08 (m, 1H),
5.1.8.18. N-(1-(((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-yl)
thiazole-2-carboxamide (11a). Compound 11a was obtained by the
general procedure D from 7a and 10a. Yield:53%; white soild;
.01 (m, 1H), 2.06 (m, 1H), 2.01e1.93 (m, 1H), 1.30 (d, J ¼ 7.0 Hz, 3H);
þ
ESI-MS: m/z ¼ 408 [M þ H] .
ꢁ
1
5
.1.8.13. Methyl(S)-3-(4-methoxyphenyl)-2-((R)-2-(4-
mp:153e154 C; H NMR (500 MHz, Chloroform-d) d 11.64 (s, 1H),
methoxypiperidine-1-carboxamido)propanamido)propanoate (18j).
Compound 18j was obtained by the general procedure D from 13j
and methyl (S)-2-amino-3-(4-methoxyphenyl)propanoate hydro-
8.97 (s, 1H), 7.51 (d, J ¼ 8.0 Hz, 1H), 7.45 (d, J ¼ 3.5 Hz, 1H), 7.29 (d,
J ¼ 8.0 Hz, 2H), 7.18e7.13 (m, 3H), 7.11 (t, J ¼ 7.3 Hz, 2H), 7.04 (d,
J ¼ 7.5 Hz, 1H), 7.01 (d, J ¼ 3.5 Hz, 1H), 6.96e6.92 (m, 2H), 6.90 (s,
1H), 5.30 (s, 1H), 4.78e4.67 (m, 2H), 4.37e4.29 (m, 1H), 3.87 (d,
J ¼ 12.5 Hz, 1H), 3.75 (d, J ¼ 12.5 Hz, 1H), 3.24 (dd, J ¼ 15.0, 5.5 Hz,
1
chloride. Yield: 83%; Colorless oil; H NMR (500 MHz, Chloroform-
d)
d
7.02 (d, J ¼ 8.5 Hz, 2H), 6.85 (d, J ¼ 7.5 Hz,1H), 6.80 (d, J ¼ 8.5 Hz,
11

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
1
H), 3.18 (d, J ¼ 5.0 Hz, 1H), 3.09 (dd, J ¼ 15.0, 6.0 Hz, 1H), 2.93 (dd,
59.11, 53.64, 52.51, 52.4, 51.20, 49.84, 49.56, 43.24, 42.14, 37.85,
34.15, 33.74, 31.95, 31.84, 27.88, 27.79, 27.34, 26.30, 26.21, 26.09,
25.89, 17.83, 16.76; ESI-MS: m/z ¼ 706 [MþH] .
J ¼ 14.0, 5.5 Hz, 1H), 2.76 (d, J ¼ 5.0 Hz, 1H), 2.69 (dd, J ¼ 23.8,
þ
11.8 Hz, 2H), 2.61e2.53 (m, 2H), 1.80e1.67 (m, 2H), 1.66e1.54 (m,
13
2
H), 1.37 (s, 3H), 1.27 (d, J ¼ 7.0 Hz, 3H); C NMR (126 MHz,
207.55, 173.72, 172.92, 171.26, 159.29, 156.81,
36.70, 136.21,135.88, 129.23, 128.40, 127.36,126.88,123.23, 122.05,
19.43, 118.60, 114.02, 111.42, 109.80, 59.14, 53.35, 52.37, 50.63,
Chloroform-d)
1
1
d
5.1.8.22. N-(1-(((S)-1-(((S)-1-(((S)-3-cyclohexyl-1-((R)-2-
methyloxiran-2-yl)-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-
1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-
yl)thiazole-2-carboxamide (11e). Compound 11e was obtained by
the general procedure D from 7a and 10c. Yield:65%; white soild;
4
3.30, 43.07, 42.16, 36.93, 27.82, 27.30, 18.24, 16.39; ESI-MS: m/
þ
z ¼ 700 [MþH] .
ꢁ
1
mp:108e110 C; H NMR (500 MHz, Chloroform-d)
d 11.64 (s, 1H),
5
.1.8.19. N-(1-(((R)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-1-((R)-2-
7.56e7.42 (m, 2H), 7.18e7.07 (m,1H), 7.02 (d, J ¼ 3.5 Hz,1H), 6.98 (d,
J ¼ 8.0 Hz, 2H), 6.73 (d, J ¼ 8.0 Hz, 2H), 5.29 (s, 1H), 4.68e4.63 (m,
1H), 4.63e4.55 (m, 1H), 4.52e4.42 (m, 1H), 4.01 (d, J ¼ 13.0 Hz, 1H),
3.93 (d, J ¼ 13.0 Hz, 1H), 3.74 (s, 3H), 3.29 (d, J ¼ 5.0 Hz, 1H),
2.98e2.86 (m, 4H), 2.85e2.77 (m, 1H), 2.72e2.65 (m, 1H), 1.93e1.72
(m, 5H), 1.70e1.60 (m, 3H), 1.55e1.45 (m, 5H), 1.34 (d, J ¼ 7.0 Hz,
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-yl)
thiazole-2-carboxamide (11b). Compound 11b was obtained by the
general procedure D from 7b and 10a. Yield:53%; white soild;
ꢁ
1
mp:153e154 C; H NMR (500 MHz, Chloroform-d)
.51 (d, J ¼ 8.0 Hz,1H), 7.44 (d, J ¼ 4.0 Hz,1H), 7.39 (d, J ¼ 7.5 Hz,1H),
.33 (d, J ¼ 8.0 Hz, 1H), 7.22e7.16 (m, 3H), 7.13 (t, J ¼ 7.5 Hz, 1H),
.08e7.02 (m, 3H), 7.01e6.95 (m, 2H), 6.81 (s, 1H), 5.92 (s, 1H),
.74e4.68 (m, 2H), 4.12e4.05 (m,1H), 4.01e3.87 (m, 2H), 3.29e3.23
m, 2H), 3.11 (dd, J ¼ 15.0, 6.0 Hz, 1H), 2.96 (dd, J ¼ 13.5, 4.5 Hz, 1H),
.87e2.65 (m, 5H), 1.91 (d, J ¼ 11.5 Hz, 1H), 1.84 (d, J ¼ 11.0 Hz, 1H),
d 8.66 (s, 1H),
13
7
7
7
4
(
2
3H), 1.24e1.10 (m, 5H), 0.95e0.81 (m, 2H); C NMR (126 MHz,
Chloroform-d) 208.46, 173.87, 172.68, 171.06, 159.42, 158.45,
d
156.96, 136.72, 130.24, 128.41, 113.85, 113.83, 59.01, 55.20, 54.49,
52.34, 50.14, 49.32, 43.34, 43.15, 42.05, 38.37, 36.64, 34.24, 33.82,
31.85, 27.81, 27.76, 26.29, 26.17, 25.92, 18.68, 16.68; ESI-MS: m/
þ
z ¼ 697 [MþH] .
