Retro hydrazino-azapeptoids as peptidomimetics of proteasome inhibitors

Article abstract of DOI:10.1021/jm049455f

Several groups of proteasome inhibitors are widely used to study the role of the ubiquin proteasome pathway in various cellular processes or as anticancer drugs. Peptidomimetics have been developed to circumvent problems inherent in peptides such as poor

Full text of DOI:10.1021/jm049455f

330
J. Med. Chem. 2005, 48, 330-334
Brief Articles
Retro Hydrazino-azapeptoids as Peptidomimetics of Proteasome Inhibitors
Sandrine Aubin,† Be´ne´dicte Martin,‡ Jean-Guy Delcros,‡ Yannick Arlot-Bonnemains,‡ and
Miche`le Baudy-Floc’h*,†
Laboratoire de Synthe`se et Electrosynthe`se Organiques (SESO), UMR CNRS 6510, Universite´ Rennes 1,
Av. du Ge´ne´ral Leclerc, F-35042 Rennes Ce´dex, France, and Groupe Cycle Cellulaire, UMR CNRS 6061 Ge´ne´tique et
De´veloppement, IFR 97 Ge´nomique Fonctionnelle et Sante´, Faculte´ de Me´decine, Universite´ Rennes 1,
2 Av. du Professeur Le´on Bernard, F-35043 Rennes Ce´dex, France
Received July 9, 2004
Several groups of proteasome inhibitors are widely used to study the role of the ubiquin
proteasome pathway in various cellular processes or as anticancer drugs. Peptidomimetics have
been developed to circumvent problems inherent in peptides such as poor bioavailability and
protease-mediated degradation, while retaining biological activity. In this study, we introduce
new pseudopeptides, the retro hydrazino-azapeptoids, designed as proteasome inhibitor
peptidomimetics. Their proteasome inhibitory activity and antiproliferative properties are
reported here.
Introduction
The ubiquitin proteasome pathway constitutes the
most important system for protein degradation in eu-
karyotic cells.1 It prevents the accumulation of nonfunc-
tional, potentially toxic proteins and allows elimination
of normal proteins that are no longer required. This
pathway depends on the central role of a complex
macromolecular structure, the 26S proteasome, which
is composed of one 20S and two 19S subunits. The 20S
proteasome exhibits multiple proteolytic activities with
strict specificities. Three major proteolytic activities can
Figure 1. Synthetic peptide aldehyde and boronic acid
be distinguished: trypsin-like (T-L), chymotrypsin-like
proteasome inhibitors.
(CT-L), and peptidyl-glutamyl peptide hydrolase (PGPH)
activities which cleave peptide bonds on the carboxyl
of the different sites. Selective inhibitors of the protea-
side of basic, hydrophobic, and acidic amino acid resi-
some are also important tools to study proteasome
dues, respectively.2 These proteolytic activities rely on
function in cells. Because proteasome malfunctions are
an unusual mechanism involving the N-terminal threo-
often associated with diseases, proteasome inhibitors
nine residue of particular â-subunits as the catalytic
constitute a novel class of therapeutic agents. These
nucleophile.3 The proton donor/acceptor role in this
inhibitors have a potential for treating cancer and
catalytic process may be performed either by a con-
neurodegenerative diseases and can also be used to
served lysine residue or a water molecule bonded to the
prevent cancer- or AIDS-associated muscle cachexia.6
Peptide aldehyde inhibitors (e.g. ALLN and MG132,
Figure 1) are still commonly used but they are relatively
nonspecific.7,8 Furthermore, due to the acidity of the
R-proton, the substituent adjacent to the aldehyde is not
configurationally stable. More recently, peptidyl boronic
acids (e.g. PS-341, Figure 1) have been shown to be
stable and highly potent inhibitors of the proteasome.9,10
Nevertheless, the activity of proteasome peptide-
based inhibitors is often hampered by poor bioavailabil-
ity and protease-mediated degradation. The desire to
remedy these disadvantages, while retaining the bio-
logical activity of the peptide, has led to the development
of peptidomimetics. A particular interesting class of
peptidomimetics is formed by peptoids11-14 and ana-
logues such as azapeptides and azapeptoids,15-21 urea-
peptoids,22,23 amino-oxypeptoids,24 â-peptoids,25 and
R-amino group of the N-terminal threonine.4
The exact substrate specificity of the different cata-
lytic sites is still not well understood. Although the
distinct activities are generally defined by the amino
acid in the P1 position of a synthetic peptide substrate,
the specificity determinants go well beyond the P1
position. In fact, the catalytic sites must overlap in
specificity to some extent since there are more activities
described than there are different catalytic â-subunits.5
Therefore, characterization of the effects of novel pro-
teasome inhibitors is useful in unraveling the specificity
* To whom correspondence should be addressed. Phone: 33 (0)2
23 23 69 33. Fax: 33 (0)2 23 23 67 38. E-mail: michele.baudy-floch@
univ-rennes1.fr.
