Preliminary studies of new proteasome inhibitors in the tumor targeting approach: Synthesis and in vitro cytotoxicity

Article abstract of DOI:10.1021/jm050181l

The proteasome is a multicatalytic protease that plays a critical role in the cell. The control of proteasomes could, thus, provide a weapon for the treatment of cancer. Therefore, we have synthesized six new peptide aldehyde inhibitors of the proteasome

Full text of DOI:10.1021/jm050181l

J. Med. Chem. 2005, 48, 6731-6740
6731
Preliminary Studies of New Proteasome Inhibitors in the Tumor Targeting
Approach: Synthesis and in Vitro Cytotoxicity
Magali Vivier,*,† Anne-Sophie Jarrousse,† Bernadette Bouchon,† Marie-Josephe Galmier,† Philippe Auzeloux,†
Jacques Sauzieres,‡ and Jean-Claude Madelmont*,†
UMR 484 INSERM-Universite´ d’Auvergne-Centre Jean Perrin, Rue Montalembert, B.P. 184,
63005 Clermont-Ferrand Cedex, France, and OTL Pharma, 15 Rue de Turbigo, 75002 Paris, France
Received February 25, 2005
The proteasome is a multicatalytic protease that plays a critical role in the cell. The control of
proteasomes could, thus, provide a weapon for the treatment of cancer. Therefore, we have
synthesized six new peptide aldehyde inhibitors of the proteasome linked to the N-(2-
diethylaminoethyl)benzamide (BZA-CO) structure, in order to target the cytotoxic activity to
malignant melanoma cells. Biological studies demonstrated the influence of length and
composition of the amino acid chain on the cytotoxicity of our compounds. Among them,
compound 19 presents the highest cytotoxicity (IC50 ) 0.64 ( 0.07 µmol): this cytotoxicity was
maintained in the presence of BZA-CO but decreased 8-fold compared to the control MG132.
Fluorescence activated cell sorter (FACS) and cytotoxic activity analysis demonstrated the
selectivity of compound 19 for melanoma cells. Finally, western blottings of ubiquitinated
proteins in IPC227F cells as well as proteasome assays confirmed that the cytotoxicity was
linked to an inhibition of the proteasome activity.
Introduction
interesting new target for anticancer drugs.10-14 Many
natural or synthetic inhibitors of the proteasome have
been developed (lactacystin,15-17 TMC95,18 peptide
aldehydes,19-22 peptide boronates,23,24 peptide vinyl
sulfones,25,26 epoxyketones,27 peptide R-keto aldehydes28
and R-ketoamides, 5-methoxy-1-indanone-3-acetic pep-
tide derivatives,29 and bivalent inhibitors30,31). All syn-
thetic inhibitors present a peptide structure necessary
for the specific recognition by the proteasome site and
a cytotoxic moiety such as an aldehyde, boronate, or
vinyl sulfone residue. Crystallography and site-directed
mutagenesis studies demonstrated that these inhibitors
interact with the N-terminal threonine (Thr 1) of the
three proteolytically active â-subunits.32-36 Additional
residues located close to the active site participate in
the catalysis mechanism, but their precise function has
not yet been determined. Among the proteasome inhibi-
tors that are under development, the dipeptidyl boronic
acid PS-341 (Bortezomib VELCADE, Figure 1)13,37,38 is
commercialized. This compound appears to be particu-
larly interesting for the treatment of several hemato-
logic malignancies (particularly multiple myeloma) and
solid tumor indications. Peptide aldehyde inhibitors also
represent a very important class among the proteasome
inhibitors. According to Kisselev et al.,11 these inhibitors
are an essential family for studies in cell culture and
tissues. Indeed, MG132 (Figure 1) was one of the first
synthetic inhibitors to be described and used in the
inhibition of proteasomes. As a peptide aldehyde inhibi-
tor, MG132 has the advantage of dissociating from the
proteasome, conferring rapid reversibility of its action.
Moreover, MG132 has mainly been studied for its
significant cytotoxic activity.39 Nevertheless, the ubiq-
uitous properties of the proteasome limit the use of the
proteasome inhibitors in any therapy. The alternative
The 26S proteasome is a multicatalytic protein com-
plex which is implicated in the degradation of the
majority of cellular proteins. It consists of a stack of two
R and two â rings. The seven different R-type subunits
form the two outer rings of the proteasome 20S cylinder,
whereas the seven â-type subunits are components of
the two inner rings of the complex. In mammalian cells,
this 20S proteolytic component is capped by a regulatory
19S complex at each extremity whose role is to unfold
the protein substrates and to stimulate proteolytic
activity. This complex exhibits at least five endopepti-
dase activities. The three principal activities are re-
ferred to as chymotrypsin-like (ChT-L) when cleavage
occurs on the carboxyl side of hydrophobic residues,
trypsin-like (T-L) when cleavage occurs on the carboxyl
side of basic amino acids, and post-glutamyl peptide-
hydrolyzing (PGPH) when cleavage occurs on the car-
boxyl side of acidic residues. The two minor peptidase
activities cleave peptide bonds after branched-chain
(BrAAP) or small neutral amino acids (SNAAP) prefer-
ring activity.1 These proteolytic activities confer on
proteasomes more of a recycler function for damaged
or misfolded proteins in the cell as well as a critical role
in a large panel of events including cell immune
surveillance,2,3 inflammatory response,4 muscle atro-
phy,5 metabolic pathway regulation,6 circadian rhythm
regulation,7 and transcriptional regulation or cell cycle
control.8,9 The latter is very important, since transcrip-
tional factors modulate cell division and could contribute
to tumor growth. These features make proteasomes an
* Authors for correspondence. Phone: (33) 4 73 15 08 00. Fax: (33)
73 15 08 01. E-mail: vivier@inserm484.u-clermont1.fr or
4
madelmont@inserm484.u-clermont1.fr.