13
1
(
1
1
.70e1.59 (m, 2H), 1.39 (s, 3H), 1.28 (d, J ¼ 7.0 Hz, 3H); C NMR
126 MHz, Chloroform-d) 207.72, 174.64, 173.16, 171.54, 160.08,
57.35, 136.55, 136.09, 133.42, 129.43, 128.41, 127.42, 126.69, 123.22,
22.05, 119.49, 118.43, 113.82, 111.39, 109.64, 59.23, 53.56, 53.19,
d
5.1.8.23. N-(1-(((R)-1-(((S)-1-(((S)-3-cyclohexyl-1-((R)-2-
methyloxiran-2-yl)-1-oxopropan-2-yl)amino)-3-(4-methoxyphenyl)-
1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-
yl)thiazole-2-carboxamide (11f). Compound 11f was obtained by
the general procedure D from 7b and 10c. Yield:65%; white soild;
5
2.47, 51.34, 43.29, 43.09, 42.10, 36.49, 27.95, 27.60, 27.04, 17.57,
þ
16.50; ESI-MS: m/z ¼ 700 [MþH] .
ꢁ
1
mp: 134e136 C; H NMR (500 MHz, Chloroform-d)
d 11.77 (s, 1H),
5
.1.8.20. N-(1-(((S)-1-(((S)-1-(((S)-3-cyclohexyl-1-((R)-2-
7.38 (d, J ¼ 3.0 Hz, 1H), 7.08 (d, J ¼ 8.5 Hz, 2H), 6.98 (d, J ¼ 3.5 Hz,
1H), 6.88 (d, J ¼ 8.0 Hz, 1H), 6.81 (d, J ¼ 8.5 Hz, 2H), 5.78 (s, 1H),
4.71e4.62 (m, 1H), 4.60e4.53 (m, 1H), 4.21e4.13 (m, 1H), 4.00 (d,
J ¼ 12.5 Hz, 1H), 3.93 (d, J ¼ 12.5 Hz, 1H), 3.77 (s, 3H), 3.34 (d,
J ¼ 5.0 Hz,1H), 3.12e2.97 (m, 2H), 2.93e2.80 (m, 3H), 2.74e2.63 (m,
1H),1.93e1.82 (m, 2H),1.80e1.65 (m, 4H),1.64e1.50 (m, 4H),1.45 (s,
methyloxiran-2-yl)-1-oxopropan-2-yl)amino)-3-(1H-indol-3-yl)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-yl)
thiazole-2-carboxamide (11c). Compound 11c was obtained by the
general procedure D from 7a and 10b. Yield:65%; white soild;
mp:162e163 C; H NMR (500 MHz, Chloroform-d)
9
1
ꢁ
1
d
11.60 (s, 1H),
.14 (s, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.48 (d, J ¼ 3.5 Hz, 1H), 7.42 (s,
H), 7.30 (d, J ¼ 8.0 Hz, 1H), 7.16 (s, 1H), 7.10 (t, J ¼ 7.5 Hz, 1H),
13
3H), 1.34e1.20 (m, 5H), 1.16e1.04 (m, 3H), 0.95e0.80 (m, 2H);
C
NMR (126 MHz, Chloroform-d) 208.64, 174.29, 172.82, 171.20,
d
7.04e6.98 (m, 3H), 5.20 (s, 1H), 4.80e4.72 (m, 1H), 4.57e4.49 (m,
159.57, 158.61, 157.55, 136.41, 131.30, 130.37, 128.30, 114.04, 59.13,
55.24, 54.08, 52.43, 51.00, 49.67, 43.32, 43.28, 42.23, 37.91, 36.64,
34.20, 33.88, 31.86, 27.87, 26.35, 26.17, 25.91, 17.98, 16.76; ESI-MS:
1
1
H), 4.40e4.32 (m, 1H), 3.90 (d, J ¼ 12.5 Hz, 1H), 3.74 (d, J ¼ 12.5 Hz,
H), 3.28 (dd, J ¼ 15.0, 6.0 Hz, 1H), 3.24 (d, J ¼ 5.0 Hz, 1H), 3.16 (dd,
þ
J ¼ 15.0, 6.0 Hz, 1H), 2.79 (d, J ¼ 5.0 Hz, 1H), 2.76e2.60 (m, 3H),
m/z ¼ 697 [MþH] .
1
5
d
.82e1.52 (m, 9H), 1.45 (s, 5H), 1.34 (d, J ¼ 7.0 Hz, 3H), 1.17e1.01 (m,
13
H), 0.87e0.75 (m, 2H);
208.48, 173.69, 172.96, 171.65, 159.23, 156.75, 136.74, 136.28,
27.38, 123.09, 122.04, 119.41, 118.59, 114.03, 111.39, 109.94, 59.06,
3.33, 52.40, 50.63, 49.60, 43.35, 43.04, 42.21, 38.17, 34.13, 33.75,
C NMR (126 MHz, Chloroform-d)
5.1.8.24. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20a). Compound 20a was obtained by the general
1
5
3
1.83, 27.83, 27.18, 26.27, 26.10, 25.89, 18.30, 16.74; ESI-MS: m/
procedure
D from 8a and 19a. Yield: 68%; White solid;
þ
ꢁ
1
z ¼ 706 [MþH] .
