Development of a new class of proteasome inhibitors with an epoxyketone warhead: Rational hybridization of non-peptidic belactosin derivatives and peptide epoxyketones

Article abstract of DOI:10.1016/j.bmc.2014.04.032

Proteasome inhibitors are currently a focus of increased attention as anticancer drug candidates. We recently performed systematic structure-activity relationship studies of the peptidic natural product belactosin A and identified non-peptidic derivative 2 as a highly potent proteasome inhibitor. However, the cell growth inhibitory effect of 2 is only moderate, probably due to the biologically unstable β-lactone warhead. Peptide epoxyketones are an important class of proteasome inhibitors exhibit high potency in cellular systems based on the efficient α,β-epoxyketone warhead. Importantly, belactosin derivatives bind primarily to the primed binding site, while peptide epoxyketones bind only to the non-primed binding site of proteasome, suggesting that hybridization of them might lead to the development of a new class of proteasome inhibitors. Thus, we successfully identified a novel chemotype of proteasome inhibitors 3 and 4 by rational structure-based design, which are expected to bind to both the primed and non-primed binding sites of proteasome.

Full text of DOI:10.1016/j.bmc.2014.04.032

Bioorganic & Medicinal Chemistry 22 (2014) 3091–3095
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a new class of proteasome inhibitors with
an epoxyketone warhead: Rational hybridization of non-peptidic
belactosin derivatives and peptide epoxyketones
Shuhei Kawamura a, Yuka Unno c, Akira Asai c, Mitsuhiro Arisawa d, Satoshi Shuto a,b,
⇑
a Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
b Center for Research and Education on Drug Discovery, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
c Graduate School of Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
d Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteasome inhibitors are currently a focus of increased attention as anticancer drug candidates. We
recently performed systematic structure–activity relationship studies of the peptidic natural product bel-
actosin A and identified non-peptidic derivative 2 as a highly potent proteasome inhibitor. However, the
cell growth inhibitory effect of 2 is only moderate, probably due to the biologically unstable b-lactone
warhead. Peptide epoxyketones are an important class of proteasome inhibitors exhibit high potency
Received 27 March 2014
Revised 15 April 2014
Accepted 15 April 2014
Available online 24 April 2014
in cellular systems based on the efficient a,b-epoxyketone warhead. Importantly, belactosin derivatives
Keywords:
bind primarily to the primed binding site, while peptide epoxyketones bind only to the non-primed bind-
ing site of proteasome, suggesting that hybridization of them might lead to the development of a new
class of proteasome inhibitors. Thus, we successfully identified a novel chemotype of proteasome inhib-
itors 3 and 4 by rational structure-based design, which are expected to bind to both the primed and non-
primed binding sites of proteasome.
Proteasome inhibitor
Epoxyketone
Belactosin A
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Belactosin A, comprising
pyl)- -alanine (trans-3,4-methano-
boxy-b-lactone moiety, is naturally occurring tripeptide
L
-alanine, 3-(trans-2-aminocyclopro-
L
L
-ornithine), and a chiral car-
The major pathway for systematic degradation of intracellular
proteins is the ubiquitin–proteasome system,1 which is involved
in many physiologically important cellular processes, such as sig-
nal transduction,2 immune responses,3 the unfolded protein
response (UPR),4 and cell cycle progression.5 Proteasome inhibition
causes cell cycle arrest and induces apoptosis, making proteasomes
attractive target molecules for drugs to fight cancer and autoim-
mune disorders.6 In fact, the US Food and Drug Administration
recently approved the proteasome inhibitors bortezomib and car-
filzomib (Fig. 1) for the treatment of multiple myeloma,7 and sev-
eral other proteasome inhibitors are currently in clinical trials.8
a
metabolite produced by Streptomyces sp. (Fig. 1),9 It inhibits pro-
teasome chymotrypsin-like (ChT-L) activity10 by acylating the
active site Thr residue via its strained b-lactone-opening, as con-
firmed by X-ray crystallographic analysis of belactosin derivatives
in complex with proteasome (Fig. 2a).11 Importantly, belactosin A
and its derivatives are the only known proteasome inhibitors that
bind to both the primed and non-primed substrate binding sites of
proteasome11 and they are thus attractive lead compounds for the
development of unique proteasome inhibitors.
As shown in Figure 3, we performed systematic structure–activ-
ity relationship studies of belactosin A and developed the highly
potent derivative 1.11a Furthermore, by the topology-based scaffold
hopping of 1 based on our structure–activity relationship (SAR) and
binding-mode analyses results,12 we identified significantly simpli-
fied non-peptide inhibitor 2.13 Despite its significant proteasome
inhibitory activity, however, the cell growth inhibitory effects were
only moderate. We hypothesized that these contradictory results
were due to its unstable b-lactone warhead under biological
Abbreviations: AMC, aminomethylcoumarin; Boc, t-butoxycarbonyl; ChT-L,
chymotrypsin-like; DIEA, N,N-diisopropylethylamine; HBTU, N,N,N0,N0-tetra-
methyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HOBt, 1-hydroxy-
benzotriazole; Piv, pivaloyl; Suc, succinyl; TFA, trifluoroacetic acid; UPR, unfolded
protein response.
⇑
Corresponding author. Tel./fax: +81 11 706 3769.
E-mail address: shu@pharm.hokudai.ac.jp (S. Shuto).
http://dx.doi.org/10.1016/j.bmc.2014.04.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3092
S. Kawamura et al. / Bioorg. Med. Chem. 22 (2014) 3091–3095
O
H
N
N
O
H2N
N
H
H
N
N
OH
O
CO2H
O
N
H
B
O
O
OH
belactosin A
ChT-L: IC50 = 1440 nM
bortezomib
systematic SAR studies
O
O
H
N
O
multi-chiral
N
O
N
peptidic scaffold
N
N
H
H
O
O
O
O
H
OH
H
O
N
N
O
O
epoxomicin
H
O
O
O
H
H
O
N
N
N
N
H
N
H
1
O
O
O
O
ChT-L: IC50 = 5.7 nM
HCT116 cell growth: IC50 = 1.82 µM
topology-based
scaffold hopping
carfilzomib
O
H
N
achiral non-peptidic
scaffold
H2N
N
H
O
CO2H
O
O
O
O
belactosin A
Figure 1. Known proteasome inhibitors.
N
H
O
O
O
conditions,14 and therefore, planned to replace it with a more stable
and irreversible warhead to develop potent cell growth inhibitors.
2
Peptide proteasome inhibitors with an
a,b-epoxyketone as the
ChT-L: IC50 = 29 nM
HCT116 cell growth: IC50 > 10 µM
reactive warhead comprise one of the most extensively studied
classes of proteasome inhibitors, which inhibit proteasome cova-
lently by forming a morpholino adduct (Fig. 2b).6a,15 This class of
proteasome inhibitors binds only to the non-primed binding site
of the proteasome and exhibits remarkable cell growth inhibitory
effects, as represented by the clinical drug carfilzomib.16,14 We
planned to produce a new class of non peptidic proteasome inhib-
itors by replacing the b-lactone moiety of the non-peptidic belact-
Figure 3. Belactosin A derivatives previously developed by us.
2. Results and discussion
2.1. Design of compounds
Figure 4 is a superposition of the two X-ray crystal structures of
proteasome in complex with epoxomicin,17 a peptidic proteasome
osin A derivatives with an a,b-epoxyketone residue. These hybrid
compounds were expected to bind to both the primed and non-
primed binding sites of proteasomes like belactosin A and to exhi-
bit potent cell growth inhibitory effects like carfilzomib due to the
inhibitor with an
a,b-epoxyketone, and belactosin A derivative
1,11a which clearly shows that epoxomicin binds to the non-primed
binding site and its P2 side-chain is directed to the vacant primed
binding site of proteasome. Therefore, we assumed that elongating
a,b-epoxyketone warhead. Here, we describe the design, synthesis,
and biological activity of this new class of proteasome inhibitors.
Thr
(a)
O
O
Thr
O
O
O
O
N
H
HO
NH2
HO
O
NH2
O
HN
belactosin derivatives
proteasome
covalent complex
Thr
Thr
(b)
O
O
Thr
O
O
O
O
N
HO
HO
HO
NH2
NH
H
O
NH2
NH
covalent complex
O
NH
OH
peptide epoxyketones
proteasome
hemiacetal intermediate
Figure 2. Inhibitory mechanism of covalent proteasome inhibitors: (a) belactosin derivatives (b) peptide epoxyketones.