† UMR 484 INSERM-Universite´ d’Auvergne-Centre Jean Perrin
‡ OTL Pharma.
10.1021/jm050181l CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/27/2005

6732 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Vivier et al.
tively), probably due to the poor formation of the lithium
derivative, we favored route b, reaction of 2-diethylami-
noethylamine with dimethylterephthalate in the pres-
ence of trimethylaluminum. The monosubstituted de-
rivative 5 (70% from 3) and the disubstituted derivative
6 (2% from 3) obtained by this route can be easily
separated by flash chromatography. The ester 5 was
transformed into the lithium salt 7 by saponification at
room temperature with lithium hydroxide (method B
in the Experimental Section) in quantitative yield.50
General synthetic pathways to obtain peptide struc-
tures are presented in Scheme 2. The final leucine
derivatives 13, 16, and 19 were prepared by following
two different routes. Whenever possible, commercially
available protected amino acids were employed. In the
first step, leucine-derivative-structured peptides 11, 14,
and 17 (route A) were prepared using standard peptide
synthesis procedures, with DCC and HOBt as catalytic
coupling agents.51-59 Protecting groups were removed
by treatment with lithium hydroxide for the methyl
group or with trifluoroacetic acid (TFA) for the tert-butyl
carbamate (Boc) group. In the next step, the coupling
reactions between the acid 7 and amines 8, 11, 14, and
17 were undertaken in the presence of coupling reagents
DCC or HOBt according to method C. An example of
synthetic routes A and B is provided by the preparation
of compound 12. According to route A, methyl leucine
ester hydrochloride 8 was coupled to compound 7 to give
compound 9 with acceptable yield (51%, method C in
the Experimental Section). The methyl-protective group
of compound 9 was removed by saponification with
lithium hydroxide (method B)50 to give compound 10 in
good yield (89%). However, the coupling reaction be-
tween 10 and N,O-dimethylhydroxylamine hydrochlo-
ride (method C) gives compound 12 in very weak yield.
This may be due to the steric hindrance of the N-
(diethylaminoethyl) chain. Consequently, we decided to
investigate route B. Using this route, the coupling
reaction between acid 7 and compound 11 allowed us
to prepare compound 12 with sufficient yield (12%). The
Weinreb amide thus obtained was reduced with LiAlH4
(method D in the Experimental Section) to give corre-
sponding aldehyde 13 with quantitative yield.39
In summary, since route B gave better yield for the
synthesis of 13, 16, and 19, we used this synthetic route
to prepare compounds 22, 25, and 28 (Scheme 3). The
pure peptide aldehyde products 13, 16, 19, 22, 25, and
28 were isolated as white solids by recrystallization from
diethyl ether and were tested as hydrochloride salts.
Biological Studies. Attached human ocular mela-
noma IPC227F cells were cultured for 24 h on a
microplate and then exposed for 48 h to increasing
concentrations of the peptide aldehydes 13, 16, 19, 22,
25, and 28 or MG132 as a control. The effect of these
compounds on cell outgrowth was evaluated by assay
with Hoechst dye 33342. First, we wanted to demon-
strate that the link from peptide aldehydes to a mela-
noma vector preserved a cytotoxicity. We also investi-
gated the effects on cytotoxicity of the substitution of
the Cbz protected group in MG132 by BZA-CO (com-
pound 19). As shown in Figure 3A, the cytotoxicity of
compound 19 was maintained (IC50 ) 0.64 ( 0.07 µM)
but has decreased 8-fold compared to that of the MG132
control (IC50 ) 0.077 ( 0.009 µM). The influence of
Figure 1. Pharmacomodulated proteasome inhibitors:
a
coupling between 123I-BZA and proteasome inhibitors.
way to overcome this limit would be to carry these drugs
selectively to the target tumoral tissue.
On this basis, our laboratory has had great experience
in targeting melanoma cells: Moreau et al.40 have
developed in our laboratory a new radiopharmaceutic
[
123I]-N-(2-diethylaminoethyl)-4-iodobenzamide (123I-
BZA, Figure 1) which binds to melanin pigmented cells
with high affinity. This radiopharmaceutic has been
used in previous scintigraphic studies in humans and
presents a sensitivity of over 80% and a specificity of
100% localization on malignant melanomas and meta-
stases.40-47
Therefore, along with other studies we initiated on
potent and selective cytotoxic antitumor agents, we
decided to synthesize new peptide aldehyde inhibitors
of the proteasome linked to the N-(2-diethylaminoethyl)-
benzamide structure (BZA-CO, Figure 1) in order to
target the cytotoxic activity to malignant melanoma
cells.
This is the first report of the synthesis and pharma-
cological characterization of six peptide aldehydes linked
to the benzamide derivative structure (Figure 2). The
effects of the introduction of BZA-CO into the peptide
aldehyde structure on the cytotoxicity, length, and
composition of the amino acid chain are also discussed.
The measurement of the three main proteasome activi-
ties is also reported.
Results and Discussion
Synthesis of Peptide Inhibitors. Two methods
were investigated to synthesize the vector structure
precursor 7 (Scheme 1). First, we used the iodine and
bromine precursors 1 and 2 which were directly trans-
formed into the carboxylic acid derivative 4 by CO2
carbonation48 through the lithium reagent.49 As these
reactions gave insufficient yields (21% and 9%, respec-

Preliminary Studies of New Proteasome Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6733
Figure 2. Synthesized peptide aldehydes.
