Novel fluorinated pseudopeptides as proteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.012

We have designed novel small inhibitors of rabbit 20S proteasome using a trifluoromethyl-β-hydrazino acid scaffold. Structural variations influenced their inhibition of the three types of active sites. Proteasome inhibition at the micromolar level was sel

Full text of DOI:10.1016/j.bmcl.2008.11.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 83–86
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel fluorinated pseudopeptides as proteasome inhibitors
Lucia Formicola a, Xavier Maréchal b, Nicolas Basse b, Michelle Bouvier-Durand b, Danièle Bonnet-Delpon a,
Thierry Milcent a, Michèle Reboud-Ravaux b, Sandrine Ongeri a,
*
a UMR-CNRS 8076, Molécules Fluorées et Chimie Médicinale, IFR 141, Faculté de Pharmacie, Université Paris-Sud 11, 5, rue J.B. Clément, 92296 Châtenay-Malabry cedex, France
b Enzymologie Moléculaire et Fonctionnelle, FRE2852, CNRS-UPMC (Université Paris 6), IJM, T43, 2 Place Jussieu, 75251 Paris Cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed novel small inhibitors of rabbit 20S proteasome using a trifluoromethyl-b-hydrazino
acid scaffold. Structural variations influenced their inhibition of the three types of active sites. Protea-
some inhibition at the micromolar level was selective, calpain I and cathepsin B were not inhibited.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 3 October 2008
Revised 2 November 2008
Accepted 4 November 2008
Available online 7 November 2008
Keywords:
Proteasome inhibitors
Pseudopeptide
Fluorine
Trifluoromethyl hydrazino
The proteasome is a large, multisubunit, proteolytic complex
that progressively degrades ubiquitinylated proteins to small pep-
tides.1 The proteasome is composed of a 20S catalytic core and two
19S regulatory caps which are responsible for the recognition,
unfolding and translocation of protein substrates into the 20S cat-
alytic core cavity. The eukaryotic 20S proteasome is formed by four
stacked rings, and each of the two inner rings is composed of seven
different b subunits.2 The b1, b2 and b5 subunits contain the post-
acid (PA), trypsin-like (T-L) and chymotrypsin-like (CT-L) active
sites where peptide bonds are cleaved on the carboxyl side of
acidic, basic and hydrophobic amino acid residues, respectively.3
The ubiquitin–proteasome system is involved in the degradation
of regulatory proteins that are crucial for many intracellular pro-
drawbacks often associated with reactive groups, such as a lack
of specificity, excessive reactivity and instability.12 However, they
have been less extensively investigated. They include ritonavir,13
aminobenzylstatine derivatives,12 lipopeptides,14 macrocyclic15,16
or linear TMC-95 derivatives.17 This report describes the first rep-
resentatives of a new class of non-covalent 20S inhibitors based on
a central fluorinated pseudopeptide.
Fluorine has become a fundamental tool in the development of
drugs.18–22 Trifluoromethylated compounds are particularly
important, as shown by the number of CF3-containing drugs and
drug-candidates in clinical use or in development. The trifluoro-
methyl group is often used in medicinal chemistry to improve met-
abolic stability and/or biological activity.23 It is hydrophobic,
electron-rich, bulky and it can mimic functional groups such as
methyl, isopropyl and phenyl. Consequently, incorporating a tri-
fluoromethyl group into peptides and peptidomimetics can greatly
alter their structural properties and thus their ability to interact
with receptors or enzymes. The incorporation of a trifluoromethyl
group into peptidomimetics to produce potent inhibitors of various
enzymes has been extensively studied.24
cesses, including cell progression, apoptosis and NF-jB activation.
Proteasome inhibitors cause selective apoptosis of malignant cells
and are therefore a new class of antineoplastic agents.4 Bortezomib
(VelcadeÒ) has been approved for treating incurable multiple mye-
loma and mantle lymphoma.5 Carfilzomib (PR-171),6 salinospora-
mide A (NPI-0052)7 and CEP-187708 are in phases I and II clinical
trials. Most proteasome inhibitors, such as epoxyketones, peptide
aldehydes, peptide vinyl sulfones and peptide boronic acids, are
covalent binding inhibitors because they bear a reactive group that
c
forms a covalent bond with the catalytic O -Thr in the three cata-
lytic sites.9–11 Non-covalent inhibitors should have smaller side
effects in therapeutics because they should not have the inherent
R2
R1
CF3 O
H
N
N
H
N
H
O
1
* Corresponding author. Tel.: +33 1 46 83 57 37; fax: +33 1 46 83 57 40.
E-mail address: Sandrine.Ongeri@u-psud.fr (S. Ongeri).
