Discovery of carboranes as inducers of 20S proteasome activity

Article abstract of DOI:10.1002/cmdc.201000112

(Chemical Equation Presented) The carborane framework gives analogues able to induce 20S proteasome activities. A series of ortho-carboranylphenoxy derivatives were synthesized as 20S proteasome agonists, and carborane derivatives 11 a and 11 b were found

Full text of DOI:10.1002/cmdc.201000112

MED
DOI: 10.1002/cmdc.201000112
Discovery of Carboranes as Inducers of 20S Proteasome Activity
Hyun Seung Ban, Hidemitsu Minegishi, Kazuki Shimizu, Minako Maruyama, Yuka Yasui, and
Hiroyuki Nakamura*[a]
In eukaryotes, proteasomes play a critical role in the degrada-
tion of most intracellular proteins.[1–4] Degradation by ubiqui-
tin-proteasome pathway controls many fundamental cellular
functions. These include cell differentiation, cell-cycle regula-
tion, signal transduction pathways, antigen processing in
immune responses, stress signaling, inflammatory responses,
and apoptosis. The first step in the pathway is activation of
ubiquitin-activating enzyme (E1), followed by ubiquitin transfer
to an ubiquitin-conjugating enzyme (E2). The second step is
transfer of ubiquitin from E2 to the substrate by ubiquitin
ligase (E3). The polyubiquitinated protein is then degraded by
a 26S proteasome.[5] The 26S proteasome comprises of a 20S
proteasome cylinder capped by a regulatory 19S complex at
each extremity. The 20S proteasome has three major proteolyt-
ic activities: caspase-like (b1), trypsin-like (b2), and chymotryp-
sin-like (b5).[6] The 19S complex (PA700) activates proteasome
degradation of ubiquitin-conjugated proteins. In addition to
PA700, 20S proteasome can separately interact with other reg-
ulatory complexes such as PA28[7] or PA200.[8] Both complexes
can activate the 20S proteasome using peptide substrates.
Many natural and synthetic inhibitors of the proteasome
have been developed, such as peptide aldehydes, peptide ep-
oxyketones, peptide vinyl sulfones, lactacystins, and peptide
boronates.[9–11] The peptide boronate PS-341 (bortezomib), an
inhibitor of the chymotrypsin-like activity of 20S proteasome,
has been approved for the treatment of multiple myelo-
ma.[12–14] Small-molecule proteasome activators have been de-
veloped to a lesser extent. Several biological lipids, including
sodium dodecyl sulfate (SDS),[15] fatty acids,[16] ceramide sul-
fates,[17] lysophosphatidylinositol,[18] and cardiolipin[19] have
been observed to activate the 20S proteasome. In general, pro-
teasomal degradation of synthetic substrates by these mole-
cules is more variable but not remarkable. Dramatic activation
of proteasome by peptide-based activators has been investi-
gated.[20] Recently, betulinic acid (BA) has attracted attention
for its anti-HIV-1 and antitumor activities.[21] BA and its dime-
thylsuccinyl derivative (DSB) were found to be preferential acti-
vators of the b5 and b1 activities of the 20S proteasome, re-
spectively.[22]
a hydrophobic pharmacophore in biologically active molecules
that interact hydrophobically with target proteins.[23–25] In this
study, we discovered that ortho-carborane derivatives have the
potential to induce the b1, b2, and b5 activities of the 20S pro-
teasome in the absence of PA28.
The synthesis of ortho-carboranyl phenol[23] and its ester de-
rivatives is shown in Scheme 1. Benzylation was performed by
treating 4-iodophenol (1) with benzyl bromide in the presence
of K2CO3. The resulting benzyl ether 2 underwent the Sonoga-
shira coupling reaction with ethynyltrimethylsilane in the pres-
ence of catalysts, PdCl2(PPh3)2 and CuI. The reaction was per-
formed in a sealed vial under microwave-irradiated (1208C).
