Synthesis and biological evaluation of boron peptide analogues of Belactosin C as proteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.103

A series of boron peptides 11, 13, 15 and 17 were designed and synthesized as proteasome inhibitors based on the structure of Belactosin C. Matteson homologation was a key step in the synthesis of the boron peptides. Compounds 11a and 13 showed significant inhibition of 20S proteasome chymotrypsin-like (β5) activity (IC₅₀ = 0.28 and 0.51 μM, respectively). Furthermore, like PS-341, compound 11a increased the G2/M cell distribution. A biparametric cytofluorimetric analysis with FITC-labeled annexin V and propidium iodide showed induction of apoptosis by compound 11a at >1 μM concentrations of compound.

Full text of DOI:10.1016/j.bmcl.2009.04.103

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3220–3224
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of boron peptide analogues of Belactosin
C as proteasome inhibitors
a
a
a
b
Hiroyuki Nakamura a, , Mizuyoshi Watanabe , Hyun Seung Ban , Wataru Nabeyama , Akira Asai
*
a Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan
b Center of Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of boron peptides 11, 13, 15 and 17 were designed and synthesized as proteasome inhibitors
based on the structure of Belactosin C. Matteson homologation was a key step in the synthesis of the
boron peptides. Compounds 11a and 13 showed significant inhibition of 20S proteasome chymotryp-
Received 12 February 2009
Revised 20 April 2009
Accepted 22 April 2009
Available online 3 May 2009
sin-like (b5) activity (IC50 = 0.28 and 0.51
lM, respectively). Furthermore, like PS-341, compound 11a
increased the G2/M cell distribution. A biparametric cytofluorimetric analysis with FITC-labeled annexin
V and propidium iodide showed induction of apoptosis by compound 11a at >1 lM concentrations of
compound.
Keywords:
Proteasome
Boron peptides
Chymotrypsin-like activity
Ó 2009 Elsevier Ltd. All rights reserved.
The ubiquitin–proteasome pathway plays a critical role in the
degradation of a majority of cellular proteins in eukaryotes.1–4
The first step in the pathway is activation by ubiquitin activating
enzyme (E1) followed by ubiquitin transfer to a ubiquitin-conju-
gating enzyme (E2). The second step involves specific binding of
E2 conjugated with substrate to ubiquitin ligase (E3). The poly-
ubiquitinated protein is then degraded by a 26S proteasome.5
According to the X-ray crystal structures, the 26S proteasome con-
sists of the 20S proteasome cylinder capped by a regulatory 19S
complex at each extremity.6 The 20S proteasome has three princi-
ple catalytic activities known as the caspase-like (b1), trypsin-like
(b2) and chymotrypsin-like (b5) activities. The hydroxy group of
the N-terminal threonine in each active site reacts as a nucleophile
to initiate amide bond cleavage of the substrate.7,8
has been approved for the clinical treatment of patients with mul-
tiple myeloma.12,13 The X-ray co-crystal structure revealed that PS-
341 binds to the N-terminal threonine residues of three catalytic
active sites, b1, b2 and b5, at high compound concentrations
through a B–O covalent linkage, which generates an sp3 hybridized
orbital complex.5 Epoxomicin reacts primarily with the chymo-
trypsin-like active site and the X-ray co-crystal structure showed
a six-membered morpholine ring formed by the N-terminal threo-
nine and epoxomicin, which results in irreversible inhibition.14
NLVS has been developed as a vinyl sulfone analogue of MG-132.
It seems likely that the hydroxyl group of the N-terminal threonine
residues reacts with the double bond of the vinyl sulfone moiety by
Michael addition.15
In 2000, Belactosin A and C were isolated from a Streptomyces
sp. by Asai and co-workers as shown in Scheme 1.16 They found
that Belactosin A and C showed in vitro antiproliferative activity
The ubiquitin–proteasome pathway regulates levels of proteins
critical for cell cycle control, including p53, p27 and cyclin B. Fur-
thermore, the proteasome degrades I
j
B
a
to generate the activated
B has
against HeLa S3 cells with IC50 values of 51 and 200 lM, respec-
transcription factor NF- B. Uncontrolled regulation of NF-
j
j
tively, after 72 h exposure. Furthermore, both Belactosin A and C
similarly inhibited the chymotrypsin-like activity of rabbit 20S
proteasome.17 Here, we focus on the b-lactone moiety of the
belactosins. The inhibitory potency of belactosins toward the pro-
teasome appears to be due to the b-lactone moiety, which under-
goes a nucleophilic ring opening by the hydroxyl group of the
N-terminal threonine residue to form a covalent bond.18 We rea-
soned that replacement of the b-lactone moiety with boronic acid
would give candidate analogues of belactosins that act reversibly.