† Laboratoire de Synthe`se et Electrosynthe`se Organiques.
‡ Groupe Cycle Cellulaire.
10.1021/jm049455f CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/24/2004

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 331
Table 1. Inhibitory Effect on 20S Proteasome CT-L Activity
and on Cell Growth
IC50 (µM)
inhibition of
proteasome
CT-L activity
cell growth inhibition
B16
Figure 2. General structure of hydrazino azapeptoids.
compd
ALLN
L1210
Mel-A
ND
Scheme 1a
15
3.0 ( 0.01
14.5 ( 2.6
PS-341 Ki ) 0.62 nM9,10
5a
>1000
>1000
>1000
>1000
>1000
>1000
>1000
50
>1000
>1000
91
53
>1000
350
6.5 ( 2.2
1.3 ( 0.8
0.6 ( 0.2
24.1 ( 2.9
30.0 ( 2.6
45.0 ( 5.5
7.6 ( 0.6
16.2 ( 2.3
27.9 ( 3.1
18.4 ( 1.8
21.2(0.6
16.2 ( 1.5
16.9 ( 0.6
41.6 ( 2.3
75.0 ( 7.8
1.3 ( 0.05
1.4 ( 0.1
0.7 ( 0.02
11.4 ( 5.0
>80
23.4 ( 0.4
2.5 ( 0.1
26.3 ( 0.9
65.5 ( 1.2
33.5 ( 1.2
53.9 ( 1.6
20.4 ( 0.8
20.5 ( 0.8
21.1 ( 0.9
74.9 ( 1.5
>80
>80
32.6 ( 0.6
2.2 ( 1.1
3.3 ( 0.4
1.8 ( 0.7
1.5 ( 0.2
14.8 ( 1.3
15.8 ( 1.9
33.3 ( 0.6
2.3 ( 1.6
17.3 ( 0.4
45.1 ( 1.1
25.1 ( 1.9
nd
18.4 ( 0.6
19.9 ( 0.8
40.3 ( 1.2
60.9 ( 2.8
16.3 ( 3.9
33.7 ( 6.0
39.3 ( 2.5
4.5 ( 0.4
5b
5f
5h
6b
6h
a Reagents and conditions: (i) BrCH2COBr, C5H5N/CH2Cl2,
9a
0 °C; (ii) PG1NHNHR (1b), CHCl3, ∆, 24 h.
10a
10b
10c
11a
11b
11c
11d
11e
11g
11h
12b
14
hydrazino-azapeptoids,26 in which the side-chains are
linked to nitrogen atoms.
