α- and β-hydrazino acid-based pseudopeptides inhibit the chymotrypsin-like activity of the eukaryotic 20S proteasome

Article abstract of DOI:10.1016/j.ejmech.2013.09.059

We describe the synthesis of a library of new pseudopeptides and their inhibitory activity of the rabbit 20S proteasome chymotrypsin-like (ChT-L) activity. We replaced a natural α-amino acid by an α- or a β-hydrazino acid and obtained inhibitors of proteasome up to a submicromolar range (0.7 μM for molecule 24b). Structural variations influenced the inhibition of the ChT-L activity. Models of inhibitor/20S proteasome complexes corroborated the inhibition efficacies obtained by kinetic studies.

Full text of DOI:10.1016/j.ejmech.2013.09.059

European Journal of Medicinal Chemistry 70 (2013) 505e524
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
a- and
b-hydrazino acid-based pseudopeptides inhibit the
chymotrypsin-like activity of the eukaryotic 20S proteasome
,
,
Andrea Bordessa a 1, Massaba Keita a 1, Xavier Maréchal b, Lucia Formicola a,
Nathalie Lagarde a, Jordi Rodrigo a, Guillaume Bernadat a, Cyril Bauvais a,
Jean-Louis Soulier a, Laure Dufau b, Thierry Milcent a, Benoit Crousse a,
,
a,
*
Michèle Reboud-Ravaux b , Sandrine Ongeri
**
a Molécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, LabEx LERMIT, Université Paris-Sud 11, Faculté de Pharmacie, 5 rue Jean-Baptiste
Clément, 92296 Châtenay-Malabry Cedex, France
b Enzymologie Moléculaire et Fonctionnelle, UR4, UPMC, Case 256, 7 Quai St Bernard, 75252 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 describe the synthesis of a library of new pseudopeptides and their inhibitory activity of the rabbit
Received 20 June 2013
Received in revised form
23 September 2013
Accepted 24 September 2013
Available online 17 October 2013
20S proteasome chymotrypsin-like (ChT-L) activity. We replaced a natural
a
-amino acid by an
a- or a b-
hydrazino acid and obtained inhibitors of proteasome up to a submicromolar range (0.7
m
M for molecule
24b). Structural variations influenced the inhibition of the ChT-L activity. Models of inhibitor/20S pro-
teasome complexes corroborated the inhibition efficacies obtained by kinetic studies.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Proteasome inhibitors
Hydrazino acid
Chymotrypsin
Pseudopeptide
Trifluoromethyl
Fluorine
1. Introduction
6
b
subunits and are classified as chymotrypsin-like (ChT-L,
subunit), trypsin-like (T-L, 2 subunit) and caspase-like or post-
acid (PA, 1 subunit) activities since peptide bonds are cleaved on
b5
b
The 26S proteasome is a large, multicatalytic threonine protease
complex that processively degrades ubiquitinylated proteins to
small peptides [1]. The ubiquitineproteasome pathway plays a
central role in the degradation of regulatory proteins that are
crucial for many intracellular processes including cell progression,
b
the carboxyl side of hydrophobic, basic and acidic amino acid res-
idues, respectively [3]. The ChT-L activity has been the focus of drug
development [4]. Inhibition of ChT-L activity induces cell cycle ar-
rest and selective apoptosis of malignant cells leading to a new
category of antineoplastic agents [5]. The dipeptide boronic acid
bortezomib (VelcadeÒ) and the epoxyketone carfilzomib (PR-171)
[6] have been approved for treating incurable multiple myeloma
(both compounds) [7] and mantle lymphoma (bortezomib) [8]. The
lactone salinosporamide A (NPI-0052) entered into clinical trials for
advanced solid and hematological malignancies [9]. Most natural
and synthetic proteasome inhibitors, such as epoxyketones, pep-
tide aldehydes, peptide vinyl sulfones and peptide boronic acids,
bear a reactive group that forms a transient or irreversible covalent
bond with the catalytic Og atom of Thr1 of the active sites [10,11].
Although these reactive groups contribute to the inhibitory activity,
they can also cause a lack of specificity, excessive reactivity and
instability which may increase adverse effects and limit efficacy of
proteasome inhibitors in vivo. Therefore, non covalent inhibitors
apoptosis and NF-kB activation. The 26S proteasome is composed of
a 20S catalytic core particle that is capped at each end by the 19S
regulatory complex which is 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]. Three proteolytic activities are localized in
* Corresponding author. Tel.: þ33 146835737; fax: þ33 146835740.
** Corresponding author. Tel.: þ33 144275078; fax: þ33 144275140.
E-mail
addresses:
michele.reboud@upmc.fr
(M.
Reboud-Ravaux),
sandrine.ongeri@u-psud.fr (S. Ongeri).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.059

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A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
(Fig. 1) [24]. These structures can mimic the typical secondary
structure of native -peptides, preserving biological activity and
enhancing proteolytic stability [25,26] making them useful tools to
design new protease inhibitors. Furthermore, the additional ni-
trogen may allow the formation of additional H-bonds [25,27].
R1
O
R2
O
O
a
H2N
H2N
OH
H2N
OH
OH
R2
R
R1
-amino acid
R3
-amino acid
-amino acid
However, to our knowledge,
rarely used in medicinal chemistry and particularly very scarcely
introduced within protease inhibitors. -Hydrazino peptides were
a-hydrazino acid scaffolds have been
a
R1
O
O
H
reported to inhibit the serine protease leukocyte elastase [25] and
retro hydrazinoeazapeptoids were recently described as covalent
N
H2N
OH
OH
N
H
H2N
R2
R
proteasome inhibitors with IC50 up to 0.7
m
M [28]. Finally, whereas
-hydrazino acid
-hydrazino acid
b-amino acids are well documented [29e31] almost nothing is
known about the b-hydrazino acid based peptidomimetics.
Fig. 1. General structure of amino acids and hydrazino acids. For clarity, each amino
acid and hydrazine acid is represented by the residue formula of only one
configuration.
We previously described preliminary results concerning the first
synthesis of CF3- -hydrazino acid and the biological evaluation of
few compounds based on a central fluorinated -hydrazino acid
scaffold that inhibit the ChT-L activity with IC50 values up to 1.6
b
b
m
M
that bind reversibly to the active sites may provide an alternative
mechanism of inhibition and offer therapeutic advantages. Non
covalent inhibitors have been less extensively investigated [12,13].
They include peptidic inhibitors such as ritonavir [14], amino-
(compounds 1a, 1b, 2, 3a, 3b, 4 and 6, Table 1) [32]. Proteasome
inhibition was selective; cytosolic calpain I and lysosomal cathepsin
B were not inhibited by compounds 1b, 3b and 4 [32]. Using a cell-
based chemiluminescent assay, compound 1b behaved as an in-
hibitor of the ChT-L activity in human HeLa cells (20% inhibition at
benzylstatine [15], and 3,4,5-trimethoxy-L-phenylalanine de-
rivatives [16], lipopeptides [17], macrocyclic [18], and linear TMC-
50 mM after 1 h 30 min incubation) [32]. In this present report, we
95 derivatives [19e22] and sulfonamide compounds [23]. We
made several changes in the structures of the fluorinated
hydrazino acid derivatives and we introduced non fluorinated
and b-hydrazino acid scaffolds. We performed pharmacomodula-
tions around the four moieties of the molecules (Fig. 2) in order to
establish structureeactivity relationships of this new class of pro-
teasome inhibitors. A library of 40 molecules was designed, syn-
thesized and evaluated on the 20S rabbit proteasome (Fig. 2). At
first, we kept in the third moiety the b-hydrazino acid scaffold (C.1.
Fig. 2; compounds 5a, 5b, 7, 8, 9, 10a, 10b, 11a, 11b, 12, 13, Table 1).
We then evaluated the influence of the length of the hydrazino
b
a
-
-
describe here a new class of non covalent 20S inhibitors based on
or -hydrazino acid scaffolds. We postulated that these peptido-
mimetic elements would mimic the natural -peptides which are
the only elements encountered so far in peptidic proteasome in-
hibitors. - and -hydrazino acid scaffolds are peptidomimetic
building blocks that have two nitrogen atoms. They can be
considered as analogs of and -amino acids, respectively, in which
the amine group has been replaced by a hydrazine (Fig. 1). - and
hydrazino acid scaffolds can also be considered as analogs of and
-amino acids where the Cb or Cg-atom is replaced by a nitrogen
a-
b
a
a
b
a
b
a
b-
b
g
scaffold by replacing the CF3-b-hydrazino acid scaffold by the
(24)
CF3- -hydrazino acid
retro hydrazino
CF3- -hydrazino acid
P = Cbz or Boc
O
-hydrazino acid
-hydrazino acid
NH2
Fig. 2. Schematic representation of hydrazino acid based pseudopeptides. The number of compounds with different A-D groups is indicated in brackets with the numbers of these
groups.

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
507
Fig. 3. Inhibition of the ChT-L activity of rabbit 20S proteasome by compounds 10b, 19a, 22b and 24b at pH 7.5 and 37 ꢀC. The experimental data were fitted to equation 1 or
equation 2.
longer retro hydrazino CF3-
pseudopeptides C.2. Fig. 2; compounds 14a, 14b, 15, 16a, 16b,
Table 1). We also evaluated the influence on the inhibition of the
b
-hydrazino acid (third moiety of the
9, 10a, 10b, 11a, 11b, and 12 is described in Scheme 1. The inter-
mediate 27 was obtained in good yield (94%) by a Michael addition
of tert-butyl carbazate on 26 [32]. Saponification of the ester 27
ChT-L activity, of the trifluoromethyl group on the
scaffold when eliminating the fluorinated group on the
zino acid (C.3. Fig. 2; compounds 17a, 17b, 18a, 18b, 19a, 19b, 20,
21a, 21b, 22a, 22b, Table 2). The shorter -hydrazino acid scaffold
b
-hydrazino acid
followed by peptide coupling with L-phenylalanine methyl ester
b
-hydra-
hydrochloride or 3,4,5-trimethoxybenzylamine using HBTU and
HOBT as coupling agent, and the consecutive acidic cleavage of tert-
butyl carbamate gave the trifluoroacetic salt of 28 [32] and 29
a
(C.4. Fig. 2) was also introduced as the third moiety (compounds 23,
24a, 24b, 25a, 25b, Table 3). In parallel, we evaluated the influence
of the nature of the amino acid residue (second moiety, Fig. 2):
protected or deprotected lysine (B.2. and B.1. respectively), aspar-
agine (B.3.) or 3,4-dimethoxyphenylalanine (B.4.). We also modu-
lated the first part of the molecule (Fig. 2) by introducing the tert-
butyloxycarbonyl (A.1.), 9-fluorenylmethoxycarbonyl (A.2.), 3-
phenoxyphenylacetyl (A.3.) and 3,4-dimethoxyphenylacetyl (A.4.)
moieties. In the fourth part (Fig. 2), we kept the phenylalanine
residue (D.1.) or replace the amino acid by introducing a 3,4,5-
trimethoxybenzylamine moiety (D.2.), a benzyloxycarbonyl (D.3.)
or a tert-butyloxycarbonyl (D.4.). We proposed a binding mode of
this new class of non covalent proteasome inhibitors to ChT-L
subunit by molecular modeling studies and we found that the ob-
tained inhibitory activities corroborated the docking calculations
performed for inhibitor/proteasome complexes.
respectively (91% and 76%). Coupling of 28 with N
a
-Boc-N -Z-L-
3
Lysine followed by acidic cleavage afforded 4 [32], that was sub-
sequently coupled with 2,5-dimethoxyphenylacetic acid to give 5a.
Hydrogenolysis of the benzyl carbamate of 5a gave 5b in good yield
(82%). The intermediate amine 28 was coupled with 2,5-
dimethoxyphenylacetic acid and N-Boc-Asparagine to afford 7
and 8 respectively, in good yield (77% and 66%). The amine resulting
from the tert-butyl carbamate (Boc) cleavage of 8, was coupled with
2,5-dimethoxyphenylacetic acid to yield 9. The peptide coupling of
29 with N
a
-Boc-N -Z-L-Lysine or N-Boc-Asparagine afforded 10a
3
and 13 respectively. The cleavage of Boc of 10a gave 10b that was
coupled with 2,5-dimethoxyphenylacetic acid to give 11a. The
benzyl carbamate hydrogenolysis of 11a gave 11b. Compound 10b
was coupled with 3-phenoxyphenylacetic acid following by
hydrogenolysis of the benzyl carbamate to afford 12 as indicated in
Scheme 1.
The synthesis of compound 14a, 14b, 15, 16a and 16b is
described in Scheme 2. Treatment of 27 with hydrazine hydrate
afforded the intermediate 30 which was coupled with N-Fmoc-3,4-
diOMe-Phe-OH in the coupling conditions previously described, to
give 15 in satisfactory yield (68%). It is noticed that 2,4,6-collidine
was used instead of DIPEA to avoid the cleavage of the Fmoc pro-
tecting group. The basic cleavage of the Fmoc group using 10%
2. Results and discussion
2.1. Chemistry
Compounds 1a, 1b, 2, 3a, 3b, 6 were prepared according to our
published methods [32]. The synthesis of compounds 4, 5a, 5b, 7e

508
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
q
11a
11b
p
g
10a
10b
m
n, o
12
CF3
O
O
O
H2N
r
N
H
N
H
13
29
O
b, k, d
7
e
O
CF3
O
CF3
O
b, c, d
a
h
i
f, g
BocHN
H2N
5b
F3C
OEt
4
5a
N
H
OEt
N
H
N
H
COOMe
28
27
26
j
d, l
8
9
Scheme 1. Synthesis of molecules 4, 5a, 5b, 7e9, 10a, 10b, 11a, 11b, and 12. Reagents and conditions: a) NH2NHBocMeOH, 70 ꢀC, 94%; b) 2N aq. NaOH, THF/MeOH, rt, 98%; c) L-
phenylalanine methyl ester hydrochloride, HBTU, HOBT, DIPEA, DCM/DMF, rt, 91%; d) TFA, DCM, rt, 100%; e) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, rt, 77%; f)
Na
-Boc-N 3 -Z-L-Lysine, HBTU, HOBT, DIPEA, DMF, rt, 57%; g) TFA, DCM, rt, 100%; h) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, rt, 84%; i) H2, 10% Pd/C, rt, DCM/TFA, rt,
82%; j) N-Boc-Asparagine, HBTU, HOBT, DIPEA, DMF, rt, 63%; k) 3,4,5-trimethoxybenzylamine, HBTU, HOBT, DIPEA, DMF, rt, 76%; l) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT,
DIPEA, DMF, rt, 77%; m) N -Boc-N 3 -Z-L-Lysine, HBTU, HOBT, DIPEA, DMF, rt, 84%; n) 3-phenoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, 24h, 71%; o) H2, 10% Pd/C, rt, 89%; p) 2,5-
dimethoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, rt, 77%; q) H2, 10% Pd/C, rt, 77%; r) N-Boc-Asparagine, HBTU, HOBT, DIPEA, DMF, rt, 55%.
a
O
CF3
O
CF3
c, d
NHBoc
a
b
H2N
NHBoc
16b
15
EtO
N
N
H
N
H
H
30
27
e, f
O
CF3
g
O
CF3
h
i
j
H
H
N
14a
14b
16a
N
O
O
H2N
EtO
N
N
N
H
H
H
O
O
31
32
Scheme 2. Synthesis of molecules 14a, 14b, 15, 16a and 16b. Reagents and conditions: a) NH2NH2.H2O, EtOH, 100%; b) N-Fmoc-3,4-diOMe-Phe-OH, HBTU, HOBT, 2,4,6-collidine, DMF,
21 h, rt, 68%; c) 10% piperidine/DMF, 12 h, rt, 88%; d) 3-phenoxyphenylacetic acid, HBTU, HOBT, 2,4,6-collidine, DMF, 24h, 84%; e) TFA, DCM, rt, 100%; f) Cl-Cbz, DIPEA, DCM, 81%; g)
NH2NH2.H2O, EtOH, reflux, 100%; h) Fmoc-3,4-diOMe-Phe-OH, HBTU, HOBT, 2,4,6-collidine, DMF, 40 h, rt, 67%; i) 10% piperidine/DMF, 12 h, rt, 88%; j) 3-phenoxyphenylacetic acid,
HBTU, HOBT, DIPEA, DMF, 24h, 85%.
piperidine in DMF followed by peptide coupling with 3-
phenoxyphenylacetic acid afford 16b. After acidic cleavage of the
Boc protecting group of 27, the amine obtained was protected with
benzyl chloroformate to afford 31 in good yield (81%). The next
reaction with hydrazine hydrate gave 32 which was coupled with
Fmoc-3,4-diOMe-Phe-OH to afford 14a. Removal of the Fmoc pro-
tecting group with 10% piperidine in DMF gave 14b. Coupling of 14b
with 3-phenoxyphenylacetic acid using HBTU, HOBT, DIPEA in dry
DMF, afforded 16a in good yield (85%) (Scheme 2).
The synthesis of molecules 17a, 17b, 18a, 18b, 19a, 19b, 20, 21a,
21b, 22a and 22b is described in Scheme 3. The intermediate 34 was
obtained in modest yield (43%) by nucleophilic substitution of ethyl
bromopropionate 33 by tert-butyl carbazate. This modest yield can
be explained by the formation of side product resulting from
O
O
O
b, c
a
f
h
g
d, e
BocHN
BocHN
18b
18a
Br
OEt
17a
17b
N
H
OEt
N
H
N
H
COOMe
34
33
35
i
b, k
j
19a
19b
O
O
BocHN
f
m, n
d, l
N
H
N
H
20
22b
22a
O
O
36
m, o
f
21b
21a
Scheme 3. Synthesis of molecules 17a, 17b, 18a, 18b, 19a, 19b, 20, 21a, 21b, 22a and 22b. Reagents and conditions: a) BocNHNH2, DIPEA, toluene, 80 ꢀC, 43%; b) 2N NaOHaq, THF/
MeOH, rt, 93%; c) L-phenylalanine methyl ester hydrochloride, HBTU, HOBT, DIPEA, DCM/DMF, rt, 82%; d) TFA, DCM, rt, 100%; e) N -Boc-N 3 -Z-L-Lysine, HBTU, HOBT, DIPEA, DMF, rt,
62%; f) TFA, DCM, rt, 100%; g) 3-phenoxyphenylacetic acid, HBTU, HOBT, 2,4,6-collidine, DMF, 81%; h) H2, 10% Pd/C, MeOH, rt, 89%; i) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT,
DIPEA, DMF, rt, 37%; j) H2, 10% Pd/C, MeOH, rt, 81%; k) 3,4,5-trimethoxybenzylamine, HBTU, HOBT, DIPEA, DMF, rt, 52%; l) N -Fmoc-N 3 -Z-L-Lysine, HBTU, HOBT, 2,4,6-collidine, DMF,
rt, 30%; m) 10% piperidine/DMF, rt, 100%; n) 3-phenoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, 24h, 52%; o) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, rt, 50%.
a
a

