Synthesis and biological activity of peptide α-ketoamide derivatives as proteasome inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00233

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudo

Full text of DOI:10.1021/acsmedchemlett.9b00233

Subscriber access provided by ALBRIGHT COLLEGE
Letter
Synthesis and biological activity of peptide #-
ketoamide derivatives as proteasome inhibitors.
Salvatore Pacifico, Valeria Ferretti, Valentina Albanese, Anna Fantinati, Eleonora Gallerani,
Francesco Nicoli, Riccardo Gavioli, Francesco Zamberlan, Delia Preti, and Mauro Marastoni
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00233 • Publication Date (Web): 06 Jun 2019
Downloaded from http://pubs.acs.org on June 11, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Synthesis and biological activity of peptide α-ketoamide derivatives
as proteasome inhibitors.
Salvatore Pacifico#, Valeria Ferretti#, Valentina Albanese#, Anna Fantinati#, Eleonora Gallerani#,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Francesco Nicoli#, Riccardo Gavioli#, Francesco Zamberlan§, Delia Preti*# and Mauro Marastoni#.
#Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari 46, 44121 Ferrara, Italy.
§School of Chemistry, University of Nottingham, University Park, NG7 2RD, United Kingdom.
KEYWORDS: Proteasome, Pseudopeptides, α-ketoamides, β subunits inhibition.
ABSTRACT: Proteasome activity affects cell cycle progression as well as the immune response and it is largely recognized as an
attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudo-
di/tripeptides bearing at the C-terminal position different α-ketoamide moieties at the C-terminal position as pharmacophoric units
for the interaction with the catalytic threonine residue that sustains the proteolytic action of proteasome. Among these, we identified
the 2-naphtyl derivative 13c as a potent and selective inhibitor of the β5 subunit of the 20S proteasome, exhibiting nanomolar potency
in vitro (β5 IC50 = 7 nM, β1 IC50 = 60 µM, β2 IC50 > 100 µM). Furthermore, it significantly inhibited proliferation and induced
apoptosis of the human colorectal carcinoma cell line HCT116.
The 26S proteasome is a sophisticated multicatalytic enzymatic
complex of key importance for intracellular protein degradation
clinical employment have emerged.15 In particular, a percentage
of patients does not respond to the treatment and a high
relapsing frequency has been observed probably due to the
development of resistances over time. In addition, dose limiting
toxicity has been witnessed after administration of proteasome
inhibitors currently in use. Peripheral neuropathy is one of the
most recurrent side effects which has been attributed to off-
target interactions.15 Thus, the design of a new generation of
inhibitors with high selectivity for the active site of the target
enzyme continues to be an active field of research since it could
overcome some of the typical side effects described for the first
generation of drugs.8,15,16
In this context, our research efforts have been aimed at the
design, synthesis and biological characterization of new classes
of peptides able to inhibit the proteasome activity.17-21 Each of
these is characterized by a distinct pharmacophoric unit,
consisting of different electrophilic groups potentially able to
interact with the hydroxyl group of the side chain of the Thr1
residue of the enzyme responsible for the proteolytic action.
Herein, we present a new contribution in this field consisting in
the development of a series of peptide-based derivatives bearing
at the C-terminal residue an α-ketoamide pharmacophoric unit
as electrophilic substrate. This moiety has already been
successfully introduced in the structure of various
pseudopeptidic enzyme inhibitors.22,23 Of note, some examples
of proteasome inhibitors bearing an α-ketoamide function have
also been reported in literature such as the cyclic polypeptide
TMC-95A and its diastereoisomers TMC-95B-D.24,25
Moreover, this electrophilic function has been inserted in the
backbone of linear peptides targeting proteasome.26 According
to these and more recent studies, the α-ketoamide motif is
emerging as the most promising group with possible therapeutic
2
and homeostasis in eukaryotic organisms.1, It appears as a
hollow cylinder consisting of a central 20S proteolytic core
(CP) capped by two 19S regulatory particles, which are
responsible for the recognition of polyubiquitinated substrates
and their guiding and transport within the CP.3 The 20S
proteasome is formed by four stacked heptameric rings of α-
and β-type subunits with the typical stoichiometry α7β7β7α7. In
eukaryotic cells, each β ring hosts three catalytic active sites
