Discovery of novel non-covalent inhibitors selective to the β5-subunit of the human 20S proteasome

Article abstract of DOI:10.1016/j.ejmech.2015.05.023

A series of linear peptides (6a-6o) were designed based on the known non-covalent 20S proteasome inhibitors TMC-95A and compound 5 via a fragment-based approach. These compounds were synthesized and evaluated against the chymotrypsin-like activity of the

Full text of DOI:10.1016/j.ejmech.2015.05.023

European Journal of Medicinal Chemistry 98 (2015) 61e68
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of novel non-covalent inhibitors selective to the
of the human 20S proteasome
b5-subunit
Kai Xu a, Ke Wang a, Ying Yang a, Ding-An Yan a, Li Huang b, Chin-Ho Chen b,
,
*
Zhiyan Xiao a
a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of linear peptides (6a-6o) were designed based on the known non-covalent 20S proteasome
inhibitors TMC-95A and compound 5 via a fragment-based approach. These compounds were synthe-
sized and evaluated against the chymotrypsin-like activity of the human 20S proteasome. Three of them
(6d, 6e and 6k) were potent inhibitors with IC50 values at the submicromolar level. These three com-
Received 14 August 2014
Received in revised form
14 May 2015
Accepted 14 May 2015
Available online 16 May 2015
pounds were selective to the
caspase-like activities of the human 20S proteasome. Docking study of the most potent compound 6e
revealed its key interactions with the 5-subunit of the 20S proteasome. These findings have provided a
b5-subunit and showed no obvious inhibition against trypsin-like and
b
Keywords:
Proteasome inhibitors
Non-covalent
Linear peptides
Selectivity
new chemical template for non-covalent proteasome inhibitors, which is ready for further structural
optimization to improve both potency and subunit selectivity.
© 2015 Elsevier Masson SAS. All rights reserved.
Fragment-based approach
1. Introduction
mainstream in this field [5], although peptidomimetic [6],
pseudopeptide-based [7] and non-peptidic [8e10] inhibitors have
The ubiquitin-proteasome system (UPS) is a proteolytic pathway
precisely regulating the cellular protein homeostasis via enzymatic
cascades [1]. The 20S proteasome is the main proteolytic compo-
been recently reported.
TMC-95A (Fig. 1a) is a natural cyclic tripeptide isolated from
fermentation broth of the ascomycete fungus Apiospora montagnei
[11]. It is a very potent inhibitor of the 20S proteasome and inhibits
the chymotrypsin-like (ChT-L) activity of the 20S proteasome with
an IC50 value of 5.4 nM [11,12]. In contrast to bortezomib and car-
filzomib, TMC-95A is a non-covalent inhibitor of the 20S protea-
some [13], which may have a better pharmacological profile with
reduced side effects. In spite of its high potency and unique inter-
action mode, its structural complexity impedes the development of
efficient total synthesis. Instead, TMC-95A has become a prototype
molecule for the discovery of novel non-covalent proteasome
inhibitors.
The crystal structure of 20S proteasome: TMC-95A complex
reveals the binding mode of TMC-95A in the active sites of the 20S
proteasome [13]. Besides hydrogen bonds formed with the peptide
backbone, TMC-95A interacts with the 20S proteasome mainly by
the (Z)-prop-1-enyl group and the asparagine residue, which pro-
trude into the S1 and S3 pockets of the active sites, respectively
(Fig. 1b). In contrast, only weak interaction between the tyrosine
residue and the shallow hydrophobic S4 pockets is observed, and
nent of the UPS. In eukaryotes, the b5, b2 and b1 subunits of the 20S
proteasome are responsible for chymotrypsin-like, trypsin-like, and
caspase-like proteolytic activities, respectively [2]. The 20S pro-
teasome has been regarded as a potential therapeutic target for the
treatment of various pathological conditions. The successful
development of 20S proteasome inhibitors, bortezomib [3] and
carfilzomib [4], into anti-cancer drugs has made the 20S protea-
some a validated target for cancer chemotherapy. However, both
bortezomib and carfilzomib are covalent proteasome inhibitors and
are associated with the problems of severe side effects, acquired
drug resistance, and unsatisfactory pharmacokinetic profiles.
Therefore, it is imperative to search for novel non-covalent pro-
teasome inhibitors as potential chemotherapeutic agents. Up to
now, peptide-based proteasome inhibitors remain to be the
* Corresponding author.
E-mail address: xiaoz@imm.ac.cn (Z. Xiao).
http://dx.doi.org/10.1016/j.ejmech.2015.05.023
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

62
K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
Fig. 1. The structure of TMC-95A (a) and its interaction mode with the b2 subunit of 20S proteasome (b).
the 3-methyl-2-oxopentanoyl group seems to contribute little to
the binding affinity [13]. Such interaction modes have manifested
the importance of the moieties interacting with S1 and S3 pockets
and insights from the crystallographic analysis have eventually led
to the discovery of several synthetically less challenging analogs of
TMC-95A (Fig. 2). Danishefsky et al. synthesized a series of
simplified TMC-95A analogs by replacing the cis-propylene residue
on the C8 side chain of TMC-95A with more readily available
groups, and eliminating the chiral center on the C14 side chain.