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
509
NHCbz
O
O
H
H
N
d, e
f
a
b, c
N
OEt
23
24a
24b
O
N
H
N
H
H2N
OEt
O
38
O
37
d, g
f
25a
25b
Scheme 4. Synthesis of molecules 23, 24a, 24b, 25a and 25b. Reagents and conditions: a) Na-Boc-N 3 -Z-L-Lysine, HBTU, HOBT, DIPEA, DMF, rt, 76%; b) 2N NaOHaq, THF/MeOH, rt,
88%; c) L-phenylalanine methyl ester hydrochloride, HBTU, HOBT, DIPEA, DCM/DMF, rt, 56%; d) TFA, DCM, rt, 100%; e) 3-phenoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, 48%; f)
H2, 10% Pd/C, MeOH/DMF, rt, 98%; g) 2,5-dimethoxyphenylacetic acid, HBTU, HOBT, DIPEA, DMF, rt, 55%.
nucleophilic attack of the same nitrogen atom of hydrazine on a
second molecule of ethyl bromopropionate 33. Saponification of 34
and successive coupling with L-phenylalanine methyl ester hydro-
chloride or 3,4,5-trimethoxybenzylamine, using HBTU, HOBT,
deleterious for the inhibitory activity (molecules 8 and 9 compared
to molecules 3b and 5b respectively). The results showed also that
shortening the pseudopeptide by connecting directly the phenox-
yphenylacetyl moiety A.3. or the 3,4-dimethoxyphenylacetyl A.4. to
DIPEA in dry DMF, gave 35 and 36 respectively. The acidic treatment
the trifluoromethyl-b-hydrazino acid scaffold (no second moiety B)
of 35 followed by peptide coupling with N
a
-Boc-N
3
L
-Lysine gave
totally suppressed the ChT-L activity inhibition by molecules 6
(compared to 1b and 2) and 7 (compared to 5b). Concerning the
first moiety of the trifluoromethyl-b-hydrazino acid based pseu-
dopeptides, the 3-phenoxyphenylacetyl A.3. (1b) was slightly more
favorable than the 3,4-dimethoxyphenylacetyl A.4. and than the
tert-butyloxycarbonyl A.1.1b vs. 3b (factor of 20); 1b vs. 5b (factor of
17a in satisfactory yield (62%). 17b, obtained after acidic cleavage
of tert-butyl carbamate of 17a was coupled with 3-
phenoxyphenylacetic acid and 2,5-dimethoxyphenylacetic acid to
afford 18a and 19a respectively. In both cases, hydrogenolysis of the
benzyl carbamate with Pd/C 10% in MeOH provided 18b and 19b
respectively, in good yield (89% and 81%). 20 was obtained after
acidic cleavage of the tert-butyl carbamate of 36, followed by
3.7); 12 (IC50 of 6.2
devoid of the first moiety A and having the lysine
whereas the NH was protected by a Cbz group were also good ChT-
L activity inhibitors (IC50 of 6.0 and 10.1 M). Inhibition was poorly
mM) vs.13 (no inhibition). Compounds 4 and 10b
a
NH deprotected
coupling with N
a
-Fmoc-N
3
L-Lysine, using HBTU, HOBT, 2,4,6-
3
collidine in dry DMF. Basic cleavage of Fmoc protecting group of
the lysine residue and successive coupling with 2,5-
dimethoxyphenylacetic acid and 3-phenoxyphenylacetic acid
gave 21a and 22a respectively. The trifluoroacetic salts 21b and 22b
were obtained in quantitative yield, by acidic cleavage of tert-butyl
carbamate of 21a and 22a respectively, with TFA in DCM.
m
affected by replacing in the fourth moiety the phenylalanine methyl
ester D.1. by the 3,4,5-trimethoxybenzylamine D.2. (molecules 12/
1b, 11b/5b and 10b/4).
However, the introduction of the retro hydrazino CF3-b-hydra-
zino acid scaffold C.2 totally suppressed the inhibitory activity of
The synthesis of molecules 23, 24a, 24b, 25a and 25b is outlined
in Scheme 4. The intermediate 38 was obtained in good yield (76%)
molecules 14a, 14b, 15, 16a and 16b (both the direction of the tri-
fluomethyl-b-hydrazino acid scaffold and the length of the third
by coupling N
37, using HBTU, HOBT DIPEA in dry DMF. Saponification of the
ester of 38 and successive coupling with -phenylalanine methyl
a
-Boc-N
3
L
-Lysine with ethyl 2-hydrazinylacetate
moiety were modified).
Similar structureeactivity relationships were observed for
pseudopeptides displaying the non fluorinated b-hydrazino acid
L
ester hydrochloride gave compound 23 in moderate yield (56%).
Subsequent Boc cleavage of 23 with TFA in DCM, followed by
coupling the trifluoroacetic salt of the amine with 3-
phenoxyphenylacetic acid and 2,5-dimethoxyphenylacetic acid
yielded 24a and 25a respectively (in moderate yield, 48% and 55%).
In both cases, the carbamate moiety hydrogenolysis afforded 24b
and 25b respectively in good yield (Scheme 4).
scaffold C.3. within their third moiety (molecules 17e22, Table 2).
For comparison with the fluorinated molecules, the favorable lysine
residue B.1. was conserved in this new series. Conversely to what
was observed with the fluorinated molecules (Table 1), protecting
the lysine NH with a Cbz or a Boc group (B.2.) was not deleterious
3
for the ChT-L inhibitory activity (molecules 18b, 19b, 21b and 22b
compared to molecules 18a, 19a, 21a and 22a). As previously
observed with the fluorinated molecules, compound 17b devoid of
the first moiety of the molecule and whose lysine
deprotected whereas the NH was protected by a Cbz group, had
also ChT-L inhibition activity (Table 2). In this series of
non fluorinated -hydrazino acid molecules, the 3-
phenoxyphenylacetyl moiety A.3. was slightly more favorable
than the 3,4-dimethoxyphenylacetyl A.4. (molecules 18b and 22b
compared to molecule 19b and 21b respectively) and the 9-
fluorenylmethoxycarbonyl A.2 (molecule 22a compared to mole-
cule 20). A more noticeable effect was observed for tert-butylox-
ycarbonyl A.1. (molecule 18a compared to molecule 17a). As already
observed for the fluorinated molecules, the deprotection of the
aNH was
2.2. Biological results
3
a
The inhibition of the ChT-L activity of rabbit 20S proteasome by
molecules 1e25 was investigated using appropriate fluorogenic
substrate Suc-LLVY-AMC (Fig. 3) [17,19]. The aldehyde proteasome
inhibitor MG132 (Z-LLL-H) was used as standard [17].
b
For molecules with a trifluoromethyl-b-hydrazino acid scaffold
C.1. as pseudopeptide third moiety (molecules 1e13, Table 1), the
results indicated that the nature of the second moiety influenced
the inhibitory potency. Replacing the 3,4-dimethoxyphenylalanine
(lateral chain B.4.) by the more hydrophilic lysine (lateral chain B.1.)
favored the inhibitory potency by a factor of 53 (molecule 1b
compared to molecule 2). The same tendency was observed for
compounds 3b and 5b compared to molecule 2 (factors of 2.6 and
14, respectively). Protecting the lysine amino group (B.2.) sup-
pressed the inhibitory activity or decreased it (molecules 1a vs.1b,
3a vs. 3b and 5a vs. 5b). Replacing the lysine residue (lateral chain
B.1.) by an asparagine residue (lateral chain B.3.) was completely
aNH of the lysine residue (molecule 17b versus molecule 17a)
whereas the NH remained protected by a Cbz group led only to a
3
decrease of ChT-L activity inhibition by a factor of 2.6. The inhibi-
tory potency was poorly affected by the nature of fourth moiety D:
molecule 22b (phenylalanine methyl ester moiety D.1.) compared
to molecule 18b (3,4,5-trimethoxybenzylamine D.2.), and molecule
21b (D.2.) compared to molecule 19b (D.1.). The comparison of the

510
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
Table 1
Chymotrypsin-like (ChT-L) proteasome inhibition potency of molecules including the trifluoromethyl-
b
-hydrazino acid scaffold in the third part. IC50
(m
M) or % inhibition at
ChT-L
100
m
M of chymotrypsin-like (ChT-L) activity of rabbit 20S proteasome at pH 7.5 and 37 ꢀC. x: activation factor.
Compound
Structure
1a (P ¼ CBz)
ni
1b (P ¼ H) racemic
1b1 (P ¼ H) Dia 1a
1b2 (P ¼ H) Dia 2a
1.6 ꢁ 0.1
8.4 ꢁ 0.5
5.9 ꢁ 0.1
2
85 ꢁ 15
3a (P ¼ CBz)
3b (P ¼ H)
4
ni
32 ꢁ 2
6.0 ꢁ 0.5
5a (P ¼ CBz)
5b (P ¼ H)
6
30%
5.9 ꢁ 0.3
x 2
7
ni
8
9
30%
ni
NHCbz
H
CF
O
10a (P0 ¼ Boc)
10b (P0 ¼ H)
35%
N
N
O
O
P'NH
N
H
H
O
O
10.1 ꢁ 0.4
NHP
O
O
O
CF O
11a (P ¼ CBz)
11b (P ¼ H)
ni
H
N
O
N
H
N
H
N
H
O
O
O
97 ꢁ 3
12
6.2 ꢁ 0.3

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
511
Table 1 (continued )
Compound
Structure
ChT-L
ni
13
14a (P0 ¼ Fmoc)
14b (P0 ¼ H)
15
x 3.6
ni
ni
16a (P00 ¼ Cbz)
ni
ni
16b (P00 ¼ Boc)
a
The two diastereoisomers of 1b (1b1 and 1b2) were separated and evaluated separately.
Table 2
Chymotrypsin-like (ChT-L) proteasome inhibition potency of molecules including
the -hydrazino acid scaffold in the third part. IC50 M) of chymotrypsin-like (ChT-
L) activity of rabbit 20S proteasome at pH 7.5 and 37 ꢀC.
results obtained for both series (Tables 1 and 2) indicated that the
presence of the trifluoromethyl group in the -hydrazino acid
b
b
(m
scaffold poorly influenced the inhibition of ChT-L activity (compare
for example, 1b to 18b, 5b to 19b, 12 to 22b, 11b to 21b). This poor
effect was also in agreement with the similar inhibitory activity
observed for compounds 1b1 and 1b2 (Table 1). The two di-
astereoisomers of 1b were separated and evaluated separately
demonstrating that the absolute configuration of the carbon
bearing the CF3 group did not influenced significantly the inhibition
Compound
Structure
ChT-L
17a (P0 ¼ Boc)
17b (P0 ¼ H)
18a (P ¼ CBz)
18b (P ¼ H)
48.5 ꢁ 2.4
18.2 ꢁ 0.4
2.8 ꢁ 0.1
1.4 ꢁ 0.1
3.6 ꢁ 0.3
(IC50 of 8.4
Shorten the third moiety C of the molecules by replacing the
hydrazino scaffold C.3. by the -hydrazino acid scaffold C.4. affor-
m m
M for 1b1 and 5.9 M for 1b2).
b
-
a
ded also good ChT-L activity inhibitors with similar or slightly
Table 3
19a (P ¼ CBz)
Chymotrypsin-like (ChT-L) proteasome inhibition potency of molecules including
the
a-hydrazino acid scaffold in the third part. IC50 (mM) of chymotrypsin-like (ChT-
L) activity of rabbit 20S proteasome at pH 7.5 and 37 ꢀC.
Compound
Structure
ChT-L
19b (P ¼ H)
8.8 ꢁ 0.3
6.0 ꢁ 0.2
23
11.8 ꢁ 1.1
20
24a (P ¼ CBz)
24b (P ¼ H)
1.1 ꢁ 0.1
21a (P ¼ Boc)
21b (P ¼ H)
31.4 ꢁ 0.7
11.8 ꢁ 1
0.7 ꢁ 0.03
NHP
O
O
O
O
22a (P ¼ Boc)
22b (P ¼ H)
4.7 ꢁ 0.5
3.3 ꢁ 0.1
25a (P ¼ CBz)
25b (P ¼ H)
7.1 ꢁ 0.2
H
N
H
N
N
N
H
O
H
O
O
1.0 ꢁ 0.03

512
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
increased efficiency (Table 3). Molecule 23 (C.4.; A.1. ¼ Boc), was 4
times more efficient than molecule 17a (C.3.; A.1. ¼ Boc). Molecules
docking studies with models in which we observed a binding mode
within bovine proteasome active site as similar as possible to the
one described by Furet et al.
24a (IC50 ¼ 1.1
mM) and 24b (IC50 ¼ 0.7
mM) bearing the phenox-
yphenylacetyl moiety A.4. had quite similar inhibitory activity than
that of the corresponding molecules 18a (IC50 ¼ 2.8 M) and 18b
M). Concerning the molecules bearing the dimethox-
We first evaluated if what we were observing could be explained
by an intrinsic different size of S1 pocket in the ChT-L active site in
bovine proteasome compared to the one in yeast proteasome (Unno
et al. reported differences between yeast and bovine proteasome
20S structure [41]). However, when we superimposed coordinates
of the bovine protein structure we chose with those of a compa-
rable structure (without co-crystallized ligands) obtained for yeast
proteasome, we observed identical conformation of the S1 pocket.
We therefore decided to carry out a detailed analysis of all the
proteasome structures available in the PDB, both proteins devoid
of ligand and co-crystals with ligands. This task allowed us to
establish a hypothetical mapping of residues defining every sub-
pocket thanks to the probing work performed by the various li-
gands already cocrystallised (see Table 1S in the Supporting
information). More importantly, our structural analysis, and in
particular comparison of cocrystals with unbound form of yeast
proteasome, revealed a joint movement of side chains of Met45
(directly exposed to the solvent) and Ile35 in order to accom-
modate “P1” side chain from the ligand upon complexation. We
hypothesized that this induction phenomenon could be extrap-
olated to bovine proteasome, and that the smaller size of S1
pocket in the “apo” form we were using might at the origin of
exclusion of bulky substituents, typically benzyl groups in the
kind of ligands we are studying (see Fig. 2S in Supporting
information). We hypothesized that taking into account adapt-
ability of the size of S1 pocket in the bovine proteasome would
be a key element to place ourselves under conditions suitable for
in silico evaluation of our inhibitors.
Indeed, when we included conformational sampling of the side
chains of both aforementioned Met45 and Ile35 residues in our
docking protocol, repeating the experiment with the compound
described by Furet et al. resulted in solutions where the “P1” side
chain of the pseudopeptide was positioned in the S1 pocket of the
ChT-L active site, in a similar manner to what was reported by these
authors [16]. Moreover, in the best pose, S3 and AS2 pockets were
also occupied, and 4 hydrogen bonds were formed with Thr21,
Gly47 and Asp125 (see Supporting information, Fig. 4S). With these
results, we estimated that the docking procedure had reached the
level of reliability required to be used in the simulation of the
binding mode of synthesized compounds 1e25 to the ChT-L active
site of bovine 20S proteasome.
Docking poses retained for compounds that were experimen-
tally good proteasome inhibitors showed a network of hydrogen
bond interactions with some or all key residues known to be
important for the binding of compounds in the 20S proteasome
(Thr1, Thr21, Gly23, Gly47, Ala49 and Asp125) as well as occupation
of S1, S3 and AS1 or AS2 pockets. One of the most potent com-
pounds (24b) established 6 hydrogen bonds with the protein
(contributing to anchor it in the binding site), out of which 3
involved key residues (Fig. 4). First, two NH groups belonging to the
phenylalanine and the hydrazide moieties donated hydrogen bonds
to the backbone oxygen of Gly47. Then the NH of the second amide
group donated another hydrogen bond to the side chain of Asp125.
Within the S3 pocket, the NH3 group of the lysine side chain of
compound 24b shared 2 hydrogen bonds with Ser123 and Ser129
residues sitting at the bottom of the pocket. The phenoxyphenyl
oxygen also established a hydrogen bond with Tyr107, delimitating
the boundary between AS1 and AS2 pockets. Both S3 and AS1
pocket were filled respectively with the lysine side chain and the
m
(IC50 ¼ 1.4
m
yphenylacetyl A.4., 25b (B.1.) was 8.8 times more efficient than 19b
(B.1.) whereas 25a (B.2. P ¼ Cbz) was two-fold less efficient than 19a
(B.2. P ¼ Cbz). As observed for
b-hydrazino acid compounds (C.3.)
(Table 2), protecting the lysine NH with a Cbz group (B.2.) in the a-
3
hydrazino compounds 23, 24a and 25a was not deleterious for the
inhibition. Again in this series, the 3-phenoxyphenylacetyl A.3. was
more favorable than the 3,4-dimethoxyphenylacetyl A.4. and tert-
butyloxycarbonyl A.1. in two cases: molecule 24a versus 25a (factor
of 6.5) and 23 (factor of 10.7) and had no effect for molecule 24b
versus 25b (factor of 1.4).
2.3. Molecular modeling
In order to find a plausible explanation for the different activ-
ities of these compounds to inhibit ChT-L activity, we engaged their
structures into computational docking experiments. To date several
theorical modeling studies have been carried out in different lab-
oratories to describe the interaction between structurally diverse
inhibitors and 20S proteasome. These studies have been mostly
conducted on the basis of crystal structures of ligand and
(eukaryotic) yeast proteasome complexes [33e37] even if differ-
ences in amino acid sequences of these proteins with that of their
homologs in mammals are noticeable. The bovine protein has been
less used for docking studies though it exhibits a sequence close to
the human one [38e40] (sequence alignments are shown in Fig. 1S
in the Supporting information). Our structural analysis and docking
studies are therefore based on subunits b5 and b6 (defining the
ꢀ
ChT-L active site) from the 2.75 A resolution crystal structure of
bovine proteasome 20S available from the Protein Data Bank [41]
(see experimental part for details of the computational procedure).
During analysis of the multiple solutions obtained after a
docking run, we estimated the quality of an inhibitor according to
its ability to reproduce the previously putative binding mode of non
covalent inhibitors based on three criteria. The first one was the
tightness of the superimposition of its structure with that of the
non covalent pseudopeptidic inhibitor published by Furet et al. (see
the structure in Fig. 3S in the Supporting information) [16]. The
second criteria was the number of hydrogen bonds contracted with
the key residues Thr1, Thr21, Gly23, Gly47, Ala49 and Asp125. Poses
maximizing the number of these hydrogen bonds were considered
the best. The third criterion was the ability to occupy S1, S3, AS1 and
AS2 pockets of proteasome 20S (the accessory pockets AS1 and AS2
are called S4 and S5 in the classical naming scheme for pockets
recognizing the corresponding Pn chains from the substrate).
Before focusing on the compounds synthesized for this study,
we tested our docking protocol. We tried to identify a binding mode
converging with the result reported by Furet et al. for their non
covalent pseudopeptidic inhibitor (see the structure in Fig. 3S in the
Supporting information) with the ChT-L active site of their ho-
mology model of the human proteasome 20S based on X-ray
structure of yeast proteasome [16]. Our first result in this validation
phase looked satisfactory overall, but analysis in depth revealed
that proper occupation of S1 subpocket was actually lacking in
every solution found, which was a major mismatch with the ac-
tivity criteria we listed. Results recently published by Maréchal
et al. [38] demonstrated that the strong non covalent inhibition of
the ChT-L activity of human proteasome by 1,2,4-oxadiazole de-
rivatives could also be due to the inhibitor binding within the
subsite S5 instead of the S1 one. However, we preferred to start our
phenoxyphenylacetyl moieties. Compound 18b, which is the
hydrazino acid homolog of 24b, showed a similar ChT-L inhibition
activity (IC50 ¼ 1.4 and 0.7 M for 18b and 24b respectively). Its
b-
m