with distinct proteolytic specificity: the β1 subunit promotes the
so-called caspase-like (C-L) or post acidic (PGPH) activity
responsible for the processing of the substrates after acid
residues; the β2 subunit displays the trypsin-like (T-L) activity
with hydrolysis after basic residues; the β5 subunit undertakes
the chymotrypsin-like activity (ChT-L) cutting substrates after
hydrophobic and aromatic residues. Despite the different
specificity, the three active sites share a common catalytic
mechanism that involves a key N-terminal threonine residue
(Thr1) responsible for the nucleophilic attack at the specific
peptide bond of the substrate.4,5
Proteasome activity affects cell cycle progression as well as the
immune response and it is largely recognized as a very
attractive pharmacological target for potential therapies against
a series of diseases.6,7 In particular, several classes of both
specific and non-specific inhibitors of proteasome activity have
been developed.7-9 The translational potential of this system has
been well established in cancer therapy with the clinical success
of the three proteasome inhibitors bortezomib, carfilzomib and
ixazomib, approved for the treatment of hematological
malignancies such as multiple myeloma.10-14 Nonetheless,
despite the efficacy of these drugs, several limitations in their
1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
application against the proteasome because of its ability to
Page 2 of 7
substituted with different (cyclo)alkyl/(hetero)aryl moieties, we
designed the alternative synthetic pathway reported in Scheme
2. This novel approach has the advantage of not requiring the
use of isocyanides that are in some cases toxic and mostly
characterized by a well-known aggressive and extremely
unpleasant smell. Firstly, Boc-protected leucinal 7 was reacted
with vinylmagnesium bromide to give the vinyl derivative 8 as
a diastereomeric mixture.31 The following protection with 2,2-
dimethoxypropane led to a mixture of cis-trans oxazolidine 9,
whose double bond was oxidized with RuO2 and NaIO4.
1
2
3
4
5
6
7
8
induce a potent but reversible inhibition of the enzyme’s
activity if compared to other investigated C-terminus warheads
(i.e. α-ketoaldehyde, α,β-epoxy ketone, boronic acid, vinyl
sulfone).27,28 Thus, we focused our attention on the tri-leucine
derivative 1a (shown in Figure 1) that has been previously
reported to inhibit the proteasome catalytic subunits at
nanomolar concentrations,29 and, with the above evidence in
mind, we designed two series of derivatives modeled on its
structure, as depicted in Figure 1.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HN
O
Scheme 1. Synthesis of the first series of ketoamide
dipeptide derivatives.
O
O
O
H
N
O
N
N
O
O
O
H
H
O
a, b
c, d
HO
R
e, f
H
OH
R
R
N
1a
H
NHFmoc
NHFmoc
NH2
First series
Second series
2a
2b
2c
2d
2e
2f
3a
3b
4a
4b
R= Benzyl
R= Benzyl
R= 4-(tert-butoxy)Bn
R= Benzyl
R= 4-(tert-butoxy)Bn
R
N
R= 4-(tert-butoxy)Bn
R= 1-Naphthyl
R= CH2CONHTrt
R= Isopropyl
O
O
R
O
O
H
R'
3c R= 1-Naphthyl
4c R= 1-Naphthyl
H
H
3d
3e
3f
4d
4e
4f
O
N
O
N
N
N
R= CH2CONHTrt
R= Isopropyl
R= Isobutyl
R= CH2CONHTrt
R= Isopropyl
R= Isobutyl
N
N
H
O
H
H
O
O
O
O
R= Isobutyl
6a-f
13a-e
Figure 1. New α-ketoamide peptide derivatives as potential
proteasome inhibitors.
g
O
R
O
O
R
O
H
H
O
N
O
N
Bn
N
N
Bn
N
N
The first series (6a-f) was conceived with the aim of simplifying
the tripeptide structure of 1a to shorter dipeptide analogues in
which the α-keto benzylamide pharmacophoric unit of the
parent compound was maintained but combined with different
C-terminal residues. In the second series (13a-e), the tri-leucine
sequence of 1a was functionalized with different α-keto
(cyclo)alkyl/(hetero)arylamides. A novel convergent synthetic
approach has been designed and applied for the obtainment of
the latter derivatives. The available strategies for the synthesis
of α-ketoamide derivatives were recently described in an
exhaustive review.25 The synthesis of the first series of target
dipeptides (6a-f) was performed as depicted in Scheme 1 and in
analogy to procedures previously reported by Stein et al.27
Briefly, different Fmoc protected L-α-amino acids (2a-f), were
initially converted into the respective aldehydes (3a-f) by a
known two-step method reported by Fehrentz and Castro.30 This
was based on the initial conversion of 2a-f into the
corresponding N,O-dimethyl hydroxamates via activation of
the carboxyl group with 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimmide (WSC) and N-hydroxybenzotriazole (HOBt)
and subsequent treatment with N,O-dimethylhydroxylamine. In
the next step, the hydroxamate derivatives were efficiently
reduced to the corresponding aldehydes 3a-f with LiAlH4.