Most of the analogs (e.g. compound 1) retained inhibitory potency
against the 20S proteasome [14]. Further simplification of the
functionalities on the TMC-95A core resulted in the identification of
compound 2 and its analogs. Crystallographic study on complex
structures between the 20S proteasome and these analogs revealed
a binding mode similar to that of TMC-95A [15]. To simplify the core
skeleton of TMC-95A, endocyclic biphenyl-ether derivatives were
synthesized, which had the complex biaryl moiety in TMC-95A
substituted with a biphenyl ether. Such structural alteration
resulted in the preservation of key interactions with the active site
so as to retain the proteasome inhibition. Compound 3 is a repre-
sentative structure of this compound series [16,17]. Various linear
analogs of TMC-95A were also synthesized to further simplify the
core skeleton of TMC-95A [18,19]. As illustrated by the structure of
compound 4, the tripeptide backbone of TMC-95A was maintained,
however, the cyclic core scaffold was dissected at the covalent bond
between the two aryl groups. Compound 4 retained the key in-
teractions with the active sites of the 20S proteasome, though due
to the loss of rigidity, it assumed a more extending conformation in
the active site [19]. Notably, the linear construct seemed to provide
a versatile skeleton for structural optimization to modulate inhib-
itory potency toward different
b subunits [18].
Other linear peptides have also been previously reported as
potent proteasome inhibitors and interestingly, most of them
showed apparent selectivity to the b5-subunit of the 20S protea-
some, which is believed to be a potential advantage over simulta-
neous inhibition of all the three subunits [20]. 5-Methoxy-1-
Fig. 2. Representative simplified TMC-95A analogs.

K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
63
indanone capped di- and tripeptides were the first structural class
reported as selective inhibitors for the ChT-L activity of the 20S
proteasome [21,22]. In addition, a novel series of capped di- and
tripeptides originated from high-throughput screening were also
identified as non-covalent proteasome inhibitors with selectivity
1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU) and
N,N-diisopropylethylamine (DIEA) to give a dipeptide scaffold. The
dipeptide was then treated with trifluoroacetic acid to free the
amino group, which was further coupled with 3-
phenoxyphenylacetic acid to give compound 6g. 6g was subjected
to hydrogenation in the presence of 10% Pd/C to yield compound 6c.
6c was subsequently coupled with the corresponding amines to
afford 6a, 6b, 6d, 6e and 6h-6n. 6f and 6o were obtained by hy-
drogenation of 6d and 6n, respectively.
toward the b5-subunit [23,24]. A series of 2-aminobenzylstatine
derivatives, which were originally designed to target the HIV-1
proteinase, were found to be non-covalent inhibitors specifically
inhibiting the ChT-L activity of the 20S proteasome [25]. Molecular
docking [26] and subsequent structure-based optimization [27,28]
led to the discovery of compound 5, which is a dipeptide-based
4. Results and discussion
proteasome inhibitor selective to the
cellular assays [28]. Molecular modeling study [28] reveals the
interaction mode between the 5 subunit of the 20S proteasome
and compound 5 (Fig. 3), which underscores the hydrophobic in-
teractions between the AS1 and AS2 pockets of the active site and
the 3-phenoxyphenylacetyl moiety in compound 5 (Fig. 3). The
structure of compound 5 provides a new chemical template for
further design of non-covalent proteasome inhibitors selective to
b5-subunit and active in
All synthesized compounds were assayed for their inhibitory
potency against the ChT-L activity of the human 20S proteasome as
described previously [29], and lactacystin was used as a reference
compound. As shown in Table 1, most compounds showed signifi-
cant inhibitory potency against the ChT-L activity of the human 20S
proteasome. Among them, 6d, 6e, 6k and 6m exhibited IC50 values
at lower micromolar or even submicromolar levels. These results
suggested that our molecular design strategy is effective in iden-
tifying novel linear peptides as potent proteasome inhibitors.
Preliminary SAR could be deduced from the data listed in
Table 1. When the terminal methyl in R2 of compound 6a was
replaced with a hydroxyl, the inhibition potency was slightly
decreased (6a vs 6b). While truncation of the side chain at R2 could
result in dramatic loss of potency (6b vs 6c). Incorporation of
additional substituents on the n-propyl group of 6a could lead to
significant improvement in potency (6a vs 6d/6e), however,
appropriate steric or electronic properties of the substituents might
be required (6e vs 6f). Variation at R2 could cause substantial dif-
ference in inhibitory potency, presumably due to the different af-
finity to the S1 pocket (6g-6l). Among the “S1 fragments”
investigated, the (2-hydroxy-4-methoxybenzyl)amino group (cf.
compound 6k) was identified as the optimal S1 moiety for further
SAR exploration on R2. Extension of the R1 side chain in compound
6k by one carbon (compound 6m) could slightly decrease the po-
tency. Replacing the R1 side chain with a basic residue (compound
6o) could lead to significant loss of inhibitory potency. However,
extending the terminal amino with benzyloxycarbonyl (compound
6n) could partially restore the potency.
b
the b5-subunit of the 20S proteasome.