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
513
Fig. 5. Docking pose of compound 18b positioned into the ChT-L binding site pocket of
the bovine proteasome X-ray structure. Key residues are shown and hydrogen bonds
formed are in orange dashes. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Fig. 4. Docking pose of compound 24b positioned into the ChT-L binding site pocket of
the bovine proteasome X-ray structure. Key residues are shown and hydrogen bonds
formed are in yellow dashes. (Color code for SAS: cyan is polar, purple is hydrophobic.)
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
other with Asp125 (Fig. 6S, Supporting informations). These ob-
servations could explain while both protected and deprotected
lysine (NH -wise) in non-fluorinated a and b-hydrazino acid mol-
3
ecules afforded inhibitors with similar efficiencies.
binding mode was also quite similar to that of 24b (Figs. 5 and 6).
18b contracted 4 hydrogen bonds in total, out of which two were
with key residues Gly47 and Asp125. Additionally, occupation of
pocket S3 was very similar (again with two hydrogen bonds
donated by the ammonium group of the lysine side chain with
Ser123), and AS2 was occupied by the terminal phenyl group
(instead of AS1 for 24b). When superimposed within the active site,
compounds 18b and 24b were tightly aligned for most of their
structure (see Fig. 6), the difference lying mainly in occupation of
AS2 instead of AS1 (which, expectedly, is not highly significant in
terms of biological activity).
In analogs 18a and 24a, protection of the side chain of the lysine
moiety by a Cbz group was found to be compatible with S3 pocket
occupation, whereas no hydrogen bond could be observed with this
part of the molecule (See Figs. 5S and 6S in Supporting
informations). The overall b-sheet structure remained intact, with
its underpinning network of hydrogen bonds with key residues. In
particular, for 18a, Gly47 exchanged two hydrogen bonds with the
NH and CO groups of the phenylalanine moiety, in addition to two
hydrogen bonds donated to Thr21 and Asp125 (respectively by one
NH group of the hydrazide function and the lysine aNH) (Fig. 5S,
Supporting informations). In 24a, the two amide groups were
Fig. 6. Superposition of docking poses of compound 24b (orange) and 18b (green)
positioned into the ChT-L binding site pocket of the bovine proteasome X-ray structure.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
responsible for the hydrogen bonds, one with Gly47 and Thr21, the

514
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
In the fluorinated series, remarkably, the two diastereoisomers
4. Experimental
of mixture 1b were able to fit nicely into the ChT-L active site with a
slightly different binding mode while they showed a similar inhi-
bition activity (IC50 ¼ 8.4 and 5.9 M for 1b1 and 1b2, Table 1). Di-
astereoisomers SRS and SSS of 1b presented 7 hydrogen bonds with
Thr1, Thr21, Gly47, Phe124 and Asp125 and 5 hydrogen bonds with
Gly47, Tyr107, Ser123 and Asp125 respectively. A difference in the
orientation of the lysine side chain and the methyl ester moiety was
also noted, but activity-critical pockets were occupied in both cases.
(see Figs. 7S and 8S in Supporting information).
Furthermore, during our molecular modeling studies, we
observed a supplementary and interesting way of binding for
compound 17b, where the protected lysine side chain aligns with
the rest of the molecule instead of occupying S3 subsite (see Fig. 9S
in Supporting information). The same phenomenon could be
observed for both diastereoisomers of compound 4.
4.1. Chemistry
The usual solvents were purchased from commercial sources.
Dimethylformamide (DMF) was distilled on CaSO4, tetrahydrofuran
(THF) was distilled on sodium/benzophenone, acetonitrile was
distilled on CaCl2. TLC was performed on silica gel, 60F-250 (0.26 mm
thickness) plates. The plates were visualized with UV light (254 nm)
or with a 3.5% solution of phosphomolybdic acid in ethanol or with a
solution of ninhydrin in ethanol. Liquid chromatography was per-
formed on Merck 60 silica gel (230e400 mesh). Separation of di-
astereoisomers of 1b was performed by HPLC using a WATERS
gradient system (pump þ controller E600, UV detector PDA 2996,
autosampler 717) and
a
column SUNFIRE (C18,
5
mm,
150 mm ꢂ 19 mm). Protected amino acids, O-benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU), 1-
hydroxybenzotriazole (HOBT) and 3-bromo-propionic acid ethyl
ester 33 were purchased from commercial sources. 2-[3-(N0-{6-
Benzyloxycarbonylamino-2-[2-(3-phenoxy-phenyl)acetylamino]
hexanoyl}-hydrazino)-4,4,4-trifluoro-butyrylamino]-3-phenyl-pro-
pionic acid methyl ester 1a, 2-[3-(N0-{6-Amino-2-[2-(3-
phenoxyphenyl)acetylamino]hexanoyl}hydrazino)-4,4,4-trifluoro-
butyrylamino]-3-phenyl-propionic acid methyl ester 1b, 2-[3-(N0-{3-
(3,4-Dimethoxyphenyl)-2-[2-(3-phenoxyphenyl)acetylamino]pro-
pionyl} hydrazino)-4,4,4-trifluorobutyrylamino]-3-phenylpropionic
acid methyl ester 2, 2-{3-[N0-(2-Benzyloxycarbonylamino-6-tert-
butoxycarbonylaminohexanoyl)hydrazino]-4,4,4-trifluorobutyrylam
ino} -3-phenylpropionic acid methyl ester 3a, 2-{3-[N0-(6-Amino-2-
tert-butoxycarbonylaminohexanoyl)hydrazino]-4,4,4-trifluorobuty
rylamino}-3-phenylpropionic acid methyl ester 3b, 2-{3-[N0-(2-
Amino-6-benzyloxycarbonylaminohexanoyl)hydrazino]-4,4,4-triflu
oro-butyrylamino}-3-phenylpropionic acid methyl ester 4, 3-Phenyl-
2-(4,4,4-trifluoro-3-{N0-[2-(3-phenoxyphenyl)acetyl]hydrazino} but
yrylamino) propionic acid methyl ester 6, Ethyl 4,4,4-
trifluorocrotonate 26, 3-(N0-tert-butoxycarbonylhydrazino)-4,4,4-
trifluorobutyric acid ethyl ester 27, the trifluoroacetic salt of 3-
phenyl-2-(4,4,4-trifluoro-3-hydrazinobutyrylamino) propionic acid
methyl ester 28, were prepared according to published methods [32].
Melting points were determined on a Kofler melting point apparatus.
Unless otherwise stated, NMR spectra were performed on a Bruker
AVANCE400(1H, 400MHz;13C,100MHz). Ifstated, NMRspectrawere
performed on a BRUKER AMX 200 (1H, 200 MHz; 13C, 50 MHz;
188 MHz, 19F) or an Ultrafield AVANCE 300 (1H, 300 MHz; 13C,
75 MHz). Unless otherwise stated, CDCl3 was used as solvent.
On the contrary, satisfying poses (ie. completing the 3 criteria:
hydrogen bonding with key residues, superposition with the
pseudopeptide of Furet et al. [16] and occupation of at least S1 and
S3 simultaneously) could not be observed for compounds that were
experimentally not able to inhibit 20S proteasome. For example, in
the case of compound 16a, even if the dimethoxyphenylalanine and
phenoxyphenylacetyl moieties were accurately orientated in the
pockets S3 and AS2 respectively, compound 16a did not share
enough hydrogen bonds with the key residues. The comparison of
16a with its structurally analog 2 which showed inhibition activity
(IC50 of 85 mM) is of particular interest. Compound 2 was correctly
bound in the active site with 5 hydrogen bonds contracted with the
key residues (Thr1, Thr21, Gly47, Asp125). The detailed analysis of
docking experiments provided some clues to explain the differ-
ences observed between our compounds for their inhibitory ac-
tivities of 20S proteasome.
3. Conclusions
To our knowledge, a-hydrazino acid scaffolds have only rarely
been used in medicinal chemistry and particularly very scarcely
introduced in protease inhibitors. Moreover, little was known about
the
confirmed that
typical secondary structure of native
concept, - and -hydrazino acid scaffolds were introduced in a
b
-hydrazino acid based peptidomimetics. In this work, we
- and -hydrazino acid structures can mimic the
-peptides. As a proof of
a
b
a
a
b
library of 40 pseudopeptides that were tested for their ability to
inhibit the 20S proteasome ChT-L activity. The peptidic proteasome
inhibitors reported in the literature commonly have borne
acid moieties [12,13]. We described here the easy preparation of
pseudopeptides bearing - and -hydrazino acid scaffolds that
inhibit the ChT-L activity of 20S proteasome up to a submicromolar
range (0.7 M for molecule 24b). Thus, the ability of preserving
biological activity by introducing in peptides, - and -hydrazino
acid scaffolds is demonstrated. By performing pharmacomodula-
tions around these hydrazino acid scaffolds, we established struc-
tureeactivity-relationships. We have shown herein the slightly
superiority of the phenoxyphenylacetyl moiety in the first part of
the pseudopeptides, as previously observed by Furet et al. [16], and
also the dramatic superiority of a lysine residue in the second part,
and finally the possibility of the nearly complete suppression of the
peptidic character of the molecules, by introducing 3,4,5-
trimethoxybenzylamine in place of the phenylalanine residue in
the fourth part. Finally, we proposed a binding mode of this new
class of non covalent proteasome inhibitors to ChT-L subunit by
molecular modeling and developed a rational explanation about
the different activities of the compounds against the ChT-L activity
thanks to the docking calculations.
a
-amino
Chemical shifts d are in ppm, and the following abbreviations are
used : singlet (s), doublet (d), doublet doublet (dd), triplet (t), quin-
tuplet (quint), multiplet (m), broad multiplet (bm), and broad singlet
(bs). Mass spectra were obtained using a Bruker Esquire electrospray
ionization apparatus at the SAMM (Facultyof Pharmacyat Châtenaye
Malabry). Element analyses (C, H, N) were performed on a Perkine
Elmer CHN, Analyser 2400 at the Microanalyses Service of the Faculty
of Pharmacy at ChâtenayeMalabry (France).
a
b
m
a
b
4.1.1. Synthesis of the target molecules including the
trifluoromethyl-b-hydrazino acid scaffold 1b, 5a, 5b, and 7e16b
4.1.1.1. 2-[3-(N0-{6-Amino-2-[2-(3-phenoxyphenyl)acetylamino]hex-
anoyl} hydrazino)-4,4,4-trifluoro-butyrylamino]-3-phenyl-propionic
acid methyl ester, trifluoroacetic acid salt 1b. Compound 1b has
been synthesized as a mixture of 2 diasteroisomers (ratio: 1/1) and
described previously.32 The two diastereoisomers were separated
by HPLC (column SUNFIRE, C18, 5
m
m, 150 mm ꢂ 19 mm; mobile
phase: a mixture of A: water (0.1% formic acid) and B: CH3CN
(mixture A/B 73/27 to 68/32 in 15 min); room temperature; flow
rate 1 mL/min; detection at 235 nm).

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
515
4.1.1.1.1. Diastereoisomer 1. TR ¼ 11.75 min 1H NMR (CD3OD):
¼ 8.44 (br s, 1H), 7.25e7.02 (m, 8H), 6.96e6.72 (m, 6H), 4.62 (m,
1.41e1.36 (m, 2H), 1.28e1.21 (m, 2H); 13C NMR (CD3OD):
174.41, 155.5, 153.5, 153.4, 152.0, 138.5, 130.6, 129.9, 129.6, 128.3,
126.3, 118.7, 114.3, 114.2, 113.1, 67.3, 60.7, 60.2, 59.9, 59.7, 56.9, 56.4,
56.0, 53.2, 53.1, 40.9, 39.0, 38.7, 34.6, 33.0, 28.4, 23.8, 15.8; 19F
d
¼ 174.42,
d
1H), 4.17 (m, 1H), 3.62 (m, 1H), 3.58 (s, 3H), 3.39 (s, 2H), 2.92 (dd
J ¼ 5.8 and 13.8 Hz, 1H), 2.83 (dd, J ¼ 8.4 and 13.8, 1H), 2.68 (m, 2H),
2.42 (m, 2H), 1.60e1.18 (m, 6H). 19F (188 MHz, CD3OD):
d
¼ ꢃ76.53).
(188 MHz,CD3OD):
d
¼ ꢃ76.67 (d, 0.3F, J ¼ 7.3 Hz), ꢃ76.62 (d, 0.7F,
Anal. Calcd for C34H41ClF3N5O6$H2O: C, 56.23; H, 5.98; N 9.65;
found C, 56.12; H, 5.75; N, 9.45.
J ¼ 7.5 Hz), ꢃ77.34 (s, 1F); IR (cmꢃ1): 3324, 1639, 1503, 1126, 700;
ESIþ MS m/z: 640 [M þ H]þ; Anal. Calcd for C32H41F6N5O9$0.5 H2O:
C, 50.39; H, 5.56; N, 9.18; found C, 50.04; H, 5.37; N, 8.73.
4.1.1.1.2. Diastereoisomer 2. TR ¼ 14.13 min 1H NMR (CD3OD):
d
¼ 8.51 (br s,1H), 7.37e7.18 (m, 8H), 7.07 (m, 2H), 6.97 (d, J ¼ 7.6 Hz,
3H), 6.84 (dd, J ¼ 8.13 and 1.9 Hz, 1H), 4.74 (dd, J ¼ 8.5 and 5.9, 1H),
4.62 (m, 1H), 3.81 (m, 1H), 3.74 (s, 3H), 3.59 (s, 2H), 3.17 (dd, J ¼ 5.9
and 13.8 Hz, 1H), 3.06 (dd, J ¼ 8.5 and 13.8 Hz, 1H), 3.01 (m, 2H),
2.56 (dd, J ¼ 6.8 Hz, 2H), 1.80e1.65 (m, 4H), 1.35 (m, 2H). 19F
4.1.1.4. 2-(3-{N0-[2-(2,5-Dimethoxy-phenyl)-acetyl]-hydrazino}-
4,4,4-trifluoro-butyrylamino)-3-phenyl-propionic acid methyl ester 7.
To a solution of the trifluoroacetic salt of 28 [32] (392 mg,
0.88 mmol, 1.0 eq.) in DMF (3 mL), were successively added DIPEA
(188 MHz, CD3OD):
34H41ClF3N5O6$H2O: C, 56.23; H, 5.98; N 9.65; found C, 56.08; H,
d
¼
ꢃ76.46. Anal. Calcd for
(730 ml, 4.4 mmol, 5.0 eq.) and HOBT (179 mg, 1.32 mmol, 1.5 eq.).
C
Meanwhile a solution of 2,5-dimethoxyphenylacetic acid (259 mg,
1.32 mmol, 1.5 eq.) and HBTU (499 mg, 1.32 mmol, 1.5 eq.) in DMF
(3 mL) was stirred for half an hour at room temperature. The so-
lutions were combined and the resulting mixture was stirred at
room temperature overnight. After evaporation under reduced
pressure, the resulting yellow oil was taken up in EtOAc (10 mL) and
successively washed with 10% aqueous citric acid (2 ꢂ 10 mL), 10%
aqueous K2CO3 (2 ꢂ 10 mL) and brine (15 mL). The organic layer
was dried over Na2SO4, filtered and evaporated under reduced
pressure to give a slightly yellow solid which was purified by col-
umn chromatography (EtOAc) to give 7 as a white solid (347 mg,
5.90; N, 9.36.
4.1.1.2. 2-[3-(N0-{6-Benzyloxycarbonylamino-2-[2-(2,5-dimethoxy-
phenyl)-acetylamino]-hexanoyl}-hydrazino)-4,4,4-trifluoro-butyr-
ylamino]-3-phenyl-propionic acid methyl ester 5a. To a solution of 4
(319 mg, 0.45 mmol, 1.0 eq.) in DMF (2 mL), DIPEA (370
ml,
2.25 mmol, 5.0 eq.) and HOBT (90 mg, 0.67 mmol, 1.5 eq.) were
added. Meanwhile a solution of 2,5-dimethoxyphenylacetic acid
(132 mg, 0.67 mmol, 1.5 eq.) and HBTU (254 mg, 0.67 mmol, 1.5 eq.)
in DMF (2 mL) was stirred for half an hour at room temperature. The
two solutions were then combined and the resulting mixture was
stirred at room temperature overnight. After concentration under
reduced pressure, the resulting yellow oil was taken up in EtOAc
(10 mL) and successively washed with 10% aqueous citric acid
(2 ꢂ 10 mL), 10% aqueous K2CO3 (2 ꢂ 10 mL) and brine (15 mL). The
organic layer was dried over Na2SO4, filtered and evaporated under
reduced pressure to give a slightly yellow solid which after pre-
cipitation in a mixture of cyclohexane/EtOAc afforded 5a as a white
solid (298 mg, 0.38 mmol, 84%). mp 140e142 ꢀC; 1H NMR (DMSO-
0.68 mmol, 77%). mp 100e102 ꢀC; 1H NMR (DMSO-d6):
d
¼ 9.37 (d,
1H, J ¼ 5.1 Hz), 8.60 (d, 1H, J ¼ 7.4 Hz), 8.03 (t, 1H, J ¼ 8.8 Hz), 7.34e
7.22 (m, 5H), 6.88e6.80 (m, 3H), 4.58e4.50 (m, 2H), 3.68 (s, 3H),
3.67 (s, 3H), 3.58 (s, 3H), 3.40 (s, 2H), 3.05e2.88 (m, 2H), 2.50e2.4
(m, 2H); 13C NMR (DMSO-d6):
d
¼ 172.3, 171.6, 168.4, 168.2, 153.7,
151.0, 136.4, 129.2, 129.1, 128.4, 126.9, 123.1, 117.1, 113.4, 11.5, 55.7,
53.8, 53.7, 52.4, 37.3, 37.1, 33.3; 19F (188 MHz, DMSO-d6):
d
¼ ꢃ74.99 (d, 0.7F, J ¼ 7.3 Hz) I diastereomer ꢃ75.37 (d, 0.3F,
J ¼ 7.1 Hz) II diastereomer; IR (cmꢃ1): 3301, 1648, 1502, 1229,
d6):
d
¼ 9.53 (t, 1H, J ¼ 5.6 Hz), 8.64 (dd, 1H, J ¼ 7.3 Hz, 8.7 Hz), 7.95
1122 698; ESIþ MS m/z: 534 [M
C
þ
Na]þ; Anal. Calcd for
(m, 1H), 7.36e7.20 (m, 10H), 6.87e6.76 (m, 3H), 5.01 (s, 2H), 4.50
(m, 1H), 4.20 (m, 1H) 3.79 (m, 1H), 3.69 (s, 3H), 3.68 (s, 3H), 3.59 (s,
3H), 3.42 (d, 2H, J ¼ 3.2 Hz), 3.06e2.90 (m, 4H), 2.46 (m, 2H), 1.57e
1.53 (m, 2H), 1.41e1.36 (m, 2H), 1.28e1.21 (m, 2H); 13C NMR
24H28F3N3O6: C, 56.36; H, 5.52; N, 8.22: found C, 56.08; H, 5.43; N,
8.01.
4.1.1.5. 2-{3-[N0-(2-tert-Butoxycarbonylamino-3-carbamoyl-pro-
pionyl)-hydrazino]-4,4,4-trifluoro-butyrylamino}-3-phenyl-propionic
acid methyl ester 8. To a solution of the trifluoroacetic salt of 28
(DMSO-d6):
d
¼
d 171.7, 171.6, 171.4, 169.8, 168.1, 156.0, 152.9, 151.3,
137.2, 137.0, 136.9, 129.0, 128.9, 128.3, 128.2, 127.7, 126.5, 116.7, 111.9,
111.6, 79.4, 78.9, 78.5, 65.1, 55.8, 55.2, 53.7, 53.6, 51.8, 51.0, 50.9,
36.7, 36.6, 32.9, 31.7, 29.0, 22.4; 19F (188 MHz, DMSO-d6):
(163 mg, 0.49 mmol, 1.0 eq.) [32] in DMF (2 mL), DIPEA (400 ml,
2.45 mmol, 5.0 eq.) and HOBT (100 mg, 0.74 mmol, 1.5 eq.) were
successively added. Meanwhile a solution of Na-Boc-Asn (170 mg,
0.74 mmol, 1.5 eq.) and HBTU (280 mg, 0.74 mmol, 1.5 eq.) in DMF
(2 mL) was stirred for half an hour at room temperature. The two
solutions were then combined and the resulting mixture was
stirred at room temperature overnight. The solvent was evapo-
rated under reduced pressure and the resulting yellow oil was
taken up in EtOAc (10 mL) and successively washed with 10%
aqueous citric acid (2 ꢂ 10 mL), 10% aqueous K2CO3 (2 ꢂ 10 mL)
and brine (15 mL). The organic layer was dried over Na2SO4,
filtered and evaporated under reduced pressure to yield a white
solid which was purified by column chromatography (EtOAc:-
MeOH 9:1) to give 8 as a white solid (168 mg, 0.30 mmol, 63%). mp
d
¼ ꢃ73.74 (d, 0.4F, J ¼ 7.3 Hz) I diastereomer ꢃ73.91 (d, 0.6F,
J ¼ 7.5 Hz) II diastereomer; IR (cmꢃ1): 3283, 2943, 1739, 1684, 1637,
1531, 1502, 1443, 1360, 1261, 1227, 1207 739; ESIþ MS m/z: 796
[M þ Na]þ; Anal. Calcd for C38H46F3N5O9.0.75$H2O: C, 57.95; H,
6.09; N, 8.90; found C, 58.04; H, 6.02; N 9.01.
4.1.1.3. 2-[3-(N0-{6-Amino-2-[2-(2,5-dimethoxy-phenyl)-acetyla-
mino]-hexanoyl}-hydrazino)-4,4,4-trifluoro-butyrylamino]-3-
phenyl-propionic acid methyl ester trifluoroacetic salt 5b. To a so-
lution of 5a (461 mg, 0.60 mmol, 1.0 eq.) in dry methanol (8 mL)
was added Pd/C 10% (46 mg). The mixture was stirred overnight
under hydrogen atmosphere at room temperature and filtered on a
celite pad. The yellowish solid obtained after concentration under
reduced pressure was dissolved in DCM/TFA 3:1 (8 mL) and stirred
for half an hour. The crude residue obtained after concentration
under reduced pressure was dissolved in DCM/Et2O with just few
drops of MeOH and precipitated at ꢃ20 ꢀC. The yellowish solid
obtained was finally washed with Et2O to afford 5b (461 mg,
150e152 ꢀC; 1H NMR (DMSO-d6):
J ¼ 7.6 Hz), 7.35e7.17 (m, 5H), 6.90 (d, 2H, J ¼ 7.6 Hz), 5.43 (m, 1H),
4.49 (m, 1H), 4.19 (m, 1H), 3.74 (m, 1H), 3.59 (s, 3H), 3.05e2.85 (m,
2H), 2.60e2.40 (m, 2H), 2.40e2.31 (m, 2H), 1.37 (s, 1H); 13C NMR
d
¼ 9.44 (m, 1H), 8.60 (d, 1H,
(DMSO-d6):
d
¼ 171.6, 171.3, 171.1, 171.0, 168.0, 154.9, 137.0, 129.0,
128.2, 126.5, 78.1, 53.7, 51.7, 50.0, 37.1, 36.7, 32.8, 28.1; 19F
0.49 mmol, 82%). mp 120e122 ꢀC; 1H NMR (CD3OD):
(m, 5H), 6.86e6.75 (m, 3H), 4.65 (m, 1H), 4.29 (m, 1H), 3.74 (s, 3H),
3.69 (s, 3H), 3.65 (s, 3H), 3.51e3.44 (m, 3H), 3.15e3.08 (m, 1H), 3.00
(m, 1H), 2.88e2.82 (m, 2H), 2.52e2.46 (m, 2H), 1.57e1.53 (m, 2H),
d
¼ 7.23e7.17
(188 MHz, DMSO-d6):
d
¼ ꢃ73.71 (d, 1H, J ¼ 7.4 Hz); IR (cmꢃ1):
3477, 2149, 1639, 1524, 1165; ESIþ MS m/z: 548 [M þ H]þ; Anal.
Calcd for C23H32F3N5O7$0.15 H2O: C, 50.20; H, 5.93; H, 12.74; found
C, 49.75; H, 5.75; N, 13.61.