These were then treated with benzyl isocyanide in the presence
of acetic acid according to the multicomponent Passerini
reaction. The resulting N-Fmoc-O-acetyl hydroxyamide
intermediates were fully deprotected with a mild basic
treatment to furnish derivatives 4a-f. The desired α-
hydroxyamides 5a-f were then obtained as inseparable
diastereomeric mixtures by standard coupling with Z-Leu-OH
followed by side chain deprotection with TFA when necessary
(5b and 5d). Finally, the oxidation with 2-iodoxybenzoic acid
gave the ketoamides 6a-f.
H
H
H
H
O
OH
O
O
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
R= Benzyl
R= Benzyl
R= 4-Hydroxy benzyl
R= 1-Naphthyl
R= CH2CONH2
R= Isopropyl
R= 4-Hydroxy benzyl
R= 1-Naphthyl
R= CH2CONH2
R= Isopropyl
R= Isobutyl
R= Isobutyl
Reagents and conditions
: a) N,O-Dimethylhydroxylamine hydrochloride,
WSC, HOBt, Et3N, DMF; b) LiAlH4, THF; c) Benzyl Isocyanide, CH3COOH;
5b
d) LiOH, THF/MeOH; e) Z-Leu-OH, WSC, HOBt, DMF; f) TFA (for
5d
and
); g) 2-Iodoxybenzoic acid, DMSO.
Scheme 2. Synthesis of the second series of ketoamide
tripeptide derivatives.
O
HO
O
a
b
c
N
H
Boc
NHBoc
NHBoc
7
8
9
OH
R
O
d, e
f
O
H2N
O
N
Boc
OH
10
11a-e
O
O
O
O
H
H
Bn
N
O
R
Bn
N
OH
g
O
N
N
O
N
N
H
H
H
H
O
O
O
O
R
13a-e
12a-e
Reagents and conditions
: a) Vinylmagnesium bromide, CH2Cl2; b) p-
Toluensulfonic acid, 2,2-dimethoxypropane, CH2Cl2; c) NaIO4,
a-e
Ruthenium(IV) oxide hydrate, Acetone; d) Appropriate amine
, HATU,
DIPEA, DMF; e) TFA; f) Z-Leu-Leu-OH, HATU, DIPEA, DMF; g) 2-
11-13a
11-13b
Iodoxybenzoic acid, DMSO;
R= 4-Fluorobenzyl-NH;
R=
For the synthesis of the second series of tripeptide analogues
13a-e, in which the nitrogen atom of the α-ketoamide group was
11-13c
11-13d
11-
Morpholin-4-yl;
13e
R= 2-Naphthyl-NH;
R= Isobutyl-NH;
R= Tetrahydroisoquinolin-2-yl.
2
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
The resulting carboxylic intermediate 10 was coupled with
different amines in the presence of HATU and DIPEA. The
susequent deprotection with trifluoroacetic acid gave 11a-e as
diastereomeric mixtures. These were then coupled under
standard conditions with the N-protected dipeptide Z-Leu-Leu-
OH to give the α-hydroxyamides 12a-e that were successively
oxidized to the corresponding α-ketoamides 13a-e with 2-
iodoxybenzoic acid. The α-hydroxyamides 5a-f/12a-e and the
α-ketoamides 6a-f/13a-e were purified via preparative HPLC
and characterized by ESI-MS and 1H-NMR spectroscopy.
1
2
3
4
5
6
7
8
9
Table 1. Inhibition of the proteasome subunits by α-hydroxy/ketoamide peptides and their effect on cell proliferation (the values
reported are the mean ± SEM of three independent experiments). aIC50 proliferation values against HCT116 cells for the most potent
compounds in the enzyme inhibition assays.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
R
X
HN
O
R
O
O
H
H
H
X
O
N
O
N
N
O
O
O
N
N
H
N
N
H
H
H
O
X
O
O
O
N
H
N
H
O
1a
: X =
1b
: X =
O
OH
OH
O
5a-f
6a-f
X =
X =
OH
O
12a-e
13a-e
X =
X =
IC50 (µM)
PGPH (β1)
IC50 (µM)
T-L (β2)
IC50 (µM)
ChT-L (β5)
IC50 (µM)a
prolif.