mp ¼ 74e75 C. H NMR (500 MHz, Chloroform-d)
d 7.26e7.20 (m,
3
H), 7.08 (d, J ¼ 7.0 Hz, 2H), 7.04 (d, J ¼ 8.5 Hz, 3H), 6.78 (d,
5
.1.8.21. N-(1-(((R)-1-(((S)-1-(((S)-3-cyclohexyl-1-((R)-2-
J ¼ 7.0 Hz, 1H), 6.74 (d, J ¼ 8.5 Hz, 2H), 4.81e4.73 (m, 1H), 4.59e4.45
(m, 2H), 4.32e4.24 (m, 1H), 3.75 (s, 3H), 3.30 (d, J ¼ 5.0 Hz, 1H),
3.27e3.17 (m, 4H), 3.05 (dd, J ¼ 14.0, 5.0 Hz, 1H), 2.95 (d, J ¼ 7.0 Hz,
2H), 2.87 (d, J ¼ 5.0 Hz, 1H), 2.74 (dd, J ¼ 14.0, 8.0 Hz, 1H), 1.95e1.82
methyloxiran-2-yl)-1-oxopropan-2-yl)amino)-3-(1H-indol-3-yl)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)piperidin-4-yl)
thiazole-2-carboxamide (11d). Compound 11d was obtained by the
general procedure D from 7b and 10b. Yield:60%; white soild;
13
(m, 4H), 1.45 (s, 3H), 1.28 (d, J ¼ 7.0 Hz, 3H); C NMR (126 MHz,
ꢁ
1
mp:190e191 C; H NMR (500 MHz, Chloroform-d)
.54 (d, J ¼ 8.0 Hz,1H), 7.40 (d, J ¼ 3.0 Hz,1H), 7.37 (d, J ¼ 7.0 Hz,1H),
.35 (d, J ¼ 8.0 Hz, 1H), 7.17 (d, J ¼ 6.0 Hz, 1H), 7.13 (t, J ¼ 7.5 Hz, 1H),
.09 (s, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.98 (d, J ¼ 3.0 Hz, 1H), 5.88 (s,
H), 4.81e4.73 (m, 1H), 4.55e4.48 (m, 1H), 4.19e4.11 (m, 1H), 3.96
d, J ¼ 12.0 Hz, 1H), 3.90 (d, J ¼ 12.0 Hz, 1H), 3.36e3.18 (m, 3H),
.82e2.72 (m, 3H), 2.71e2.61 (m, 1H), 1.88e1.76 (m, 2H), 1.75e1.60
m, 5H), 1.60e1.53 (m, 2H), 1.46e1.41 (m, 5H), 1.29e1.15 (m, 6H),
d
8.92 (s, 1H),
Chloroform-d) d 207.20, 173.35, 170.79, 158.45, 156.19, 135.97,
7
7
7
1
(
2
(
130.28, 129.30, 128.65, 128.46, 126.97, 113.84, 59.19, 55.20, 54.13,
52.42, 49.92, 45.63, 37.04, 36.37, 25.46, 18.13, 16.46; ESI-MS: m/
z ¼ 551 [M þ H] .
þ
5.1.8.25. N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20b). Compound 19b was obtained by the general
13
1.12e1.01 (m, 2H), 0.88e0.72 (m, 2H); C NMR (126 MHz, Chlo-
roform-d)
d
208.77, 174.52, 173.02, 171.77, 159.61, 157.31, 136.26,
procedure
D from 8a and 18b. Yield: 65%; White solid;
ꢁ
1
135.56, 127.43, 123.39, 122.04, 119.50, 118.51, 113.85, 111.40, 110.02,
mp ¼ 181e182 C. H NMR (500 MHz, Chloroform-d)
d 7.25e7.17
12

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
(
(
4
5
m, 3H), 7.12e7.08 (m, 2H), 7.03 (m, 3H), 6.84 (d, J ¼ 8.5 Hz,1H), 6.75
d, J ¼ 8.5 Hz, 2H), 4.78e4.71 (m, 1H), 4.66 (d, J ¼ 7.0 Hz, 1H),
carboxamide (20f). Compound 20f was obtained by the general
procedure
D from 8a and 19f. Yield: 62%; White solid;
ꢁ
1
.58e4.51 (m, 1H), 4.21e4.13 (m, 1H), 3.76 (s, 3H), 3.38e3.20 (m,
H), 3.02 (dd, J ¼ 13.5, 5.0 Hz, 1H), 2.98e2.87 (m, 2H), 2.85 (d,
mp ¼ 120e121 C. H NMR (500 MHz, Chloroform-d)
d 8.06 (d,
J ¼ 8.5 Hz, 2H), 8.02 (t, J ¼ 7.5 Hz, 2H), 7.51e7.45 (m, 2H), 7.35 (t,
J ¼ 7.5 Hz, 2H), 7.12 (t, J ¼ 7.5 Hz, 2H), 7.09e7.04 (m, 3H), 6.96 (d,
J ¼ 7.0 Hz, 2H), 6.77e6.70 (m, 3H), 6.61 (d, J ¼ 7.0 Hz, 1H), 6.32 (d,
J ¼ 7.0 Hz, 1H), 4.72e4.66 (m, 1H), 4.66e4.62 (m, 1H), 4.62e4.57 (m,
1H), 3.72 (s, 3H), 3.21 (d, J ¼ 5.0 Hz, 1H), 3.04e2.96 (m, 2H), 2.91
(dd, J ¼ 14.0, 7.0 Hz, 1H), 2.85 (d, J ¼ 5.0 Hz, 1H), 2.59 (dd, J ¼ 14.0,
J ¼ 5.0 Hz,1H), 2.76 (dd, J ¼ 14.0, 8.5 Hz,1H),1.89 (s, 4H),1.42 (s, 3H),
1
3
1
1
1
3
.23 (d, J ¼ 7.0 Hz, 3H); C NMR (126 MHz, Chloroform-d)
d 207.37,
73.47, 170.90, 158.51, 156.49, 136.32, 130.31, 129.38, 128.57, 128.41,
26.87, 113.98, 59.19, 55.23, 54.13, 52.73, 52.42, 50.29, 45.67, 36.91,
þ
6.47, 25.49, 17.97, 16.46; ESI-MS: m/z ¼ 551 [M þ H] .
13
8.5 Hz, 1H), 1.45 (d, J ¼ 7.0 Hz, 3H), 1.41 (s, 3H); C NMR (126 MHz,
5
.1.8.26. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-((R)-2-
Chloroform-d) d 206.92, 171.96, 170.25, 158.73, 152.33, 138.25,
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)-3,4-dihydroisoquinoline-
135.47, 130.41, 129.19, 128.49, 128.02, 127.13, 127.08, 125.27, 122.54,
120.14, 114.07, 113.91, 59.22, 55.22, 54.25, 52.83, 52.44, 50.30, 37.03,
36.98, 19.11, 16.45; ESI-MS: m/z ¼ 647 [M þ H] .