S. Kawamura et al. / Bioorg. Med. Chem. 22 (2014) 3091–3095
3093
non-primed
binding site
primed
binding site
P3 Ile P2 Thr
S1
S1
O
O
Thr
O
O
O
HO
P1
O
P1
non-primed
binding site
NH
OH
NH2
Thr
O
H
N
non-primed
binding site
NH
HO
primed
binding site
primed
binding site
OH
P2
HN
O
S3
P3
S3
HN
O
NH
epoxomicin
1
NH
O
N
O
O
P4
O
S4
S4
Figure 4. Superposition of the X-ray crystal structures of epoxomicin (gray tube; PDB code, 1G65)17 and belactosin A derivative 1 (green tube; PDB code, 4J70)11a in complex
with proteasome.
the P2 side-chain would allow us to design unique hybrid com-
pounds of the peptide epoxyketones and the non-peptide belacto-
sin A derivatives that could bind to both the primed and non-
primed biding sites. Although all of the peptide epoxyketone pro-
teasome inhibitors known to date have a P3 side-chain accommo-
dated in the proteasome S3 pocket,18 we postulated that
interactions with the primed binding site would compensate for
the lack of interaction with the S3 pocket, and thus designed
hybrid compounds 3 and 4 without a P3 side-chain, as shown in
Figure 5.
To confirm the molecular design, we performed computational
simulations to predict binding modes of the designed hybrids 3
and 4. As shown in Figure 6, in the simulated binding mode, the
moiety derived from the non-peptidic belactosin A derivative
ligated with the P2 side-chain is effectively accommodated in the
primed binding site, as expected. Therefore, we synthesized these
molecules and evaluated their biological effects.
2.2. Synthesis
Designed compounds 3 and 4 were synthesized as shown in
Scheme 1. Boc-Asp-OBn 5 was condensed with 613 or 719 by a
mixed anhydride method to yield compounds 8 or 9, respectively.
The Boc group of 8 and 9 was removed by treatment with TFA/CH2-
Cl2, and subsequent acetylation of the products with acetic anhy-
dride gave 10 and 11, respectively. The Bn group of 10 and 11
was removed under hydrogen transfer conditions using 1,4-cyclo-
hexadiene, and the resulting carboxylic acids were subsequently
condensed with the known epoxyketone unit prepared by depro-
tection of 1220 to yield the target compounds 3 and 4, respectively.
O
O
O
HN
O
O
O
N
N
H
O
HN
O
O
O
HN
O
design of hybrids
O
O
N
H
O
NH
3
O
O
O
OH HN
O
O
HN
O
O
N
H
O
HN
O
2
epoxomicin
O
4
Figure 5. Design of hybrids of non-peptide belactosin A derivative and peptide epoxyketone.