Compd
R
MG132
1a
1.45± 0.31
0.73± 0.01
17.82± 0.90
4.59± 0.06
0.43± 0.01
>100
0.007± 0.001
0.014± 0.001
0.53± 0.04
0.82± 0.03
0.78± 0.01
ND
1b
Dipeptide analogues
5a
Bn
Bn
>100
57.98 ± 4.38
17.61 ± 1.18
51.63± 5.06
21.54± 2.05
>100
>100
51.09 ± 3.34
70.45± 5.93
12.43± 0.92
>100
ND
ND
6a
82.36 ± 6.72
>100
5b
4-OH-Bn
4-OH-Bn
1-Naphthyl
1-Naphthyl
CH2CONH2
CH2CONH2
Isopropyl
Isopropyl
Isobutyl
ND
6b
50.11± 3.84
>100
>100
ND
5c
6c
73.22± 5.91
53.15± 4.27
89.47± 8.17
30.57± 2.76
15.45± 1.21
20.44± 1.76
1.09± 0.12
>100
>100
ND
5d
>100
>100
ND
6d
>100
>100
ND
5e
>100
82.07± 7.55
63.34± 6.13
50.21± 4.33
0.92± 0.08
ND
6e
2.11± 0.24
>100
ND
5f
ND
6f
Isobutyl
1.32± 0.11
8.6± 0.34
Tripeptide analogues
12a
13a
12b
13b
12c
13c
12d
13d
12e
13e
4-F-Bn-NH-
4-F-Bn-NH-
>100
11.5± 0.81
>100
>100
0.59± 0.01
>100
>100
0.056± 0.003
>100
ND
0.93± 0.13
ND
Morpholin-4-yl
Morpholin-4-yl
2-Naphtyl-NH
2-Naphtyl-NH
Isobutyl-NH
0.56± 0.02
>100
85.4± 3.12
>100
0.48± 0.02
8.1
ND
ND
57.5± 4.06
>100
>100
0.007± 0.001
>100
<0.10
ND
>100
Isobutyl-NH
5.9± 0.32
>100
0.43± 0.03
>100
0.012± 0.002
>100
0.81± 0.12
ND
Tetrahydroisoquinolin-2-yl
Tetrahydroisoquinolin-2-yl
>100
38.9± 0.98
0.77± 0.04
ND
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
1
2
3
4
5
6
7
8
The capability of the synthetized compounds of inhibiting each
ranging from 7 to 770 nM), with various level of selectivity over
the β1 and β2 subunits. Among these, derivative 13c, in which
the α-keto benzylamide of 1a was replaced by the 2-
naphtylamide moiety, displayed the highest potency with an
IC50 value of 7 nM against the chymotryptic activity, and very
low or none activity against the β1 and β2 subunits (IC50 values
of 57 and >100 µM, respectively). Thus, the introduction of a
bulky and lipophilic aromatic bicycle at the c-terminal portion
of the linear peptide structure led to a slight improvement of the
β5 activity inhibition (2-fold) associated with a remarkable
increase of selectivity (selectivity ratios: β1/β5 = 8.200, β2/β5
>14.200) in comparison with the parent compound 1a. In this
series of molecules, the investigated ketoamide substitutions
determined the following order of potency: 2-naphtyl-
NH>Isobutyl-NH≈Bn-NH>4-F-benzyl-NH>morpholin-4-
yl>tetrahydroisoquinolin-2-yl. These data would suggest that
the presence of a more flexible primary amide, compared to a
secondary one, would enhance the inhibition of the ChT-L
activity. This is particularly evident when comparing the
activities of 1a and 13e, since the latter’s isoquinoline
derivative can be considered a constrained analogue of the
parent benzylamide compound.
of the three proteasome activities was measured through an in
vitro assay which is based on the employment of Suc-LLVY-
AMC (for the ChT-L), Boc-LRR-AMC (for the T-L) and Z-
LLE-AMC (for the PGPH) as specific fluorogenic substrates.32
Briefly, semi-purified proteasomes were pre-treated with
increasing concentrations (0.01-100 µM) of the new pseudo-
peptides (5a-f, 6a-f, 12a-e and 13a-e) in an activity buffer. The
trileucine derivatives 1a, 1b (Table 1, synthetized according to
reported procedures 28, 29) have been evaluated under the same
conditions for comparison reasons along with the known
proteasome inhibitor MG132 (Z-LLL-CHO)33 as internal
standard. The inhibitory activity of all compounds is reported in
Table 1 and expressed as IC50 value in µM concentration.