Scheme 1. Synthesis of BZA-CO Precursora
Scheme 2. General Methods for the Preparation of
Leucinal Derivatesa
a Reagents: (8) HCl, Leu-OMe; (11) TFA-Leu-NOMeMe; (14)
TFA-Leu-Leu-NOMeMe; (17) TFA-Leu-Leu-Leu-NOMeMe. (a)
DCC, HOBt, TEA, CH2Cl2 or CH2Cl2/DMF, rt; (b) LiOH, THF/
H2O, rt; (c) DCC, HOBt, TEA, CH2Cl2 or CH2Cl2/DMF or BOP,
DMF, TEA, rt; (d) AlLiH4, THF, -80 °C. b Route B is preferred to
route A.
compound 19, the compounds with one or two leucine
residues (13 and 16) were inefficient at inhibiting cell
growth. Thus, it appears that the length of the amino
acid chain could play a major role in cytotoxic activity.
These results corroborate those previously obtained with
epoxide proteasome inhibitors.27
Since the dipeptidyl inhibitor PS-341 contains leucine
and phenylalanine residues,23 the effect of amino acid
chain composition on cytotoxicity was investigated. The
comparison of cytotoxicities between compounds 16 and
22 that contained two leucine residues or a leucine and
a phenylalanine residue, respectively. Figure 3B and
Table 1 showed that the presence of phenylalanine at
a Reagents: (a) BuLi, CO2, Et2O or Et2O/THF, -70 °C; (b)
NH2CH2CH2NEt2, Al(CH3)3, CH2Cl2, reflux; (c) LiOH, THF/H2O,
b
room temperature (rt). Compounds 1 and 2 are synthesized as
already published.45 c Route b is preferred to route a.
pharmacomodulation on the cytotoxicity of the peptide
chain was then estimated. According to different previ-
ous optimization studies,23,25,27-29 all the peptide alde-
hydes synthesized here contained a leucine at the P1
position: this leucine should increase the chymotrypsin-
like activity of these inhibitors.27 Comparisons were
made of the cytotoxicity of compounds 13, 16, and 19
that contained one, two, or three leucine residues,
respectively. As shown in Figure 3A, in contrast to
the P2 position dramatically enhanced potency (IC50
)

6734 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Scheme 3. Synthesis of Compounds 22, 25, and 28a
Vivier et al.
a Reagents: (20) TFA-Phe-Leu-NOMeMe; (23) TFA-Leu-Phe-
Leu-NOMeMe; (26) TFA-Phe-Leu-Leu-NOMeMe. (a) DCC, HOBt,
TEA, CH2Cl2 or CH2Cl2/DMF, rt; (b) AlLiH4, THF, -80 °C.
20.88 ( 1.96 µM for compound 22 versus no inhibition
observed for compound 16).
Figure 3. Cytotoxicity assay. Attached IPC227F cells were
incubated with increasing concentrations of (A) MG132 (9),
13 (b), 16 ([), or 19 (2); (B) 16 ([) or 22 (]); or (C) 19 (2), 25
(O), or 28 (0) for 48 h, washed with 1X PBS, and then frozen
at -80 °C. Cell survival data were determined using fluores-
cent Hoechst dye 33342. The data are expressed as mean
percentages ( standard deviation (SD) of the untreated control
(n ) 3).
Conversely, the comparison of the tripeptidyl com-
pounds 19, 25, and 28 that contained, respectively, three
leucine residues, a phenylalanine at the P2 position, and
a phenylalanine at the P3 position showed that the
presence of a phenylalanine did not increase cytotoxicity
(Figure 3C and Table 1). Indeed, the IC50 values of 25
and 28 were, respectively, 4.5-fold and 18-fold higher
than that of 19. However, the migration of the phenyl-
alanine from the P2 to the P3 position (compounds 25
and 28) increased cytotoxicity by 4-fold. These results
are in accordance with the study on epoxycetones
already published by Eloffson et al.27 In summary, at
equal chain lengths, amino acid composition plays a key
role in obtaining sufficient toxicity.
We attempted then to correlate the cytotoxic activity
to the proteasome inhibition. To confirm that these
newly synthesized molecules inhibited proteasome, we
observed the accumulation of ubiquitinated proteins in
IPC227F cells. The proteins were tagged with several
ubiquitin molecules in order to be recognized and
degraded by the proteasomes. Therefore, the inhibition
of the proteasome was able to lead to the accumulation
of ubiquitinated proteins (Figure 4).8,9,21,38,60,61 IPC227F
cells were then treated at various time intervals with
10 µM cytotoxic drugs 19, 22, 25, and 28, and following
extraction, the accumulation of polyubiquitinated pro-
teins was visualized as a dark smear of proteins by
Western blotting using anti-ubiquitin antibody. As
shown in Figure 5, under the conditions used, only
compound 22 did not lead to the accumulation of
ubiquitinated proteins, even after 48 h of incubation.
This could indicate that the cytotoxic effect of compound
22 leads to some result other than proteasome inhibi-
tion. However, incubation of IPC227F cells with 19, 25,
or 28 produced an accumulation of ubiquitin-tagged
proteins in a time-dependent manner, since a smear
appeared after 24 h of incubation. These observations
are perfectly comparable to those obtained with the
reference product MG132.