Figure 1. New trifluoromethyl-b-hydrazino acid scaffold.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.012

84
L. Formicola et al. / Bioorg. Med. Chem. Lett. 19 (2009) 83–86
As part of ongoing investigations into the design of proteasome
Compounds 2–8 were prepared as outlined in Scheme 1. The
common intermediate 12 was obtained in four steps from ethyl
4,4,4-trifluorocrotonate 9.36 Michael addition of tert-butyl-carbaz-
ate 10 on 9 gave the N-protected trifluoromethyl-b-hydrazino es-
inhibitors and the development of new trifluoromethyl peptidom-
imetics,25–30 we have developed new trifluoromethyl inhibitors
based on the trifluoromethyl-b-hydrazino acid scaffold 1 (Fig. 1).
Retro hydrazino-azapeptoids were recently described as cova-
ter 11 which was deprotected and coupled to the L-phenylalanine
lent proteasome inhibitors with IC50s of 50À500
l
M,31 whereas
methyl ester. The cleavage of the Boc group of the hydrazine moi-
ety gave compound 12. This intermediate 12 was then used to ob-
tain compounds 2–8 using standard solution phase coupling
chemistry. A simple coupling with 3-phenoxyphenylacetic acid
gave compound 3. Coupling 12 with Fmoc-L-3,4-dimethoxyphenyl-
alanine, cleavage of the Fmoc group, and coupling with
hydrazinopeptides inhibited the serine protease leukocyte elas-
tase.32 b-Hydrazino acids are peptidomimetic building blocks that
have two nitrogen atoms. They can be considered to be analogs of
b-amino acids in which the amine group has been replaced by a
hydrazine. But while the b-amino acids are well documented,
33–35 almost nothing is known about these peptidomimetics. Obvi-
ously, these structures can mimic the typical secondary structure
3-phenoxyphenylacetic acid gave compound 2. Coupling 12 with
Na
-Boc-N
e
-Z-L-lysine gave compound 6, which was N
e-Z deprotec-
of native
a
-peptides, making them useful tools with which to de-
ted to give compound 7, or Na-Boc deprotected to give compound
sign new protease inhibitors. To our knowledge, the synthesis of
CF3-b-hydrazino acid has not been reported. This scaffold was cho-
sen because it has a trifluoromethyl group, which can greatly im-
prove the acidity of the neighbouring hydrazine functional group
and thus increase its hydrogen bond donor ability.22 The non-cova-
lent interactions of several peptidic and peptidomimetic protea-
some inhibitors are mainly mediated by hydrogen bonds
implicating in particular residues 21, 47 and 49 with the formation
of an antiparallel b-sheet between the inhibitor and the amino acid
residues of the binding pockets.9 Occupancy of the S1 and S3 sub-
sites appears to be essential for efficient inhibitor binding.9,12 The
elongation of the backbone may introduce flexibility, thus facilitat-
ing the putative interaction of substituents R1 and R2 with the
enzyme S1 and S3 pockets. Compounds 2–8 (Fig. 2) were designed
and synthesized. They have a phenylalanine amino acid at the
C-terminal end whose phenyl group is assumed to occupy the S1
pocket. The N-terminus of our starting molecule 2 was inspired
by aminobenzylstatine derivatives,12 which interact with the S3
pocket and the phenoxysubstituted benzylic N-terminal group
with the AS1 and AS2 accessory hydrophobic pockets. We intro-
duced several structural variations in the pseudopeptide N-termi-
nus in order to evaluate their influence on specific binding and
affinity for the three types of active sites.
8. Compound 8 was then coupled to 3-phenoxyphenylacetic acid to
give compound 4 which was N-deprotected to provide product 5.
The coupling of N-protected trifluoromethyl-b-hydrazino acid (ob-
tained from 11) to L-phenylalanine methyl ester gave a 1:1 mixture
of diastereoisomers that could not be separated by flash chroma-
tography or by crystallisation. All the compounds progressed then
as 1:1 diastereoisomer mixes (except for compound 2 which was
obtained with a diastereiosomeric ratio of 2/3, probably because
of the loss of one diastereiosomer during purification). The ratio
was evaluated by NMR 1H and 19F.
The capacities of molecules 2–8 (diastereoisomeric mixtures) to
inhibit the three activities of rabbit 20S proteasome were assayed
using appropriate fluorogenic substrates (Fig. 3).14,17,37 The
aldehyde proteasome inhibitor MG132 (Z-LLL-H) was used as
standard.17
Compound 2 inhibited the CT-L and PA activities of rabbit 20S
proteasome (IC50 = 85 and 72 lM, respectively) (Table 1). Shorten-
ing the pseudopeptide by connecting directly the phenoxy benzyl
moiety on the trifluoromethyl-b-hydrazino acid scaffold and elim-
inating the 3,4-dimethoxyphenylalanine totally removed the
capacity of molecule 3 to inhibit all three active sites (Table 1).