The reaction reached completion within 25 min to give the
corresponding coupling product in 98% yield. The TMS group
was removed by treatment with LiOH in aqueous THF solution
to afford alkyne 3 in quantitative yield. The reaction of alkyne
3 with decaborane proceeded in the presence of acetonitrile
(5 equiv) in toluene at reflux to give the corresponding carbor-
ane 4 in 25% yield. The benzyl group was removed by hydro-
Carboranes (dicarba-closo-dodecaboranes; C2B10H12) exhibit
remarkable thermal stability, and their icosahedral geometry
and exceptional hydrophobic property may allow their use as
[a] Dr. H. S. Ban, H. Minegishi, K. Shimizu, M. Maruyama, Y. Yasui,
Prof. Dr. H. Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University
1-5-1, Mejiro, Toshima-ku, Tokyo, 171-8588 (Japan)
Fax: (+81)3-5992-1029
Scheme 1. Reagents and conditions: a) BnBr, K2CO3, DMF, RT, 95%; b) ethynyl-
trimethylsilane, PdCl2(PPh3)2, CuI, DIEA, microwave (1208C), 98%;
c) LiOH·H2O, THF/H2O (1:1), >99%; d) B10H14, CH3CN, toluene, reflux, 25%;
e) H2, Pd/C, CH3OH, >99%; f) Ac2O or BzCl, DMAP, Py, CH2Cl2, 6a: 97%, 6b:
33%.
E-mail: hiroyuki.nakamura@gakushuin.ac.jp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000112.
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ChemMedChem 2010, 5, 1236 – 1241

genolysis to afford 5 in quantitative yield. Acetate 6a and ben-
zoate 6b were obtained from 5 in 97% and 33% yields, re-
spectively.
borane coupling gave the carborane benzyl ester 12, which
was treated under hydrogenation conditions to give carboxylic
acid 13. The phenoxy ester derivative 14 was then synthesized
from carboxylic acid 13 using N,N’-dicyclohexylcarbodiimide
(DCC) coupling with phenol in 23% yield.
The synthesis of ortho-carboranyl phenoxyacetic acid 13 and
its derivatives 8, 11 a–b, 12, and 14 is shown in Scheme 2. The
reaction of 4-iodophenol (1) with ethyl chloroacetate proceed-
The effects of the prepared carboranes 5, 6a–b, 8, 11–14,
ortho-carborane and BA on the caspase-like (b1), trypsin-like
(b2) and chymotrypsin-like (b5) activities of the 20S protea-
some were evaluated using fluorogenic substrates Z-LLE-AMC,
Boc-LRR-AMC, and Suc-LLVY-AMC, respectively. PA28 was used
as a positive control for comparison. The results are summar-
ized in Table 1. Although ortho-carborane, carboranylphenol 5,
and its acetate 6a did not affect b1, b2, and b5 activities of
the 20S proteasome at 10 mm, significant induction of b5 activ-
ity was observed for compound 6b (358%) along with slight
induction of b1 (176%) and b2 (160%) activities. Conversely,
compounds 11 a and 11 b significantly induced b1 (350%) and
b2 (373%) activities, respectively.
The profiles of these compounds toward the b5 activity of
the 20S proteasome and HeLa cell growth inhibition were dif-
ferent. Compound 11 a weakly induced b2 (205%) and inhibit-
ed b5 (87.6%) activities at 10 mm without inhibiting cell
growth (GI50 ꢀ100 mm), while compound 11 b significantly in-
hibited b5 activity (4.5%) at the same concentration, and its
GI50 value was 20.3 mm. The phenoxy ester derivative 14
showed weak biological activity compared to compound 6b,
and significant induction of b1 and b2 activities was not ob-
served.
Furthermore, we tested the adamantine derivative 15, which
is considered to be a mimic of the carborane derivative 11 b.