Finally, to confirm that the accumulation of ubiquitin-
tagged proteins observed is due to proteasome inhibi-

Preliminary Studies of New Proteasome Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6735
Table 1. Concentration Inhibiting 50% of Cell Growth (IC50) in
inefficient at inhibiting cell growth. The presence of the
phenylalanine at the P2 position appeared to increase
the cytotoxicity of the dipeptidyl aldehydes (compound
22) but, surprisingly, did not increase the cytotoxicity
of the tripeptidyl aldehydes (compounds 25 and 28
versus 19). In summary, among all the compounds
tested, the derivative 19 presented the highest cytotox-
icity (IC50 ) 0.64 ( 0.07 µmol): although cytotoxicity
decreased 8-fold in comparison to that of the control
MG132, the substitution of the Cbz protective group by
the BZA-CO cluster maintained a significant cytotox-
icity on human ocular melanoma IPC227F cells.
The accumulation of polyubiquitinated proteins in
IPC227F cells and proteasomes assays confirmed that
the cytotoxicity observed after incubation in the pres-
ence of pharmacomodulated compounds was due to
proteasome activity inhibition.
IPC227F Cellsa
cpd
Gb
Cbz
BZA-CO
BZA-CO
BZA-CO
BZA-CO
BZA-CO
BZA-CO
P3
P2
P1
IC50 (µM)
MG132
13
leu
leu
leu
leu
leu
leu
leu
leu
leu
0.077 ( 0.009
NIc
16
leu
leu
phe
phe
leu
NIc
19
leu
0.64 ( 0.07
20.88 ( 1.96
2.84 ( 0.34
11.92 ( 1.20
22
25
leu
phe
28
a IC50 values are determined from the survival curves. Values
are the means ( SD from three independent experiments, each
of which was performed in triplicate. All the compounds are tested
as chlorhydrate salts. b Cbz ) Ph-CH2-O-CO-; BZA ) Et2-
NH-(CH2)2-NH-CO-C6H4-. c No inhibition observed.
tion, the three main proteasome activities, i.e., ChT-L,
T-L, and PGPH activities, were measured on 20S
proteasomes incubated with compounds 19, 25, and 28.
As shown in Figure 6A, the three compounds tested
reduced the ChT-L activity (70-95% inhibition) in a
dose-dependent manner (0.1-10 µM), whereas we did
not observe any significant decrease of the T-L activity
(Figure 6B). These results are in agreement with results
published by Eloffson et al.27 and Stein et al.39 We
wanted to increase the ChT-L activity of pharmaco-
modulated inhibitors by the introduction of leucine at
the P1 position. Concerning the PGPH activity (Figure
6C), only compounds 19 and 25 were efficient in a dose-
dependent manner, similar to the MG132 control.
However, these percentages of inhibition measured for
PGPH activity were less important than the one ob-
served for the ChT-L activity (approximately 40-80%).
Compound 28 appeared to be less effective with a global
inhibition rate of 35%. These results confirmed that the
cytotoxic activity toward melanoma cells and the ac-
cumulation of ubiquitinated proteins after treatment
with compounds 19, 25, and 28 are indeed correlated
to the inhibition of proteasomes by these new molecules.
At last, we observed that compound 19 exhibited a
high cytotoxic activity in IPC227 F cells (IC50 ) 0.64 (
0.07µM) but failed to exhibit a cytotoxic activity in
normal melanocytes, with IC50 values higher than 100
µM (Figure 7). These preliminary results are consistent
with the selectivity of the compounds for melanoma
cells. Furthermore, FACS analysis did not demonstrate
any changes in percentages in the different phases of
the cell cycle in the normal melanocytes treated by
compound 19 (Figure 8A). Conversely, the percentages
in the M4Beu cells (human melanoma cells) treated by
compound 19 increased in phase G2M and decreased in
phase S (Figure 8B). These results of the cell cycle
profile confirm the hypothesis of selectivity of our
compounds in favor of melanoma cells.
The preliminary results concerning the selectivity of
our compounds for melanoma cells have demonstrated
their low cytotoxicity in normal melanocytes. The drug
biodistribution of the 125[I] derivative of compound 19
was also studied in mice bearing subcutaneous implan-
tation of malignant melanoma cells. These experiments
showed an accumulation of 2.7% injected dose per gram
(DI/g) of this compound in B16 tumors at 15 min, with
1.2% of this aldehyde remaining in the tumor at 3 and
6 h after administration, thus giving a tumor/muscle
ratio of over 2.3 (data not shown). These two results are
in accordance with the selectivity for melanoma cells of
our pharmacomodulated derivatives.
This compound conjugation approach will be entirely
validated after in vivo experiments on tumor-bearing
animals with radiolabeled compounds in order to verify
the selectivity of the compounds to malignant melano-
mas and metastases.
Experimental Section
Chemistry. All reagents and solvents were from com-
mercial suppliers and used with no further purification.
Tetrahydrofuran (THF) was distilled over sodium-benzophe-
none. All other reaction solvents were anhydrous or high-
performance liquid chromatography (HPLC) commercial grades
(Carlo Erba Reagenti, Milan, Italy). Compound purity was
checked by thin layer chromatography (TLC) on precoated
silica gel plates (plastic sheet 60 F254, layer thickness 0.25 mm;
SDS, Pepin, France), aluminum oxide plates (60 F254, neutral
type E, layer thickness 0.20 nm; Merck, Darmstadt, Germany),
or RP-18 plates (RP-18 F254S; Merck) using DCM/MeOH 98/
2% (solvent mixture A) or H2O/CH3CN/TFA 40/60/0.1% (sol-
vent mixture B). Melting points were determined with a
Reichert-Jung-Koffler apparatus. Infrared spectra were
recorded in KBr pellets or in CCl4 on an FT Vector 22
instrument (ν expressed in cm-1; Bruker, Bremen, Germany,
developed in the Supporting Information). Proton and carbon
magnetic resonance spectra (1H and 13C NMR) were performed
in CDCl3 or DMSO-d6 on a Bruker AM 200 (4.7 T) or a Bruker
DRX 500 (11.7 T) spectrometer (developed in the Supporting
Information). Chemical shifts (δ) are reported in parts per
million relative to the internal standard (CH3)3Si or relative
Conclusion
1
In this study, we report the synthesis and pharma-
cological characterization of six leucine peptides linked
to the benzamide structure. It appears that the intro-
duction of the BZA-CO cluster to the peptidyl Weinreb
amide (route B) gives a better yield than the formation
of the Weinreb amide at the last step (route A).