In this Letter, we report the synthesis and biological evaluation
of boron peptide analogues of Belactosin C.
been linked to cancer, inflammatory disease and immune system
dysfunction.8,9 Therefore, development of proteasome inhibitors
is considered an attractive approach for new therapeutic agents.10
Many natural and synthetic inhibitors of the proteasome, such
as peptide aldehyde (MG-132), peptide boronate (PS-341, Bortezo-
mib), peptide epoxyketone (Epoxomicin), and peptide vinylsulfone
(NLVS), have been developed as shown in Figure 1.11 MG-132,
which was one of the first synthetic inhibitors, interacts reversibly
with the N-terminal threonine residue of the b5 active site.9 PS-341
The boron peptides can be constructed from three components:
1, 2 and 3. The components 1 are N-Boc protected glycine (1a),
* Corresponding author. Tel.: +81 339860221; fax: +81 359921029.
E-mail address: hiroyuki.nakamura@gakushuin.ac.jp (H. Nakamura).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.103

H. Nakamura et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3220–3224
reversible inhibitors
3221
N
N
O
O
O
H
N
H
N
OH
N
H
B
O
N
H
N
H
CHO
O
OH
O
MG-132
PS-341
irreversible inhibitors
I
HO
O
O
H
N
O
O
O
H
N
N
H
N
H
N
O2N
N
H
N
H
S
O
O
O
O
O
OH
O
Epoxomicin
NLVS
Figure 1. Structures of proteasome inhibitors.
O
O
H
H
N
N
H2N
N
H2N
N
H
H
O
CO2H
O
O
CO2H
O
O
O
M
Belactosin A
Belactosin C
β
μ
5: IC50 = 0.21
O
R1
O
H
N
OH
O
N
N
B
n
H
H
O
CONHR2
OH
Boron peptides
n = 1~3
O
R1
O
n
OH
H2N
OH
+
+
O
N
OBn
H2N
HCl
B
H
O
CO2H
OH
1a: R1 = H
b: R1 = CH3
c: R1 = i-Bu
d: R1 = Bn
2a: n = 0
b: n = 1
c: n = 2
3
Scheme 1. Design and retro synthesis of boron peptides as Belactosin analogues.
Figure 2. Flow cytometer analysis of cell cycle and apoptosis.

3222
H. Nakamura et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3220–3224
alanine (1b), leucine (1c) and phenylalanine (1d). Components 2
were prepared by selective benzyl esterification of -aspartic acid
(2a), -glutamic acid (2b), and -2-aminoadipic acid (2c). Compo-
O
R1
R1
O
O
a
L
O
OH
O
N
N
O
b
N
L
L
H
H
O
O
nent 3 was synthesized from isobutyl boronic acid 4 as shown in
O
Scheme 2.19 Matteson homologation20 of the pinanediol ester 5
1a-d
7a-d
gave the a-chloro boronic ester 6 in 81% yield with >98% diastere-
O
R1
O
H
N
oselectivity. Treatment of 6 with lithium hexamethyldisilazide
afforded the corresponding silylated amino boronic ester 7, which
was treated with hydrochloric acid at À78 °C to give aminoboronic
ester 3 as the HCl salt. Because of its moisture sensitivity, com-
pound 3 was used without further purification.
O
N
OBn
H
O
O
NH
8a-d
We next synthesized the boron peptides (n = 2) from the com-
ponents 1a–d and benzyl L-glutamate 2b as shown in Scheme 3.