516
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
4.1.1.6. 2-[3-(N0-{3-Carbamoyl-2-[2-(2,5-dimethoxy-phenyl)-acety-
lamino]-propionyl}-hydrazino)-4,4,4-trifluoro-butyrylamino]-3-
phenyl-propionic acid methyl ester 9. A solution of 8 (355 mg,
0.64 mmol, 1.0 eq.) in DCM/TFA 2:1 (9 mL) was stirred for 2 h at
room temperature. After removal of the solvent under reduced
pressure, the excess of trifluoroacetic acid was coevaporated with
methanol and the resulting yellowish solid oil precipitated with
Et2O. The resulting white solid was dissolved in DMF (3 mL), then
4.1.1.8. [5-Amino-5-(N0-{2,2,2-trifluoro-1-[(3,4,5-trimethoxy-benzyl-
carbamoyl)-methyl]-ethyl}-hydrazinocarbonyl)-pentyl]-carbamic
acid benzyl ester trifluoroacetic salt 10b. A solution of 10a (741 mg,
1.04 mmol, 1.0 eq.) in DCM/TFA 3:1 (15 mL) was stirred for 2 h at
room temperature. After removal of the solvent under reduced
pressure, the excess of TFA was coevaporated with methanol. The
crude was then precipitated with diethyl ether and washed with
cyclohexane (3 ꢂ 15 mL) to afford 10b as a white solid (745 mg,
1.04 mmol, quantitative). mp: 136e138 ꢀC; 1H NMR (DMSO-d6):
DIPEA (540
ml, 3.25 mmol, 5.0 eq.) and HOBT (129 mg,
0.95 mmol, 1.5 eq.) were successively added. Meanwhile a solu-
tion of 2,5-dimethoxyphenylacetic acid (187 mg, 0.95 mmol, 1.5
eq.) and HBTU (361 mg, 0.95 mmol, 1.5 eq.) in DMF (3 mL) was
stirred for half an hour at room temperature. The two solutions
were combined and the resulting mixture was stirred at room
temperature overnight. The solvent was evaporated under
reduced pressure and the resulting yellow oil was taken up in
EtOAc (15 mL). The organic layer was successively washed with
10% aqueous citric acid (2 ꢂ 10 mL), water (15 mL), 10% aqueous
K2CO3 (2 ꢂ 10 mL) and brine (15 mL). The organic layer was dried
over Na2SO4, filtered and evaporated under reduced pressure to
afford a white solid which after washing several times with
EtOAc, cyclohexane and Et2O gave 9 as a white solid: mp 168e
d
¼ 9.98 (m, 1H), 8.59 (m, 1H), 8.14 (bs, 3H), 8.02 (m, 1H) 7.38e7.29
(m, 5H), 7.21 (m, 1H), 6.57 (s, 2H), 5.00 (s, 2H), 4.20e4.30 (m, 2H),
3.89 (m, 1H), 3.74 (s, 6H), 3.65 (m, 1H), 3.62 (s, 3H), 2.98 (m, 2H),
2.58 (m, 2H), 1.65e1.69 (m, 2H), 1.38e1.42 (m, 2H), 1.25e1.29 (m,
2H); 13C NMR (DMSO-d6):
d
¼ 171.0, 169.9,169.8,159.0,138.4,138.2,
135.8, 129.5, 129.0, 128.8, 106.0, 67.4, 61.1, 60.1, 59.7, 56.6, 53.2, 53.1,
44.5, 41.4, 34.7, 34.6, 32.2, 30.5, 23.0; 19F (188 MHz, DMSO-d6):
d
¼ ꢃ76.51 (m, 1F), ꢃ77.33 (s, 1F); IR (cmꢃ1): 2924, 1667, 1594, 1123;
ESIþ MS m/z: 614 [M þ Hþ]; Anal. Calcd for C30H39F6N5O9$1.5 H2O:
C, 47.74; H, 5.62; N, 9.28; found C, 47.52; H, 5.04; N, 8.80.
4.1.1.9. [5-[2-(2,5-Dimethoxy-phenyl)-acetylamino]-5-(N0-{2,2,2-
trifluoro-1-[(3,4,5-trimethoxy-benzylcarbamoyl)-methyl]-ethyl}-
hydrazinocarbonyl)-pentyl]-carbamic acid benzyl ester 11a. To a
solution of 12b (563 mg, 0.77 mmol, 1.0 eq.) in DMF (4 mL), DIPEA
170 ꢀC (259 mg, 0.42 mmol, 65%); 1H NMR (DMSO-d6):
d
¼ 9.35
(m, 1H), 8.63 (m, 1H), 8.02 (m, 1H), 7.34e7.22 (m, 5H), 6.92e6.82
(m, 3H), 5.42 (bs, 1H), 4.59 (m, 2H), 3.78 (m, 4H), 3.75 (s, 3H),
3.68 (s, 3H), 3.46 (s, 2H), 3.18e3.11 (m, 1H), 3.04e2.97 (m, 1H),
(640
ml, 3.85 mmol, 5.0 eq.) and HOBT (157 mg, 1.16 mmol, 1.5 eq.)
were successively added. Meanwhile
a
solution of 2,5-
2.52 (m, 4H); 13C NMR (DMSO-d6):
d
¼ 171.8, 171.7, 171.1, 171.0,
dimethoxyphenylacetic acid (227 mg, 1.16 mmol, 1.5 eq.) and
HBTU (440 mg,1.16 mmol,1.5 eq.) in DMF (4 mL) was stirred for half
an hour at room temperature. The two solutions were combined
and the resulting mixture was stirred at room temperature over-
night. After evaporation of the solvent under reduced pressure, the
resulting yellow oil was taken up in EtOAc (20 mL) and washed
with 10% aqueous citric acid (2 ꢂ 15 mL), 10% aqueous K2CO3
(2 ꢂ 15 mL), brine (20 mL), and distilled water. The organic layer
was dried over Na2SO4, filtered and evaporated under reduced
pressure to give a slightly yellow solid which was purified by pre-
cipitation in a hot mixture of methanol/EtOAc to give 11a as a white
solid (434 mg, 0.59 mmol, 77%). mp: 138e140 ꢀC; 1H NMR (DMSO-
170.6, 169.8, 168.2,152.9, 151.1, 137.0, 129.0, 128.2, 126.5, 116.6,
112.1, 55.9, 55.3, 53.7, 53.6, 51.7, 48.4, 37.0, 36.7, 32.8; 19F (DMSO-
d6):
d
¼ ꢃ73.72 (d, 0.7F, J ¼ 7.4 Hz) I diastereomer ꢃ73.85 (d, 0.3,
J ¼ 7.8 Hz) II diastereomer; IR (cmꢃ1): 3286, 1639, 1537, 1406,
1226, 1124; ESIþ MS m/z: 626 [M þ Na]þ; Anal. Calcd for
C
28H34F3N5O8: C, 53.76; H, 5.48; N, 11.19; found C, 53.79; H, 5.30;
N, 10.75.
4.1.1.7. [5-tert-Butoxycarbonylamino-5-(N0-{2,2,2-trifluoro-1-
[(3,4,5-trimethoxy-benzylcarbamoyl)-methyl]-ethyl}-hydrazino-
carbonyl)-pentyl]-carbamic acid benzyl ester 10a. The trifluoro-
acetic salt of 29 (930 mg, 1.99 mmol, 1.0 eq.) was dissolved in
DMF (5 mL), then DIPEA (1.6 mL, 10.0 mmol, 5.0 eq.) and HOBT
(323 mg, 2.39 mmol, 1.2 eq.) were successively added. Mean-
while a solution of Na-Boc-N -3 Z-Lys (910 mg, 2.39 mmol, 1.2
eq.) and HBTU (906 mg, 2.39 mmol, 1.2 eq.) in DMF (5 mL) was
stirred for half an hour at room temperature. The two solutions
were then combined and the resulting mixture was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the resulting yellow oil was taken up in
EtOAc (30 mL). The resulting organic phase was successively
washed with 10% aqueous citric acid (2 ꢂ 20 mL), 10% aqueous
K2CO3 (2 ꢂ 20 mL) and brine (30 mL). The organic layer was
dried over Na2SO4, filtered and evaporated under reduced
pressure to give a slightly yellow solid which was purified by
column chromatography (cyclohexane:EtOAc 1:1) to give 10a as
a white solid (1.19 g, 1.67 mmol, 84%): mp 112e114 ꢀC; 1H
d6):
d
¼ 9.57 (d, 1H, J ¼ 5.4 Hz), 8.56 (t, 1H, J ¼ 5.8 Hz), 7.95 (d, 1H,
J ¼ 8.0 Hz), 7.37e7.22 (m, 5H), 7.22 (t, 1H, J ¼ 5.4 Hz), 6.86e6.76 (m,
1H), 6.70e6.65 (m, 2H), 6.57 (s, 2H), 5.54 (t, 1H, J ¼ 4.4 Hz), 5.00 (s,
2H), 4.24 (m, 3H), 3.86 (m, 1H), 3.73 (s, 6H), 3.67 (s, 6H), 3.62 (s, 3H),
3.40 (m, 2H), 2.99e2.81 (m, 2H), 2.50e2.40 (m, 2H), 1.57e1.49 (m,
2H), 1.46e132 (m, 2H), 1.30e1.20 (m, 2H); 13C NMR (DMSO-d6):
d
¼ 171.3, 169.8, 168.0, 156.5, 152.9, 152.7, 151.3, 137.2, 136.3, 134.7,
128.3, 127.9, 127.7, 125.6, 125.5, 116.7, 111.9, 111.6, 104.5, 65.1, 59.9,
55.8, 55.7, 55.2, 51.1, 51.0, 42.4, 36.7, 36.6, 33.2, 31.8, 29.0, 22.4; 19
F
(188 MHz, DMSO-d6):
d
¼ ꢃ76.51 (m, 1F), ꢃ77.33 (s, 1F); IR (cmꢃ1):
3298,1633, 1503, 1228, 1126, 698; ESIþ MS m/z: 793 [M þ Hþ]; Anal.
Calcd for C38H48F3N5O10$H2O: C, 56.34; H, 6.23; N, 8.65; found C,
56.55; H, 6.38; N, 8.41.
4.1.1.10. 3-[2-[(2S)-6-Amino-2-[[2-(2,5-dimethoxyphenyl)acetyl]
amino] hexanoyl]hydrazino]-4,4,4-trifluoro-N-[(3,4,5-
trimethoxyphenyl) methyl]butanamide 11b. To a solution of 11a
(119 mg, 0.15 mmol, 1 eq.) in dry DMF/MeOH (5:30 mL, v/v) was
added 10% Pd/C (11 mg). The mixture was stirred overnight under
hydrogen atmosphere at room temperature. The catalyst was fil-
trated on a celite pad and the solvent was evaporated under
reduced pressure to afford 11b as a white solid (70 mg, 0.10 mmol,
NMR:
d
¼ 9.48 (d, 1H, J ¼ 5.2 Hz), 8.57 (m, 1H), 7.28e7.36 (m,
5H), 7.23 (t, 1H, J ¼ 5.3 Hz), 6.85 (m, 1H), 6.58 (s, 2H), 5.54 (m,
1H), 5.00 (s, 2H), 4.20e4.27 (m, 2H), 3.85 (m, 2H), 3.74 (s, 6H),
3.62 (s, 3H), 2.97 (m, 2H), 2.50 (m, 2H), 1.52 (m, 2H), 1.42 (m,
9H), 1.22 (m, 4H); 13C NMR:
d
¼ 167.9, 163.2, 163.0, 155.9, 152.6,
137.1, 136.1, 134.6, 128.2, 127.6, 104.3, 104.3, 77.8, 65.0, 59.8,
55.6, 28.9, 28.0, 22.6; 19F (188 MHz):
d
¼
ꢃ74.62 (d, 1F,
75%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.57 (t, 1H, J ¼ 5.4 Hz), 7.97
J ¼ 7.5 Hz), ꢃ74.89 (d, 1F, J ¼ 6.3 Hz); IR (cmꢃ1): 3312, 1637,
(m, 1H), 6.86 (d, 1H, J ¼ 8.7 Hz), 6.76 (m, 2H), 6.57 (s, 2H), 5.54 (d,
1H, J ¼ 3.6 Hz), 4.28 (m, 2H), 4.20 (m, 1H), 3.86 (m, 1H), 3.74 (s, 9H),
3.67 (s, 6H), 3.41 (m, 2H), 2.73 (m, 2H), 2.51 (m, 2H), 1.56 (m, 1H),
1.50 (m, 1H), 1.35 (m, 2H), 1.21 (m, 2H). 13C NMR (75 MHz, DMSO-
1525, 1250, 1126; ESIþ MS m/z: 714.4 [M þ H]þ; Anal. Calcd for
C
33H46F3N5O9$H2O: C, 54.19; H, 6.63; N, 9.58; found C, 54.17; H,
6.19; N, 9.43.