The effects of 5a-f and 12a-e against the three catalytic
activities of proteasomes were evaluated in order to establish
the actual importance of the ketone’s carbonyl of the
α‐ketoamide pharmacophore in the interaction with Thr1. It has
been reported that such interaction leads to a reversible
hemiacetal adduct resulting from the nucleophilic attack of the
Thr1 hydroxyl group on the ketone carbonyl of the α‐ketoamide
portion.28 Interestingly, although devoid of electrophilic
properties, the α-hydroxyamide moiety of 5a-f and 12a-e,
mimics the tetrahedral intermediate which is supposed to follow
the nucleophilic addition of Thr1 to the α‐ketoamide. However,
our SAR study strongly confirms that the presence of the
electrophilic α‐ketoamide moiety is mandatory for the
inhibitory activity towards all the three catalytic sites as its
conversion to the corresponding α‐hydroxyamide led to a
marked loss of potency (compare 1a with 1b, 5a-f with 6a-f and
12a-e with 13a-e). Likewise, shortening of the trileucine motif
of 1a to the dileucine analogue 6f resulted in a general decrease
of potency, particularly evident against the chymotryptic-like
activity (β5 IC50 = 0.014 and 0.92 nM for 1a and 6f,
respectively). Nonetheless, we explored about the possibility of
restoring the activity by the introduction of C-terminal amino
acids other than the leucine residue of 6f (i.e. Phe, Tyr, 1-Nal,
Asn and Val for compounds 6a-e, respectively). This led to a
further reduction of potency with IC50 values that reached the
high micromolar range against the three investigated activities.
Thus, compound 6f (with the sequence Z-Leu-Leu-CONHBn)
was confirmed as the most active of this first series as able to
inhibit the three activities of the enzyme complex with IC50
values of about 1µM, without significant selectivity in the
biological response. Most of the analyzed dipeptide derivatives
had a mild selectivity for the post-acidic and/or chymotryptic
activities, with the exception of 6e, characterized by a C-
terminal valine, that inhibited the β2 tryptic subunit (IC50 = 2.11
µM) with significant selectivity over the β1 (7-fold) and β5 (30-
fold) subunits. These preliminary results suggested that both the
length of the peptide sequence and the c-terminal leucine
residue would contribute in eliciting a better inhibition. Thus,
we designed the second series of tripeptide α‐ketoamides
(compounds 13a-e in Table 1) in which the Z-LLL template of
1a was maintained but combined with different substituents on
the nitrogen of the α‐ketoamide moiety. The resulting
compounds exhibited from submicromolar to low nanomolar
potency in inhibiting the β5 chymotryptic activity (β5 IC50
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Effect of compounds 13c and 13d on apoptosis induction.
HCT116 tumor cells cultured for 48 or 72 hours in the presence or
absence of the indicated compounds at the concentration of 1 µM.
Numbers in the dot plots represent percentages of cells. One
representative experiment out of three is shown.
The anti-proliferative activity of selected compounds (6b, 6f,
1a, 13a, 13c and 13d) was evaluated at concentration ranging
from 0.1 to 100 µM against the human colorectal carcinoma cell
line HCT116 and compared to that of the reference inhibitor
MG132. The effect of the compounds on cell viability was
measured at 72 hours and reported in Table 1 expressed as IC50.
Interestingly, a significant anti-proliferative activity was
observed for all the examined molecules with variable degree
of potencies, basically reflecting their capability to inhibit the
β5
4
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
1
2
A
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B
Figure 3. Molecules 13c (A) and 1a (B) in the β5 binding pocket with a schematic diagram of the inhibitor–protein interactions.
subunit of the proteasome, mediating the chymotryptic activity
(ChT-L). Indeed, the Leu-Leu derivative 6f (IC50 ChT-L= 920
nM and IC50 proliferation=8.6 µM, Table 1) seems to be more
potent than the Leu-Tyr analogue 6b (IC50 ChT-L= 12.43 µM
and IC50 proliferation > 100 µM ), while both compounds were
shown to have lower activity than the reference
pseudotripeptide 1a (IC50 ChT-L= 14 nM and IC50
proliferation= 0.78 µM) and MG132 (IC50 ChT-L = 7nM and
IC50 proliferation= 0.82 µM). Notably, the tripeptide analogues
13a, 13c and 13d, which all showed IC50 for ChT-L <100nM,
induced a strong anti-proliferative effect, comparable or even
superior to that of the reference pseudotripeptide 1a and of
MG132, as all the three compounds displayed an IC50
proliferation <1 µM. In particular, the compound 13c, which
showed the highest activity inhibiting the β5 subunit of the
proteasome, also exerted the strongest anti-proliferative effect.
Finally, to determine if this effect was associated to cell death
induction, apoptosis levels were measured in HCT116 cells
treated for 48 and 72 hours with two selected compounds, i.e.
13c and 13d. The positivity to Annexin V and negativity to
propidium iodide (P.I.) identifies early apoptotic cells, while the
double positivity to both Annexin V and P.I., late apoptotic
cells. As shown in Figure 2, 13c exhibited increased levels of
late apoptotic cells already at 48 hours, and this effect was even
higher at 72 hours, consistently with the lower IC50 proliferation
shown in comparison to 13d.