O
R1
O
c
d
e
H
N
Carboxylic acids 1a–d were activated with N-hydroxysuccinimide
and the resulting esters 7a–d were treated with 2b to afford the
corresponding dipeptides. The dipeptides were again activated
with N-hydroxysuccinimide and the resulting esters were treated
with 2-naphtylmethylamine under basic conditions to give 8a–d
in 36–51% yield. Hydrogenation of 8a–d proceeded in the presence
of 10% Pd/C under an atmosphere of hydrogen. The corresponding
carboxylic acids 9a–d were activated by treatment with isobutyl
chloroformate (IBCF) using N-methylmorpholine (NMM) as a base
and the resulting acid anhydrides were reacted with the aminobo-
ronic ester 3 to give the corresponding boron esters 10a–d. Finally,
hydrolysis of pinanediol esters 10a–d was carried out by two-
phase transesterification with isobutyl boronic acid under acidic
conditions to afford 11a–d in 18–25% yield. In a similar manner,
the benzylamide derivative 13 was also synthesized from 7a
(Scheme 4).
O
N
H
OH
O
O
NH
9a-d
O
R1
O
H
N
O
O
N
N
H
B
H
O
O
O
NH
10a-d
R1
O
Boron peptides derived from L-2-aminoadipic acid 2c were also
O
H
N
synthesized. Scheme 5 shows the synthesis of 15a and 15b from
the activated esters 7a and 7b. We also synthesized compounds
17a–c, which have a Boc group at the terminal end of component
2 instead of component 1, as shown in Scheme 6.
OH
O
N
H
N
H
B
O
OH
O
NH
We first examined cell growth inhibition of the various com-
pounds using the HeLa cell line. The cells were incubated with dif-
ferent concentrations of each compound for 72 h. Cell viability was
then determined using the MTT assay. As shown in Table 1, com-
pound 11a displayed significant cell growth inhibition (IC50 value
11a-d
Scheme 3. Reagents and conditions: (a) HOSu, DCC, dioxane, 45–99%; (b) (i) 2b,
KHCO3 aq, THF, rt; (ii) HOSu, DCC, dioxane; (iii) 2-naphtylmethylamine, aqueous
KHCO3, THF, rt, 36–51%; (c) H2, 10% Pd/C, EtOH, rt, 74–78%; (d) (i) IBCF, NMM, THF,
À20 °C; (ii) 3, TEA, CHCl3, À20 °C, 16–37%; (e) i-BuB(OH)2, 1 N HCl aq, MeOH/
hexane, rt, 18–25%.
was 0.35
growth inhibition (0.67–5.11
significant inhibitory activity even at a concentration of 10
l
M). Although 11b–d, 13 and 15b gave moderate cell
M), 15a and 17a–c did not display
M.
l
l
Next, we examined in vitro inhibition of the chymotrypsin-like,
caspase-like and trypsin-like activities of the 20S proteasome
according to the literature protocol.17 Briefly, compounds were
was quenched with stop buffer [20 mM Tris (pH 8.9), 0.5% SDS].
Fluorescence of AMC cleaved from substrates by the proteolytic
activity was then measured (365 nm excitation/460 nm detection).
The results are shown in Table 1. The boron peptides selectively
inhibited the chymotrypsin-like activity (b5) of 20S proteasome.
Among the compounds synthesized, compound 11a possessed
the greatest inhibition of the chymotrypsin-like activity, although
the IC50 value was 10-fold higher than that of PS-341. The substi-
tuent at the R1 position (compounds 11b–d) did not increase inhib-
mixed with 75 lM of a fluorogenic substrate Z-LLE-AMC (cas-
pase-like), Boc-LRR-AMC (trypsin-like), or Suc-LLVY-AMC (chymo-
trypsin-like) in assay buffer [20 mM Tris (pH 8.0), 0.5 mM EDTA,
0.035% SDS] in a 96-well plate. Hydrolysis was initiated by the
addition of 50 ng of 20S proteasome isolated and purified from hu-
man erythrocytes. After incubation at 37 °C for 1 h, the reaction
a
b
O
OH
O
B
Cl
B
B
O
O
OH
4
5
6
HCl
H2N
c
d
O
O
(TMS)2N
B
B
O
O
7
3
Scheme 2. Reagents and conditions: (a) (+)-pinanediol, Et2O, 97%; (b) (i) n-BuLi, CH2Cl2, THF, À100 °C; (ii) ZnCl2, À100 °C, 81%; (c) LiN(TMS)2, THF, À78 °C; (d) 1 N HCl in Et2O.