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
517
d6):
d
¼ 171.3, 169.9, 168.1, 152.8, 151.3, 136.3, 134.9, 125.7, 111.9,
C23H34F3N5O8$0.5H2O: C, 48.12; H, 6.16; N, 12.20; found C, 48.27; H,
5.88; N, 11.86.
111.6,104.4, 67.1, 59.9, 57.8, 55.7, 51.1, 42.3, 36.7, 33.4, 33.2, 31.9, 31.4,
22.4. 19F (188 MHz, DMSO-d6):
d
¼ ꢃ71.34 (s), ꢃ72.71 (s): 2 di-
astereoisomers: 75/25. APCIþ MS m/z: 658 [M þ H]þ. Anal. Calcd for
4.1.1.13. N0-(3-{N0-[3-(3,4-Dimethoxyphenyl)-2-(9H-fluoren-9-
ylmethoxycarbonylamino) propionyl]hydrazino}-3-oxo-1-
trifluoromethylpropyl)hydrazinecarboxylic acid benzyl ester 14a.
A solution of the 32 (359 mg, 1.12 mmol) and Fmoc-L-3,4-
dimethoxyphenylalanine (501 mg, 1.12 mmol) in dry DMF
(13 mL) was stirred for 5 min at room temperature. Then were
C
30H42F3N5O8$4 H2O: C, 49.37; H, 6.92; N, 9.60; found: C, 48.83; H,
6.35; N, 9.76.
4.1.1.11. 3-[2-[(2S)-6-Amino-2-[[2-(3-phenoxyphenyl)acetyl]amino]
hexanoyl]hydrazino]-4,4,4-trifluoro-N-[(3,4,5-trimethoxyphenyl)
methyl]butanamide 12. To a solution of 10b (376 mg, 0.61 mmol, 1.0
eq.) in dry DMF (10 mL) was successively added 3-
phenoxyphenylacetic acid (166 mg, 0.73 mmol, 1.2 eq.), DIPEA
(556 mg, 4.30 mmol, 7.0 eq.), HBTU (277 mg, 0.73 mmol, 1.2 eq.),
and HOBT (99 mg, 0.73 mmol, 1.2 eq.). The mixture was stirred
overnight at room temperature under argon atmosphere. After
removal of the solvent under reduced pressure, the residue was
taken up in EtOAc (15 mL), washed with 10% aqueous citric acid
(2 ꢂ 10 mL), water (10 mL), 10% aqueous K2CO3 (2 ꢂ 10 mL) and
brine (10 mL). The organic layer was dried over Na2SO4, filtered and
evaporated under reduced pressure to give a crude product which
was purified by column chromatography (EtOAc) to afford the Cbz
protected amine (355 mg, 4.30 mmol, 71%) as a white solid. 1H NMR
added successively 2,4,6-collidine (447
mL, 3.36 mmol), HOBT
(166 mg, 1.23 mmol) and HBTU (467 mg, 1.23 mmol). The mixture
was stirred at room temperature under argon atmosphere for
40 h. After removal of the solvent under reduced pressure, the
residue was triturated in EtOAc (15 mL) to afford 14a as a white
solid (560 mg, 0.75 mmol, 67%). mp: 202e204 ꢀC; 1H NMR
(DMSO-d6):
d
¼ 10.23 (bs, 1H), 10.15 (bs, 1H), 8.90 (bs, 1H), 7.90
(d, J ¼ 7.4 Hz, 2H), 7.70 (d, J ¼ 7.6 Hz, 1H), 7.43e7.26 (m, 11 H),
7.01 (bs, 1H), 6.89e6.80 (m, 2 H), 5.36 (bs, 1H), 5.08 (s, 2H), 4.32
(m, 1H), 4.19e4.10 (m, 3H), 3.93 (m, 1H), 3.72 (s, 3H), 3.68 (s, 3H),
2.99 (m, 1H), 2.76 (m, 1H), 2.52 (m, 2H). 13C NMR (DMSO-d6):
d
¼ 170.6; 170.4, 166.9, 157.0, 155.8, 148.4, 147.4, 143.8; 143.7,
140.6, 136.8, 130.3, 128.4, 127.9, 127.7, 127.6, 127.0, 125.3, 120.1,
113.2, 121.2, 111.6, 65.7, 65.6, 59.7, 55.4, 55.3, 54.9, 46.5, 37.3, 31.5.
(300 MHz):
d
¼ 8.10 (s, 1H), 7.92 (m, 1H), 7.35e7.18 (m, 6H), 7.02 (m,
2H), 6.92e6.71 (m, 7H), 6.49 (s, 2H), 6.30 (bs, 1H), 5.01 (s, 2H), 4.90
(m, 1H), 4.32 (m, 2H), 4.15 (m, 1H), 3.79 (m, 1H), 3.73 (s, 9H), 3.07
(m, 2H), 2.41 (m, 2H), 2.23 (m, 2H), 1.70e1.41 (m, 2H), 1.32 (m, 2H),
19F (188 MHz, DMSO-d6) :
d
¼ ꢃ73.6, ꢃ73.8 (m). IR (cmꢃ1): 3284,
1696, 1605, 1171, 1124, 692. ESIþ MS m/z: 772 [M þ Na]þ. Anal.
Calcd for C38H38F3N5O8$0.4 H2O: C, 60.33; H, 5.18; N 9.26. Found:
C, 60.57; H, 4.97; N, 9.01.
1.15 (m, 2H); 13C NMR (75 MHz):
d
¼ 171.1, 169.7, 168.1, 156.6, 156.4,
152.8, 138.6, 136.3, 136.0, 134.7, 129.9, 129.6, 128.3, 127.7, 124.1,
123.3,119.2,118.5, 116.0 (q, J ¼ 283.0 Hz),104.4, 67.1, 66.6, 59.9, 57.5,
55.7, 51.9, 50.9, 42.3, 41.8, 33.3, 32.0, 26.5, 23.1; 19F (188 MHz):
4.1.1.14. N0-(3-{N0-[2-Amino-3-(3,4-dimethoxyphenyl)propionyl]
hydrazino}-3-oxo-1-trifluoromethyl-propyl)hydrazinecarboxylic acid
benzyl ester 14b. To compound 14a (70 mg, 0.10 mmol) was added a
solution of piperidine in DMF (10% v/v, 2 mL). The mixture was
stirred at room temperature for 1.30 h. After removing the solvent
under reduced pressure, the residue obtained was triturated in
Et2O to yield 14b as a white powder (37 mg, 0.07 mmol, 78%). mp:
d
¼ ꢃ76.51 (m, 1F), ꢃ77.33 (s, 1F). To a solution of the Cbz protected
amine (183 mg, 0.22 mmol, 1.0 eq.) in dry DMF/MeOH 1/15 (48 mL)
was added 10% Pd/C (18 mg). The mixture was stirred overnight
under hydrogen atmosphere at room temperature and was filtered
on a celite pad. After removal of the solvent under reduced pressure
12 was obtained as a white solid (89%). 1H NMR (DMSO-d6):
102e104 ꢀC; 1H NMR (300 MHz, DMSO-d6):
7.31 (m, 5 H), 6.85e6.70 (m, 3 H), 5.36 (bs, 1H), 5.06 (s, 2H), 3.90 (m,
d
¼ 8.87 (bs, 1H), 7.40e
d
¼ 9.62 (s,1H), 8.53 (m,1H), 8.21 (m,1H), 7.37 (m, 2H), 7.27 (m, 2H),
7.11 (m, 2H), 7.02 (m, 1H), 6.99 (m, 1H), 6.94 (m, 1H), 6,57 (s, 2H),
5,54 (m, 1H), 4.24 (m, 2H), 4.19 (m, 1H), 3.85 (m, 1H), 3.73 (s, 6H),
3.62 (s, 3H), 3.45 (m, 2H), 2.51 (m, 2H), 2.23 (m, 2H), 1.52 (m, 2H),
1H), 3.73 (s, 3H), 3.71 (s, 3H), 3.45 (m, 1H), 2.90 (m, 1H), 2.85 (m,
1H), 2.50 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
d
¼ 172.8, 166.6,
156.9, 148.4, 147.2, 136.7, 130.7, 128.3, 127.8, 127.7, 113.1, 121.3, 111.6,
1.29 (m, 2H), 1.19 (m, 2H); 13C NMR (75 MHz, DMSO-d6):
d
¼ 171.1,
65.6, 55.0, 54.9, 54.3, 40.8, 31.4. 19F (188 MHz, DMSO-d6):
d
¼ ꢃ73.7
169.7, 168.1, 156.6, 156.4, 152.8, 138.6, 136.3, 134.7, 129.9, 129.6,
124.1, 123.3, 119.2, 118.5, 104.4, 115.8 (q, J ¼ 283.0 Hz), 59.9, 57.5,
55.7, 51.9, 50.9, 42.3, 41.8, 33.3, 32.0, 26.5, 23.1. 19F (188 MHz,
(d, J ¼ 7.3 Hz). IR (cmꢃ1): 3229, 1709, 1602, 1158, 1118, 696. ESIþ MS
m/z: 550 [M þ Na]þ. Anal. Calcd for C23H28F3N5O6: C, 52.37; H, 5.35;
N 13.28. Found: C, 52.40; H, 5.30; N, 13.51.
DMSO-d6):
75/25; ESIþ MS m/z: 690 [M
34H42F3N5O7$0.75 H2O: C, 58.66; H, 5.97; N, 10.36; found. C, 58.38;
H, 6.09; N, 9.40.
d
¼ ꢃ71,35 (s, 1F), ꢃ72,75 (s, 1F): 2 diastereoisomers:
þ
H]þ; Anal. Calcd for
4.1.1.15. N0-(1-{N0-[3-(3,4-Dimethoxyphenyl)-2-(9H-fluoren-9-
C
ylmethoxycarbonylamino)
2,2,2-trifluoroethyl)hydrazinecarboxylic acid tert-butyl ester 15.
solution of 30 (100 mg, 0.35 mmol) and Fmoc-L-3,4-
propionyl]hydrazinocarbonylmethyl}-
A
4.1.1.12. [2-Carbamoyl-1-(N0-{2,2,2-trifluoro-1-[(3,4,5-trimethoxy-
benzylcarbamoyl)-methyl]-ethyl}-hydrazinocarbonyl)-ethyl]-carba-
mic acid tert-butyl ester 13. Compound 13 was synthesized
following the same procedure described for 12a using the tri-
fluoroacetic salt of 29 (548 mg, 1.18 mmol, 1.0 eq.) and Na-Boc-Asn
(328 mg,1.41 mmol,1.2 eq.), except that the resulting colorless solid
was successively washed with Et2O, EtOAc and petroleum ether to
give 13 as a white solid (367 mg, 0.65 mmol, 55%). mp: 174e176 ꢀC;
dimethoxyphenylalanine (157 mg, 0.35 mmol) in dry DMF (3 mL)
was stirred for 5 min at room temperature. Then 2,4,6-collidine
(140
mL, 1.05 mmol), HOBT (53 mg, 0.39 mmol) and HBTU
(148 mg, 0.39 mmol) were successively added. The mixture was
stirred at room temperature under argon atmosphere for 21 h. After
removal of the solvent under reduced pressure, the residue was
triturated with EtOAc to give 15 as a white solid (171 mg,
0.24 mmol, 68%). mp: 208e210 ꢀC; 1H NMR (DMSO-d6)):
d
¼ 10.18
1H NMR (DMSO-d6):
d
¼ 9.41 (dd, 1H, J ¼ 4.9 Hz, J ¼ 16.2 Hz), 8.51
(br s, 2H), 8.41 (br s, 1H), 7.81e7.86 (m, 2H), 7.60 (d, J ¼ 7.2 Hz, 2H),
7.23e7.40 (m, 5H), 6.98 (d, J ¼ 2.8 Hz, 1H), 6.80 (br s, 2H), 5.13 (br s,
1H), 4.28 (m, 1H), 4.10e4.14 (m, 3H), 3.84 (br s, 1H), 3.70 (s, 3H),
(d, 1H, J ¼ 7.8 Hz), 7.25 (s, 1H), 6.88 (s, 2H), 6.58 (bs, 2H), 5.53 (m,
1H), 4.24 (m, 3H), 3.83 (m, 1H), 3.74 (s, 6H), 3.62 (s, 3H), 2.50 (m,
2H) 2.39 (m, 2H), 1.36 (s, 9H); 13C NMR (DMSO-d6):
d
¼ 171.0, 168.0,
3.66 (s, 3H), 2.94 (m, 1H), 2.72 (m, 1H), 2.50 (m, 2H), 1.37 (s, 9H). 13
C
154.9, 152.7, 137.2, 137.0, 135.7, 134.8, 128.3, 125.4, 78.9, 59.9, 57.72
NMR (DMSO-d6)):
d
¼ 170.8; 170.7, 167.4, 155.7, 148.9, 147.9, 144.1e
(dd, 1H, J ¼ 7.8 Hz, J ¼ 27.1 Hz), 53.6, 50.0, 37.6, 33.6, 28.1; 19F
144.3, 141.1, 130.8, 128.0, 127.5, 125.7, 121.8, 120.5, 113.9, 112.3, 79.3,
(188 MHz, DMSO-d6):
d
¼ ꢃ73.5 (m); IR (cmꢃ1): 3319, 2361,1637,
66.2, 55.9e56.0, 55.3, 47.0, 31.9, 28.6. 19F (188 MHz, DMSO-d6)):
1127; ESIþ MS m/z: 566 [M
þ
H]þ; Anal. Calcd for
d
¼ ꢃ73.7. IR (cmꢃ1): 3284, 1696, 1159, 1124, 738. ESIþ MS m/z:

518
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
738 [M þ Na]þ. Anal. Calcd for C35H40F3N5O8: C, 58.73; H, 5.63; N
(390 mg, 2.89 mmol, 1.2 eq.) were successively added. Meanwhile a
solution of N BocN ZLysine (1.10 g, 2.89 mmol, 1.2 eq.) and HBTU
9.79. Found: C, 58.35; H, 5.55; N, 9.76.
a
3
(1.09 mg, 2.89 mmol, 1.2 eq.) in DMF (5 mL) was stirred for half an
hour at room temperature. The solutions were then combined and
the resulting mixture was stirred at room temperature overnight.
The solvent was evaporated under reduced pressure and the
resulting yellow oil was taken up in EtOAc (15 mL) and successively
washed with 10% aqueous K2CO3 (2 ꢂ 20 mL), brine (30 mL) and
water (2 ꢂ 20 mL). The organic layer was dried over Na2SO4, filtered
and evaporated under reduced pressure to give a slightly yellow oil
which was purified by column chromatography (EtOAc) to give 17a
as a white solid (940 mg, 1.50 mmol, 62%). mp: 106e108 ꢀC; 1H
4.1.1.16. N0-[3-(N0-{3-(3,4-Dimethoxyphenyl)-2-[2-(3-
phenoxyphenyl) acetylamino] propionyl}hydrazino)-3-oxo-1-
trifluoromethyl-propyl]hydrazinecarboxylic acid benzyl ester 16a.
A solution of 14b (178 mg, 0.34 mmol) and 3-phenoxyphenylacetic
acid (84 mg, 0.37 mmol) in dry DMF (11 mL) was stirred for 5 min at
room temperature. Then DIPEA (112 mL, 0.68 mmol, 2.0 eq.), HOBT
(50 mg, 0.37 mmol, 1.1 eq.) and HBTU (140 mg, 0.37 mmol, 1.1 eq.)
were successively added. The mixture was stirred at room tem-
perature under argon atmosphere for 38 h. After removal of the
solvent under reduced pressure, the residue obtained was tritu-
rated in EtOAc (20 mL) to yield 16a as a white solid (213 mg,
NMR (300 MHz):
7.15 (s, 1H), 5.20 (m, 1H), 5.09 (s, 2H), 4.85 (m,1H), 4.15 (m,1H), 3.60
d
¼ 8.07 (bs, 1H), 7.20e7.41 (m, 10H), 7.17 (s, 1H),
0.29 mmol, 85%). mp: 178e180 ꢀC; 1H NMR (DMSO-d6):
d
¼ 10.22
(s, 3H), 3.05e3.28 (m, 6H), 2.35 (t, 2H, J ¼ 6.8 Hz), 1.31e1.88 (m, 6H),
(bs,1H),10.11 (bs,1H), 8.85 (bs,1H), 8.33 (m,1H), 7.40e7.32 (m, 7H),
7.22 (t, J ¼ 8.1 Hz, 1H), 7.14 (t, J ¼ 7.1 Hz, 1H), 6.98 (d, J ¼ 7.9 Hz, 2H),
6.90e6.71 (m, 6 H), 5.35 (bs, 1H), 5.07 (s, 2H), 4.58 (m, 1H), 3.91 (m,
1H), 3.70 (s, 6H), 3.40 (m, 2H), 2.96 (m, 1H), 2.71 (m, 1H), 2.55 (m,
1.50 (s, 9H); 13C NMR (75 MHz):
d
¼ 172.7, 171.5, 157.8, 156.3, 137.3,
136.6, 136.0, 128.8, 127.7, 126.7, 80.4, 67.5, 66.7, 65.8, 52.9, 52.2, 47.8,
40.2, 38.2, 37.2, 34.4, 32.4, 30.3, 28.0, 23.0; IR (cmꢃ1): 3309, 1686,
1653, 1524, 1248, 1167; ESIþ MS m/z: 650 [M þ Na]þ; Anal. Calcd for
2H). 13C NMR (100 MHz, DMSO-d6):
d
¼ 170.2, 170.0, 169.5, 166.9,
C32H45N5O8 C, 61.23; H, 7.23; N, 11.16; found C, 61.03; H, 7.14; N,
156.9, 156.6, 156.3, 148.3, 147.4, 138.4, 136.8, 130.0, 129.5, 128.3,
127.9, 127.7, 124.0, 123.3, 121.2, 119.4, 118.5, 116.4, 113.1, 111.5, 65.6,
10.94.
55.4, 55.3, 52.5, 41.7, 37.6, 31.49. 19F (188 MHz, DMSO-d6):
d
¼ ꢃ73.7
4.1.2.2. 2-{3-[N0-(2-Amino-6-benzyloxycarbonylamino-hexanoyl)-
hydrazino]-propionylamino}-3-phenyl-propionic acid methyl ester
trifluoroacetic salt 17b. A solution of 17a (99 mg, 0.16 mmol, 1.0 eq.)
in DCM/TFA 3:1 (4 mL) was stirred for 2 h at room temperature. The
solvent was evaporated under reduced pressure and the excess of
TFA was coevaporated with methanol. The crude product was then
precipitated with diethyl ether and washed with cyclohexane
(3 ꢂ 15 mL) to afford 17b as a white solid (101 mg, 0.16 mmol,
quantitative). mp: 96e98 ꢀC; 1H NMR (300 MHz, DMSO-d6):
(d, J ¼ 4.9 Hz). IR (cmꢃ1): 3271, 1704, 1601, 1161, 1121, 690. ESIþ MS
m/z: 760 [M þ Na]þ. Anal. Calcd for C37H38F3N5O8$0.25 H2O: C,
59.91; H, 5.24; N 9.44. Found: C, 59.93; H, 5.32; N, 9.57.
4.1.1.17. N-[1-(N-{3-(3,4-Dimethoxyphenyl)-2-[2-(3-Phenoxyphenyl)
acetylamino] propionyl}-hydrazinocarbonylmethyl)-2,2,2-
trifluoroethyl]hydrazinecarboxylic acid tert-butyl ester 16b. To
compound 15 (382 mg, 0.53 mmol) was added a solution of
piperidine in DMF (10% v/v, 5 mL). After stirring at room temper-
ature for 1.30 h, the solvent was removed under reduced pressure
and the residue obtained was triturated in Et2O to yield the free
amine as a white solid (231 mg, 88%) that was used without further
purification. To a solution of the crude amine (76 mg, 0.15 mmol)
and 3-phenoxyphenylacetic acid (38 mg, 0.17 mmol) in dry DMF
(4 mL) was added, after stirring for 5 min at room temperature,
d
¼ 9.89 (bs, 1H), 8.41 (s, 1H), 8.20 (s, 3H), 7.95 (s, 1H), 7.17e7.40 (m,
10H), 6.51 (bs, 1H), 5.02 (s, 2H), 4.51 (m, 1H), 3.55 (m, 1H), 3.50 (s,
3H), 3.10e2.82 (m, 6H), 2.23 (t, 2H, J ¼ 6.8 Hz), 1.22e1.70 (m, 6H);
13C NMR (DMSO-d6, 75 MHz)
d 174.5, 174.1, 169.5, 159.4, 138.8,
138.5, 130.8, 130.2, 129.8; 129.2, 128.3, 67.8, 55.7, 53.6, 53.1, 41.6,
51.6, 38.7, 35.3, 32.5, 30.8, 23.5; IR (cmꢃ1): 2929, 1668, 1531, 1199,
1129; ESIþ MS m/z: 528 [M
þ
H]þ; Anal. Calcd for
2,4,6-collidine (60
mL, 0.45 mmol), HOBT (23 mg, 0.17 mmol) and
C29H38F3N5O8$1.5H2O: C, 52.09; H, 6.19; N, 10.48; found C, 52.48; H,
HBTU (64 mg, 0.17 mmol). After stirring at room temperature under
argon atmosphere for 24 h, the solvent was removed under reduced
pressure, the residue was triturated in EtOAc (15 mL) to afford 16b
as a white solid (88 mg, 0.12 mmol 84%). mp: 184e186 ꢀC; 1H NMR
6.28; N, 10.13.
4.1.2.3. 2-[3-(N0-{6-Benzyloxycarbonylamino-2-[2-(4-phenoxy-
phenyl)-acetylamino]-hexanoyl}-hydrazino)-propionylamino]-3-
phenyl-propionic acid methyl ester 18a. Compound 18a was syn-
thesized following the same procedure described for 17a. Depro-
tection of 17b in DCM/TFA gave the trifluoroacetic salt of the amine
(491 mg, 0.76 mmol, 1.0 eq.) which was coupled with 2-
phenoxyphenylacetic acid to give 18a (452 mg, 0.61 mmol, 81%)
as a white solid. Mp: decomposition at 70e80 ꢀC; 1H NMR
(DMSO-d6):
d
¼ 10.20, 10.10 (2s, 2H), 8.41 (s, 1H), 8.30 (m, 1H), 7.35
(t, J ¼ 7.8 Hz, 2H), 7.20 (t, J ¼ 8.3 Hz, 1H), 7.12 (t, J ¼ 7.8 Hz, 1H), 7.00
(d, J ¼ 7.8 Hz, 2H), 6.87e6.69 (m, 6H), 5.13 (s, 1H), 4.55 (m, 1H), 3.85
(br s, 1H), 3.67 (s, 6H), 3.40 (m, 2H), 2.93 (m, 1H), 2.70 (m, 1H), 2.49
(m, 2H), 1.38 (s, 9H). 13C NMR (DMSO-d6):
d
¼ 170.0, 170.1, 167.4,
157.1, 156.8,148.9, 147.9, 138.8,130.4,129.9,123.8, 119.0, 124.5, 121.7,
119.8, 116.8, 113.7, 112.1, 79.3, 55.9, 55.8, 52.9, 42.2, 38.0, 31.9, 28.6.
(300 MHz):
d
¼ 8.45 (s, 1H), 7.41e7.10 (m, 15H), 7.14 (bs, 1H), 7.01e
19F (188 MHz, DMSO-d6):
d
¼ ꢃ73.7 (m). IR (cmꢃ1): 3216 (NH),
6.80 (m, 4H), 6.38 (d, 1H, J ¼ 7.3 Hz), 5.25 (m, 1H), 5.10 (s, 2H), 4.83
(dd, 1H, J ¼ 7.5 and 12.2 Hz), 4.28 (dd, 1H, J ¼ 7.2 et 14.0 Hz), 3.79 (s,
3H), 3.52 (s, 2H), 3.16 (m, 1H), 3.13 (m, 2H), 3.03 (m, 1H), 3.02 (m,
2H), 2.22 (m, 2H), 1.75 (m, 1H), 1.55 (m, 1H), 1.46 (m, 2H), 1.24 (m,
1699, 1653, 1159, 1121, 691. ESIþ MS m/z: 726 [M þ Na]þ. Anal. Calcd
for C34H40F3N5O8: C, 57.85; H, 5.73; N 9.95. Found: C, 57.01; H, 5.68;
N, 9.86.
2H); 13C NMR (75 MHz):
d
¼ 172.9, 171.8, 171.4, 171.1, 156.6, 153.8,
4.1.2. Synthesis of the target molecules including the
b-hydrazino
151.1,136.6,136.1,135.8,129.1; 128.6,128.5,128.0,127.0,124.2,117.2,
113.2, 111.8, 66.6, 56.0, 53.3, 52.5, 51.6, 47.7, 40.5, 37.5, 34.3, 31.1,
29.3, 22.3; IR (cmꢃ1): 3264, 1645, 1529, 1210; ESIþ MS m/z: 760
[M þ Na] þ, 776 [M þ K] þ; Anal. Calcd for C41H47N5O8$0.75 H2O: C,
65.58; H, 6.52; N, 9.53; found C, 65.95; H, 6.40; N, 8.87.
acid scaffold 17ae22b
4.1.2.1. 2-{3-[N0-(6-Benzyloxycarbonylamino-2-tert-butox-
ycarbonylamino-hexanoyl)-hydrazino]-propionylamino}-3-phenyl-
propionic acid methyl ester 17a. A solution of 35 (914 mg,
2.41 mmol, 1.0 eq.) in DCM/TFA 3:1 (12 mL) was stirred for 2 h at
room temperature. The solvent was evaporated under reduced
pressure and the excess of trifluoroacetic acid was coevaporated
with methanol. The resulting slightly yellow solid was dissolved in
DMF (5 mL), then DIPEA (2.0 mL, 12.0 mmol, 5.0 eq.) and HOBT
4.1.2.4. Methyl (2S)-2-[3-[2-[(2S)-6-amino-2-[[2-(3-phenoxyphenyl)
acetyl]amino]
panoate 18b. To a solution of 18a (114 mg, 0.15 mmol, 1 eq.) in dry
methanol (5 mL) was added 10% Pd/C (11 mg). The mixture was
hexanoyl]hydrazino]propanoylamino]-3-phenyl-pro