Biological studies demonstrated that the compounds
with one or two leucine residues (13 and 16) were
to solvent signals (CDCl3, δ ) 7.26 ppm for H NMR and δ )
77.0 ppm for 13C NMR, or DMSO-d6, δ ) 2.49 ppm for 1H NMR
and δ ) 39.0 ppm for 13C NMR). Electrospray ionization mass
spectra (ESI-MS) were obtained on a ESQUIRE-LC spectrom-
eter in positive or negative mode (solvent: CH3CN or CH3-
CN/H2O 1:1; Bruker). Main fragmentations of [M + H]+ ions
derived from the electrospray of synthesized derivatives have
been determined (developed in the Supporting Information).
The amino acids required for the preparation of inhibitors 11,

6736 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Vivier et al.
Figure 4. Ubiquitin-proteasome protein degradation. The process starts with the ATP-dependent activation of ubiquitin (Ub-
COOH) by an ubiquitin-activating enzyme (E1), followed by transfer to an ubiquitin-conjugating enzyme (E2) and, finally, attachment
of the ubiquitin to the protein substrate with or without the help of the ubiquitin-protein ligases (E3). The polyubiquitinated
substrate is recognized and subsequently degraded by the 26S proteasome. The ubiquitin monomers are reclaimed by the action
of deubiquinylating enzymes (DUBs). These last enzymes are responsible for the recycling of the ubiquitin back into the pool of
free ubiquitin in the cell.
at 0-5 °C for 2 h and was transferred to a solution of
dimethylterephthalate (53.02 g, 273 mmol) in 600 mL of
dichloromethane. The reaction mixture was refluxed for 77 h,
and then, the reaction was quenched with water (40 mL). The
white precipitate obtained was filtrated and extracted with
CH2Cl2 (4 mL × 100 mL). The combined organic layers were
washed with brine, dried over MgSO4, and concentrated in part
under vacuum. After addition of methanol, the precipitate of
excess of dimethylterephthalate was eliminated by filtration.
The filtrate was evaporated under vacuum. Purification was
achieved by flash chromatography (aluminum oxide, CH2Cl2/
MeOH 99.5/0.5) to afford 17.7 g of 5 (yield: 70%) and 0.69 g of
6 (yield: 2%). 5: white solid; mp 62-64 °C; TLC Rf 0.4
(aluminum oxide, CH2Cl2/MeOH 98/2); Anal. (C15H22N2O3, 0.3
H2O) C, H, N. 6: white solid; mp 138-140 °C; TLC Rf 0.2
(aluminum oxide, solvent mixture A).
General Method B. Lithium 4-(((Diethylamino)ethyl)-
carbamoyl)benzoate (7). To a solution of lithium hydroxide
monohydrate (3.91 g, 93.3 mmol, 1.5 equiv) in water (100 mL)
was added dropwise a solution of compound 5 (17.3 g, 62.2
mmol) in THF (150 mL) at 0 °C. The mixture was allowed to
reach room temperature and then stirred for 1 h. The reaction
mixture was evaporated under reduced pressure, and the crude
product was washed with acetone and diethyl ether to afford
16.8 g of 7 (yield: 100%): white solid; mp > 200 °C; TLC Rf
0.7 (RP-18, solvent mixture B); Anal. (C14H19LiN2O3, 0.75 H2O,
1.0 LiOH) C, H, N.
Figure 5. Accumulation of polyubiquitinated proteins in
IPC227F cells. IPC227F cells were treated for different time
intervals with 10 µM MG132 or compound (Cpd) 19, 22, 25,
or 28. Accumulation of ubiquitin-tagged proteins was evalu-
ated by Western blotting.
General Method C: Methyl 2-(4-(((Diethylamino)eth-
yl)carbamoyl)benzamido)-4-methylpentanoate (9). To a
solution of acid salt 7 (1.80 g, 6.69 mmol) in CH2Cl2 at 0 °C
were added successively hydroxybenzotriazole (HOBt, 0.16 g,
1.16 mmol, 0.3 equiv), dicyclocarbodiimide (DCC, 1.24 g, 5.99
mmol, 1.15 equiv) and triethylamine (1.60 mL, 16.7 mmol, 2.5
equiv). The mixture was stirred at 0 °C for 1 h. A solution of
amine 8 (1.04 g, 5.76 mmol, 1 equiv) in CH2Cl2 was added
dropwise at 0 °C, and the reaction mixture was then stirred
overnight at room temperature. A white precipitate of dicy-
clohexylurea (DCU) was eliminated by filtration, and the
filtrate was washed with a saturated sodium bicarbonate
aqueous solution (100 mL) and brine (100 mL), dried over
MgSO4, and concentrated under vacuum. The pure compound
(9) was obtained by recrystallization in diethyl ether (yield:
51%): white solid; mp 162-164 °C.
14, 17, 20, 23, and 26 were synthesized by standard peptide
chemistry methods and/or literature synthesis.