Compound 15 also induced b1 (149%) and b5 (192%) activi-
ties of the 20S proteasome without affecting b2 (114%) activity
at 10 mm. Under the assay conditions used here, BA showed
significant, but nonselective, induction of b1 (526%), b2
(206%), and b5 (128%) activities. This is in contrast to the se-
lective activation of b5 was reported by Chen and co-work-
ers.[22] These differing results may be caused by variations in
the assay conditions. Because the detailed assay conditions
were not described in their report, the current assay was car-
ried out based on the protocol reported by Asai and co-work-
ers.[26] Regarding the 20S proteasome activities induced by
PA28, the results described here are consistent with reported
results.[22]
Scheme 2. Reagents and conditions: a) ClCH2CO2Et, K2CO3, DMF, RT, >99%;
b) ethynyltrimethylsilane, PdCl2(PPh3)2, CuI, Et3N, microwave (1208C), 91%;
c) TBAF, THF, RT, 60%; d) B10H14, CH3CN, toluene, reflux, 48%; e) LiOH·H2O,
THF/H2O (1:1), >99%; f) aniline or 3-aminobenzophenone, EDCI, HOBt,
DIPEA, DMF, 10a: quant; 10b: 70%; g) B10H14, CH3CN, toluene, reflux, 11 a:
61%, 11 b: 36%; h) BnCl, Na2CO3, DMF, 97%; i) B10H14, CH3CN, toluene, reflux,
49%; j) H2, Pd/C, CH3OH, 45%; k) phenol, DCC, DMAP, CH2Cl2, 23%.
ed quantitatively by treatment with K2CO3 in DMF. The result-
ing ester underwent the Sonogashira coupling reaction with
ethynyltrimethylsilane in the presence of PdCl2(PPh3)2 and CuI
under microwave irradiation to give 7 in 97% yield. Compound
7 was treated with tetrabutylammonium fluoride to remove
the TMS group. The resulting acetylene was reacted with deca-
borane, under the same conditions used to synthesize carbor-
ane 4, to give the corresponding carborane 8 in 48% yield.
When compound 7 was treated with LiOH in aqueous THF so-
lution, deprotection of both TMS and ester groups occurred to
afford carboxylic acid 9 in quantitative yield. Carboxylic acid 9
was reacted with aniline or 3-benzoylaniline to give the ani-
lides 10a and 10b, respectively, which were then treated with
decaborane under the established conditions to give the corre-
sponding carboranes 11 a and 11 b in 61% and 36% yields, re-
spectively. The protection of carboxylic acid 9 with benzyl chlo-
ride in the presence of sodium bicarbonate followed by deca-
We next investigated the dose- and time-dependent nature
of the effects of compounds 11 a and 11 b on proteasome ac-
tivity. As shown in Figure 1, compound 11 a increased b1 and
b2 activities and slightly inhibited b5 activity of the 20S protea-
some at 10 and 30 mm (Figure 1). For compound 11 b, b1 and
b2 activities were increased and b5 activity was significantly in-
hibited at 10 mm (Figure 2).
According to the results obtained in this study, both carbor-
anylphenoxy and phenyl moieties are important structural fea-
tures for activation of the 20S proteasome. The ester deriva-
tives 6b and 14 showed selective induction of b5, whereas the
amide derivatives 11 a and 11 b preferentially activated b1 and
b2, respectively. The adamantine derivative 15 only weakly in-
duced the b1 and b5 activities of the 20S proteasome, sug-
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column chromatography (silica gel;
hexane/EtOAc, 10:1) to give the
title compound as a white solid
(1.47 g, 4.8 mmol, 96%): 1H NMR
(400 MHz, CDCl3): d=7.57 (2H, d,
J=8.8 Hz), 6.69 (2H, d, J=9.2 Hz),
4.59 (2H, s), 4.27 (2H, q, J=7.2 Hz),
1.29 ppm (3H, t, J=7.2 Hz);
13C NMR (100 MHz, CDCl3): d=
168.3, 157.6, 138.2, 116.9, 83.9,
65.2, 61.3, 14.1 ppm; MS (ESI+):
m/z 328.9 [M+Na]+.