2. Molecular design
Based on the chemical templates of both TMC-95A and com-
pound 5, we report herein the discovery of a novel series of non-
covalent proteasome inhibitors via a fragment-based drug design
approach. Compound 6a (Fig. 4 and Table 1) was first designed by
incorporating the S1/S3 fragments in TMC-95A analogs and the
AS1/AS2 fragments in compound 5. As expected, compound 6a
exhibited significant inhibition against the ChT-L activity of the 20S
proteasome with an IC50 of 12.97 mM. This result encouraged us to
further modify the P1 and P3 areas of compound 6a to improve its
potency. A series of compounds with the general formula of 6 were
thereby designed and synthesized. Compound 6b and 6c intro-
duced a hydroxyl group in R2 to examine the possibility of forming
additional hydrogen bonds with the active sites. Compounds 6d-6f
further furbished the n-propyl group in 6a with various sub-
stituents. Compounds 6g-6l were designed following the fragment-
based strategy by incorporating at R2 various “S1 fragments” from
previously reported non-covalent proteasome inhibitors to explore
SAR in this molecular area [24,28]. Compounds 6m-6o were
intended to check the effects of the S3 moiety on proteasome
inhibitory activity by altering the asparagine residue in R1.
Three compounds 6d, 6e and 6k with the most potent inhibitory
activity against ChT-L activity were further evaluated against
and 2-subunits. The compounds were tested in a series of four-
fold dilutions (10, 2.5, 0.625, 0.156 and 0.039 g/mL) and none of
b1-
b
m
them showed percentage inhibition above 50% against trypsin-like
and caspase-like activities even at the highest tested concentration.
The results suggested that this novel series of proteasome in-
hibitors could be selective to the b5-subunit of the 20S proteasome,
which is consistent with the previous observations on linear pep-
tide proteasome inhibitors.
3. Chemistry
The synthesis of the desired compounds is shown in Scheme 1.
The corresponding Boc protected amino acids and H-L-Val-OBn
were coupled in the presence of 2-(1H-7-azabenzotriazol-1-yl)-
A docking study on the most potent compound 6e indicated that
the compound could occupy the active site between
b5 and b6
subunits of the 20S proteasome. The 2D- and 3D-plots of key in-
teractions between compound 6e and the 20S proteasome were
illustrated in Fig. 5. As expected, the tripeptide backbone formed
several hydrogen bonds with the active site residues (e.g. Thr 21,
Gly 47 and Ala 49 in the b5 subunit and Asp 114 in the b6 subunit).
The R1 and R2 side chains of compound 6e extended to the S3 and
S1 pockets of the active site, respectively. Hydrogen bonds were
also observed between the hydroxyl group in R2 and Thr1 of the
b5
subunit, as well as the carbonyl oxygen atom in R2 and Gly 47. This
could at least partly explain a more than 40-fold increase in potency
of 6e as compared with compound 6a. Furthermore,
and -cation interactions were also detected between residues Tyr
96, His 98 and the phenoxyphenyl moiety, which is in consistence
p-p stacking
p
Fig. 3. Schematic representation of the interaction mode between compound 5 and
the b5-subunit of the 20S proteasome.

64
K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
Fig. 4. Molecular design of the target compounds.
Scheme 1. Synthesis of compound 6a-6o.* Reagents and conditions: (a) HATU, DIEA, DMF, r.t., overnight; (b) CF3COOH, DCM, r.t., 4 h; (c) 3-phenoxyphenylacetic acid, HATU, DIEA,
DMF, r.t., overnight; (d) 10% Pd/C, MeOH, H2, overnight; (e) appropriate amines, HATU, DIEA, DMF, r.t., overnight. *R1, R2 are defined in Table 1.
with the molecular design to implant this “fragment” so as to
induce additional interactions with the accessory subpockets of the
20S proteasome.
6. Experimental section
6.1. Material and measurements
All reactions were monitored by thin-layer chromatography
with pre-coated silica gel F254 plates purchased from Merck, Inc.
All melting points were determined on Yanaco Melting point
apparatus and were uncorrected. Mass spectra (MS) were taken in
ESI mode on Agilent Technologies LC/MSD TOF instruments. 1H
NMR spectra were recorded on Varian Mercury 300-MHz or 400-
MHz spectrometer. 13C NMR spectra were recorded on Bruker
AVeIIIe500 spectrometer. Chemicals were obtained from local
suppliers and were used without further purification.
5. Conclusion
A series of linear peptides were designed based on the chemical
templates of TMC-95A and compound 5 via a fragment-based
design strategy. The N-terminal of the peptides was extended
with the 3-phenoxyphenylacetyl moiety to form additional in-
teractions with accessory subpockets in the active sites. For most
compounds, the asparagine residue in TMC-95A was preserved as
the “S3 fragment”, while the (Z)-prop-1-enyl group was modified
or replaced with various more readily available “S1 fragments”
from known proteasome inhibitors. New linear peptides with IC50
values at submicromolar concentrations against the ChT-L activity
of the human 20S proteasome have been identified. Notably, these
compounds exhibited significant selectivity toward the
of the human 20S proteasome. Docking study on the most active
compound 6e revealed key interactions between 6e and the 5-
subunit of the 20S proteasome. These novel linear peptide pro-
teasome inhibitors provides a versatile chemical template ready for
further structural optimization to modulate potency and selectivity.