H. Nakamura et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3220–3224
3223
Table 1
O
O
a,b
H
Cell-growth and proteasome inhibitions of boron peptides
N
7a
O
N
H
OH
a
Compound
IC50
b5c
(lM)
O
Cell growth inhibitionb
b1c
b2c
O
NH
12
11a
11b
11c
11d
13
15a
15b
17a
17b
17c
MG-132
PS-341
0.35 0.02
2.35 0.59
0.79 0.03
0.66 0.09
5.11 0.31
>10
>10
>10
>10
0.28 0.04
0.84 0.04
1.50 0.42
1.47 0.30
0.54 0.14
2.74 0.32
4.28 0.08
4.51 1.31
>10
8.54 1.97
9.84 1.17
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
O
O
c,d
H
N
OH
O
N
N
B
H
H
O
OH
O
NH
>10
4.12 0.81
0.07 0.03
0.02 0.01
>10
7.23 1.16
0.68 0.09
NDd
0.02 0.01
13
a
Concentration required to inhibit cell growth or proteasome activity by 50%.
The values are mean sd of triplicate samples.
Scheme 4. Reagents and conditions: (a) (i) 2b, KHCO3 aq, THF, rt; (ii) HOSu, DCC,
dioxane; (iii) benzylamine, aqueous KHCO3, THF, rt, 44%; (b) H2, 10% Pd/C, EtOH, rt,
41%; (c) (i) IBCF, NMM, THF, À20 °C; (ii) 3, TEA, CHCl3, À20 °C, 45%; (d) i-BuB(OH)2,
1 N HCl aq, MeOH/hexane, rt, 15%.
b
Growth inhibition in HeLa cells determined by MTT assay.
Inhibition of chymotrypsin-like (b5), caspase-like (b1) and trypsin-like (b2)
c
activity of human 20S proteasome.
d
Not determined.
O
R1
O
2
a, b
H
N
framework, such as compounds 15a and 15b (n = 3), and without
component 1 (i.e., compounds 17a–c) were poor inhibitors in both
the cell growth and proteasome assays.
It has been reported that proteasome inhibitors induce cell cy-
cle arrest and apoptosis.21 Therefore we carried out flow cytometry
analysis using compounds 11a and 13.22,23 As shown in Table 2,
compound 11a increased the G2/M cell distribution in a similar
manner to that of PS-341. Furthermore, a biparametric cytofluori-
metric analysis with FITC-labeled annexin V and propidium iodide
7a b
-
O
N
H
OH
O
O
NH
1
14a
b
: R = H
: R = CH3
1
O
R1
O
2
c
H
N
(PI) (Fig. 2) revealed that compound 11a (1–10
lM) and compound
OH
O
N
H
N
H
B
13 (10 M) induces apoptosis of HeLa cells in over 10% of the pop-
l
O
OH
ulation. These results indicate that the inhibition of cell growth by
boron peptides is mediated by induction of G2/M cell cycle arrest
and apoptosis.
O
NH
15a-b
In conclusion, we have designed a new class of boron peptides
as inhibitors of the chymotrypsin-like activity of the 20S protea-
some based around the structure of Belactosin C. Inhibitory activity
is dependent on the length of the boron peptide. Compounds 11a
and 13 were found to possess significant inhibition of 20S protea-
some chymotrypsin-like (b5) activity, and inhibitory potency of
both compounds is similar to that of Belactosin C. Furthermore,
compound 11a increased the G2/M cell distribution in a similar
Scheme 5. Reagents and conditions: (a) (i) 2c, KHCO3 aq, THF, rt; (ii) HOSu, DCC,
dioxane; (iii) 2-naphtylmethylamine, aqueous KHCO3, THF, rt, 37–64%; (b) H2, 10%
Pd/C, EtOH, rt, 74–77%; (c) (i) IBCF, NMM, THF, À20 °C, (ii) 3, TEA, CHCl3, À20 °C,
33%; (d) i-BuB(OH)2, 1 N HCl aq, MeOH/hexane, rt, 25–43%.
O
H
N
a, b
O
2a-c
OH
manner to that of PS-341, and induced apoptosis at >1 lM concen-
n
tration of compound in HeLa cells. The findings presented herein
represent a significant step towards the development of protea-
some inhibitors with therapeutic utility.