General Method A: 4-((Diethylamino)ethyl)carbam-
oylbenzoic Acid (4). To a solution of 1 (2.01 g, 5.81 mmol)
in a THF/Et2O mixture (5 mL/200 mL) at -70 °C was added
a solution of n-butyllithium 2.5 M/THF (7 mL, 17.5 mmol, 3
equiv) under nitrogen atmosphere. In another ball of the
vacuum system, a solution of H2SO4 (50 mL) was added to
barium carbonate (1.27 g, 6.39 mmol, 1.1 equiv). CO2 gas
formed and was transferred on the reaction mixture at 20 Pa.
The mixture was allowed to reach room temperature and then
was stirred overnight. The reaction was quenched by water.
The aqueous layer (pH ) 2) was washed with CHCl3 (3 mL ×
50 mL) and then concentrated under reduced pressure to afford
0.3 g of 4 in the form of a hydrochloride salt (yield: 21%): beige
solid; mp 186-188 °C; TLC Rf 0.7 (RP-18, solvent mixture B).
Methyl 4-(((Diethylamino)ethyl)carbamoyl)benzoate
(5). To a solution of 2-diethylaminoethylamine (10.56 g, 91.0
mmol) in 60 mL of CH2Cl2 was added dropwise a solution of
trimethylaluminum 2 M in hexane (50 mL, 100 mmol) at 0 °C
under a nitrogen atmosphere. The reaction mixture was stirred
Lithium 2-(4-(((Diethylamino)ethyl)carbamoyl)benz-
amido)-4-methylpentanoate (10). Compound 10 was pre-
pared according to method B. Compound 9 (1.63 g, 4.18 mmol)
gives after purification 1.2 g of compound 10 (yield: 89%):
white solid; mp 179-181 °C; TLC Rf 0.7 (RP-18, solvent
mixture B); Anal. (C20H30LiN3O4, 1.5 H2O, 0.5 LiOH) C, H, N.

Preliminary Studies of New Proteasome Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6737
Figure 7. Cytotoxicity assay in normal melanocytes. Normal
melanocytes cells (MshL1) were incubated with increasing
concentrations of (A) compound 19 or (B) MG132 for 48 h,
washed with 1X PBS, and then frozen at -80 °C. Cell survival
data were determined using fluorescent Hoechst dye 33342.
The data are expressed as mean percentages ( SD of the
untreated control (n ) 3).
Figure 6. Proteasome inhibition assays. Proteasomes were
incubated for 30 min at room temperature in the absence
(control, dark gray bars) or presence of 0.1 µM (black bars), 1
µM (light gray bars), and 10 µM (white bars) 19, 25, 28,
MG132, or epoxomicin. Assays were then incubated 1 more
hour at 37 °C with (A) 100 µM Suc-LLVY-AMC (ChT-L
activity), (B) 100 µM Boc-LRR-AMC (T-L activity), or (C) 200
µM Z-LLE-AMC (PGPH activity). Peptidic activities were
determined by measuring the liberated fluorophore AMC using
a fluorescence microplate reader at 360/460 nm. Values are
means ( SD (n ) 3).
Figure 8. Flow cytometric analysis of human melanoma cells
and normal melanocytes. Distribution in a cell cycle of (A)
normal melanocytes (MshL1) or (B) M4Beu cells (human
melanoma cells) incubated with 10 mM compound 19 or
MG132 for 24 h was performed using an Epics XL (Coulter,
Hialeah, FL) after propidium iodide labeling of cells and
normalized by the ratio of compound 19/control (light gray
bars) or MG132/control (white bars).
N1-((Diethylamino)ethyl)-N4-(1-(methoxy(methyl)ami-
no)-4-methyl-1-oxopentan-2-yl)terephthalamide (12). The
synthesis of 12 was realized according to method C from
compound 7 (1.0 g, 3.7 mmol) and compound 11 (1.1 g, 3.7
mmol). The crude product (2.4 g) was purified by flash
chromatography (aluminum oxide, CH2Cl2/gradient of MeOH
0.5 up to 2%) to afford 0.45 g of 12 (yield: 12%): white solid;
mp 120-122 °C; TLC Rf 0.3 (aluminum oxide, solvent mixture
A); Anal. (C22H36N4O4, 1.2 H2O) C, H, N.
lithium aluminum hydride was eliminated by the Mihailovic
method. The resulting crude reaction mixture was extracted
with CH2Cl2 (3 mL × 20 mL). The combined organic layers
were washed with brine (10 mL), dried over MgSO4, and
concentrated under vacuum to afford 87 mg of compound 13
(yield: 100%). The corresponding hydrochloride was obtained
by stirring 13 in ether/HCl 2 N as a very hygroscopic salt. (13,
HCl): white solid; mp 112-114 °C; TLC Rf 0.4 (aluminum
oxide, CH2Cl2/MeOH 95/5).
General Method D: Synthesis of N1-((Diethylamino)-
ethyl)-N4-(4-methyl-1-oxopentan-2-yl)terephthalamide
(13). A solution of lithium aluminum hydride in ether (0.8 mL,
0.8 mmol, 3 equiv) was added dropwise at -80 °C under a
nitrogen atmosphere to a solution of compound 12 (101 mg,
0.24 mmol) in anhydrous THF (4 mL). The mixture was stirred
for 90 min and then was allowed to warm to 0 °C. The reaction
was quenched by the addition of water (20 mL), and excess
N1-((Diethylamino)ethyl)-N4-(1-(1-(methoxy(methyl)-
amino)-4-methyl-1-oxopentan-2-ylamino)-4-methyl-1-ox-
opentane-2-yl)terephthalamide (15). To a solution of com-
pound 7 (0.77 g, 2.84 mmol) in DMF (10 mL) were added
benzotriazoyloxytris[diethylamino]phosphonium hexafluoro-
phosphate (BOP) (1.38 g, 3.12 mmol) and triethylamine (0.4

6738 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Vivier et al.
mL). The mixture was stirred at room temperature for 15 min,
and then, a solution of 14 (1.13 g, 2.83 mmol) in DMF (10 mL)
was added. The mixture reaction was stirred overnight at room
temperature and evaporated under vacuum. The resulting
crude reaction (1.12 g) was purified by flash chromatography
(aluminum oxide, CH2Cl2/MeOH 0 up to 2%). The pure
compound 15 was isolated by recrystallization from ether
(yield: 41%): beige solid; mp 57-59 °C; TLC Rf 0.6 (aluminum
oxide, CH2Cl2/MeOH 96:4); Anal. (C28H48ClN5O5, 1.5 H2O) C,
H, N.