6.2. Preparation and characterization of compounds 6a-6o
A 125 ml round bottom flask was charged with a magnetic stir
bar, Boc-Asn-OH (3.032 g, 13 mmol), H-L-Val-OBzl$TosOH (4.955 g,
13 mmol), HATU (4.965 g, 13 mmol), DIEA (4.200 g, 32.5 mmol) and
DMF (40 ml) were then added. The mixture was stirred under ice
bath for 1 h and then stirred overnight at room temperature. After
removal of the solvent under reduced pressure, the residue was
dissolved in ethyl acetate and sequentially washed by 1 M
b5-subunit
b

K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
65
Table 1
washed by 1 M hydrochloride acid, saturated sodium bicarbonate
and brine. The organic layer was dried over anhydrous sodium
sulfate. After concentrated, 1.366 g 6g (yield: 36.0% for steps a-c in
Scheme 1) was afforded by column chromatography
(DCM:MeOH ¼ 80:1). 6g (1.366 g) was suspended in methanol
(100 ml) with 10% Pd/C (130 mg), and stirred overnight at room
temperature under hydrogen atmosphere. After filtration, the
filtrate was concentrated to give 1.040 g 6c (yield: 91.7%). 6c
(0.220 g, 0.5 mmol), n-propylamine (0.030 g, 0.5 mmol), HATU
(0.190 g, 0.5 mmol) and DIEA (0.162 g, 1.25 mmol) were dissolved in
DMF (15 ml), the mixture was then stirred overnight at room
temperature. The reaction mixture was concentrated and the
resulting residue was purified by column chromatography
(DCM:MeOH ¼ 20:1) to give the desired compound 6a (54 mg,
yield: 22.5%).
Inhibition against the ChT-L activity of the human 20S proteasome by compounds
6a-6o.
Compd
R1
R2
IC50 (m
M)a
6a
6b
6c
6d
CH2C(O)NH2
CH2C(O)NH2
CH2C(O)NH2
CH2C(O)NH2
NH(CH2)2CH3
NH(CH2)2OH
OH
12.97b
17.19b
NAd
0.92c
6e
6f
CH2C(O)NH2
CH2C(O)NH2
0.29c
Compounds 6b, 6d, 6e and 6h-6n were synthesized with the
experimental protocols described for compound 6a.
13.88b
Compound 6d (0.632 g, 1 mmol) was dissolved in methanol and
10% Pd/C (0.060 g) was added to the solution. The reaction mixture
was stirred at room temperature overnight. After filtration, the
filtrate was concentrated to give 0.541 g 6f (yield: 99.0%).
Compound 6o was synthesized from 6n with the protocols
described for 6f.
6g
6h
6i
6j
6k
CH2C(O)NH2
CH2C(O)NH2
CH2C(O)NH2
CH2C(O)NH2
CH2C(O)NH2
OBz
17.31b
NA
NA
NH(CH2)2Ph
(R)-NHCH(CH3)Ph
(S)eNHCH(CH3)Ph
NA
0.66c
6.2.1. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-NH(CH2)2CH3
(6a)
Yield: 22.5%; mp: 236.0e238.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
d
7.29e7.17 (m, 3H), 7.05e6.96 (m, 2H), 6.92e6.89 (m, 3H),
6l
CH2C(O)NH2
(CH2)2C(O)NH2
(CH2)4NHCbz
(CH2)4 NH2
NA
6.79e6.75 (dd, 1H, J ¼ 1.5, 8.4 Hz), 4.71e4.66 (t, 1H, J ¼ 6.6 Hz),
4.06e4.04 (d, 1H, J ¼ 6.0 Hz), 3.46 (s, 2H), 3.08e3.02 (m, 2H),
2.74e2.66 (m, 1H), 2.57e2.50 (m, 1H), 2.08e1.99 (m, 1H), 1.50e1.38
(m, 2H), 0.85e0.76 (m, 9H); 13C NMR (DMSO-d6, 125 Hz):
d 172.1,
6m
6n
6o
1.63b
11.73b
NA
171.3, 170.8, 170.3, 157.1, 156.9, 138.8, 130.5, 130.1, 124.7, 123.8, 119.9,
119.0, 117.0, 58.1, 50.1, 42.2, 40.7, 37.3, 30.7, 22.7, 19.6, 17.9, 11.9; MS-
ESI m/z: 483 [MþH]þ.
6.2.2. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-NH(CH2)2OH (6b)
Yield: 22.8%; mp: 162.0e166.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
d
7.27 (t, 2H, J ¼ 5.7 Hz), 7.21 (t, 1H, J ¼ 6.0 Hz), 7.05e6.97 (m, 2H),
6.92e6.90 (m, 3H), 6.78 (d, 1H, J ¼ 6.0 Hz), 4.70 (t, 1H, J ¼ 5.1 Hz),
4.10 (d,1H, J ¼ 4.2 Hz), 3.52 (t, 2H, J ¼ 4.5 Hz), 3.47 (s, 2H), 3.28e3.19
(m, 2H), 2.76e2.70 (m, 1H), 2.58e2.52 (m, 1H), 2.13e2.05 (m, 1H),
0.79 (t, 6H, J ¼ 4.5 Hz); 13C NMR (DMSO-d6, 150 Hz):
d 172.2, 171.2,
171.1, 170.3, 157.1, 156.9, 138.7, 130.5, 130.1, 124.7, 123.8, 119.9, 119.0,
117.0, 60.2, 58.0, 50.1, 42.2, 41.9, 37.2, 30.7, 19.6, 17.9; MS-ESI m/z:
485 [MþH]þ.
Lactacystin
10.09c
a
The IC50 values are for inhibition against the ChT-L activity of the human 20S
6.2.3. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-OH (6c)
proteasome.
Yield: 91.7%; mp: 155.0e158.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
b
c
The IC50 values are the averages from two independent experiments.
The IC50 values are the averages from three independent experiments.