Docking studies were performed for a subset of compounds (6a,
6b, 5f, 6f and 13c) selected in respect to their biological profile,
and the best binding poses of each molecule in the β1, β2 and
β5 binding sites were compared to those of the known α-
ketoamide tripeptide 1a.29 Figure 3A shows compound 13c, the
most potent and selective β5 inhibitor identified in this work,
docked in the β5 binding pocket alongside the reference
compound 1a (Figure 3B). Moreover, the best binding poses for
compounds 1a and 13c in the β1 and β2 catalytic subunits have
been illustrated in figures S1 and S2 of the supplementary
material. For each proposed binding pose, a schematic diagram
of the inhibitor–protein interactions has been also supplied. For
both molecules, the α-ketoamide group was found to interact in
all binding sites with the active Thr1 residue via hydrogen
bonding interactions, with donor-acceptor distances in the range
of 2.6-3.3 Å, typical of medium/strong H-bonds. Furthermore,
1a and 13c were found to be surrounded by (or interacting with)
several residues which the structural analyses indicated as
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
involved in the formation of the receptor pockets 5. For
molecule 13c, the important residues Thr21, Gly47 and His108
were directly interacting with the ligand (Figure 3A). As a
reference, for the ChT-L active center, residues Thr21, Gly47,
Ala49, Ala50 were found to be involved in the binding of known
inhibitors such as Bortezomib.5 Moreover, docking results at
the β5 subunit show that the distal aromatic regions of 13c,
unlike 1a, were able to establish a significant number of π/π
stacking and CH/π interactions with the residues His108, Trp25,
Tyr170 and Gly47. Of note, these interactions involve both the
benzyl and naphtyl terminal groups, favoring the anchorage of
the molecule to the proteasome active site. In our models, a
lower number of residues of the β1 (Gly47) and β2 (Gly47, Gln22)
subunits would make contact with the distal aromatic portions
of 13c and this could account for the overall selectivity profile
of the molecule.
S6 from docking studies are available as Supporting Information
free of charge from the ACS Publications website.
1
2
3
4
5
6
7
8
AUTHOR INFORMATION
Corresponding Author
* Phone: +39-532-455501. E-mail address: prtdle@unife.it
Author Contributions
S.P., V.A. and A.F. performed the chemical synthesis. V.F.
performed and interpreted the docking study. E.G., F.N. and R.G.
performed the in vitro molecular pharmacology studies. F. Z.
drafted the manuscript. M.M. and D.P. oversaw and developed the
project. All authors have given approval to the final version of the
manuscript.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Funding Sources
For what is concerning the docking results obtained for the
dipeptide derivative 6f (Figure S3) in comparison with the
tripeptide analogue 1a, both molecules show their capability to
fit well all the three investigated binding sites taking contact
with key residues of the binding pockets. In β1, for instance, the
conserved residues are Lys33, Thr21, Ser118, Arg45. In β2, it is
worth mentioning the presence of the residue Cys118 of subunit
β3, which is responsible for the character of the S3 specificity
pocket for the selective β2 inhibitor Mal-βAla-val-Arg-al5. It is
also worth noting that the tripeptide 1a, with a longer sequence
by one non-polar alkyl amino acid, is able to establish van der
Waals interactions with further residues that insist around the
binding pocket, something that the shorter dipeptide 6f
presented in this work is not able to do.
The docking simulation of the other 6a, 6b and 5f molecules did
not give good results in comparison with those presented above,
as far as the mutual Thr1-ketoamide group position is
concerned. The five best poses for each molecule in the three
binding pockets are reported in the Supplementary material
(Figures S4-S6).
In summary, this paper describes the synthesis, biological
evaluation and docking analysis of two series of pseudo-
di/tripeptides as proteasome inhibitors. The entire investigated
molecules feature at the C-terminal portion an α-ketoamide as
the pharmacophoric unit able to interact with and block the
catalytic threonine of the active subunits of the 26S proteasome.
Our stepwise SAR optimization work led to the identification
of 13c as a potent and selective inhibitor of the β5 subunit of the
20S proteasome with nanomolar potency in vitro. The
compound significantly inhibited proliferation and induced
apoptosis of the human colorectal carcinoma cell line HCT116
confirming the potential of β5-selective proteasome inhibitors
in cancer therapy. Recent findings would also suggest that the
inhibition of the β5 activity by selective ligands could have
some therapeutic perspectives in the cardiovascular area since
promoting beneficial effects in rat models of ischemia
reperfusion injury.34
DP is supported by the funds FAR 2017 (Fondo di Ateneo per la
Ricerca Scientifica) and FFABR 2017 (Finanziamento delle attività
base di ricerca) of the University of Ferrara. MM is supported by
the funds FAR 2017 of the University of Ferrara. The FACSCanto
II was funded by Ferrara University Grant “Bando per
l’acquisizione di strumenti per la ricerca di ateneo-2015”.