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
519
stirred overnight under hydrogen atmosphere at room temperature
and filtered on a celite pad. After the removal of the solvent under
reduced pressure, 18b was obtained as yellow resinous oil (81 mg,
d
¼ 172.0, 158.3, 152.7, 137.1, 134.7, 104.7, 59.9, 55.9, 48.6, 43.8, 34.2.
To a solution of the hydrochloride salt of the amine (1.04 g,
3.28 mmol, 1 eq.) in dry DMF (25 mL) was successively added N
Fmoc-N -Z- -Lys (1.7 g, 3.68 mmol, 1.1 eq.), collidine (2.5 g,
a
-
0.13 mmol, 89%). 1H NMR:
d
¼ 7.55 (bs,1H), 7.23e6.85 (m,15H), 4.80
3
L
(m, 1H), 4.33 (m, 1H), 3.67 (m, 3H), 3.51 (m, 2H), 3.10 (m, 1H), 3.02
(m, 1H), 3.00 (m, 2H), 2.70 (m, 2H), 2.24 (m, 2H), 1.70 (m, 1H), 1.59
21.1 mmol, 7 eq.), EDC (694 mg, 3.68 mmol, 1.1 eq.), and HOBT
(489 mg, 3.68 mmol, 1.1 eq.). The mixture was stirred overnight at
room temperature under argon atmosphere. After removal of the
solvent under reduced pressure, the residue was taken up in EtOAc
(20 mL) and successively washed with 10% aqueous citric acid
(2 ꢂ 20 mL), water (20 mL), 10% aqueous K2CO3 (2 ꢂ 20 mL) and
brine (20 mL). The organic layer was dried over Na2SO4, filtered and
evaporated under reduced pressure to give a crude product that
was purified by column chromatography (EtOAc) to afford 20
(672 mg, 0.92 mmol, 30%) as a white solid. mp: 140e142 ꢀC; 1H
(m, 1H), 1.49 (m, 2H), 1.33 (m, 2H). 13C NMR:
d
¼ 172.9, 171.9, 171.2,
171.0, 157.54, 156.9, 136.7, 136.3, 130.0, 129.8, 129.1, 128.5, 127.0,
124.0, 123.4, 119.7, 118.9, 117.4, 53.5, 52.4, 51.8, 47.5, 43.0, 40.5, 37.5,
34.1, 31.7, 30.0, 22.3. ESIþ MS m/z: 604 [M þ H]þ. Anal. Calcd for
C
33H41N5O6$2.5 H2O: C, 61.13; H, 7.17; N, 10.90; found C, 61.36; H,
6.50; N, 10.07.
4.1.2.5. Methyl
(2S)-2-[3-[2-[(2S)-6-(benzyloxycarbonylamino)-2-
[[2-(2,5-dimethoxyphenyl)acetyl]amino]hexanoyl]hydrazino]prop-
anoylamino]-3-phenyl-propanoate 19a. Compound 19a was syn-
thesized following the same procedure described for 17a.
Deprotection of 17b in DCM/TFA gave the trifluoroacetic salt of the
amine (490 mg, 0.93 mmol, 1 eq.) which was coupled with 2,5-
dimethoxyphenylacetic acid (235 mg, 1.2 mmol, 1.2 eq.). Except,
the crude product was purified by column chromatography (EtOAc/
MeOH, 9:1) to give 19a as a white solid (247 mg, 0.35 mmol, 37%).
NMR:
d
¼ 7.74 (d, 2H, J ¼ 7.6 Hz), 7.55 (d, 2H, J ¼ 7.5 Hz), 7.38 (t, 2H,
J ¼ 7.3 Hz), 7.29 (t, 2H, J ¼ 7.4 Hz), 7.13 (t, 1H, J ¼ 5.3 Hz), 6.52 (s, 2H),
5.58 (m, 1H), 4.71 (m, 1H), 4.37 (m, 2H), 4.35 (m, 2H), 4.17 (m, 1H),
4.06 (m, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 3.11 (m, 2H), 3.06 (m, 2H),
2.36 (m, 2H),1.79 (m,1H),1.66 (m,1H),1.45 (m, 2H),1.41 (s, 9H),1.35
(m, 2H). 13C NMR:
d
¼ 171.6, 171.5, 156.3, 153.3, 143.6, 141.3, 137.1,
134.2, 127.8, 127.1, 124.9, 120.0, 104.9, 80.2, 67.0, 60.1, 56.0, 53.6,
48.1, 47.1, 43.7, 39.7, 34.7, 31.6, 29.5, 28.4, 22.4. IR (cmꢃ1): 2926,
1682, 1650, 1592. ESIþ MS m/z: 756 [M þ 23]þ. Anal. Calcd for
mp: 171e173 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 7.20e7.33 (m,10H),
7.14 (d, 1H, J ¼ 7.3 Hz), 6.82e6.73 (m, 3H), 6.48 (d, 1H, J ¼ 7.3 Hz),
5.10 (s, 2H), 4.83 (dd, 1H, J ¼ 7.5 and 12.2 Hz), 4.28 (dd, 1H, J ¼ 7.2
and 14.0 Hz), 3.72 (s, 6H), 3.79 (s, 3H), 3.52 (s, 2H), 3.16 (m, 1H), 3.13
(m, 2H), 3.03 (m, 1H), 3.02 (m, 2H), 2.21 (t, 2H, J ¼ 5.4 Hz), 1.75 (m,
1H), 1.55 (m, 1H), 1.46 (m, 2H), 1.24 (m, 2H). 13C NMR (75 MHz,
C39H51N5O9 C. 62.71, H. 7.10, N. 9.38 found C. 62.86, H. 7.20, N. 9.24.
4.1.2.8. tert-Butyl
N-[(5S)-5-[[2-(2,5-dimethoxyphenyl)acetyl]ami
no]-6-oxo-6-[2-[3-oxo-3-[(3,4,5-trimethoxyphenyl)methylamino]
propyl] hydrazino]hexyl] carbamate 21a. Compound 21a was syn-
thesized according to the procedure described for the synthesis of
20a from 22 (307 mg, 0.42 mmol, 1 eq.), which was successively
deprotected and coupled with 2,5-dimethoxyphenylacetic acid
(99 mg, 0.50 mmol, 1.2 eq.). 21a was obtained as a white solid
(142 mg, 0.20 mmol, 50%). mp: 138e140 ꢀC; 1H NMR (300 MHz):
CDCl3):
d
¼ 172.9, 171.8, 171.4, 171.1, 156.6, 153.8, 151.1, 136.6, 136.1,
129.1; 128.6, 128.5, 128.0, 127.0, 124.2, 117.2, 113.2, 111.8, 66.6, 56.0,
53.3, 52.5, 51.6, 47.7, 40.5, 38.9, 37.5, 34.3, 31.1, 29.3, 22.3. IR (cmꢃ1):
3283, 1742, 1682, 1638, 1535. APCIþ MS m/z: 706 [M þ H]þ. Anal.
Calcd for C37H47N5O9$1.5 H2O: C, 60.64; H, 6.89; N, 9.56; found C,
60.48; H, 6; 71; N, 9.31.
d
¼ 7.23 (t, 1H, J ¼ 5.0 Hz), 7.18 (m, 1H), 6.79 (m, 1H), 6.78 (m, 1H),
6.77 (m, 1H), 6.54 (s, 2H), 6.41 (d, 1H, J ¼ 6.9 Hz), 4.96 (m, 1H), 4.36
(m, 2H), 4.23 (m, 1H), 3.82 (s, 6H), 3.81 (s, 3H), 3.78 (s, 6H), 3.49 (s,
2H), 3.05 (t, 2H, J ¼ 6.3 Hz), 3.01 (m, 2H), 2.28 (t, 2H, J ¼ 6.3 Hz), 1.78
(m,1H),1.55 (m,1H), 1.42 (s, 9H), 1.41 (m, 2H),1.25 (m, 2H). 13C NMR
4.1.2.6. Methyl (2S)-2-[3-[2-[(2S)-6-amino-2-[[2-(2,5-
dimethoxyphenyl)acetyl]amino] hexanoyl]hydrazino]propanoylami
no]-3-phenyl-propanoate di(hydrochloride) salt 19b. To a solution of
19a (175 mg, 0.24 mmol, 1 eq.) in dry methanol (5 mL) was added
10% Pd/C (17.5 mg). The mixture was stirred overnight under
hydrogen atmosphere at room temperature and filtered on a celite
pad. After removal of the solvent under reduced pressure and
addition of HCl/MeOH, 19b was obtained as a white hygroscopic
solid (110 mg, 0.17 mmol, 73%). 1H NMR (300 MHz, CD3OD):
(75 MHz):
d
¼ 171.7, 171.6, 171.2, 156.2, 153.9, 153.3, 151.1, 137.1,
134.4, 124.2, 117.4, 113.2, 104.8, 79.2, 60.8, 56.1, 55.7, 51.9, 48.2, 43.7,
40.0, 39.0, 34.6, 31.1, 29.6, 28.4, 22.5. IR (cmꢃ1): 2926, 1680, 1591,
1459, 1225. ESIþ MS m/z: 712 [M
þ
23]þ. Anal. Calcd for
C34H51N5O10: C. 57.69, H. 7.56, N. 9.90 found C. 57.73, H. 7.17, N. 9.87.
d
¼ 7.28e7.19 (m, 5H), 6.83 (m, 3H), 4.67 (dd, 1H, J ¼ 5.73 and
4.1.2.9. 3-[2-[(2S)-6-amino-2-[[2-(2,5-dimethoxyphenyl)acetyl]
amino]hexanoyl] hydrazino]-N-[(3,4,5-trimethoxyphenyl)methyl]
8.71 Hz), 4.34 (dd, 1H, J ¼ 5.82 and 8.30), 3.79 (s, 3H), 3.74 (s, 3H),
3.69 (s, 3H), 3.52 (d, 2H, J ¼ 5.06 Hz), 3.17 (dd, 1H, J ¼ 5.63 and
13.82), 3.01e2.88 (m, 5H), 2.31 (m, 2H), 1.39e1.04 (m, 6H). 13C NMR
propanamide di(trifluoroacetic) salt 21b. A solution of 21a (93 mg,
0.13 mmol,1.0 eq.) in DCM/TFA 3:1 (4 mL) was stirred for 2 h at room
temperature. The solvent was evaporated under reduced pressure
and the excess of TFA was coevaporated with methanol to afford 21b
as a white solid (81 mg, 0.13 mmol, quantitative).1H NMR (300 MHz,
(75 MHz, CD3OD):
d
¼ 174.7, 173.4, 172.4, 171.9, 155.0, 153.1, 138.0,
130.3, 129.6, 128.0, 126.0, 118.3, 113.7, 112.7, 68.2, 56.7, 56.2, 55.5,
52.8, 50.0, 40.5, 38.4, 38.2, 32.0, 30.4, 28.0, 23.4. IR (cmꢃ1): 2926,
1682, 1650, 1592. ESIþ MS m/z: 572 [M þ H]þ. Anal. Calcd for
CD3OD):
d
¼ 6.87 (d,1H, J ¼ 8.8 Hz), 6.80 (s,1H), 6.79 (m,1H), 6.62 (s,
C
29H43Cl2N5O7$2.5 H2O: C. 50.50, H. 7.03, N. 10.16 found C. 50.58, H.
2H), 4.32 (m, 1H), 4.31 (m, 2H), 3.82 (s, 6H), 3.77 (s, 3H), 3.73 (s, 6H),
3.60 (s, 2H), 3.44 (m, 2H), 2.91 (m, 2H), 2.68 (m, 2H),1.88 (m,1H),1.78
(m, 1H), 1.68 (m, 2H), 1.44 (m, 2H). 13C NMR (75 MHz, CD3OD):
7.26, N. 9.63.
4.1.2.7. 9H-fluoren-9-ylmethyl
N-[(1S)-5-(tert-butoxycarbonylami
d
¼ 173.2, 170.9, 153.7, 153.2, 151.7, 136.9, 134.4, 124.5, 117.0, 112.2,
no)-1-[[[3-oxo-3-[(3,4,5-trimethoxyphenyl)methylamino]propyl]
amino] carbamoyl]pentyl]carbamate 20. A solution of 36 (1.2 g,
3.28 mmol, 1 eq.) in HCl 4 M in dioxane (25 mL) was stirred for 2 h
at room temperature. The solvent was evaporated under reduced
pressure to afford the hydrochloride salt of the amine (1.14 g,
3.57 mmol, quantitative) as a colorless oil, which was used without
111.3, 104.8, 59.7, 55.3, 55.2, 54.7, 51.9, 47, 43.0, 39.0, 36.8, 30.4, 30.0,
26.6, 22.3. IR (cmꢃ1): 2926, 1682, 1650, 1592. ESIꢃ MS m/z: 624
[M þ 2H2OeH]- and 588 [M ꢃ H]ꢃ. Anal. Calcd for C33H45F6N5O12$4
H2O: C. 44.54, H. 6.02, N. 7.87 found C. 44.17, H. 5.66, N. 7.37.
4.1.2.10. tert-Butyl N-[(5S)-6-oxo-6-[2-[3-oxo-3-[(3,4,5-
trimethoxyphenyl)methylamino] propyl]hydrazino]-5-[[2-(3-
phenoxyphenyl)acetyl] amino]hexyl]carbamate 22a. To 20 (613 mg,
0.84 mmol, 1 eq.) was added a solution of piperidine in DMF (10% v/
further purification. 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.51 (s, 3H),
6.52 (s, 2H), 4.21 (d, 2H, J ¼ 5.5 Hz), 3.79 (s, 6H), 3.61 (s, 3H), 3.15 (t,
2H, J ¼ 6.6 Hz), 2.60 (t, 2H, J ¼ 6.6 Hz). 13C NMR (75 MHz, DMSO-d6):