NA, not active. No significant inhibition was observed at a series of four-fold
d
7.28e7.17 (m, 3H), 7.04e6.96 (m, 2H), 6.92e6.89 (m, 3H), 6.77 (d,
d
1H, J ¼ 8.1 Hz), 4.71 (t,1H, J ¼ 6.6 Hz), 4.21 (d,1H, J ¼ 4.5 Hz), 3.46 (s,
dilutions (10, 2.5, 0.625, 0.156 and 0.039 mg/mL).
2H), 2.69e2.62 (m, 1H), 2.57e2.49 (m, 1H), 2.10e1.99 (m, 1H), 0.79
(t, 6H, J ¼ 6.6 Hz); 13C NMR (DMSO-d6, 125 Hz):
d 173.2, 171.9, 171.5,
170.5, 157.1, 156.9, 138.7, 130.5, 130.2, 124.7, 123.8, 119.9, 119.0, 117.0,
hydrochloride acid, saturated sodium bicarbonate and brine. The
organic layer was dried over anhydrous sodium sulfate. 5.2 g white
solid was obtained after removal of the solvent. 3.006 g white solid
was dissolved in TFA/DCM (18 ml/20 ml), the mixture was stirred
overnight at room temperature. Then the solvent was removed
under reduced pressure to give a light yellow oil which is used for
the next step without further purification. DMF (40 ml), 3-
phenoxyphenylacetic acid (1.628 g, 7.13 mmol), HATU (2.715 g,
7.13 mmol) and DIEA (9.228 g, 7.13 mmol) were sequentially added
to the oil obtained. The mixture was stirred overnight at room
temperature then the solvent was removed under reduced pres-
sure. The residue was dissolved in ethyl acetate and sequentially
57.5, 49.9, 42.2, 37.2, 30.4, 19.5, 18.0; MS-ESI m/z: 442 [MþH]þ.
6.2.4. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-L-allo-Thr-OBn
(6d)
Yield: 84.7%; mp: 180.0e183.0 ꢀC 1H NMR (CD3OD, 300 Hz):
d
7.29e7.17 (m, 8H), 7.05e6.96 (m, 2H), 6.92e6.89 (m, 3H), 6.77 (d,
1H, J ¼ 8.7 Hz), 5.14e5.05 (m, 2H), 4.67 (t, 1H, J ¼ 6.6 Hz), 4.36 (d,
1H, J ¼ 6.0 Hz), 4.16 (d, 1H, J ¼ 6.0 Hz), 4.00e3.93 (m, 1H), 3.47 (s,
2H), 2.70e2.63 (m, 1H), 2.56e2.49 (m, 1H), 2.04e1.98 (m, 1H),
1.17e1.13 (m, 3H), 0.78e0.72 (m, 6H); 1H NMR (DMSO-d6, 400 Hz):
d
8.40 (d, 1H, J ¼ 8.0 Hz), 8.26 (d, 1H, J ¼ 8.0 Hz), 7.54 (d, 1H,

66
K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
Fig. 5. The interaction mode of compound 6e proposed by docking study. (a) 2D-plots: Residues marked with the letter “K” represents amino acids from the
proteasome, while those with the letter “L” are from the 6 subunit. (b) 3D-plots: The red dotted lines represents hydrogen bonding interactions. (For interpretation of the ref-
erences to color in this figure caption, the reader is referred to the web version of this article.)
b5 subunit of the 20S
b
J ¼ 8.0 Hz), 7.40e7.31 (m, 8H), 7.28 (t, 1H, J ¼ 8.0 Hz), 7.13 (t, 1H,
J ¼ 8.0 Hz), 7.04e6.99 (3H), 6.94 (s, 1H), 6.89e6.83 (m, 2H), 5.10 (s,
2H), 5.05 (t,1H, J ¼ 4.0 Hz), 4.60 (d,1H, J ¼ 8.0 Hz), 4.25 (m, 2H), 3.93
(m, 1H), 3.46 (s, 2H), 2.55 (dd, 1H, J ¼ 16.0, 16.0 Hz), 2.35 (dd, 1H,
J ¼ 8.0, 16.0 Hz), 1.90 (m, 1H), 1.10 (d, 3H, J ¼ 8.0 Hz), 0.73 (d, 3H,
J ¼ 8.0 Hz), 0.65 (d, 3H, J ¼ 8.0 Hz); 13C NMR (DMSO-d6, 125 Hz):
[MþH]þ.
6.2.5. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-L-allo-Thr-OMe
(6e)
Yield: 64.6%; mp: 203.0e206.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
d
7.27 (t, 2H, J ¼ 6.0 Hz), 7.21 (t, 1H, J ¼ 6.0 Hz), 7.05e6.98 (m, 2H),
d
172.0, 171.4, 171.2, 170.8, 170.4, 157.1, 156.9, 138.7, 136.4, 130.5,
6.92e6.90 (m, 3H), 6.78 (d, 1H, J ¼ 6.0 Hz), 4.68 (t, 1H, J ¼ 5.1 Hz),
4.31 (d, 1H, J ¼ 4.2 Hz), 4.17 (d, 1H, J ¼ 4.8 Hz), 4.01e3.95 (m, 1H),
3.64 (s, 3H), 3.47 (s, 2H), 2.72e2.66 (m, 1H), 2.58e2.52 (m, 1H),
130.1, 128.8, 128.4, 128.2, 124.7, 123.8, 119.9, 119.0, 117.0, 66.8, 66.2,
59.2, 57.2, 50.1, 42.2, 37.2, 31.3, 20.3, 19.5, 17.8; MS-ESI m/z: 633

K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
67
2.08e1.99 (m, 1H), 1.16 (d, 3H, J ¼ 4.8 Hz), 0.83e0.78 (m, 6H); 13C
6.2.11. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-ValeNHe(2-
NMR (DMSO-d6, 125 Hz):
d
172.0, 171.4, 171.2, 170.4, 157.1, 156.9,
hydroxy-4-methoxybenzyl) (6k)
138.7, 130.5, 130.1, 124.7, 123.8, 119.9, 119.0, 117.0, 66.8, 59.1, 57.3,
Yield: 31.5%; mp: 201.0e203.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
d 7.29e7.17 (m, 3H), 7.05e6.96 (m, 3H), 6.92e6.89 (m, 3H), 6.77 (d,
52.0, 50.1, 42.2, 37.2, 31.2, 20.4, 19.4, 17.9; MS-ESI m/z: 557 [MþH]þ.