Notes
The authors declare no competing financial interest.
REFERENCES
(1) Glickman, M. H.; Ciechanover A. The ubiquitin-proteasome
proteolytic pathway: destruction for the sake of construction. Physiol.
Rev. 2002, 82, 373-428.
(2) Ciechanover, A. Proteolysis: from the lysosome to ubiquitin and the
proteasome. Nat. Rev. Mol. Cell. Biol. 2005, 6, 79-87.
(3) Finley, D. Recognition and processing of ubiquitin-protein
conjugates by the proteasome. Annu. Rev. Biochem. 2009, 78, 477-513.
(4) Groll, M.; Heinemeyer, W.; Jager, S.; Ullrich, T.; Bochtler, M.;
Wolf, D. H.; Huber, R. The catalytic sites of 20S proteasomes and their
role in subunit maturation: a mutational and crystallographic study.
Proc. Natl. Acad. Sci. USA 1999, 96, 10976-10983.
(5) Borissenko, L; Groll, M. 20S proteasome and its inhibitors:
crystallographic knowledge for drug development. Chem Rev. 2007,
107, 687-717.
(6) Sijts, E. J.; Kloetzel, P. M. The role of the proteasome in the
generation of MHC class I ligands and immune responses. Cell. Mol.
Life Sci. 2011, 68, 1491-1502.
(7) Kisselev, A. F.; Van der Linden, W. A.; Overkleeft, H. S.
Proteasome inhibitors: an expanding army attacking a unique target.
Chem. Biol. 2012, 19, 99-115.
(8) Groll, M.; Huber, R.; Moroder, L. The persisting challenge of
selective and specific proteasome inhibition. J. Pept. Sci. 2009, 15, 58-
66.
(9) Micale, N.; Scarbaci, K.; Troiano, V.; Ettari, R.; Grasso, S.;
Zappalà, M. Peptide-based proteasome inhibitors in anticancer drug
design. Med. Res. Rev. 2014, 34, 1001-1069.
(10) Moreau, P.; Richardson, P. G.; Cavo, M.; Orlowski, R. Z.; San
Miguel, J. F.; Palumbo, A.; Harousseau, J. L. Proteasome inhibitors in
multiple myeloma: 10 years later. Blood 2012, 120, 947-959.
(11) Adams, J. The development of proteasome inhibitors as anticancer
drugs. Cancer Cell 2004, 5, 417-421.
(12) Moreau, P. The emerging role of carfilzomib combination therapy
in the management of multiple myeloma. Expert Rev. Hematol. 2014,
7, 265-290.
(13) Shirley, M. Ixazomib: first global approval. Drugs 2016, 76, 405-
411.
Supporting Information
Detailed synthetic procedures, spectroscopic data and full
characterizations of the described compounds, procedures for
biological experiments, computational procedures and Figures S1-
(14) Allegra, A.; Alonci, A.; Gerace, D.; Russo, S.; Innao, V.; Calabrò,
6
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
L.; Musolino, C. New orally active proteasome inhibitors in multiple
myeloma. Leuk. Res. 2014, 38, 1-9.
(25) De Risi, C.; Pollini, G. P.; Zanirato, V. Recent developments in
general methodologies for the synthesis of α-ketoamides. Chem. Rev.
2016, 116, 3241-3305.
(26) Lynch, S. M.; Narayanan, A.; Steiner, S. Ketoamide
immunoproteasome inhibitors. WO 2014056748.
(27) Stein, M. L.; Cui, H.; Beck, P.; Dubiella, C.; Voss, C.; Krüger, A.;
Schmidt, B.; Groll, M. Systematic comparison of peptidic proteasome
inhibitors highlights the α-ketoamide electrophile as an auspicious
reversible lead motif. Angew. Chem. Int. Ed. Engl. 2014, 53, 1679-
1683.
1
2
3
4
5
6
7
(15) Park, J.E.; Miller, Z.; Jun, Y.; Lee, W.; Kim, K.B. Next-generation
proteasome inhibitors for cancer therapy. Transl Res. 2018, 198:1-16.
(16) Gozzetti, A.; Papini, G.; Candi, V.; Brambilla, C. Z.; Sirianni, S.;
Bocchia, M. Second generation proteasome inhibitors in multiple
myeloma. Anticancer Agents Med. Chem. 2017, 17, 920-926.
(17) Marastoni, M.; Baldisserotto, A.; Canella, A.; Gavioli, R.; De Risi,
C.; Pollini, G. P.; Tomatis, R. Arecoline tripeptide inhibitors of
proteasome. J. Med. Chem. 2004, 47, 1587-1590.