520
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
v, 12 mL). The mixture was stirred at room temperature for 2 h. The
solvent was evaporated under reduced pressure to afford the free
amine (quantitative) as a colorless oil, which was used without
MgSO4, filtered and concentrated under reduced pressure to give
23 (1.65 g, 3.60 mmol, 88%) as a white foam which was used in the
next step without further purification. 1H NMR (300 MHz):
d
¼ 8.81
further purification. 1H NMR (300 MHz, DMSO-d6):
d
¼ 6.74 (m,
(s, 1H), 7.34 (m, 5H), 5.61 (m, 1H), 5.10 (s, 2H), 4.25 (m, 1H), 3.40 (s,
1H), 6.58 (s, 2H), 4.98 (m, 1H), 4.30 (d, 2H, J ¼ 5.8 Hz), 4.05 (m, 1H),
3.76 (s, 6H), 3.62 (s, 3H), 3.15 (m, 2H), 3.07 (m, 2H), 2.30 (m, 2H),
1.62e1.28 (m, 6H), 1.38 (s, 9H). 13C NMR (75 MHz, DMSO-d6):
2H), 3.15 (m, 2H), 1.65e1.28 (m, 6H), 1.41 (s, 9H). 13C NMR (75 MHz):
d
¼ 173.8, 172.1, 156.8, 156.0, 136.5, 128.5, 128.0, 80.5, 66.7, 53.2,
52.8, 40.50, 32.0, 29.3, 28.3, 22.5. IR (cmꢃ1): 2951, 1694, 1519, 1367,
d
¼ 169.7,169.5,152.7,146.2,135.2,132.1,104.3, 77.2, 60.0, 55.8, 54.3,
1246. APCIꢃ MS m/z: 451 [M ꢃ H]ꢃ
53.6, 47.8, 42.1, 35.0, 34.2, 30.7, 25.7, 24.1. ESI- MS m/z: 510 [M ꢃ H]-.
To a solution of the free amine (305 mg, 0.42 mmol, 1 eq.) in dry
DMF (10 mL) was added successively 3-phenoxyphenylacetic acid
(145 mg, 0.50 mmol, 1.2 eq.), DIPEA (380 mg, 2.94 mmol, 7 eq.),
HBTU (191 mg, 0.50 mmol, 1.2 eq.), and HOBT (68 mg, 0.50 mmol,
1.2 eq.). The mixture was stirred overnight at room temperature
under argon atmosphere. After removal of the solvent under
reduced pressure, the residue was taken up in EtOAc (10 mL) and
successively washed with 10% aqueous citric acid (2 ꢂ 10 mL),
water (10 mL), 10% aqueous K2CO3 (2 ꢂ 10 mL) and brine (10 mL).
The organic layer was dried over Na2SO4, filtered and evaporated
under reduced pressure to yield a crude product which was puri-
fied by column chromatography (EtOAc) to afford 22a (157 mg,
0.21 mmol, 52%) as a white solid. mp: 142ꢀCe144 ꢀC; 1H NMR
To a solution of the carboxylic acid (1.20 g, 2.64 mmol, 1 eq.) in
dry DMF (20 mL) was added successively H-Phe-OMe (690 mg,
3.16 mmol, 1.2 eq.), DIPEA (1.36 g, 10.56 mmol, 4 eq.), HBTU (1.20 g,
3.16 mmol, 1.2 éq.), and HOBT (430 mg, 3.16 mmol, 1.2 éq.). The
mixture was stirred overnight at room temperature under argon
atmosphere. After removal of the solvent under reduced pressure,
the residue was taken up in EtOAc (20 mL) and washed with 10%
aqueous citric acid (2 ꢂ 15 mL), water (15 mL), 10% aqueous K2CO3
(2 ꢂ 15 mL) and brine (20 mL). The organic layer was dried over
Na2SO4, filtered and evaporated under reduced pressure to give a
crude product which was purified by column chromatography
(EtOAc) to afford 23 (907 mg, 1.48 mmol, 76%) as a white foam. 1H
NMR (300 MHz):
d
¼ 8.08 (m, 1H), 7.37e7.15 (m, 10H), 7.05 (d, 1H,
J ¼ 5.6 Hz), 6.99 (d, 1H, J ¼ 5.5 Hz), 5.08 (s, 2H), 4.79 (m, 1H), 3.90
(300 MHz):
d
¼ 8.31 (t,1H, J ¼ 5.5 Hz), 8.21 (d,1H, J ¼ 8.0 Hz), 7.31 (t,
(m, 1H), 3.65 (s, 3H), 3.38 (s, 2H), 3.03 (m, 4H), 1.47 (m, 6H), 1.34 (s,
1H, J ¼ 8.2 Hz), 7.25 (m, 1H), 7.17 (t, 1H, J ¼ 5.6 Hz), 7.10 (t, 1H,
J ¼ 7.0 Hz), 6.95 (t, 1H, J ¼ 9.0Hz), 6.93 (m, 1H), 6.90 (m, 2H), 6.87
(m, 2H), 6.52 (s, 2H), 6.44 (m, 1H), 4.74 (m, 1H), 4.34 (m, 2H), 4.28
(m,1H), 3.80 (s, 9H), 3.46 (s, 2H), 3.07 (m, 2H), 3.02 (m, 2H), 2.32 (m,
2H),1.75 (m,1H),1.58 (m,1H),1.42 (m, 2H),1.41 (s, 9H),1.25 (m, 2H).
9H). 13C NMR (75 MHz):
d
¼ 172.4, 172.2, 171.5, 156.7, 155.8, 136.6,
135.9, 129.1, 128.7, 128.0, 128.5, 80.7, 66.6, 53.2, 53.0, 52.4, 37.5, 31.4,
29.3, 28.2, 22.3. IR (cmꢃ1): 3303, 235), 1664, 1517, 1455. APCIþ MS
m/z: 614 (M þ H)þ. Anal. Calcd for C31H43N5O8$0.25H2O: C, 60.67;
H, 7.06; N, 11.41; found: C, 60.25; H, 7.13; N, 10.95.
13C NMR (75 MHz):
d
¼ 171.6, 171.1, 171.0, 157.7, 156.8, 156.3, 153.3,
153.2,137.1,136.4,134.3,130.2,129.8,123.9,123.5,119.5,117.5,104.8,
79.2, 60.8, 56.1, 51.8, 48.0, 43.6, 43.1, 36.9, 34.5, 31.2, 29.5, 28.4, 22.5.
IR (cmꢃ1): 3585, 2931, 2048, 1638, 1586. ESIþ MS m/z: 744
[M þ 23]þ. Anal. Calcd for C38H51N5O9 C. 61.68, H. 7.24, N. 9.47
found C. 61.46, H. 7.18, N. 9.37.
4.1.3.2. Methyl (2S)-2-[[2-[2-[(2S)-6-(benzyloxycarbonylamino)-2-
[[2-(3-phenoxyphenyl)
acetyl]amino]hexanoyl]hydrazino]acetyl]
amino]-3-phenyl-propanoate 24a. A solution of 23 (775 mg,
1.26 mmol, 1 eq.) in DCM/TFA 3/1 (19 mL) was stirred for 2 h at
room temperature. The solvent was evaporated under reduced
pressure and the excess of TFA was coevaporated with methanol to
afford the hydrochloride salt of the amine as a colorless oil, which
was used without further purification. 1H NMR (300 MHz, DMSO-
4.1.2.11. 3-[2-[(2S)-6-amino-2-[[2-(3-phenoxyphenyl)acetyl]amino]
hexanoyl]hydrazino]-N-[(3,4,5-trimethoxyphenyl)methyl]
prop-
anamide hydrochloride salt 22b. A solution of 22a (103 mg,
0.14 mmol,1 eq.) in HCl 4 M in dioxane (10 mL) was stirred for 2 h at
room temperature. The solvent was evaporated under reduced
pressure to afford the hydrochloride salt 22b (100 mg, 0.15 mmol,
d6):
d
¼ 9.75 (m, 1H), 8.30 (bs, 3H), 8.08 (m, 1H), 7.05e7.41 (m, 10H),
6.28 (bs, 2H), 5.01 (s, 2H), 4.55 (m, 1H), 3.61 (m, 1H), 3.59 (s, 3H),
3.45 (s, 2H), 2.98 (m, 4H), 1.71e1.18 (m, 6H). 13C NMR (75 MHz,
DMSO-d6):
d
¼ 171.7, 169.40, 167.8, 156.0, 137.2, 136.9, 129.0, 125.3,
quantitative) as a colorless oil. 1H NMR (free amine):
d
¼ 7.39 (bs,
65.1, 53.2, 53.3, 51.8, 37.5, 30.7, 28.8, 21.4. IR (cmꢃ1): 2944, 1664,
1528, 1439, 1157. ESIþ MS m/z: 515 [M þ H]þ
1H), 7.30 (m, 2H), 7.22 (t, 1H, J ¼ 8.1 Hz), 7.08 (t, 1H, J ¼ 7.2 Hz), 6.95
(m, 3H), 6.89 (m, 2H), 6.84 (bs, 1H), 6.50 (s, 2H), 4.31 (m, 1H), 4.30
(d, 2H, J ¼ 5.0 Hz), 3.79 (s, 6H), 3.78 (s, 3H), 3.47 (s, 2H), 3.05 (t, 2H,
J ¼ 5.5 Hz), 2.61 (t, 2H, J ¼ 6.3 Hz), 2.31 (t, 2H, J ¼ 5.5 Hz), 1.67 (m,
1H), 1.56 (m, 1H), 1.39 (m, 2H), 1.26 (m, 2H). 13C NMR (free amine):
To a solution of the trifluoroacetic salt of the amine (300 mg,
0.63 mmol, 1 eq.) in dry DMF (10 mL) was successively added 3-
phenoxyphenylacetic acid (218 mg, 0.75 mmol, 1.2 éq.), DIPEA
(871 mg, 5.30 mmol, 7 eq.), HBTU (287 mg, 0.75 mmol, 1.2 eq.), and
HOBT (102 mg, 0.75 mmol, 1.2 eq.). The mixture was stirred over-
night at room temperature under argon atmosphere. After removal
of the solvent under the reduced pressure, the residue was taken up
in EtOAc (10 mL), and washed with 10% aqueous citric acid
(2 ꢂ 10 mL), water (10 mL), 10% aqueous K2CO3 (2 ꢂ 10 mL) and
brine (10 mL). The organic layer was dried over Na2SO4, filtered and
evaporated to give a crude product which was purified by column
chromatography (EtOAc) to afford the compound 24a (227 mg,
0.38 mmol, 48%) as a white solid. mp: 156e157 ꢀC; 1H NMR
d
¼ 171.7, 171.1, 157.6, 156.8, 153.2, 137.0, 136.6, 134.2, 130.1, 129.9,
129.8, 123.9, 123.5, 119.6, 118.9, 117.3, 104.8, 60.8, 56.0, 51.9, 48.0,
43.6, 43.0, 41.0, 34.5, 31.7, 31.5, 22.4. IR (cmꢃ1): 2926, 1682, 1650,
1592. ESIþ MS m/z: 622 [M þ H]þ. Anal. Calcd for C33H43N5O7$0.75
H2O: C. 62.39, H. 7.08, N. 11.03 found C. 62.69, H. 7.29, N. 10.75 (free
amine).
4.1.3. Synthesis of the target molecules including the a-hydrazino
acid scaffold 23e25b
4.1.3.1. Methyl (2S)-2-[[2-[2-[(2S)-6-(benzyloxycarbonylamino)-2-
(tert-butoxycarbonylamino) hexanoyl]hydrazino]acetyl]amino]-3-
phenyl-propanoate 23. To solution of 38 (2.00 g, 4.16 mmol, 1 eq.)
in THF/MeOH (1/1, 30 mL) was added an aqueous 2 N NaOH (4 mL).
The reaction was stirred at room temperature over 3 h. The solvent
was removed under reduced pressure (without distilling the water)
and the remaining solution was brought at pH ¼ 5 by addition of
10% aqueous citric acid. The aqueous phase was extracted with
EtOAc (2 ꢂ 30 mL). The combined organic layers were dried over
(300 MHz):
d
¼ 8.05 (s, 1H), 7.35 (d, 1H, J ¼ 8.3 Hz), 7.24e6.82 (m,
21H), 6.25 (m, 1H), 5.07 (s, 2H), 4.75 (m, 1H), 4.24 (m, 1H), 3.26 (s,
3H), 3.42 (m, 2H), 3.38 (s, 2H), 3.10 (m, 4H), 1.74 (m, 1H), 1.53 (m,
1H), 1.42 (m, 2H), 1.19 (m, 2H). 13C NMR (75 MHz):
d
¼ 172.7, 171.4,
171.0, 169.9, 157.6, 156.8, 156.7, 136.5, 130.2, 136.0, 130.2, 129.8,
127.2, 123.9, 123.5, 119.6, 119.0, 117.2, 66.6, 54.7, 53.1, 52.5, 51.4, 43.1,
40.4, 37.4, 31.4, 29.3, 22.3. IR (cmꢃ1): 3304, 1690, 1646, 1533, 1487,
1258. APCIþ MS m/z: 724 [M þ H]þ. Anal. Calcd for C40H45N5O8: C,
66.37; H, 6.27; N, 9.68; found: C, 66.39; H, 6.37; N, 9.57.

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
521
4.1.3.3. Methyl (2S)-2-[[2-[2-[(2S)-6-amino-2-[[2-(3-
phenoxyphenyl)acetyl]amino]hexanoyl] hydrazino]acetyl]amino]-3-
phenyl-propanoate hydrochloride salt 24b. To a solution of 24a
(114 mg, 0.15 mmol, 1 eq.) in dry MeOH/DMF (4/1, 25 mL) was
added Pd/C 10% (11 mg). The reaction mixture was stirred overnight
under hydrogen atmosphere at room temperature and filtered on a
celite pad. After removal of the solvent under reduced pressure,
HCl/MeOH was added and precipitation with Et2O afforded the
hydrochloride salt of 24b as a colorless solid (81 mg, 0.13 mmol,
4.1.4. Synthesis of the intermediates 29e36 and 38
4.1.4.1. N0-{2,2,2-Trifluoro-1-[(3,4,5-trimethoxy-benzylcarbamoyl)-
methyl]-ethyl}-hydrazine carboxylic acid tert-butyl ester 29. To a
solution of 27 (1.34 g, 4.92 mmol, 1.0 eq.) and HBTU (2.23 g,
5.90 mmol, 1.2 eq.) in DMF (10 mL) were successively added HOBT
(798 mg, 5.90 mmol, 1.2 eq.), DIPEA (1.65 mL, 9.84 mmol, 2.0 eq.)
and phenylalanine hydrochloride. The reaction was performed
under argon atmosphere at room temperature overnight. The sol-
vent was evaporated over reduced pressure. The product was taken
up in EtOAc (25 mL), and the organic layer was successively washed
with 10% aqueous citric acid (2 ꢂ 15 mL), water (30 mL), 10%
aqueous K2CO3 (2 ꢂ 15 mL) and brine (20 mL). The organic layer
was dried over Na2SO4, filtered and evaporated in vacuo to give a
slightly yellow solid which was purified by column chromatog-
raphy (EtOAc:cyclohexane 1:1) to give the protected compound as a
white solid (1.70 g, 3.76 mmol, 76%): mp 138e140 ꢀC; 1H NMR
98%). mp: 118e120 ꢀC; 1H NMR (DMSO-d6):
d
¼ 8.48 (d, 1H,
J ¼ 8.0 Hz), 8.35 (d,1H, J ¼ 7.6 Hz), 8.25 (d,1H, J ¼ 8.2 Hz), 7.38 (t, 2H,
J ¼ 6.7 Hz), 7.28 (m, 4H), 7.21 (m, 2H), 7.13 (t, 1H, J ¼ 7.2 Hz), 7.03 (d,
1H, J ¼ 8.2 Hz), 6.99 (m, 2H), 6.94 (m, 2H), 5.25 (m,1H), 4.24 (m,1H),
4.14 (m, 1H), 3.56 (s, 3H), 3.40 (s, 2H), 3.26 (m, 2H), 3.11 (m, 1H),
2.95 (m, 1H), 2.52 (m, 2H), 1.55 (m, 1H), 1.49 (m, 1H), 1.33 (m, 2H),
1.22 (m, 2H). 13C NMR (DMSO-d6):
d
¼ 171.7, 171.1, 169.9, 169.7,
156.6, 156.5, 137.0, 135.6, 130.1, 130.0, 129.7, 129.0, 128.3, 126.9,
126.7, 124.2, 123.4, 119.3, 119.2, 118.6, 116.6, 55.2, 53.7, 53.4, 51.9,
41.9, 40.8, 37.0, 32.0, 31.4, 22.6. IR (cmꢃ1): 3514 2926, 2048, 1656,
d
¼ 7.90 (bs,1H), 6.64 (s, 2H), 6.29 (s, 1H), 4.45 (m, 2H), 4.34 (m, 1H),
3.89 (s, 6H), 3.86 (s, 3H), 3.79 (m, 2H), 2.57 (s, 1H), 1.67 (s, 9H); 13
C
d
¼ 168.2,156.7, 153.2, 137.1, 105.1, 81.5, 60.8, 59.0, 56.1, 44.0, 33.3,
1485. ESIþ MS m/z: 590 [M
þ
H]þ. Anal. Calcd for
28.2; 19F (188 MHz):
d
¼ ꢃ75.09 (d, 1H, J ¼ 7.0 Hz); IR (cmꢃ1): 3300,
C
32H40ClN5O6$H2O: C. 59.66, H. 6.58, N. 10.87. found C. 59.51, H.
1652, 1506, 1234, 1123; ESIþ MS m/z: 474.2 [M þ Na]þ; Anal. Calcd
for C19H28F3N3O6$H2O: C, 48.61; H, 6.45; N, 8.95; found C, 48.40; H,
5.88; N, 8.49. A solution of the Boc-protected compound (926 mg,
1.99 mmol, 1.0 eq.) in DCM/TFA 3:1 (12 mL) was stirred for 2 h at
room temperature. The solvent was evaporated under reduced
pressure to give the trifluoroacetic salt of 29 in quantitative yield, as
6.57, N. 10.57.
4.1.3.4. Methyl (2S)-2-[[2-[2-[(2S)-6-(benzyloxycarbonylamino)-2-
[[2-(2,5-dimethoxyphenyl) acetyl]amino]hexanoyl]hydrazino]acetyl]
amino]-3-phenyl-propanoate 25a. Compound 25a was synthesized
according to the procedure described for 24a from 23 that was
successively deprotected and coupled with 2,5-dimethoxy
phenylacetic acid (209 mg, 1.07 mmol, 1.2 eq.), except that the
crude product was purified by column chromatography (EtOAc/
MeOH : 9:1). 25a was obtained as a white solid (423 mg,
a
slightly yellow solid and that was used without further
purification.
4.1.4.2. N0-(2,2,2-Trifluoro-1-hydrazinocarbonylmethylethyl)hydra-
zine carboxylic acid tert-butyl ester 30. To a solution of 27 (2.0 g,
6.7 mmol) in ethanol (50 mL) was added hydrazine monohydrate
(3.34 g, 66.7 mmol, 10.0 eq.). After stirring at room temperature
overnight, the solvent was removed under reduced pressure, to
afford 30 as a white solid (2.11 g, quantitative yield). 1H NMR
0.61 mmol, 69%). mp: 143e145 ꢀC; 1H NMR:
d
¼ 8.20 (s, 1H), 7.47
(d, 1H, J ¼ 8.3 Hz), 7.24e7.33 (m, 8H), 7.14 (m, 2H), 6.78 (m, 2H),
6.77 (m, 1H), 6.52 (d, J ¼ 7.5 Hz, 1H), 5.12 (m, 1H), 5.07 (s, 2H), 4.83
(m, 1H), 4.63 (m, 1H), 4.24 (m, 1H), 3.78 (s, 3H), 3.72 (s, 3H), 3.69 (s,
3H), 3.49 (m, 2H), 3.38 (s, 2H), 3.15 (m, 2H), 3.10 (m, 2H), 1.74 (m,
(CD3OD):
(CD3OD):
d
¼ 3.60 (m, 1H), 2.32 (m, 2H), 1.32 (s, 9H); 13C NMR
1H), 1.53 (m, 1H), 1.42 (m, 2H), 1.19 (m, 2H). 13C NMR:
d
¼ 172.5,
d
¼ 173.6,129.4 (q, J ¼ 286.3 Hz), 85.0, 62.5 (q, J ¼ 27.7 Hz),
171.4, 169.9, 156.5, 153.7, 151.1, 136.7, 136.0, 129.1, 128.6, 128.5,
128.0, 127.1, 124.3,117.2, 113.1, 66.5, 56.0, 55.7, 54.8, 53.0, 52.4, 51.4,
40.5, 38.8, 37.4, 31.4, 29.3, 22.3. IR (cmꢃ1): 3285, 1731, 1688, 1649,
1501, 1223. APCIþ MS m/z: 692 [M þ H]þ. Anal. calcd for
35.4, 32.0. 19F (CD3OD):
d
¼ ꢃ75.1 (d, J ¼ 7.1 Hz).
4.1.4.3. 3-(N0-Benzyloxycarbonylhydrazino)-4,4,4-trifluorobutyric
acid ethyl ester 31. Compound 27 (200 mg, 0.66 mmol) was dis-
solved in dry CH2Cl2 (2.5 mL) and trifluoroacetic acid (2.5 mL,
33.0 mmol, 50.0 eq.) was added dropwise at room temperature.
The reaction mixture was stirred at room temperature for 2 h.
Solvent was evaporated under reduced pressure to yield light
brown solid oil (228 mg, with excess of TFA). Azeotropic removal
of excess TFA with toluene gave the deprotected product that was
used without any further purification in the course of the syn-
thesis. This compound (207 mg, 0.66 mmol) was dissolved in
C
36H45N509: C, 62.50; H, 6.56; N, 10.12; found: C, 62.45; H, 6.60; N,
10.02.
4.1.3.5. Methyl (2S)-2-[[2-[2-[(2S)-6-amino-2-[[2-(2,5-
dimethoxyphenyl)acetyl] amino]hexanoyl]hydrazino]acetyl]amino]-
3-phenyl-propanoate 25b. To a solution of 25a (100 mg, 0.14 mmol,
1 eq.) in dry MeOH/DMF (4/1 v/v 25 mL) was added 10% Pd/C
(11 mg). The mixture was stirred overnight under hydrogen at-
mosphere at room temperature and filtered on a celite pad. After
removal of the solvent under reduced pressure 25b was obtained
as a white solid (79 mg, 0.13 mmol, quantitative). mp: 84e86 ꢀC;
CH2Cl2 (6 mL) and benzylchloroformiate (104
m
L, 0.73 mmol, 1.1
L,
eq.) was added dropwise at 0 ꢀC. After was added DIPEA (287
m
1.65 mmol, 2.5 eq.) and the reaction mixture was stirred at room
temperature for 21 h. After removal of the solvent under reduced
pressure, the residue was dissolved in EtOAc (9 mL). The organic
phase was successively washed with 10% aqueous citric acid
(9 mL), water (9 mL), 10% aqueous K2CO3 (9 mL) and brine (9 mL).
The organic layer was dried over Na2SO4, filtered and concen-
trated. A purification by chromatography on silica gel, eluting
with EtOAc/cyclohexane (5/5), afforded 31 as a colorless oil
1H NMR (DMSO-d6):
d
¼ 8.48 (d, 1H, J ¼ 7.4 Hz), 8.09 (d, 1H,
J ¼ 8 Hz), 7.96 (d, 1H, J ¼ 8.0 Hz), 7.28e7.21 (m, 5H), 6.86 (d, 1H,
J ¼ 8.5 Hz), 6.75 (d, 2H, J ¼ 4.4 Hz), 5.25 (m, 1H), 4.48 (dd, 1H,
J ¼ 7.4 and 14.2 Hz), 4.16 (m, 1H), 3.69 (s, 3H), 3.67 (s, 3H), 3.57 (s,
3H), 3.42 (s, 2H), 3.26 (m, 2H), 3.04 (m, 1H), 2.92 (m, 1H), 2.53 (m,
2H), 1.59 (m, 1H), 1.49 (m, 1H), 1.34 (m, 2H), 1.25 (m, 2H). 13C NMR
(DMSO-d6):
d
¼ 171.7, 171.2, 169.8, 169.7, 152.9, 151.3, 137.0, 130.0,
129.0, 128.3, 126.6125.7, 116.8, 111.9, 111.6, 55.9, 55.3, 53.7, 53.4,
51.8, 51.2, 41.0, 36.9, 36.7, 31.9, 31.8, 22.5. IR (cmꢃ1): 2932, 1647,
1500, 1224. ESIþ MS m/z: 558 [M þ H]þ. Anal. Calcd for
(168 mg, 0.5 mmol, 76%). 1H NMR (200 MHz):
d
¼ 7.42e7.28 (m,
5 H), 6.29 (br s, 1H), 5.11 (s, 2H), 4.20 (q, J ¼ 7.2 Hz, 2H), 3.89 (m,
1H), 2.58 (m, 2H), 1.22 (t, J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz):
C
28H39N5O7$0.75H2O C. 58.91, H. 7.17, N. 12.27 found C. 58.87, H.
d
¼ 169.7, 157.2, 135.7, 128.6, 128.4, 128.2, 125.2 (q, J ¼ 279.8 Hz),
6.79, N. 12.36.
67.5, 61.5, 58.2 (q, J ¼ 27.8 Hz), 32.1, 14.0. 19F (188 MHz):