If the carbonyl group contributes to the binding of the
target molecule, the peptide bonds and the order of the
amino acids have to be reversed (“retro sequence”) in a
peptoid peptidomimetic to maintain the relative orien-
tation of the carbonyl groups to the R groups. This
improved mimicry of the parent peptide may be respon-
sible for the higher biological activity of retropeptoids.27
The hydrazino-azapeptoids, which are formed from NR-
substituted acid hydrazino-acetic monomers or aza-â3-
amino acid units (aza-â3) and aza-amino acid unit (aza)
or N-substituted azaglycine (Naza) monomers are hy-
brid pseudopeptides with nitrogen-enriched peptidic
backbones.26 As shown in Figure 2, side chains are
attached to the nitrogen atom of the analogue monomer
instead of the R-carbon atom of the amino acid monomer
in peptides. Spectroscopic studies and X-ray crystal-
lography indicated that hydrazino-azapeptoids are ca-
pable of adopting eight-membered, hydrogen-bonded
turns (N-N turns).28 In previous work, we have shown
that modified C-terminal hydrazino-azapeptoids are
potent inhibitors of cell proliferation.29 As part of an
effort to explore new peptoid analogues with potentially
useful biological properties, a series of retro hydrazino-
azapeptoids, which contain NHNRCO bonds instead of
CONRNH bonds, was synthesized and screened for
proteasome inhibition and cell cytotoxicity. In short,
these materials were designed to mimic the amino acid
derived system in Figure 1 and were expected to have
similar biological activity.
>1000
500
>1000
>1000
>1000
>80
>80
14.7 ( 3.9
5.0 ( 0.4
Table 2. Inhibitory Effect of Selected Compounds on the
Activity of Calpain I and Cathepsin B
IC50 (µM)
compd
calpain I
cathepsin B
ALLN
10a
1
>1000
>1000
12
>1000
>1000
11b
position as an electrophilic group to give irreversible
interaction with the nucleophilic threonine in position
1 of the proteasome. Analogues 5, 9, and 14 were
obtained by acylation of the terminal hydrazine of 4 or
8. Taking account of our previous work,29 we introduce
a bromo acetyl group in N-terminal position to give
target inhibitors 5, 9, and 14. Analogues such as 6 were
obtained by condensation of substituted hydrazine 4
with 3-bromobenzaldehyde followed by in situ reduction
of the hydrazone.
Dipeptidyl boronic acids are new potent proteasome
inhibitors.6,31 Boronic acids act as transition-state ana-
logues for serine proteases because the boron can accept
the oxygen lone pair of the active site serine residue. It
seems likely that these compounds react similarly with
the catalytic N-terminal threonine residue of the pro-
teasome catalytic subunits.32,33 For these reasons, we
also synthesized analogues 10, 11, and 12 with a boronic
acid in the N-terminal position by condensation of the
substituted hydrazine 4 with o, m, or p-formylben-
zeneboronic acid.
Biological Evaluation. The synthesized hydrazino-
azapeptoids were screened for proteasome inhibition
and for cytotoxicity in L1210, B16, and Mel-A cells
(Table 1).
Proteasome Inhibition. Among all the hydrazino-
azapeptoids studied, only the boronic acid derivatives
10a, 11a, 11b, 11d, and 11g, displayed an inhibitory
effect on the proteolytic activity of the 20S proteasome.
With IC50s ranging from 50 to 500 µM, their ability to
inhibit the proteasome CT-L activity remains lower than
that of ALLN (IC50 ) 15 µM), which was used as a
control. Like all the other compounds, these boronic acid
Results and Discussion
Synthesis. Initially, we examined the submonomer
synthesis of hydrazino-azapeptoids backbones of 3 which
have been already described (Scheme 1).29 Finally, we
adopted a monomer strategy, which consisted of cou-
pling aza-â3-amino acids 2 and aza or N-aza amino
esters 1 (Scheme 2). Aza-â3-amino acids 2 were prepared
as previously described,30 by nucleophilic substitution
of tert-butyl or benzyl bromoacetates with N,N′-disub-
stituted hydrazines (or N-aza amino esters) 1b, or by
reductive amination of glyoxylic acid and N-aza amino
acid 1b. In addition, dipeptoidic analogues 3 were
obtained by a coupling reaction. Tripeptoid analogues
(e.g., 7) and diaza-â3 peptides (13b) were synthesized
by the coupling of two readily accessible monomers.