þ
2(1H)-carboxamide (20c). Compound 20c was obtained by the
general procedure D from 8a and 19c. Yield: 63%; White solid;
ꢁ
1
mp ¼ 178e179 C. H NMR (400 MHz, Chloroform-d)
d
7.26e7.10
5.1.8.30. 3-Methoxy-N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-
((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20g). Compound 20g was obtained by the general
(
m, 7H), 7.08e6.99 (m, 4H), 6.91 (d, J ¼ 7.5 Hz, 1H), 6.69 (d,
J ¼ 8.5 Hz, 2H), 6.59 (d, J ¼ 7.5 Hz, 1H), 4.89 (d, J ¼ 5.0 Hz, 1H),
4
3
5
.82e4.73 (m, 1H), 4.54e4.46 (m, 3H), 4.38e4.27 (m, 1H), 3.64 (s,
H), 3.60e3.50 (m, 2H), 3.28 (d, J ¼ 5.0 Hz, 1H), 3.06 (dd, J ¼ 14.0,
.0 Hz, 1H), 2.94 (d, J ¼ 6.5 Hz, 2H), 2.90e2.81 (m, 3H), 2.72 (dd,
procedure
D from 8a and 19g. Yield: 63%; White solid;
ꢁ
1
mp ¼ 66e67 C. H NMR (500 MHz, Chloroform-d)
d
7.25e7.18 (m,
13
J ¼ 14.0, 8.0 Hz, 1H), 1.46 (s, 3H), 1.30 (d, J ¼ 7.0 Hz, 3H); C NMR
3H), 7.08e7.02 (m, 4H), 7.01e6.92 (m, 1H), 6.79e6.71 (m, 2H),
6.70e6.61 (m, 1H), 4.79e4.72 (m, 1H), 4.57 (s, 1H), 4.52e4.45 (m,
1H), 4.29e4.21 (m, 1H), 3.98e3.92 (m, 1H), 3.75 (s, 3H), 3.47e3.34
(m, 3H), 3.33e3.30 (m, 4H), 3.29e3.27 (m, 1H), 3.08e3.01 (m, 1H),
2.99e2.85 (m, 3H), 2.76e2.67 (m, 1H), 2.10e2.03 (m, 1H), 2.01e1.90
(
101 MHz, Chloroform-d)
35.79, 134.91, 132.99, 130.26, 129.26, 128.51, 128.47, 128.34, 127.00,
26.83, 126.54, 126.34, 113.92, 59.15, 55.09, 54.23, 52.41, 50.11,
d 207.07, 173.18, 170.60, 158.53, 156.97,
1
1
4
5.40, 41.24, 37.11, 36.57, 28.97, 18.24, 16.44; ESI-MS: m/z ¼ 613
þ
þ
[M þ H] .
(m, 1H), 1.45 (s, 3H), 1.26 (m, 3H); ESI-MS: m/z ¼ 581 [M þ H] .
5
.1.8.27. (S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-
yl(S)-3-(4-methoxyphenyl)-2-((R)-2-(1,2,3,4-tetrahydroisoquinoline-
-carboxamido)propanamido)propanoate (20d). Compound 20d
was obtained by the general procedure D from 8a and 19d. Yield:
5.1.8.31. 3-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-
((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20h). Compound 20h was obtained by the general
2
ꢁ
1
6
d)
6
1
0%; White solid; mp ¼ 76e77 C. H NMR (400 MHz, Chloroform-
procedure
D from 8a and 19h. Yield: 70%; White solid;
ꢁ
1
d
7.25e7.13 (m, 6H), 7.12e7.00 (m, 5H), 6.95 (d, J ¼ 7.5 Hz, 1H),
mp ¼ 167e168 C. H NMR (500 MHz, Chloroform-d)
d 7.26e7.18
.86 (d, J ¼ 8.0 Hz, 1H), 6.75 (d, J ¼ 8.5 Hz, 2H), 5.18 (d, J ¼ 7.0 Hz,
(m, 3H), 7.10 (d, J ¼ 7.0 Hz, 2H), 7.04 (d, J ¼ 8.0 Hz, 2H), 6.94 (d,
J ¼ 7.5 Hz, 1H), 6.81e46.73 (m, 3H), 4.80e4.73 (m, 1H), 4.71e4.63
(m, 1H), 4.58e4.50 (m, 1H), 4.23e4.14 (m, 1H), 4.01e3.91 (m, 1H),
3.76 (s, 3H), 3.55e3.35 (m, 4H), 3.34e3.29 (m, 4H), 3.02 (dd,
J ¼ 14.0, 5.0 Hz, 1H), 2.95e2.90 (m, 2H), 2.86 (d, J ¼ 5.0 Hz, 1H), 2.76
(dd, J ¼ 13.5, 8.5 Hz, 1H), 2.11e1.92 (m, 2H), 1.43 (s, 3H), 1.26e1.18
H), 4.81e4.71 (m, 1H), 4.63e4.48 (m, 3H), 4.30e4.18 (m, 1H), 3.74
(
s, 3H), 3.68e3.60 (m, 1H), 3.58e3.52 (m, 1H), 3.28 (d, J ¼ 5.0 Hz,
1
3
4
H), 3.01 (dd, J ¼ 14.0, 5.0 Hz,1H), 2.97e2.89 (m, 2H), 2.87e2.79 (m,
H), 2.73 (dd, J ¼ 14.0, 8.5 Hz, 1H), 1.38 (s, 3H), 1.25 (d, J ¼ 7.0 Hz,
13
H); C NMR (101 MHz, Chloroform-d)
d 207.31, 173.50, 170.77,
þ
1
1
58.56, 157.27, 136.11, 134.96, 133.14, 130.29, 129.33, 128.46, 128.41,
28.35, 126.89, 126.72, 126.43, 126.33, 114.01, 59.12, 55.19, 54.14,
(m, 3H); ESI-MS: m/z ¼ 581 [M þ H] .
5
2.59, 52.36, 50.50, 45.48, 41.28, 37.07, 36.56, 28.95, 18.17, 16.37;
5.1.8.32. 4-Methoxy-N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-
((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)piperidine-1-carboxamide
(20i). Compound 20i was obtained by the general procedure D
þ
ESI-MS: m/z ¼ 613 [M þ H] .