N1-((Diethylamino)ethyl)-N4-((4-methyl-1-oxopentan-
2-ylamino)-1-oxopentan-2-yl)terephthalamide (16). The
Weinreb amide 15 (100 mg, 0.19 mmol) reduction was achieved
according to method D to afford 50 mg of 16 (yield: 58%). The
corresponding hydrochloride was obtained by stirring 16 in
ether/HCl 2 N as a very hygroscopic salt: beige solid; mp 72-
74 °C; TLC Rf 0.5 (RP-18, solvent mixture B).
N1-((Diethylamino)ethyl)-N4-(1-(1-(methoxy(methyl)-
amino)-4-methyl-1-oxopentan-2-ylamino)-4-methyl-1-ox-
opentan-2-yl)teraphthalamide (18). The Weinreb amide 18
was synthesized according to method C with compounds 7
(1.09 g, 4.05 mmol) and 17 (1.89 g, 3.68 mmol) in DMF (30
mL). The crude product (2.13 g) was purified by flash chro-
matography (aluminum oxide, CH2Cl2/gradient of MeOH 0.5
to 2%) to afford 1.78 g of 18 (yield: 75%): white solid; mp 96-
98 °C; TLC Rf 0.4 (aluminum oxide, CH2Cl2/MeOH 95/5).
N1-((Diethylamino)ethyl)-N4-(4-methyl-1-(4-methyl-1-
(4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-ylami-
no)-1-oxopentan-2-yl)terephthalamide (19). The reduction
of Weinreb amide 18 (0.96 g, 1.5 mmol) was achieved following
method D. The pure product was isolated by recrystallization
from diethyl ether to afford 0.63 g of 19 (yield: 72%). The
hydrochloride salt was precipitated with a solution of ether/
HCl 2 N. 19: white solid; mp 118-120 °C; TLC Rf 0.6
(aluminum oxide, CH2Cl2/MeOH 95:5); Anal. (C34H58ClN5O5,
2.5 H2O) C, H.
N1-((Diethylamino)ethyl)-N4-(1-(1-(methoxy(methyl)-
amino)-4-methyl-1-oxopentan-2-ylamino)-1-oxo-3-
phenylpropan-2-yl)terephthalamide (21). The synthesis of
Weinreb amide 21 was realized according to method C with a
solution of compound 7 (0.69 g, 2.53 mmol) and a solution of
20 (1.0 g, 2.3 mmol) in a mixture of CH2Cl2 (20 mL)/DMF (10
mL). The crude product was purified by flash chromatography
(aluminum oxide, CH2Cl2/MeOH 0.5 up to 2%) to afford 0.3 g
of 21 (yield: 23%): white solid; mp 101-103 °C; TLC Rf 0.4
(aluminum oxide, CH2Cl2/MeOH 95/5); Anal. (C31H45N5O5, 0.5
H2O) C, H.
N1-((Diethylamino)ethyl)-N4-(4-methyl-1-oxo-1-(1-oxo-
3-phenylpropan-2-ylamino)pentan-2-yl)terephthala-
mide (22). Reduction of Weinreb amide 21 (0.19 g, 0.33 mmol)
was achieved according to method D to afford 0.12 g of 22
(71%). The hydrochloride salt was precipitated with a solution
of ether/HCl 2 N. 22: white solid; mp 116-118 °C; TLC Rf 0.5
(aluminum oxide, CH2Cl2/MeOH 95:5); Anal. (C29H41ClN4O4,
4.5 H2O) C, H, N.
N1-((Diethylamino)ethyl)-N4-(1-(1-(1-(methoxy(methyl)-
amino)-4-methyl-1-oxopentan-2-ylamino)-1-oxo-3-
phenylpropan-2-ylamino)-4-methyl-1-oxopentan-2-yl)-
terephthalamide (24). The synthesis of Weinreb amide 24
was realized according to method C with a mixture of
compound 7 (1.5 g, 2.53 mmol) and compound 23 (2.65 g, 4.83
mmol) in CH2Cl2 (10 mL)/DMF (10 mL). The crude product
was purified by flash chromatography (aluminum oxide, CH2-
Cl2/MeOH 0.5 up to 2%) to afford 2.07 g of 24 as a hydrochlo-
ride salt (yield: 63%): white solid; mp 92-94 °C; TLC Rf 0.5
(aluminum oxide, CH2Cl2/MeOH 95/5); Anal. (C37H56N6O6, 1.0
H2O) C, H, N.
N1-((Diethylamino)ethyl)-N4-(1-(1-(4-methyl-1-oxopen-
tan-2-ylamino)-1-oxo-3-phenylpropan-2-ylamino)-1-oxo-
pentan-2-yl)terephthalamide (25). The reduction of Wein-
reb amide 24 (0.45 g, 0.66 mmol) was achieved according to
method D to afford 0.26 g of 25 (64%). The hydrochloride salt
was precipitated with a solution of ether/HCl 2 N: white solid;
mp 127-129 °C; TLC Rf 0.5 (aluminum oxide, CH2Cl2/MeOH
95/5); Anal. (C35H52ClN5O5, 2.5 H2O) C, H, N.