Table 1. The effect of carboranes 5, 6, 8, 11–14 and adamantine 15 on caspase-like (b1), trypsin-like (b2), and
chymotrypsin-like (b5) activities of the 20S proteasome[a] and growth inhibition of HeLa cells.[b]
Compound[c]
b1 [%]
b2 [%]
b5 [%]
GI50 [mm]
o-carborane
138Æ6.4
102Æ5.6
94.0Æ1.8 103Æ7.8
>100
R=H
R=Ac
R=COPh
5
76.0Æ0.8 124Æ1.9
95.2Æ0.1 106Æ4.4
17.1Æ3.7
>100
21.9Æ1.4
6a 116 Æ18.6
6b 176Æ1.8
160Æ2.5
358Æ57.9
Ethyl 2-(4-(2-(trimethylsilyl)ethy-
nyl)phenoxy)acetate (7): Trimeth-
ylacetylene (3.4 mL, 2.4 mmol) was
added to a solution of 2-(4-iodo-
phenoxy)acetate (8 mmol), PdCl2-
(PPh3)2 (110 mg, 0.16 mmol), CuI
(76 mg, 0.4 mmol), and Et3N
(8.1 mL, 78 mmol) in DMF under Ar.
The mixture was heated by micro-
wave irradiation to 1208C for
25 min. The solvent was removed
in vacuo, and the residue was dis-
solved in EtOAc. The mixture was
washed with aq HCl (2n), neutral-
ized with saturated aq NaHCO3,
washed with brine, dried over
anhyd MgSO4, filtered and concen-
trated in vacuo. Purification by
column chromatography (silica gel;
R=OEt
R=OCH2Ph
R=OH
R=OPh
R=NHPh
R=
8
12
106Æ12.7
73.7Æ1.2
91.5Æ0.8
56.2Æ1.9
23.4Æ0.1
67.1Æ20.9 81.1Æ0.5 133Æ41.4
13 111 Æ7.3
14
97.1Æ17.3 70.0Æ3.8 145Æ8.5
11 a 350Æ28.4 205Æ26.5 87.6Æ3.5 >100
102Æ7.0
124Æ7.2
>100
85.7Æ12.1
11 b 181Æ7.7
373Æ49.8
4.5Æ1.0
20.3Æ0.8
15 149Æ15.5 114Æ11.8 192Æ15.8 >100
PA28
Betulinic acid
526Æ4.3
206Æ2.7
128Æ6.0
N.D.
15.9Æ0.4
609.5Æ24.0 522.8Æ24.9 545.2Æ39.6
[a] Human 20S proteasome activities were measured using specific fluorophore-tagged peptides (Z-LLE-AMC
for b1; Boc-LRR-AMC for b2;, Suc-LLVY-AMC for b5). [b] HeLa cells were incubated for 72 h with various concen-
trations (100 nm–100 mm) of compounds, and viable cells were determined by MTT assay. The drug concentra-
tion required to inhibit cell growth by 50% (GI50) was determined from semi-logarithmic dose–response plots,
and results represent the mean ÆSD of triplicate samples. [c] Proteasome activities were measured with com-
pounds (10 mm), PA28 (500 ng), or betulinic acid (10 mm).
100% hexane) gave
7.32 mmol, 91%):
7
(2.02 g,
1H NMR
(400 MHz, CDCl3): d=7.40 (2H, d,
J=9.2 Hz), 6.82 (2H, d, J=8.8 Hz),
4.61 (2H, s), 4.27 (2H, q, J=7.2 Hz),
1.29 (3H, t, J=7.2 Hz), 0.23 ppm
gesting that a carborane framework is more suitable for induc-
(9H, s); 13C NMR (72 MHz, CDCl3): d=168.5, 157.8, 133.5, 116.4,
114.5, 104.7, 92.9, 65.3, 61.4, 14.1 ppm; MS (ESI+): m/z 299.0
[M+Na]+, 315.0 [M+K]+.
tion of the 20S proteasome activities.