5
.1.8.28. N-((S)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-((R)-2-
ꢁ
1
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)-9H-carbazole-9-
carboxamide (20e). Compound 20e was obtained by the general
from 8a and 19i. Yield: 65%; White solid; mp ¼ 76e78 C. H NMR
(400 MHz, Chloroform-d) 7.24e7.15 (m, 3H), 7.09e6.94 (m, 5H),
d
6.79 (d, J ¼ 7.6 Hz, 1H), 6.73 (d, J ¼ 8.0 Hz, 2H), 5.10 (d, J ¼ 6.4 Hz,
1H), 4.81e4.70 (m, 1H), 4.57e4.44 (m, 1H), 4.43e4.20 (m, 1H), 3.73
(s, 3H), 3.63e3.50 (m, 2H), 3.40e3.29 (m, 4H), 3.25 (d, J ¼ 5.0 Hz,
1H), 3.18e2.96 (m, 3H), 2.94e2.87 (m, 2H), 2.84 (d, J ¼ 5.0 Hz, 1H),
2.70 (dd, J ¼ 14.0, 8.0 Hz, 1H), 1.78 (s, 2H), 1.58e1.45 (m, 2H), 1.41 (s,
procedure
D from 8a and 19e. Yield: 61%; White solid;
ꢁ
1
mp ¼ 187e188 C. H NMR (500 MHz, Chloroform-d)
d
8.04e7.99
(
7
m, 4H), 7.49e7.43 (m, 2H), 7.34 (t, J ¼ 7.5 Hz, 2H), 7.19e7.13 (m, 3H),
.08 (d, J ¼ 8.5 Hz, 2H), 6.94 (dd, J ¼ 7.0, 2.0 Hz, 2H), 6.88 (d,
13
J ¼ 7.5 Hz, 1H), 6.64e6.56 (m, 3H), 6.37 (d, J ¼ 7.5 Hz, 1H), 4.76e4.69
3H), 1.24 (d, J ¼ 7.0 Hz, 3H); C NMR (101 MHz, Chloroform-d)
(
3
1
m, 1H), 4.68e4.62 (m, 2H), 3.52 (s, 3H), 3.18 (d, J ¼ 5.0 Hz, 1H),
d 207.24, 173.40, 170.69, 158.49, 156.93, 135.85, 130.30, 129.25,
.02e2.93 (m, 3H), 2.85 (d, J ¼ 5.0 Hz, 1H), 2.61 (dd, J ¼ 14.0, 8.0 Hz,
128.49, 128.44, 126.96, 113.90, 75.28, 59.11, 55.67, 55.16, 54.26,
13
H), 1.48 (d, J ¼ 7.0 Hz, 3H), 1.44 (s, 3H); C NMR (126 MHz,
206.99, 171.94, 170.28, 158.61, 152.34, 138.20,
35.43, 130.36, 129.21, 128.53, 127.99, 127.12, 125.25, 122.56, 120.14,
13.96,113.89, 59.18, 54.97, 54.43, 52.66, 52.41, 50.08, 37.51, 37.04,
52.33, 50.24, 41.14, 37.12, 36.70, 30.27, 18.47, 16.39; ESI-MS: m/
þ
Chloroform-d)
1
1
d
z ¼ 595 [M þ H] .
5.1.8.33. 4-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-
((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)piperidine-1-carboxamide
(20j). Compound 20j was obtained by the general procedure D
þ
18.86, 16.46, 0.02; ESI-MS: m/z ¼ 647 [M þ H] .
5
.1.8.29. N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)-9H-carbazole-9-
ꢁ
from 8a and 19j. Yield: 73%; White solid; mp ¼ 78e79 C. 1H NMR
(500 MHz, Chloroform-d)
d 7.25e7.18 (m, 3H), 7.12e7.09 (m, 2H),
13

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
7
5
(
3
.02 (d, J ¼ 8.5 Hz, 2H), 6.92 (d, J ¼ 7.5 Hz, 1H), 6.78e6.66 (m, 3H),
.10 (s, 1H), 4.78e4.71 (m, 1H), 4.58e4.48 (m, 1H), 4.14 (s, 1H), 3.76
s, 3H), 3.69e3.62 (m, 1H), 3.61e3.54 (m, 1H), 3.41e3.35 (m, 1H),
.34 (s, 3H), 3.31 (d, J ¼ 5.0 Hz, 1H), 3.20e3.07 (m, 2H), 3.02 (dd,
5.1.8.37. (S)-3-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-
(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)
amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)piperidine-1-
carboxamide (20n). Compound 20n was obtained by the general
J ¼ 13.5, 5.0 Hz, 1H), 2.93e2.89 (m, 2H), 2.86 (d, J ¼ 5.0 Hz, 1H), 2.72
procedure
D from 8a and 19n. Yield: 64%; White solid;
ꢁ
1
(
(
d
dd, J ¼ 13.5, 8.5 Hz, 1H), 1.87e1.80 (m, 2H), 1.59e1.50 (m, 2H), 1.42
mp ¼ 77e78 C. H NMR (500 MHz, Chloroform-d)
d 7.26e7.18 (m,
13
s, 3H), 1.22 (d, J ¼ 7.0 Hz, 3H); C NMR (126 MHz, Chloroform-d)
3H), 7.12 (d, J ¼ 7.0 Hz, 2H), 7.03 (d, J ¼ 8.5 Hz, 2H), 6.91 (d,
J ¼ 6.5 Hz, 1H), 6.76 (d, J ¼ 8.5 Hz, 2H), 5.18 (s, 1H), 4.79e4.71 (m,
1H), 4.56e4.49 (m, 1H), 4.16 (s, 1H), 3.81e3.73 (m, 4H), 3.45e3.38
(m, 1H), 3.35 (s, 3H), 3.32 (d, J ¼ 5.0 Hz, 1H), 3.29e3.23 (m, 1H),
3.22e3.16 (m, 1H), 3.06e2.97 (m, 2H), 2.92 (d, J ¼ 6.5 Hz, 2H), 2.86
207.31, 173.58, 170.81, 158.56, 157.28, 136.22, 130.29, 129.37,
28.46, 128.39, 126.92, 114.03, 75.33, 59.19, 55.70, 55.23, 54.11,
1
5
2.71, 52.42, 50.58, 41.28, 41.21, 36.98, 36.53, 30.27, 30.24, 18.01,
þ
16.46; ESI-MS: m/z ¼ 595 [M þ H] .
(
d, J ¼ 5.0 Hz, 1H), 2.75 (dd, J ¼ 14.0, 8.5 Hz, 1H), 1.95e1.88 (m, 1H),
5
.1.8.34. (R)-3-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-
1.79e1.70 (m, 1H), 1.58e1.49 (m, 1H), 1.49e1.39 (m, 4H), 1.25 (d,
13
(
((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)
J ¼ 7.0 Hz, 3H); C NMR (126 MHz, Chloroform-d)
d 207.32, 173.51,
amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20k). Compound 20k was obtained by the general
procedure
mp ¼ 74e75 C. H NMR (500 MHz, Chloroform-d)
170.80, 158.56, 157.54, 136.21, 130.31, 129.36, 128.47, 128.38, 126.94,
114.04, 74.62, 59.19, 56.24, 55.24, 54.07, 52.71, 52.42, 50.66, 48.01,
44.46, 36.98, 36.50, 29.43, 22.43, 17.90, 16.46; ESI-MS: m/z ¼ 595
D from 8a and 19k. Yield: 69%; White solid;
ꢁ
1
þ
d
7.25e7.18 (m,
[M þ H] .