Hybrid pseudopeptoids 3 and 7 and the oligomers 13
were deprotected under standard conditions. Halo-
methyl ketone was introduced in the free N-terminal

332 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Scheme 2. Synthesis of Hydrazino Azapeptoids 11, 12, 14a
Brief Articles
a Reagents and conditions: (a) BrCH2CO2Bn, K2CO3, toluene, ∆, 24 h; H2 (1 atm), 5% Pd/C (b) OHCCO2H, methanol, NaBH3CN, HCl
(2 N); (c) HNR1NR2PG, EDCI, CH2Cl2, rt, 48 h; (d) aza-â3aa-OPG, EDCI, CH2Cl2, rt, 48 h; (e) CF3CO2H/CH2Cl2; (f) BrCH2COBr, C5H5N/
CH2Cl2, 0 °C; (g) HCOC6H4Br, methanol, NaBH3CN, HCl (2 N), (h) HCOC6H4B(OH)2, Et2O, rt.
derivatives had no inhibitory effect (up to 1 mM) on
either PGPH or T-L activity of the proteasome. Never-
theless, the active boronic acid derivatives were more
specific than ALLN, which inhibited the PGPH activity
(IC50 ) 45 µM). As shown in Table 2, the compounds
10a and 10b were also more selective than ALLN and
did not inhibit cathepsin B and calpain I, two cysteine
proteases which were very sensitive to ALLN.
Cell Growth Inhibition/Cytotoxicity. Independent
of their ability to inhibit proteasome, the effect of all
synthesized compounds on cell growth was tested on
murine leukemia L1210, melanoma B16, and normal
melanocyte Mel-A cells using the MTT assay which
reveals either a cytostatic or a cytotoxic effect of drugs
on cultured cells. Most of the compounds showed similar
Figure 3. Endogenous proteasome CT-L activity in B16 cells
cultured 24 h in the presence of 6h, 11b, and ALLN. Insert:
IC50s on the three cell lines. With the exception of 5h,
all bromoacetylated compounds (5a, 5b, 5f, 9a, 14)
showed low IC50s (from 0.6 to 7.6 µM) which could be
related to the high reactivity of their functional group.
In contrast, compounds with bromophenyl or a boronic
acid group were less effective with IC50s ranging from
15 to 75 µM. 10a and 11b, which are the most potent
proteasome inhibitors, had IC50s around 20 µM on the
three cell lines while ALLN was more active with IC50s
of 3 µM on L1210 and 14.5 µM on B16 cells. None of
the compounds displayed any marked selectivity for B16
versus their normal counterpart Mel-A. Surprisingly,
the retro analogues 11g and 11h, as well as the
Correlation between proteasome and cell growth inhibitions
in B16 cells treated with various concentrations of 11b.
bromophenyl derivative 6b, were more active on normal
melanocyte than on B16 melanoma cells.
Relation between Cell Growth and Proteasome
Inhibition. We wondered if the proteasome is an in vivo
target for the hydrazino-azapeptoids with in vitro pro-
teasome inhibitory activity. The activity of endogenous
proteasome was measured, using a fluorescent sub-
strate, in lysates of B16 cells cultured in the presence
of either 11b (10 to 40 µM), 6h (20 and 40 µM), or ALLN
(50 µM). As shown in Figure 3, endogenous proteasome

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 333
azaLeu-OBz (10a, R ) R1 ) CH2CH(CH3)2, R2 ) H). To a
stirred solution of 4a (1.8 g, 5 mmol) in diethyl ether (5 mL)
was added dropwise 2-formylbenzenzeboronic acid (0.8 g, 5.5
mmol) in a solution of diethyl ether (5 mL). A white precipitate
appeared. After stirring for 1 h, the precipitate was filtered
and washed with diethyl ether to give 10a (1.70 g, 82%): mp
142 °C.1H NMR (CDCl3) δ 0.91 (d, J ) 6.0 Hz, 6H, CH3), 1.12
(d, J ) 6.2 Hz, 6H, CH3), 1.86 (m, 1H, CH), 2.15 (m, 1H, CH),
3.31 (br, 2H, CH2), 3.81 (br, 2H, CH2), 4.50 (br, 2H, CH2), 5.40
(s, 2H, CH2), 7.01 (t, J ) 7.0 Hz, 1H, CH), 7.52 (m, 5H, C6H5),
8.21 (d, J ) 7.5 Hz, 1H, CH), 9.30 (s, 1H, NH), 11.26 (s, 2H,
OH). Anal. (C25H35N4O5B) C, H, N.