In conclusion, carborane derivatives 6b, 11 a, and 11 b were
found to be potent inducers of b5, b1, and b2 activities of the
20S proteasome, respectively. The carborane framework is es-
sential for induction of the 20S proteasome activities. Since
small-molecule proteasome activators have not been devel-
oped, the current compounds represent potential chemical
probes for the investigation of proteasome-dependent degra-
dation pathways.
2-(4-Ethynylphenoxy) acetic acid (9): A solution of 7 (1.98 g,
7.2 mmol) in THF/H2O (1:1; 40 mL) was treated with LiOH·H2O
(1.21 g, 28.8 mmol) at RT overnight. After neutralization of the reac-
tion with aq HCl (1n), the mixture was extracted with EtOAc,
washed with brine, dried over anhyd MgSO4, filtered and concen-
trated in vacuo. Purification by column chromatography (silica gel;
1
100% EtOAc) gave 9 (379 mg, 1.8 mol, quant): H NMR (400 MHz,
CDCl3): d=7.45 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 4.69 (2H,
s), 3.01 ppm (1H, s); 13C NMR (72 MHz, CD3OD): d=172.3, 159.7,
134.4, 116.6, 115.7, 84.1, 77.3, 65.7 ppm; MS (ESIÀ): m/z 175.9
[MÀNa]À.
Experimental Section
Synthesis
2-(4-Ethynylphenoxy)-N-phenylacetamide (10a): A DMF solution
of 9 (100 mg, 0.57 mmol), aniline (0.08 mL, 0.85 mmol), EDCI
(164 mg, 0.85 mmol), HOBt (131 mg, 0.86 mmol), and DIPEA
(0.146 mL, 8.56 mmol) was stirred at RT overnight. The reaction
was quenched with H2O, extracted with EtOAc, washed with brine,
dried over anhyd MgSO4, filtered and concentrated in vacuo. Purifi-
cation by column chromatography (silica gel; hexane/EtOAc, 3:1)
The experimental procedure for the synthesis of compound 11 a
from 4-iodophenol (1) is reported below. Spectral data of all tested
compounds 5, 6a–b, 8, 12–14, and 15 are described in the Sup-
porting Information.
Ethyl 2-(4-iodophenoxy)acetate: Ethyl chloroacetate (0.54 mL,
5.1 mmol) was added to a mixture of 1 (1.1 g, 5 mmol) and K2CO3
(2.1 g, 15 mmol) in DMF. The mixture was stirred at RT overnight,
and the reaction was quenched with H2O. The mixture was extract-
ed with EtOAc, washed with brine, dried over anhyd MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by
1
gave 10a (143 mg, 0.57 mmol, quant) as a colored solid: H NMR
(400 MHz, CDCl3): d=8.21 (1H, br s), 7.57 (2H, d, J=7.6 Hz), 7.48
(2H, d, J=8.8 Hz), 7.35 (2H, t, J=7.6, 8.4), 7.16 (1H, t, J=7.6, 7.6),
6.94 (2H, d, J=6.8), 4.60 (2H, s), 3.04 ppm (1H, s); 13C NMR
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(100 MHz, CDCl3): d=165.7, 157.1,
136.6, 133.9, 129.1, 125.0, 120.1,
116.2, 114.8, 82.9, 76.7, 67.5 ppm;
MS (ESI+): m/z 328.9 [M+Na]+.