3
6
1
3
2
H), 7.14e7.09 (m, 2H), 7.03 (d, J ¼ 8.5 Hz, 2H), 6.96 (d, J ¼ 7.0 Hz,1H),
.76 (d, J ¼ 8.5 Hz, 3H), 4.79e4.72 (m,1H), 4.71 (s,1H), 4.56e4.49 (m,
H), 4.20e4.13 (m, 1H), 3.97 (s, 1H), 3.77 (s, 3H), 3.48e3.38 (m, 4H),
.35e3.30 (m, 4H), 3.02 (dd, J ¼ 13.5, 5.0 Hz, 1H), 2.92 (d, J ¼ 6.5 Hz,
H), 2.87 (d, J ¼ 5.0 Hz, 1H), 2.76 (dd, J ¼ 14.0, 8.5 Hz, 1H), 2.09e2.01
5.2. In vitro assays of chymotrypsin-like, trypsin-like, and caspase-
like activities of the proteasome and immunoproteasome
13
(
m, 1H), 1.99e1.93 (m, 1H), 1.42 (s, 3H), 1.24 (d, J ¼ 7.0 Hz, 3H);
C
The human constitutive proteasome was given by Dr. Jiang-ping
Wu (Notre-Dame Hospital, Montreal, QC, Canada), derived from
human hepatic cells; the immunoproteasome was purchased from
Boston Biochem, derived from human peripheral blood mono-
nuclear cell. We used several fluorogenic substrates, the fluorogenic
substrate Suc-LLE-AMC was used to measure the Capase-Like ac-
NMR (126 MHz, Chloroform-d)
d 207.46, 173.38, 170.83, 158.54,
1
5
3
56.51, 136.26, 130.31, 129.40, 128.48, 128.44, 126.90, 114.02, 79.54,
9.19, 56.58, 55.24, 54.17, 52.70, 52.45, 50.68, 50.35, 43.81, 36.93,
6.49, 30.75, 17.99, 16.46; ESI-MS: m/z ¼ 581 [M þ H] .
þ
5
.1.8.35. (S)-3-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-
tivity of the proteasome,
the Trypsin-Like activity of the proteasome,
substrate Suc-PAL-AMC was used to measure the Capase-Like ac-
tivity of the immunoproteasome, 1i. Suc-VGR-AMC was used to
measure the Trypsin-Like activity of the immunoproteasome, 2i.
Suc-WLA-AMC was used to measure the Chymotrypsin-Like activ-
ity of 5c and Suc-ANW-AMC was used to measure the
Chymotrypsin-Like activity of 5i. All compounds were solved in
b
1c. Suc-KQL-AMC was used to measure
(
((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)
b2c. The fluorogenic
amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)pyrrolidine-1-
carboxamide (20l). Compound 20l was obtained by the general
b
procedure
mp ¼ 185e186 C. H NMR (500 MHz, Chloroform-d)
m, 3H), 7.12e7.09 (m, 2H), 7.04 (d, J ¼ 8.5 Hz, 2H), 6.94 (d,
D
from 8a and 19l. Yield: 62%; White solid;
b
ꢁ
1
d
7.26e7.17
(
b
J ¼ 7.0 Hz, 1H), 6.81 (s, 1H), 6.76 (d, J ¼ 8.5 Hz, 2H), 4.78e4.71 (m,
b
1
3
3
2
1
H), 4.68 (d, J ¼ 6.0 Hz, 1H), 4.58e4.50 (m, 1H), 4.22e4.13 (m, 1H),
.94 (s, 1H), 3.76 (s, 3H), 3.55e3.45 (m, 1H), 3.44e3.34 (m, 3H),
.33e3.28 (m, 4H), 3.02 (dd, J ¼ 14.0, 5.0 Hz,1H), 2.96e2.88 (m, 2H),
.86 (d, J ¼ 5.0 Hz, 1H), 2.76 (dd, J ¼ 14.0, 8.5 Hz, 1H), 2.10e2.03 (m,
DMSO‑d
DMSO‑d
6
, and then diluted with deionization water to be 10%
containing solution. Buffer conditions: 100 mM Tris-HCl,
6
pH 7.5. In the assay, ONX-0914 was used as Positive control, and 2%
DMSO‑d was used as negative control. The blank was no enzyme
which was replaced by buffer. Briefly, first add 20 L of the human
proteasome (40 g/mL) or immunoproteasome (1 g/mL) per well.
Subsequently add 10 L of the sample. Cover with a adhesive strip
and incubated 15 min at the at room temperature (25 C). Then
20 L of the flurogenic substrates (100 M final) were added into
6
13
H), 2.01e1.93 (m, 1H), 1.42 (s, 3H), 1.23 (d, J ¼ 7.0 Hz, 3H); C NMR
m
(
126 MHz, Chloroform-d)
d
207.39, 173.35, 170.85, 158.53, 156.50,
m
m
136.23, 130.31, 129.39, 128.52, 128.44, 126.91, 114.00, 79.25, 59.18,
5
m
ꢁ
6.60, 55.24, 54.18, 52.71, 52.44, 50.63, 50.31, 43.61, 36.95, 36.52,
þ
31.03, 17.97, 16.46; ESI-MS: m/z ¼ 581 [M þ H] .
m
m
the each well. Covered with a new adhesive strip and incubated 1 h
ꢁ
5
.1.8.36. (R)-3-Methoxy-N-((R)-1-(((S)-3-(4-methoxyphenyl)-1-
at the room temperature (25 C). Signal from cleaved AMC mole-
(
((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)
cule was detected on EnVision® Multilabel Plate Reader with a
355 nm excitation filter and a 460 nm emission filter. IC50 data was
calculated using GraphPad Prism software, and the equation'-
sigmoidal doseeresponse (variable slope)’ was chosen for curve
fitting.
amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)piperidine-1-
carboxamide (20 m). Compound 20m was obtained by the general
procedure
D from 8a and 19m. Yield: 60%; White solid;
ꢁ
1
mp ¼ 78e79 C. H NMR (500 MHz, Chloroform-d)
d 7.25e7.17 (m,
3
2
1
H), 7.13 (d, J ¼ 7.0 Hz, 2H), 7.04e6.96 (m, 3H), 6.75 (d, J ¼ 8.5 Hz,
H), 6.64 (d, J ¼ 7.0 Hz, 1H), 5.06 (d, J ¼ 5.5 Hz, 1H), 4.79e4.70 (m,
H), 4.56e4.47 (m, 1H), 4.17e4.05 (m, 1H), 3.76 (s, 3H), 3.65 (d,
5.3. Cancer cell proliferation assay
J ¼ 12.5 Hz, 1H), 3.46e3.39 (m, 1H), 3.36e3.29 (m, 4H), 3.29e3.23
m, 1H), 3.19e3.09 (m, 2H), 3.02 (dd, J ¼ 14.0, 5.0 Hz, 1H), 2.93 (d,
J ¼ 6.5 Hz, 2H), 2.85 (d, J ¼ 5.0 Hz, 1H), 2.74 (dd, J ¼ 14.0, 8.5 Hz, 1H),
.94e1.87 (m, 1H), 1.79e1.70 (m, 1H), 1.58e1.51 (m, 1H), 1.50e1.43
(
5.3.1. Cell culture
RPMI-8226 and MM.1S cell lines were obtained from the
American Type Culture Collection (ATCC) (Manassas, VA, USA) and
maintained at 37 C in a humidified atmosphere containing 5% CO
The human MM cell lines PRMI 8226 and MM.1S were cultured in
RPMI-1640 media supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100
Invitrogen (Grand Island, NY, USA).