1H, J ¼ 8.7 Hz), 6.29e6.27 (m, 2H), 4.68 (t, 1H, J ¼ 6.9 Hz), 4.18 (s,
2H), 4.12e4.06 (m, 1H), 3.64 (s, 3H), 3.46 (s, 2H), 2.72e2.64 (m, 1H),
2.57e2.50 (m, 1H), 2.07e1.98 (m, 1H), 0.75 (t, 6H, J ¼ 4.8 Hz); 13C
6.2.6. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-L-allo-Thr-OH
(6f)
Yield: 99.0%; mp: 215.0e218.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
NMR (DMSO-d6, 125 Hz):
d 172.1, 171.6, 171.4, 170.3, 159.8, 157.1,
156.9,156.2,138.8,130.5,130.1, 129.8,124.7,123.8,119.9,119.0, 117.9,
117.0, 104.6, 101.6, 57.9, 55.4, 50.1, 42.2, 37.6, 37.3, 30.8, 19.6, 17.9;
MS-ESI m/z: 577 [MþH]þ.
d
7.27 (t, 2H, J ¼ 5.7 Hz), 7.20 (t, 1H, J ¼ 6.0 Hz), 7.04e6.98 (m, 2H),
6.92e6.90 (m, 3H), 6.77 (d, 1H, J ¼ 6.0 Hz), 4.69 (t, 1H, J ¼ 4.8 Hz),
4.33 (d, 1H, J ¼ 3.9 Hz), 4.19 (d, 1H, J ¼ 4.5 Hz), 4.05e3.99 (m, 1H),
3.47 (s, 2H), 2.71e2.66 (m, 1H), 2.58e2.52 (m, 1H), 2.09e2.00 (m,
1H), 1.17 (d, 3H, J ¼ 4.8 Hz), 0.84e0.79 (m, 6H); 13C NMR (DMSO-d6,
6.2.12. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-ValeNHe(2,4-
dimethoxybenzyl) (6l)
125 Hz):
d 172.3, 172.0, 171.2, 171.1, 170.4, 157.1, 156.9, 138.7, 130.5,
Yield: 81.3%; mp: 217.0e222.0 ꢀC; 1H NMR (DMSO-d6, 300 Hz):
130.2,124.7, 123.8, 119.9, 119.0,117.0, 66.0, 58.9, 57.3, 50.1, 42.2, 37.2,
31.3, 20.1, 19.5, 17.9; HRMS-ESI m/z: 543.2440 [MþH]þ, calcd for
d
8.47 (d, 1H, J ¼ 7.5 Hz), 8.199 (m, 1H), 7.67 (d, 1H, J ¼ 9.3 Hz), 7.43
C
27H35O8N4: 543.2449.
(s, 1H), 7.37 (t, 2H, J ¼ 7.5 Hz), 7.27 (t, 1H, J ¼ 7.8 Hz), 7.11 (t, 1H,
J ¼ 7.2 Hz), 7.04e6.94 (m, 5H), 6.88 (s, 1H), 6.83 (d, 1H, J ¼ 7.2 Hz),
6.51 (d, 1H, J ¼ 2.1 Hz), 6.43 (d, 1H, J ¼ 8.1 Hz), 4.62e4.59 (m, 1H),
4.17e4.12 (m, 3H), 3.75 (s, 3H), 3.72 (s, 3H), 3.45 (s, 2H), 2.59e2.49
(m, 1H), 2.41e2.34 (m, 1H), 1.99e1.92 (m, 1H), 0.74e0.69 (m, 6H);
6.2.7. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-OBz (6g)
Yield: 36.0%; mp: 123.0e127.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
7.29e7.16 (m, 8H), 7.04e6.96 (m, 2H), 6.91e6.88 (m, 3H), 6.76 (dt,
d
13C NMR (DMSO-d6, 125 Hz):
d 172.1, 171.3, 171.0, 170.4, 160.0, 158.0,
1H, J ¼ 8.1, 1.5 Hz), 5.13e5.01 (m, 2H), 4.72 (t,1H, J ¼ 6.6 Hz), 4.26 (d,
1H, J ¼ 4.8 Hz), 3.45 (s, 2H), 2.65e2.58 (m, 1H), 2.55e2.47 (m, 1H),
2.08e1.97 (m, 1H), 0.78e0.72 (m, 6H); 13C NMR (DMSO-d6, 125 Hz):
157.1, 156.9, 138.8, 130.5, 130.1, 129.0, 124.7, 123.8, 119.9, 119.3, 119.0,
117.0, 104.6, 98.5, 58.1, 55.8, 55.6, 50.3, 42.2, 37.3, 30.8, 19.6, 17.9;
MS-ESI m/z: 591 [MþH]þ.
d
171.9, 171.8, 171.6, 170.4, 157.1, 156.9, 138.7, 136.3, 130.5, 130.1,
128.9, 128.6, 128.5, 124.7, 123.8, 119.9, 119.0, 117.0, 66.4, 57.8, 49.8,
42.2, 37.4, 30.4, 19.3, 18.3; MS-ESI m/z: 532 [MþH]þ.