2-(4-Carboranylphenoxy)-N-phe-
nylacetamide
(0.17 mL, 3.3 mmol) was added to
mixture of 10a (140 mg,
0.57 mmol) and decaborane
(11a):
CH3CN
a
(77 mg, 0.63 mmol) in toluene
under Ar. The mixture was refluxed
overnight. After the solvent was
removed in vacuo, the residue was
purified by column chromatogra-
phy (silica gel; hexane/EtOAc, 50:1)
to give 11 a as
a white solid
(130 mg, 0.35 mmol, 61%): Rf =
0.27 (n-hexane/EtOAc, 3:1); mp:
139–1408C; 1H NMR (400 MHz,
CDCl3): d=8.13 (1H, br s), 7.57
(2H, d, J=8.8 Hz), 7.49 (2H, d, J=
6.8 Hz), 7.37 (2H, t, J=8.4, 8.4),
7.17 (1H, t, J=8.8, 7.2), 6.94 (2H,
d, J=7.2), 4.61 (2H, s), 3.89 ppm
(1H, br s); 13C NMR (100 MHz,
CDCl3): d=165.4, 158.0, 136.6,
129.6, 129.1, 127.5, 125.1, 120.2,
114.9, 76.1, 67.6, 60.7 ppm; IR
(KBr): 3749.4, 3672.2, 3649.1,
3390.6, 3336.6, 3062.7, 2920.0,
2592.2, 1685.7, 1600.8, 1535.2,
1512.1, 1446.5, 1361.7, 1296.1,
1253.6, 1188.1, 1076.2, 1026.1,
906.5, 883.3, 837.0 cmÀ1
; HRMS
(ESIÀ): m/z [MÀH]À calcd for
C16H23B10NO2: 368.2654, found:
368.2653.
Biology
Cytotoxicity assays: The human cer-
vical carcinoma cell line, HeLa, was
used for the cell growth assays.
Cells (5ꢁ103 cells per well of a 96-
well plate) were incubated at 378C
for 72 h in 100 mL RPMI-1640
medium containing various con-
centrations of test compounds.
After incubation, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium
bromide (10 mL, 5 mgmLÀ1 in PBS)
was added to each well and cells
were further incubated at 378C for
4 h. After removal of the medium,
DMSO (100 mL) was added and ab-
sorbance at 595 nm was deter-
mined using a 96-well plate reader.
Activity of the 20S proteasome: The
three peptidase activities (b1, b2,
and b5) of the 20S proteasome
were assayed with specific fluoro-
phore-tagged peptides (Z-LLE-AMC
Figure 1. Effects of compound 11 a on a) trypsin-like activity, b) caspase-like activity, and c) chymotrypsin-like ac-
tivity of the 20S proteasome. The fluorescence of specific fluorophore-tagged peptides (Z-LLE-AMC for b1; Boc-
*
LRR-AMC for b2; Suc-LLVY-AMC for b5) in the absence of 20S proteasome were measured as a control (none, ).
Florescence: excitation, 365 nm; detection, 460 nm.
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for b1; Boc-LRR-AMC for b2; Suc-
LLVY-AMC for b5). Briefly, com-
pounds were mixed with 50 ng of
the 20S proteasome isolated from
human erythrocytes in assay buffer
(20 mm Tris (pH 8.0), 0.5 mm EDTA,
and 0.035% SDS). Hydrolysis was
initiated by the addition of 70 mm
of substrate. After incubation for
2 h at 378C, the reaction was
quenched with stop buffer (20 mm
Tris (pH 8.9) and 0.5% SDS). Fluo-
rescence of AMC cleaved from
substrates by each enzymatic ac-
tivity was measured (excitation,
365 nm; detection, 460 nm). Pro-
teasome–activator protein com-
plex PA28, composed of two ho-
mologous subunits: PA28a and
PA28b (Boston Biochem, Inc.,
Boston, USA), was used as a con-
trol experiment.
Acknowledgements
This work was supported in part
by a Grant-in-Aid for Scientific Re-
search on Priority Areas Cancer
Therapy from the Ministry of Edu-
cation, Culture, Sports, Science,
and Technology (Japan).
Keywords: 20S proteasome
·
activators · caspase-like activity ·
drug discovery
activity
·
trypsin-like
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Published online on May 11, 2010
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