N1-(2-(Diethylamino)ethyl)-N4-(1-(1-(1-(methoxy-
(methyl)amino)-4-methyl-1-oxopentan-2-ylamino)-4-meth-
yl-1-oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)-
terephthalamide (27). Weinreb amide 27 was synthesized
according to method C from a solution of compound 7 (1.2 g,
4.2 mmol) and a solution of 26 (2.07 g, 3.77 mmol) in a mixture
of CH2Cl2 (30 mL)/DMF (10 mL). The crude product was
purified by flash chromatography (aluminum oxide, CH2Cl2/
MeOH 0 up to 2%) to afford 1.42 g of 27 (yield: 55%). 27,
HCl: white solid; mp 107-109 °C; TLC Rf 0.5 (aluminum
oxide, CH2Cl2/MeOH 95/5); Anal. (C37H57ClN6O6, 2.5 H2O, 3.5
HCl) C, H, N.
N1-(2-(Diethylamino)ethyl)-N4-(1-(1-(4-methyl-1-oxo-
pentan-2-ylamino)-4-methyl-1-oxopentan-2-ylamino)-1-
oxo-3-phenylpropan-2-yl)terephthalamide (28). The re-
duction of Weinreb amide 27 (1.80 g, 2.64 mmol) was achieved
according to method D to afford 0.72 g of 28 (41%). The
hydrochloride salt was precipitated with a solution of ether/
HCl 2 N. 28: white solid; mp 128-130 °C; TLC Rf 0.6 (RP-18,
solvent mixture B); Anal. (C35H52ClN5O5, 3.5 H2O) C, H, N.
Cytotoxicity Assay. Attached human ocular melanoma
IPC227F cells were seeded in 96 well plates and incubated
for 24 h at 37 °C in a humidified atmosphere under 5% CO2
with a standard medium (DMEM and 10% calf fetal serum).
After the addition of fresh medium containing increasing
concentrations of drugs previously prepared in DMSO (final
concentration 0.025%), cells were incubated for 48 h for the
determination of IC50, washed with 1X PBS buffer, and then
frozen at -80 °C. After thawing at room temperature, cells
were incubated for 1 h at room temperature with a 0.01% SDS
solution, frozen again at -80 °C, and then thawed again at
room temperature. The Hoechst dye 33342 solution was added
to each sample (final concentration 15 µg/mL), and plates were
incubated for 1 h at room temperature on a plate shaker in
the dark. Fluorescence was measured using a fluorescence
microplate reader at 360/460 nm (Fluoroskan Ascent FL;
Labsystems, Farnborough, Hampshire), and cell survival rates
(percent cell survival relative to untreated control) were
calculated. The cytotoxic activity of drugs was expressed as
the concentration inhibiting cell growth by 50% (IC50) calcu-
lated from the survival curves.
Western Blot. IPC227F cells cultured in 10 cm diameter
dishes were treated with 10 µM cytotoxic drugs prepared in
DMSO and diluted in fresh culture medium (final concentra-
tion of DMSO 0.025%) for various incubation times. Control
cells were cultured with a standard medium and DMSO
0.025%. Cells were then scraped off and centrifuged for 8 min
at 440g and 4 °C. The cell pellets were lysed with 100 µL of
lysis buffer (50 mM Tris (pH ) 7.5), 120 mM NaCl, 0.5% NP40,
and 1X protease inhibitors) for 15 min on ice and sonicated
for three pulses of 15 s, at 50% output power, using a Microsom
XL ultrasonic cell disrupter from Heat Systems. Lysates were
then centrifuged for 10 min at 10000g and 4 °C to remove
cellular debris, and the supernatant protein concentration was
determined according to Bradford62 with bovine serum albumin
as the standard. Equal amounts of total cell lysates (100 µg)
were resolved on 7.5% SDS-PAGE and transferred onto
nitrocellulose membranes. The membranes were incubated for
2 h in a blocking solution (25 mM Tris (pH ) 8), 125 mM NaCl,
0.1% Tween-20, 4% nonfat dried milk) and then reacted
overnight at room temperature with a primary monoclonal
antibody raised against ubiquitin at a 1/1000 dilution (FK2;
Affiniti Research Products Limited, U.K.). Membranes were
then incubated for 1 h with a mouse peroxidase-conjugated
secondary antibody (1/5000). Ubiquitin-tagged proteins were
detected by the enhanced chemiluminescence (ECL) detection
system (Amersham Biosciences, Burckinghamshire, England).
20S Proteasome Inhibition Assays. The chymotrypsin-
like, trypsin-like, and peptidyl glutamyl peptide hydrolase
activities of 20S proteasomes were measured as follows. 0.5-1
µg of 20S mammalian proteasomes (Affiniti) were incubated
for 30 min at room temperature in the presence of 0.1, 1, and

Preliminary Studies of New Proteasome Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6739
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Acknowledgment. We thank Dr. M. Borel for the
proton and carbon magnetic resonance spectra per-
formed on the Bruker DRX 500. We also thank M. Bayle
for the synthesis of several intermediates. We are
grateful for many helpful discussions throughout the
course of this work with Dr. J. Helfenbein and Dr. M.-
F. Moreau.
Supporting Information Available: Main fragmenta-
tions of [M + H]+ ions derived from electrospray of Weinreb
amides 12, 15, 18, 21, 24, and 27 and spectroscopic data. This
information is available free of charge via the Internet at http://
pubs.acs.org.
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