Replacing the 3,4-dimethoxyphenylalanine amino acid by the more
hydrophilic lysine amino acid could facilitate the interaction of the
NHCbz
O
O
O
O
CF3 O
O
CF3 O
H
N
H
N
6
7
O
2
3
O
O
N
H
N
H
N
H
N
H
N
H
N
H
O
O
O
O
NH2
CF3
O
H
N
O
O
CF3 O
N
H
N
H
O
O
O
O
N
N
N
O
H
H
H
O
O
NHCbz
H
NHCbz
O
O
CF3 O
CF3
O
H
8
N
N
O
4
5
N
O
N
H
N
H
H2N
N
H
N
H
H
O
O
O
O
NH2
O
O
CF3 O
H
N
O
N
N
H
N
H
H
O
O
Figure 2. Synthesized trifluoromethyl-b-hydrazino compounds 2–8.

L. Formicola et al. / Bioorg. Med. Chem. Lett. 19 (2009) 83–86
85
O
CF3
O
CF3
O
a
b, c, d
OEt +H2N
BocHN
H2N
F3C
NHBoc
10
N
H
OEt
N
H
N
H
COOMe
9
11
12
O
3
e
O
CF3
O
g, h
H
f
2
N
12
FmocHN
N
N
H
COOMe
H
O
13
i
j
7
6
k
m
l
8
4
5
Scheme 1. Synthesis of compounds 2–8. Reagents and conditions: (a) MeOH, 70 °C, 94%; (b) 2 N aq NaOH, THF/MeOH, rt, 98%; (c)
HBTU, HOBt, DIPEA, DCM/DMF, rt, 91%; (d) TFA, DCM, rt, 100%; (e) 3-phenoxyphenylacetic acid, HBTU, HOBt, 2,4,6-collidine, rt, 42%. (f) Fmoc-
HBTU, HOBt, 2,4,6-collidine, rt, 22%; (g) 10% piperidine/DMF, rt, 84%; (h) 3-phenoxyphenylacetic acid, HBTU, HOBt, 2,4,6-collidine, rt, 41%; (i) N
L
-phenylalanine methyl ester hydrochloride,
-3,4-dimethoxyphenylalanine,
-Boc-N -Z- -lysine, HBTU,
L
a
e
L
HOBt, DIPEA, DMF, rt, 57%; (j) H2, 20% Pd/C, MeOH, rt then citric acid monohydrate, rt, 51%; (k) TFA, DCM, rt, 100%; (l) 3-phenoxyphenylacetic acid, HBTU, HOBt, 2,4,6-
collidine, rt, 71%; (m) H2, 20% Pd/C, MeOH, rt then TFA, rt, 100%.
solic calpain I was inhibited by compounds 5, 7 and 8.38 We used
a cell-based chemiluminescent assay39 to show that compound 5
100
inhibited the CT-L activity in human HeLa cells (20% inhibition at
80
50
l
M after incubation for 1.5 h).
In conclusion, we have identified a series of fluorinated pseudo-
peptides that incorporate a trifluoromethyl-b-hydrazino acid scaf-
fold, as new class of proteasome inhibitors. These new
60
40
20
0
a
fluorinated pseudopeptides are very easy to synthesize. A limited
SAR around the fluorinated scaffold resulted in the discovery of
compounds having differential inhibitory capacities for CT-L, PA
and T-L in micromolar range without effect on challenging proteo-
lytic enzymes such as calpain and cathepsin B. These encouraging
results have led us to further optimize the lead compounds 5, 7 and
8 using molecular modeling and continuing biological evaluation.
0
5
10
15
[I] µM
Figure 3. Inhibition of CT-L activity of rabbit 20S proteasome by compound 5 at pH
7.5 and 37 °C. The experimental data were fitted to equation% inhibition = 100[5]nH
(IC50nH + [5]nH) with nH = 4.5.
/
Acknowledgments
Table 1
We thank the European Community for the financial support
(Marie Curie Early Stage training Fellowship of the European Com-
munity’s Sixth Framework Programme: contract MEST-CT-2004-
515968) (for LF), Claire Troufflard (University Paris-Sud 11) for
the NMR experiments, and Dr. Owen Parkes for assistance with
the English text.
IC50 (lM) or % inhibition at 100 lM of rabbit 20S proteasome at pH 7.5 and 37 °C. x,
activation factor. Values are means of three experiments. CT-L, chymotrypsin-like
activity; PA, post-acid activity; T-L, trypsin-like activity; ni, non-inhibitor
Compound
CT-L
PA
T-L
2
3
4
5
6
7
8
85 15
x2
ni
1.6 0.1
ni
32
72 0.7
ni
200
2.7 0.1
ni
6
x4
ni
ni
8.4 1.3
x3
30%
Supplementary data
2
0.5
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.012.
5.9 0.5
ni
4.4 1.2
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39. Proteasome Glo Cell-Based Assay (Promega) using MG-132 as a standard.