1
13
ꢁ
(
m, 1H), 1.41 (s, 3H), 1.22 (d, J ¼ 7.0 Hz, 3H); C NMR (126 MHz,
Chloroform-d) 207.36, 173.61, 170.84, 158.49, 157.52, 136.27,
30.27, 129.36, 128.51, 128.43, 126.90, 113.98, 74.55, 59.18, 56.21,
2
.
d
1
5
2
5.22, 54.14, 52.67, 52.39, 50.74, 47.70, 44.55, 36.95, 36.34, 29.25,
2.21, 17.95, 16.43; ESI-MS: m/z ¼ 595 [M þ H] .
mg/mL streptomycin from
þ
14

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
5
.3.2. Cell proliferation assays
iCP
immunoproteasome
major histocompatibility complex class I
multiple myeloma
3
A 90
m
L aliquot of RPMI-8226 (5 ꢂ 10 cells per well) or MM.1S
MHC-I
MM
RPs
4
(
3 ꢂ 10 cells per well) was seeded into 96-well plates and then
treated with 10 L of 0.2% DMSO‑d or varying concentrations of
m
6
regulatory particles
tested compounds for 72 h. Cell viability was measured using the
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay
SAR
tCP
structureꢀactivityrelationship
thymoproteasome
(
MTS; Promega, Madison, WI). 20
m
L of the combined MTS/PMS
TFA
THF
trifluoroacetic acid
tetrahydrofuran.
solution was pipetted into each of the 96-wells plate and then
incubated for 2e4 h at 37 C in a humidified, 5% CO
ꢁ
2
atmosphere.
The optical density of each well was determined at 490 nm
background subtraction at 690 nm) using a SpectraMax 340
Appendix A. Supplementary data
(
microplate reader (Molecular Devices, Sunnyvale, CA, USA). Growth
inhibitory ratios were calculated as follows: Growth inhibitory
ratio ¼ (Acontrol - Asample)/Acontrol. IC50 values were derived from a
nonlinear regression model (curvefit) based on a sigmoidal dose
responsecurve (variable slope) and computed using GraphPad
Prism version 5.02, GraphPad Software.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113556.
References
[1] E.E. Manasanch, R.Z. Orlowski, Proteasome inhibitors in cancer therapy, Na-
ture reviews, Clin. Oncol. 14 (2017) 417e433.
[
[
2] J. Zhao, B. Zhai, S.P. Gygi, A.L. Goldberg, mTOR inhibition activates overall
protein degradation by the ubiquitin proteasome system as well as by auto-
phagy, Proc. Natl. Acad. Sci. U.S.A. 112 (2015) 15790e15797.
3] D. Voges, P. Zwickl, W. Baumeister, The 26S proteasome: a molecular machine
designed for controlled proteolysis, Annu. Rev. Biochem. 68 (1999)
5
.4. Molecular docking
The molecular docking was performed using Discovery Studio
(
version 2.1) with the default option. The X-ray crystal structure of
1015e1068.
immunoproteasome (PDB code: 5L5D) was used as the docking
template. It was prepared by adding missing hydrogen bond,
removing crystallographic waters and ligands with CHARMM force
field. Three-dimensional (3D) conformation of compound 11a, 11b,
E-83, 20l was prepared by Discovery Studio 2.1/Diverse confor-
mation generation, then whose energy minimized by DS 2.1/
Minimize molecule. For the docking calculation of compound 11a,
[4] M. Groll, L. Ditzel, J. L o€ we, D. Stock, M. Bochtler, H.D. Bartunik, R. Huber,
Structure of 20S proteasome from yeast at 2.4 A resolution, Nature 386 (1997)
463e471.
[
5] J. L o€ we, D. Stock, B. Jap, P. Zwickl, W. Baumeister, R. Huber, Crystal structure of
the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution,
Science (New York, N.Y.) 268 (1995) 533e539.
[
[
6] P.M. Cromm, C.M. Crews, The proteasome in modern drug discovery: second
life of a highly valuable drug target, ACS Cent. Sci. 3 (2017) 830e838.
7] E.M. Huber, M. Basler, R. Schwab, W. Heinemeyer, C.J. Kirk, M. Groettrup,
M. Groll, Immuno- and constitutive proteasome crystal structures reveal dif-
ferences in substrate and inhibitor specificity, Cell 148 (2012) 727e738.
8] D.A. Ferrington, D.S. Gregerson, Immunoproteasomes: structure, function, and
antigen presentation, Progress in molecular biology and translational science
11b, E-83, 20l Discovery Studio 2.1/C-DOCKER was used to generate
the conformations. The graphical image was analyzed by PyMOL
v0.99.
[
[
109 (2012) 75e112.
9] M. Basler, C.J. Kirk, M. Groettrup, The immunoproteasome in antigen pro-
cessing and other immunological functions, Curr. Opin. Immunol. 25 (2013)
Declaration of competing interest
7
4e80.
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.
[10] T. Nitta, S. Murata, K. Sasaki, H. Fujii, A.M. Ripen, N. Ishimaru, S. Koyasu,
K. Tanaka, Y. Takahama, Thymoproteasome shapes immunocompetent
repertoire of CD8þ T cells, Immunity 32 (2010) 29e40.
[
11] S. Murata, K. Sasaki, T. Kishimoto, S. Niwa, H. Hayashi, Y. Takahama, K. Tanaka,
Regulation of CD8þ T cell development by thymus-specific proteasomes,
Science (New York, N.Y.) 316 (2007) 1349e1353.
Acknowledgments
[
12] R.C. Kane, A.T. Farrell, R. Sridhara, R. Pazdur, United States Food and Drug
Administration approval summary: bortezomib for the treatment of pro-
gressive multiple myeloma after one prior therapy, Clin. Canc. Res. : Off. J. Am.
Assoc. Canc. Res. 12 (2006) 2955e2960.