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S. Kawamura et al. / Bioorg. Med. Chem. 22 (2014) 3091–3095
(a)
S1
O
O
HO
P1
O
NH
Thr
non-primed
binding site
non-primed
binding site
NH
O
primed
binding site
O
OH
primed
binding site
S3
HN
N
H
O
O
3
S4
O
(b)
S1
O
HO
P1
O
NH
Thr
non-primed
binding site
non-primed
binding site
NH
O
primed
binding site
O
OH
primed
binding site
S3
HN
N
H
O
4
S4
Figure 6. Computationally predicted binding mode of compounds 3 (a) and 4 (b).
O
NH2
n
O
O
6
: n = 1
7 : n = 0
O
O
Boc
O
O
HN
HN
O
O
Boc
O
HN
a
b or c
d,e
O
O
N
H
N
O
n
n
HO
H
OBn
OBn
OBn
5
8
10
: n = 1
: n = 1
9 : n = 0
11 : n = 0
f
O
O
O
HN
g
O
N
H
n
O
HN
O
d
O
Boc
O
N
H
O
3 : n = 1
4
12
: n = 0
Scheme 1. Synthesis of target compounds 3 and 4. Reagents and conditions: (a) PivCl, Et3N, CH2Cl2, 0 °C to rt; (b) 6, Et3N, CH2Cl2, 0 °C to rt; (c) 7, Et3N, CH2Cl2, 0 °C to rt; (d)
TFA, CH2Cl2; (e) Ac2O, Et3N, CH2Cl2, 0 °C to rt; (f) 1,4-cyclohexadiene, Pd/C, EtOH; (g) HBTU, HOBt, DIEA, THF, ꢀ5 °C, 60% for 3 (6 steps), 60% for 4 (6 steps).
2.3. Pharmacological effects
We also evaluated the cell growth inhibitory effects of these
compounds on HCT116 cells, and the results are summarized in
Table 1. Compounds 3 and 4 showed definite cell growth inhibitory
The inhibitory effects of compounds on the ChT-L activity of
human 20S proteasomes were evaluated using the chromophoric
substrate Suc-LLVY-AMC. As summarized in Table 1, compounds
effects with an IC50 value of 1.9 lM and 2.9 lM, respectively, in
spite of the lack of their P3 side-chain, which suggests that the
interactions with the proteasome primed binding site compen-
sated for the lack of the interactions with proteasome S3 pocket.
3 and 4 inhibited ChT-L activity with an IC50 of 2.3
lM and
5.7 M, respectively, despite the absence of a P3 side-chain.
l

S. Kawamura et al. / Bioorg. Med. Chem. 22 (2014) 3091–3095
3095
Table 1
Inhibitory effects of 3 and 4 on proteasome ChT-L activity and HCT116 cell growth
O
O
O
O
HN
O
HN
O
O
O
N
H
N
H
HN
O
HN
O
O
O
O
3
4
a
Compound number
IC50
[lM]
ChT-L activity
HCT116 cell growth
3
4
2.3 0.1
5.7 0.4
1.9
2.9
a
Based on three experiments.
Interestingly, compound 4 with one hydrophobic moiety showed
proteasome inhibitory activity comparable to that of compound 3
with two hydrophobic moieties. These results are consistent with
our previous results on the hybrid compounds of peptide boro-
nates and non-peptide belactosin derivatives, in which the contri-
bution of the hydrophobic interaction due to the moieties derived
from non-peptidic belactosin derivatives to their proteasome
inhibitory activity were robustly shown.19 To our knowledge, these
are the first peptide epoxyketone proteasome inhibitors without a
P3 side-chain. Importantly, as we expected, the cell growth inhib-
itory effects were comparable to the proteasome inhibition effects,
in stark contrast with the effects of their parent compound 2 that
has a biologically unstable b-lactone warhead, of which HCT116
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Supplementary data
Supplementary data (experimental details of synthesis, biologi-
cal evaluations, computational simulations, and a table listing
combustion analysis data for target compounds) associated with
this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2014.04.032.
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