activity was decreased in a dose-dependent manner in
B16 cells treated with 11b, a hydrazino-azapeptoid with
in vitro proteasome inhibitory activity. In addition, a
linear correlation (r ) 0.975) was observed between cell
growth and endogenous proteasome activity inhibitions
(see insert of Figure 3).
As expected, ALLN-treated B16 cells also displayed
a reduced proteasome activity. In contrast, 6h, a com-
pound with an antiproliferative activity similar to 11b
but devoid of any in vitro proteasome inhibitory activity,
did not induce any decrease in endogenous proteasome
activity even at a dose equivalent to 2 times its IC50.
These results suggest that the proteasome inhibitor 11b
may promote cell growth inhibition through inhibition
of endogenous proteasome.
m-B(OH)2PhCH)-aza-â3-Phe-N-azaLeu-OBz (11b R )
CH2C6H5, R1 ) CH2CH(CH3)2, R2 ) H). To a stirred solution
of 4b (1.92 g, 5 mmol) in diethyl ether (5 mL) was added
dropwise 3-formylbenzenzeboronic acid (0.8 g, 5.5 mmol) in a
solution of diethyl ether (5 mL). A white precipitate appeared.
After stirring for 1 h, the precipitate was filtered and washed
1
with diethyl ether to give 11b (2.11 g, 82%): mp 157 °C; H
Conclusion
NMR (DMSO-d6 δ 0.92 (m, 6H, CH3), 1.92 (m, 1H, CH), 3.35
(br, 2H, CH2), 4.23 (br, 2H, CH2), 4.69 (s, 2H, CH2), 5.23 (s,
2H, CH2), 7.23 (s, 1H, CH), 7.40 (m, 12H, C6H5), 7.64 (t, J )
6.0 Hz, 1H, CH), 7.70 (d, J ) 7.1 Hz, 1H, CH), 7.85 (s, 1H,
CH), 8.13 (s, 2H, NH + OH), 10.1 (s, 1H, OH); Anal.
(C28H33N4O5B) C, H, N, B.
The present study is, to our knowledge, the first
attempt to synthesize and evaluate the biological activ-
ity of peptidomimetics of known proteasome inhibitors.
The more active compounds were boronic acid deriva-
tives (e.g. 11b) that displayed both in vitro and in vivo
proteasome inhibitory activity. Despite a high selectivity
and specificity, their proteasome inhibitory activity is
very low compared to PS-341, the reference boronic acid
inhibitor which is active in the nanomolar range. In the
present hydrazino-azapeptoid series, the boronic acid is
attached to a phenyl group. This structural feature
likely causes a steric hindrance which could impair the
binding of the compound into the active site and could
be responsible for their low affinity for the proteasome.
These observations will be taken into consideration in
the design of more active peptidomimetics.
Acknowledgment. This work was supported by the
“Ligue Nationale Contre le Cancer” (LNCC), the “As-
sociation pour la Recherche sur le Cancer” (ARC) and
the “Conseil Re´gional de Bretagne”.
Supporting Information Available: Synthesis and
analytical data for 1-14 and biological techniques. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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