We thank Jianyang Pan (Research and Service Center,College of
Pharmaceutical Sciences, Zhejiang University) for performing NMR
spectrometry for structure elucidation. We also thank Huazhou
Ying (College of Pharmaceutical Sciences, Zhejiang University) for
performing mass spectrometry for structure elucidation. This work
was supported by grants from the key project of Zhejiang Provincial
Natural Science Foundation of China (LZ15H300001), the National
Science and Technology Major Project of China (2018ZX09711002-
[
13] M. Alsina, S. Trudel, R.R. Furman, P.J. Rosen, O.A. O'Connor, R.L. Comenzo,
A. Wong, L.A. Kunkel, C.J. Molineaux, A. Goy, A phase I single-agent study of
twice-weekly consecutive-day dosing of the proteasome inhibitor carfilzomib
in patients with relapsed or refractory multiple myeloma or lymphoma,
Clinical cancer research, Off. J. Am. Assoc. Canc. Res. 18 (2012) 4830e4840.
14] P. Moreau, T. Masszi, N. Grzasko, N. Bahlis, M. Hansson, L. Pour, I. Sandhu,
P. Ganly, B. Baker, S. Jackson, A.-M. Stoppa, D. Simpson, P. Gimsing,
A. Palumbo, L. Garderet, M. Cavo, S. Kumar, C. Touzeau, F. Buadi, P. Richardson,
Ixazomib, an investigational oral proteasome inhibitor (PI), in: Combination
with Lenalidomide and Dexamethasone (IRd), Significantly Extends
Progression-free Survival (PFS) for Patients (Pts) with Relapsed And/or Re-
fractory Multiple Myeloma (RRMM), The Phase 3 Tourmaline-MM1 Study,
[
0
07-001), and the Science and Technology Commission of Shanghai
Municipality (17431903000).
2015.
Abbreviations used
[
15] R. Ettari, M. Zappal aꢀ , S. Grasso, C. Musolino, V. Innao, A. Allegra, Immuno-
proteasome-selective and non-selective inhibitors: a promising approach for
the treatment of multiple myeloma, Pharmacol. Ther. 182 (2018) 176e192.
cCP
CP
DCM
DIPEA
DMSO-d
EDCI
constitutive proteasome
core particle
dichloromethane
N,N-diisopropylethylamine
dimethyl sulfoxide
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
fetal bovine serum
Food and Drug Administration
1-hydroxybenzotriazole
half maximal inhibitory concentration
[16] D.J. Kuhn, R.Z. Orlowski, The immunoproteasome as a target in hematologic
malignancies, Semin. Hematol. 49 (2012) 258e262.
17] S. Downey-Kopyscinski, E.W. Daily, M. Gautier, A. Bhatt, B.I. Florea,
C.S. Mitsiades, P.G. Richardson, C. Driessen, H.S. Overkleeft, A.F. Kisselev, An
[
inhibitor of proteasome b2 sites sensitizes myeloma cells to immunoprotea-
some inhibitors, Blood advances 2 (2018) 2443e2451.
6
[
18] D. Dytfeld, M. Luczak, T. Wrobel, L. Usnarska-Zubkiewicz, K. Brzezniakiewicz,
K. Jamroziak, K. Giannopoulos, A. Przybylowicz-Chalecka, B. Ratajczak,
J. Czerwinska-Rybak, Comparative proteomic profiling of refractory/relapsed
multiple myeloma reveals biomarkers involved in resistance to bortezomib-
based therapy, Oncotarget 7 (2016) 56726e56736.
FBS
FDA
HOBt
IC50
[19] T. Muchamuel, M. Basler, M.A. Aujay, E. Suzuki, K.W. Kalim, C. Lauer,
C. Sylvain, E.R. Ring, J. Shields, J. Jiang, P. Shwonek, F. Parlati, S.D. Demo,
15

X. Li, D. Hong, M. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113556
M.K. Bennett, C.J. Kirk, M. Groettrup, A selective inhibitor of the immuno-
proteasome subunit LMP7 blocks cytokine production and attenuates pro-
gression of experimental arthritis, Nat. Med. 15 (2009) 781e787.
from
2955e2965.
b
-amino acid as proteasome inhibitors, Bioorg. Med. Chem. 22 (2014)
[24] E.M. Huber, W. Heinemeyer, G. de Bruin, H.S. Overkleeft, M. Groll,
A humanized yeast proteasome identifies unique binding modes of inhibitors
[
[
[
[
20] A.V. Singh, M. Bandi, M.A. Aujay, C.J. Kirk, D.E. Hark, N. Raje, D. Chauhan,
K.C. Anderson, PR-924, a selective inhibitor of the immunoproteasome sub-
unit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo, Br.
J. Haematol. 152 (2011) 155e163.
21] X.W. Dong, J.K. Zhang, L. Xu, J.X. Che, G. Cheng, X.B. Hu, L. Sheng, A.H. Gao, J. Li,
T. Liu, Y.Z. Hu, Y.B. Zhou, Covalent docking modelling-based discovery of
tripeptidyl epoxyketone proteasome inhibitors composed of aliphatic-het-
erocycles, Eur. J. Med. Chem. 164 (2019) 602e614.
for the immunosubunit b5i, EMBO J. 35 (2016) 2602e2613.
[25] H.W.B. Johnson, E. Lowe, J.L. Anderl, A. Fan, T. Muchamuel, S. Bowers,
D.C. Moebius, C. Kirk, D.L. McMinn, Required immunoproteasome subunit
inhibition profile for anti-inflammatory efficacy and clinical candidate KZR-
616 ((2 S,3 R)- N-((S)-3-(Cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-
1-oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-
morpholinoacetamido)propanamido)propenamide), J. Med. Chem. 61 (2018)
11127e11143.
[26] D. Niewerth, J. van Meerloo, G. Jansen, Y.G. Assaraf, T.C. Hendrickx, C.J. Kirk,
J.L. Anderl, S. Zweegman, G.J. Kaspers, J. Cloos, Anti-leukemic activity and
mechanisms underlying resistance to the novel immunoproteasome inhibitor
PR-924, Biochem. Pharmacol. 89 (2014) 43e51.
22] G. de Bruin, E.M. Huber, B.T. Xin, E.J. van Rooden, K. Al-Ayed, K.B. Kim,
A.F. Kisselev, C. Driessen, M. van der Stelt, G.A. van der Marel, M. Groll,
H.S. Overkleeft, Structure-based design of
b1i or b5i specific inhibitors of
human immunoproteasomes, J. Med. Chem. 57 (2014) 6197e6209.
23] J. Zhang, J. Cao, L. Xu, Y. Zhou, T. Liu, J. Li, Y. Hu, Design, synthesis and bio-
logical evaluation of novel tripeptidyl epoxyketone derivatives constructed
16