522
A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
d
¼ ꢃ75.5 (d, J ¼ 7.2 Hz). IR (cmꢃ1): 3331, 1722, 1159, 1128, 699.
J ¼ 6.9 and 13.1 Hz), 3.09e2.99 (m, 3H), 2.34 (t, 2H, J ¼ 5.4 Hz), 1.45
ESIþ MS m/z: 357 [M þ Na]þ
(s, 9H); 13C NMR (75 MHz):
d
¼ 172.0, 171.1, 137.2, 129.2,129.0,128.1,
126.5, 78.3, 53.4, 51.7, 47.4, 36.7, 33.8, 28.1; IR (cmꢃ1): 2935, 1728,
4.1.4.4. N0-(2,2,2-Trifluoro-1-hydrazinocarbonylmethylethyl)hydrazi-
necarboxylic acid benzyl ester 32. To a solution of 31 (148 mg,
0.44 mmol) in EtOH (3 mL) was added hydrazine monohydrate
1529, 1156, 837; ESIþ MS m/z: 388.3 [M þ Na]þ; Anal. Calcd for
C18H27N3O5: C, 59.16; H, 7.45; N, 11.50; found C, 59.20; H, 7.20; N,
11.05.
(214 mL, 4.4 mmol) and the reaction mixture was stirred at room
temperature for 23 h. After removing the solvent under reduced
pressure, 32 was obtained as a white solid (140 mg, 0.44 mmol,
quantitative yield). mp: 146e148 ꢀC). 1H NMR (200 MHz, CD3OD):
4.1.4.7. tert-Butyl N-[[3-oxo-3-[(3,4,5-trimethoxyphenyl)methylami
no]propyl]amino] carbamate 36. Compound 34 was saponified to
give the 3-(N0-tert-butoxycarbonyl-hydrazino)-propionic acid ac-
cording the same procedure described for the synthesis of com-
pound 35. To a solution of 3-(N0-tert-butoxycarbonyl-hydrazino)-
propionic acid (1.3 g, 6.38 mmol, 1 eq.) in dry DMF (30 mL) was
successively added 3,4,5-trimethoxybenzylamine (1.5 g, 7.65 mmol,
1.2 eq.), DIPEA (3.3 g, 25.5 mmol, 7 eq.), HBTU (2.9 g, 7.65 mmol, 1.2
eq.), and HOBT (1.0 g, 7.65 mmol, 1.2 eq.). The mixture was stirred
overnight at room temperature under argon atmosphere. After
removal of the solvent under reduced pressure, the residue was
taken up in EtOAc (20 mL) and successively washed with 10%
aqueous citric acid (2 ꢂ 20 mL), water (20 mL), 10% aqueous K2CO3
(2 ꢂ 20 mL) and brine (20 mL). The organic layer was dried over
Na2SO4, filtered and evaporated under reduced pressure to give a
crude product which was purified by column chromatography
(EtOAc) to afford 36 (1.26 g, 3.28 mmol, 52%) as a white foam. 1H
d
¼ 7.33e7.23 (m, 5 H), 5.09 (s, 2H), 3.80 (m, 1H), 2.48 (dd, J ¼ 4.8
and 15.0 Hz,1H), 2.39 (m,1H). 13C NMR (75 MHz, CD3OD):
d
¼ 170.9,
159.6, 138.0, 129.5, 129.1, 129.0, 123.3 (q, J ¼ 279.8 Hz), 67.9, 59.5 (q,
J ¼ 27.8 Hz), 32.6. 19F (188 MHz, CD3OD):
¼ ꢃ76.9 (d, J ¼ 7.3 Hz). IR
d
(cmꢃ1): 3307, 1705, 1654, 1174, 1120, 696. ESIþ MS m/z: 343
[M þ Na]þ. Anal. Calcd for C12H15F3N4O3$0.4H2O: C, 44.03; H, 4.88;
N 17.12. Found: C, 43.93; H, 4.54; N, 17.57.
4.1.4.5. 3-(N0-tert-Butoxycarbonyl-hydrazino)-propionic acid ethyl
ester 34. A solution of 3-bromo-propionic acid ethyl ester 33 (2 mL,
15.7 mmol, 1.0 eq.), DIPEA (2.6 mL, 15.7 mmol, 1.0 eq.) and Boc-
hydrazine (3.1 g, 3.1 g, 23.55 mmol, 1.5) in toluene was heated at
80 ꢀC for 4 days. After removal of the solvent under reduced
pressure, the crude product was purified by column chromatog-
raphy (cyclohexane:EtOAc 6:4) to give 34 as a slightly yellow oil
NMR (300 MHz):
d
¼ 7.18 (bs, 1H), 6.51 (s, 2H), 6.45 (bs, 1H, NH),
(1.58 g, 6.80 mmol, 43%). 1H NMR:
d
¼ 6.26 (bs, 1H), 4.08 (q, 2H,
6.01 (s, 1H), 4.31 (d, 2H, J ¼ 5.7 Hz), 3.81 (s, 6H), 3.80 (s, 3H), 3.06 (t,
J ¼ 7.1 Hz), 3.06 (t, 2H, J ¼ 6.6 Hz), 2.41 (t, 2H, J ¼ 6.6 Hz),1.39 (s, 9H),
2H, J ¼ 6.1 Hz), 2.35 (t, 2H, J ¼ 6.1 Hz), 1.45 (s, 9H). 13C NMR
1.19 (t, 3H, J ¼ 7.1 Hz); 13C NMR:
d
¼ 172.2, 156.7, 80.3, 60.3, 47.3,
(75 MHz):
d
¼ 171.7, 157.3, 153.3, 136.9, 134.3, 104.8, 80.8, 60.8, 56.1,
33.0, 28.2, 14.0; IR (cmꢃ1): 2980, 1714, 1252, 1151, 1025; ESIþ MS m/
z: 233 [M þ Hþ]; Anal. Calcd for C10H20N2O4: C, 51.71; H, 8.68; N,
12.06; found C, 51.61; H, 8.25; N, 11.58.
48.5, 43.6, 34.4, 28.6. IR (cmꢃ1): 3303, 2352, 1647, 1592, 1457. ESIþ
MS m/z: 406 [M þ Na]þ
4.1.4.8. Ethyl 2-[2-[6-(benzyloxycarbonylamino)-2-(tert-butoxycarbo
nylamino)hexanoyl] hydrazino]acetate 38. To a solution of ethyl 2-
hydrazinylacetate hydrochloride 37 (1.0 g, 6.47 mmol, 1 eq.) in dry
DMF (20 mL) was successively added Na-Boc-N 3 -Z-Lys (2.95 g,
7.76 mmol, 1.2 eq.), DIPEA (5.8 g, 4.3 mmol, 7 eq.), HBTU (2.95 g,
7.76 mmol, 1.2 eq.), and HOBt (1.04 g, 7.76 mmol, 1.2 eq.). The
mixture was stirred overnight at room temperature under argon
atmosphere. After removal of the solvent under reduced pressure,
the residue was taken up in EtOAc (20 mL) and washed successively
with 10% aqueous citric acid (2 ꢂ 15 mL), water (15 mL), 10%
aqueous K2CO3 (2 ꢂ 15 mL) and brine (20 mL). The organic layer
was dried over Na2SO4, filtered and evaporated under reduced
pressure. The crude product obtained was purified by column
chromatography (EtOAc/cyclohexane, 8:2) to give 38 (2.35 g,
4.1.4.6. 2-[3-(N0-tert-Butoxycarbonyl-hydrazino)-propionylamino]-
3-phenyl-propionic acid methyl ester 35. An aqueous solution of
NaOH 2 N (4.8 mL, 9.6 mmol, 1.1 eq.) was added to a solution of 34
(2.00 g, 8.6 mmol, 1.0 eq.) in THF/MeOH (10/10 mL, v/v). The re-
action was stirred at room temperature over 3 h. After removing the
solvent under reduced pressure without distilling the water, the
remaining solution was brought at pH ¼ 5 by addition of aqueous
solution of 1 N HCl. The aqueous phase was extracted with EtOAc
(3 ꢂ 20 mL). Then, the combined organic layers were dried over
MgSO4, filtered and concentrated under reduced pressure to give
the 3-(N0-tert-Butoxycarbonyl-hydrazino)-propionic acid as a white
solid (1.64 g, 8.0 mmol, 93%) which was used in the next step
without further purification. mp: 112e114 ꢀC; 1H NMR (DMSO-d6):
d
¼ 8.17 (m, 2H), 3.30 (s, 1H), 2.87 (t, 2H, J ¼ 7.0 Hz), 2.30 (t, 2H,
4.9 mmol, 76%) as a white foam. 1H NMR (300 MHz):
d
¼ 8.30 (m,
J ¼ 7.0 Hz), 1.39 (s, 9H); 13C NMR (DMSO-d6):
d
¼ 173.2, 156.3, 78.2,
1H), 8.16 (s, 1H), 7.27 (m, 5H), 5.25 (m, 1H), 5.01 (s, 2H), 4.12 (q, 2H,
46.7, 32.5, 28.0; IR (cmꢃ1): 3350, 2981, 1679, 1437, 1156, 860; ESIþ
MS m/z: 227 [M þ H]þ, 431.1 [2M þ Na]þ; Anal. Calcd for
C8H16N2O4: C 47.05; H, 7.90; N, 13.72; found C, 47.43; H, 7.90; N,
13.33. A solution of the acid compound (0.819 g, 4.0 mmol, 1.0 eq.)
and HBTU (1.52 g, 4.0 mmol, 1.0 eq.) in DMF (10 mL) was stirred for
30 min, then were successively added HOBT (650 mg, 4.8 mmol, 1.2
eq.), DIPEA (1.4 mL, 8.0 mmol, 2.0 eq.) and phenylalanine hydrox-
ychloride (1.04 g, 4.8 mmol, 1.2 eq.). The reaction was performed
under argon atmosphere a room temperature overnight. After
removal of the solvent under reduced pressure, the crude product
was taken up in EtOAc (15 mL) and successively washed with 10%
aqueous K2CO3 (2 ꢂ 15 mL), brine (20 mL) and water (2 ꢂ 15 mL).
The organic layer was dried over Na2SO4, filtered and evaporated
under reduced pressure to give a slightly yellow solid which was
purified by column chromatography (EtOAc) to give 35 as a white
solid (1.26 g, 3.3 mmol, 82%). mp; 86e88 ꢀC; 1H NMR (300 MHz,
J ¼ 7.1 Hz), 3.90 (m, 1H), 3.56 (s, 2H), 3.10 (m, 2H), 1.47 (m, 6H), 1.34
(s, 9H), 1.18 (t, 3H, J ¼ 7.1 Hz). 13C NMR (75 MHz):
d
¼ 172.2, 171.5,
156.7, 155.8, 136.6, 128.5, 128.0, 80.3, 66.6, 53.2, 40.3, 31.4, 29.3,
28.2, 22.3,15.26. IR (cmꢃ1): 3290,1684, 1519,1367,1245. ESIþ MS m/
z: 503 [M þ Na]þ
4.2. Enzymatic studies
ChT-L activity was determined by monitoring the hydrolysis of
Suc-LLVY-AMC, for 45 min at 37 ꢀC in the absence (control) or
presence of test compounds (0.1e200 mM). The buffer (pH 7.5) was
20 mM Tris, 1 mM DTT, 10% glycerol, 0.02% (w/v). The final con-
centration of rabbit 20S proteasome and Suc-LLVY-AMC were
0.3 nM and 50 nM respectively. Substrate and inhibitors were
previously dissolved in DMSO leading to the final and constant
DMSO concentration of 2% (v/v) in the enzymatic assays. The IC50
values (inhibitor concentrations giving 50% inhibition) were ob-
tained by plotting the percent inhibition against inhibitor
DMSO-d6):
d
¼ 8.45 (s, 1H), 8.30 (s, 1H), 7.28e7.13 (m, 5H), 6.10 (bs,
1H), 4.85 (dd, 1H, J ¼ 6.9 and 13.1 Hz), 3.73 (s, 3H), 3.11 (dd, 1H,

A. Bordessa et al. / European Journal of Medicinal Chemistry 70 (2013) 505e524
523
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concentration to equation 1: % inhibition ¼ 100[I]/(IC50 þ [I]), or
nH
equation 2: % inhibition ¼ 100[I]nH/(IC þ [I]nH) where nH is the
50
Hill number. The Km value of the fluorogenic substrate Suc-LLVY-
AMC in our experimental conditions was 30 ꢁ 5
mM.
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S. Ongeri Curr. Pharm. Des. 19 (2013) 4115e4130.
4.3. Molecular modeling
Coordinates of “apo” bovine proteasome were retrieved from
the X-ray structure available in the PDB [42] (accession code 1IRU,
ꢀ
resolution 2.75 A) [41]. Hydrogen atoms were added and only
chains L and M defining the binding site at their interface were
retained.
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S. Emch, M. Groettrup, J. Biol. Chem. 274 (1999) 35734e35740.
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Initial coordinates for relevant stereoisomers of the ligands were
generated using CORINA v3.44 software [43]. Ligands were then
docked individually using GOLD v5.1 software [44] with default
parameters (but using of GoldScore scoring function) and full
torsional freedom for the side chains of Met45 and Ile35 (belonging
to the S1 subpocket) was allowed.
During docking calculations, the scoring function was succes-
sively configured to bias conformational space exploration towards
the solutions exhibiting hydrogen bonds with the maximum
number of key residues from one of the two following subsets:
Thr21, Ala49 and Asp125 or Gly47, Ala49 and Asp125 (40 solutions
were calculated for each case.)
[20] M. Groll, N. Gallastegui, X. Marechal, V. LeRavalec, N. Basse, N. Richy, E. Genin,
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Thieme:Stuttgart, New York, NY, 2003, pp. 423e457.
Solutions were analyzed and cherry-picked using Hermes v1.5
(companion program of GOLD) on the basis of criteria detailed in
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