6.2.13. N-2-(3-phenoxyphenyl)acetyl-L-Gln-L-ValeNHe(2-
hydroxy-4-methoxybenzyl) (6m)
yield: 75.6%; mp:195.0e196.0 ꢀC; 1H NMR(DMSO-d6, 300 Hz):
6.2.8. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-NH(CH2)2Ph (6h)
Yield: 83.7%; mp: 243.0e245.0 ꢀC; 1H NMR (DMSO-d6, 300 Hz):
d
d
8.29 (d, 2H, J ¼ 6.0 Hz), 7.78 (d, 1H, J ¼ 6.0 Hz), 7.36e7.24 (m, 4H),
7.11 (t, 1H, J ¼ 7.5 Hz), 7.02e6.93 (m, 5H), 6.83e6.75 (m, 2H),
6.34e6.27 (m, 2H), 4.30e4.24 (m, 1H), 4.15e4.10 (m, 3H), 3.63 (s,
3H), 3.44 (s, 2H), 2.07e2.02 (m, 2H), 1.91e1.84 (m, 2H), 1.74e1.66
8.40 (d, 1H, J ¼ 7.8 Hz), 8.02 (t, 1H, J ¼ 5.4 Hz), 7.61 (d, 1H,
J ¼ 8.7 Hz), 7.41e7.36 (m, 3H), 7.31e7.25 (m, 3H), 7.20e7.10 (m,
4H),7.05e6.95 (m, 5H), 6.86e6.82 (m, 1H), 4.66e4.59 (m, 1H), 4.05
(t, 1H, J ¼ 7.8 Hz), 3.46 (s, 2H), 3.33e3.20 (m, 2H), 2.69 (t, 2H,
J ¼ 7.8 Hz), 2.61e2.54 (m, 1H), 2.42e2.35 (m,1H), 1.96e1.87 (m, 1H),
(m, 1H), 0.75e0.72 (m, 6H); 13C NMR (DMSO-d6, 125 Hz):
d 174.3,
171.7, 170.4, 159.8, 157.1, 156.9, 139.0, 130.5, 130.1, 129.8, 124.7, 123.8,
119.8, 119.1, 117.8, 117.0, 101.7, 58.1, 55.3, 52.7, 42.3, 37.7, 32.1, 31.1,
28.4, 19.6, 18.4; HRMS-ESI m/z: 591.2800 [MþH]þ, calcd for
0.73e0.65 (m, 6H); 13C NMR (DMSO-d6, 125 Hz):
d 172.1, 171.3,
170.9,170.4,157.1, 156.9,139.8,138.8,130.5,130.1, 129.1, 128.8,126.5,
124.7, 123.8, 119.9, 119.0, 117.0, 58.0, 50.1, 42.2, 40.6, 37.3, 35.5, 30.7,
19.6, 17.8; MS-ESI m/z: 545 [MþH]þ.
C26H35N4O7: 591.2819.
6.2.14. N-2-(3-phenoxyphenyl)acetyl-L-Lys(Cbz)-L-ValeNHe(2-
hydroxy-4-methoxybenzyl) (6n)
6.2.9. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-(R)-NHCH(CH3)
Ph (6i)
Yield: 62.2%; mp: 142.0e143.0 ꢀC; 1H NMR (DMSO-d6, 300 Hz):
Yield: 88.1%; mp: 261.0e263.0 ꢀC; 1H NMR (DMSO-d6, 300 Hz):
8.38 (d, 1H, J ¼ 8.1 Hz), 8.26 (d, 1H, J ¼ 8.1 Hz), 7.69 (d, 1H,
d
9.55 (s, 1H), 8.27e8.20 (m, 2H), 7.74 (d, 1H, J ¼ 9.0 Hz), 7.39e7.21
(m, 8H), 7.19e7.09 (m, 2H), 7.03e6.92 (m, 4H), 6.82 (d, 1H,
J ¼ 7.8 Hz), 6.36e6.28 (m, 2H), 4.99 (s, 2H), 4.27e4.22 (m, 1H),
4.18e4.04 (m, 3H), 3.64 (s, 3H), 3.44 (d, 2H, J ¼ 3.3 Hz), 2.93e2.88
(m, 2H), 1.93e1.86 (m, 1H), 1.58e1.18 (m, 6H), 0.76e0.74 (m, 6H);
d
J ¼ 9.0 Hz), 7.40e7.35 (m, 3H), 7.30e7.19 (m, 5H), 7.03e6.89 (m, 5H),
6.82 (d, 1H, J ¼ 7.5 Hz), 4.91 (t, 1H, J ¼ 7.2 Hz), 4.65e4.58 (m, 1H),
4.13e4.08 (m, 1H), 4.05e3.98 (m, 1H), 3.44 (s, 2H), 2.58e2.53 (m,
1H), 2.40e2.33 (m, 1H), 2.01e1.94 (m, 1H), 1.31 (d, 3H, J ¼ 7.2 Hz),
0.76 (d, 3H, J ¼ 6.9 Hz), 0.70 (d, 3H, J ¼ 6.6 Hz); 13C NMR (DMSO-d6,
13C NMR (DMSO-d6, 125 Hz):
d 172.0, 171.7, 170.3, 159.8, 157.1, 156.9,
156.5, 156.3, 139.1, 137.7, 130.5, 130.1, 129.9, 128.8, 128.2, 124.6,
123.8, 119.7, 119.0, 117.8, 117.0, 104.5, 101.6, 65.6, 58.1, 55.3, 53.0,
42.3, 40.7, 37.6, 32.0, 31.0, 29.6, 23.1, 19.6, 18.5; MS-ESI m/z: 724
[MþH]þ.