O
O
NH
16a-c
Table 2
O
n
c, d
H
Cell cycle arrest and apoptosis induction of boron peptides
O
N
OH
N
H
B
Compound
Cell cycle distributiona (%)
Apototic cellb (%)
O
OH
O
NH
G1
S
G2/M
None
56.4 0.39
57.7 0.02
23.0 0.82
56.2 1.32
43.2 0.01
14.6 1.03
30.0 0.46
16.4 0.71
14.4 0.42
30.7 0.75
30.7 1.73
15.9 0.55
13.7 0.87
26.0 0.73
62.6 0.31
13.1 0.53
26.0 1.76
69.6 0.47
2.6 0.14
12.9 1.47
15.2 1.69
7.6 1.59
11.7 0.28
14.5 3.56
17a: n = 0
b: n = 1
c: n = 2
11a
13
1 lM
10 lM
1
lM
10
0.1
l
lM
M
Scheme 6. Reagents and conditions: (a) Boc2O, TEA, THF/H2O, rt, 78–99%; (b) (i)
HOSu, DCC, dioxane, À5 °C; (ii) 2-naphtylmethylamine, aqueous KHCO3, THF, rt,
78–83%; (b) H2, 10% Pd/C, EtOH, rt, 79–97%; (c) (i) IBCF, NMM, THF, À20 °C; (ii) 3,
TEA, CHCl3, À20 °C, 16–44%; (d) i-BuB(OH)2, 1 N HCl aq, MeOH/hexane, rt, 30–44%.
PS-341
a
HeLa cells were incubated for 24 h with the indicated concentration of com-
pounds 11a, 13 or PS-341. The cells were harvested and stained with propidium
iodide (PI).
itory potency of chymotrypsin-like activity. Interestingly, the ben-
zylamide derivative 13 also showed effective inhibition of chymo-
trypsin-like activity, despite displaying only moderate growth
inhibition in the HeLa cell assay. The boron peptides with a longer
b
After incubation of 12 h with compounds 11a, 13 or PS-341, the cells were
stained with annexin V-FITC and PI. The effect on cell cycle and apoptosis were
determined by flow cytometry. Values are mean sd of samples determined in
triplicate.

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H. Nakamura et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3220–3224
14. Groll, M.; Kim, K. B.; Kairies, N.; Huber, R.; Crews, C. M. J. Am. Chem. Soc. 2000,
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23. Spectral data for lead compounds: Compound 11a: 1H NMR (400 MHz; CD3OD) d
7.71–7.62 (m, 4H), 7.35–7.28 (m, 3H), 4.48–4.36 (m, 3H), 3.62 (s, 2H), 2.52–
2.37 (m, 3H), 2.10 (br s, 3H), 1.90 (br s, 3H), 1.53–1.17 (m, 5H), 1.29 (s, 9H),
0.82–0.79 (m, 6H); 13C NMR (75 MHz, CD3OD) d 179.1, 173.1, 172.7, 158.7,
137.0, 134.8, 134.2, 129.3, 128.8, 128.6, 127.2, 126.9, 126.8, 126.7, 80.9, 53.6,
44.8, 44.2, 41.0, 28.6, 27.8, 27.0, 27.0, 23.9, 22.1; IR(KBr) 3274, 2931, 2362,
2331, 1652, 1508, 1488, 1419, 1363, 1168, cmÀ1; MS (ESI, positive) m/z 593
([M(OMe)+Na]+), 607 ([M(OMe)2+Na]+). Compound 13: 1H NMR (400 MHz;
CD3OD) d 7.22–7.11 (m, 5H), 4.50 (br s, 1H), 4.34–4.23 (m, 3H), 3.24–3.20 (m,
3H), 2.53 (br s, 1H), 2.38 (t, J = 7.8 Hz, 2H), 2.12 (br s, 1H), 1.89 (br s, 1H) 1.53
(br s, 1H), 1.32 (s, 9H), 1.26–1.16 (m, 2H), 0.83–0.80 (m, 6H); 13C NMR
(75 MHz, CD3OD) d 179.1, 173.0, 172.8, 158.7, 139.6, 129.6, 128.5, 128.2, 80.9,
53.5, 44.9, 44.1, 41.0, 28.7, 27.8, 27.0, 27.0, 23.8, 22.2; IR(KBr) 3735, 3676,
3649, 3303, 2929, 2869, 2343, 1652, 1508, 1498, 1436, 1367, 1168, 1126,
1029 cmÀ1; MS (ESI, positive) m/z 543 ([M(OMe)+Na]+), 557 ([M(OMe)2+Na]+).
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