8
9
(18) Baldisserotto, A.; Ferretti, V.; Destro, F.; Franceschini, C.;
Marastoni, M.; Gavioli, R.; Tomatis, R. Alpha,beta-unsaturated N-
acylpyrrole peptidyl derivatives: new proteasome inhibitors. J Med
Chem. 2010, 53, 6511-6515.
(28) Voss, C.; Scholz, C.; Knorr, S.; Beck, P.; Stein, M. L.; Zall, A.;
Kuckelkorn, U.; Kloetzel, P. M.; Groll, M.; Hamacher, K.; Schmidt B.
α-Keto phenylamides as P1’-extended proteasome inhibitors.
ChemMedChem 2014, 9, 2557-2564.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Franceschini, C.; Trapella, C.; Calia, R.; Scotti, A.; Sforza, F.;
Gavioli, R.; Marastoni, M. C-terminal trans,trans-muconic acid ethyl
ester partial retro-inversopseudopeptides as proteasome inhibitors. J.
Enzyme Inhib. Med. Chem. 2013, 28, 1034-1039.
(29) Braun, H. A.; Umbreen, S.; Groll, M.; Kuckelkorn, U.;
Mlynarczuk, I.; Wigand, M. E.; Drung, I.; Kloetzel, P. M.; Schmidt, B.
Tripeptide mimetics inhibit the 20 S proteasome by covalent bonding
to the active threonines. J. Biol. Chem. 2005, 280, 28394-23401.
(30) Fehrentz, J. A.; Castro, B. An efficient synthesis of optically active
α-(t-butoxycarbonylamino)-aldehydes from α-amino acids. Synthesis
1983, 8, 676-678.
(31) DeMattei, J. A.; Leanna, M. R.; Li, W.; Nichols, P. J.; Rasmussen,
M. W.; Morton, H. E. An Efficient Synthesis of the Taxane-Derived
Anticancer Agent ABT-271. J. Org. Chem. 2001, 66, 3330-3337.
(32) Hansen, M. B.; Nielsen, S. E.; Berg, K. Re-examination and
further development of a precise and rapid dye method for measuring
cell growth/cell kill. J. Immunol. Methods 1989, 119, 203-210.
(33) Lee, D.H.; Goldberg, A.L. Proteasome inhibitors: valuable new
tools for cell biologists. Trends Cell. Biol. 1998, 8, 397-403.
(34) Sanchez G, Berrios D, Olmedo I, Pezoa J, Riquelme JA,
Montecinos L, Pedrozo Z, Donoso P. Activation of chymotrypsin-like
activity of the proteasome during ischemia induces myocardial
dysfunction and death. PLoS One. 2016, 111, e0161068.
(20) Scotti, A.; Trapella, C.; Ferretti, V.; Gallerani, E.; Gavioli, R.;
Marastoni, M. Studies of C-terminal naphthoquinone dipeptides as 20S
proteasome inhibitors. J. Enzyme Inhib. Med. Chem. 2016, 3, 456-463.
(21) Marastoni, M.; Trapella, C.; Scotti, A.; Fantinati, A.; Ferretti, V.;
Marzola, E.; Eleonora, G.; Gavioli, R.; Preti, D. Naphthoquinone
aminoacid derivatives, synthesis and biological activity as proteasome
inhibitors. J. Enzyme Inhib. Med. Chem. 2017, 32, 865-877.
(22) Chang, K.O.; Kim, Y.; Groutas, W.C.; Hua, D.; Saif, L.J. Broad-
spectrum antivirals against 3C or 3C-like proteases of picornavirus-like
supercluster: picornaviruses, caliciviruses and coronaviruses. US
20140243341.
(23) Dondapati, J. S.; Godi, S.; Babu, A.P. QSAR studies on peptide
alpha-ketoamides and alpha-ketohydroxamate derivatives as calpain I
inhibitors. J. Enzyme Inhib. Med. Chem. 2008, 23, 757-762. 18618317
(24) Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.;
Ohnuki, T.; Komatsubara, S. TMC-95A, B, C, and D, novel proteasome
inhibitors produced by Apiospora montagnei Sacc. TC 1093.
Taxonomy, production, isolation, and biological activities. J. Antibiot.
(Tokyo) 2000, 53, 105-109.
Insert Table of Contents artwork here
IC50 β1 = 60 µM
IC50 β2 > 100 µM
IC50 β5 = 7 nM
O
NH
O
O
H
H
O
N
N
N
H
O
O
Proteasome
inhibition
13c
7
ACS Paragon Plus Environment