125 Hz):
d 172.0, 171.4, 170.3, 170.1, 157.1, 156.9, 144.9, 138.8, 130.5,
130.1, 128.7, 127.1, 126.5, 124.7, 123.8, 119.9, 119.0, 117.0, 58.1, 50.1,
48.3, 42.2, 37.3, 30.8, 22.9, 19.6, 18.0; MS-ESI m/z: 545 [MþH]þ.
6.2.10. N-2-(3-phenoxyphenyl)acetyl-L-Asn-L-Val-(S)eNHCH(CH3)
Ph (6j)
6.2.15. N-2-(3-phenoxyphenyl)acetyl-L-Lys-L-ValeNHe(2-
hydroxy-4-methoxybenzyl) (6o)
Yield: 30.6%; mp: 243.0e246.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
Yield: 90.1%. mp: 165.0e166.0 ꢀC; 1H NMR (CD3OD, 300 Hz):
d
7.30e7.13 (m, 8H), 7.06e6.98 (m, 2H), 6.93e6.91 (m, 3H), 6.78 (d,
d 7.35e7.24 (m, 3H), 7.10e7.02 (m, 3H), 6.97e6.96 (m, 3H), 6.84 (d,
1H, J ¼ 7.8 Hz), 4.91 (q,1H, J ¼ 6.9 Hz), 4.71 (t,1H, J ¼ 6.6 Hz), 4.11 (d,
1H, J ¼ 5.7 Hz), 3.48 (s, 2H), 2.75e2.68 (m, 1H), 2.60e2.52 (m, 1H),
2.06e1.99 (m, 1H), 1.39 (d, 3H, J ¼ 7.2 Hz), 0.74 (d, 6H, J ¼ 6.9 Hz);
1H, J ¼ 8.0 Hz), 6.32e6.35 (m, 2H), 4.33 (t, 1H, J ¼ 4.0 Hz), 4.25 (d,
2H, J ¼ 8.0 Hz), 4.10 (d, 1H, J ¼ 8.0 Hz), 3.71 (s, 3H), 3.52 (s, 2H), 2.59
(t, 2H, J ¼ 4.0 Hz), 2.05e1.95 (m, 1H), 1.78e1.42 (m, 6H), 0.87e0.84
13C NMR (DMSO-d6, 150 Hz):
d
172.0, 171.3, 170.4, 170.1, 157.1, 156.9,
(m, 6H); 13C NMR (DMSO-d6, 125 Hz):
d 172.1, 170.9, 170.2, 161.1,
145.2,138.7,130.5,130.1,128.6,127.0,126.4,124.7,123.8,119.9,119.0,
117.0, 57.8, 50.2, 48.4, 42.2, 37.3, 31.0, 23.0, 19.6, 17.8; MS-ESI m/z:
545 [MþH]þ.
157.1, 157.0, 156.9, 139.2, 130.5, 130.1, 130.0, 124.7, 123.8, 119.7, 119.0,
118.0, 117.0, 104.2, 101.7, 58.2, 55.3, 53.0, 42.3, 42.2, 37.8, 32.5, 30.9,
30.4, 23.1, 19.6, 18.5; MS-ESI m/z: 591 [MþH]þ.

68
K. Xu et al. / European Journal of Medicinal Chemistry 98 (2015) 61e68
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6.3. Proteasome assay
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The 20S proteasome activity was determined using a Calbio-
chem proteasome assay kit following the manufacturer's protocol.
The chymotrypsin-like activity of the proteasome was assayed by
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E. Novellino, S. Grasso, A. Lavecchia, M. Zappala, Eur. J. Med. Chem. 83 (2014)
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incubating the proteasome (0.5
mg/ml) with the fluorogenic sub-
strate Suc-LLVY-AMC (20 M) in the presence of proteasome acti-
m
vators and tested compounds. The trypsin-like and caspase-like
activities of the 20S proteasome were determined using the
fluorogenic substrates Bz-VGR-AMC and (Z)-LLE-bNA, respectively.
Cleavage of the substrates by the proteasome resulted in emission
of fluorescence, which was measured and expressed as the rate of
the reaction by using a Biotek fluorometer. The rate of the cleavage
reaction is defined as dF/dt, where dF/dt is the change of fluores-
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P. Sarthou, J. Sobczak-Thepot, M. Reboud-Ravaux, J. Vidal, J. Med. Chem. 50
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Acknowledgment
Support for this research was provided by the 863 Hi-Tech
Program (2012AA020302) and the National Natural Science Foun-
dation of China (81302703).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.023.
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