Synthesis, radiosynthesis, and biological evaluation of new proteasome inhibitors in a tumor targeting approach

Article abstract of DOI:10.1021/jm701419g

Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant melanoma cells. Of these, 10 displayed the highe

Full text of DOI:10.1021/jm701419g

J. Med. Chem. 2008, 51, 1043–1047
1043
Brief Articles
Synthesis, Radiosynthesis, and Biological Evaluation of New Proteasome Inhibitors in a Tumor
Targeting Approach
Magali Vivier,*,† Maryse Rapp,† Janine Papon,† Pierre Labarre,† Marie-Josephe Galmier,† Jacques Sauzière,§ and
Jean-Claude Madelmont*,†
INSERM, UMR 484, Clermont-Ferrand, F-63005, France, Laboratoires de Chimie Minérale et Analytique, UniVersité Clermont 1,
Clermont-Ferrand, F-63001, France, Centre Jean-Perrin, F-63011, France, and OTL Pharma, 15 Rue de Turbigo, Paris, F-75002, France
ReceiVed NoVember 10, 2007
Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde
inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant
melanoma cells. Of these, 10 displayed the highest cytotoxicity (0.18 ( 0.16 µM). A radiosynthesis of the
iodine aldehyde was performed. Its drug biodistribution showed that some selectivity of the benzamide
group toward malignant melanoma tissue was conserved.
Introduction
limit the use of the proteasome inhibitors in therapy. One way
to overcome this limitation is to carry these drugs selectively
to the target tumoral tissue. A new class of radiopharmaceuticals
targeting melanic tissue has been developed by us: the N-(2-
diethylaminoethyl)benzamide derived class, e.g., BZA (Figure
1). Two members have been successfully used in humans in a
phase II clinical trial as radiopharmaceuticals in the diagnosis
of disseminated melanoma.6 We synthesized proteasome inhibi-
tors vectorized with this benzamide group to target melanic
tumoral tissue. Preliminary results showed that the tripeptide
aldehyde tested (BZA-CO-LLL-H, Figure 1) presented the
highest cytotoxicity toward human ocular melanoma IPC227F
cells of the first peptide aldehyde inhibitors of the proteasome
linked to the N-(2-diethylaminoethyl)benzamide structure (BZA-
CO, Figure 1).7 To confirm the targeting of tumoral melanic
tissue, we then synthesized the iodine leucinal derivative.
Here, we report the synthesis, radiosynthesis, and pharma-
cological characterization of the iodine peptide aldehyde linked
to the benzamide derivative structure (Figure 1). The effects
on cytotoxicity of introducing an iodine atom and substituents
on the aromatic ring into the peptide aldehyde structure are also
discussed. We also report on the pharmacokinetic study of 125I
in C57Bl/6 J mice bearing the murine B16 melanoma to
determine whether the selectivity of the benzamide group toward
malignant melanoma tissue was preserved.
The 26S proteasome is a multicatalytic protein complex that
forms the central enzyme in intracellular protein degradation.
It consists of a 20S proteolytic component capped with a
regulatory 19S complex at each end, the role of which is to
unfold the protein-substrates and stimulate proteolytic activity.
This complex exhibits at least five endopeptidase activities: post
glutamyl peptide hydrolyzing (PGPHa) activity, trypsin-like (T-
L) activity, chymotrypsin-like (ChT-L) activity, and two minor
peptidase activities that cleave peptide bonds after branched-
chain amino acid preferring (BrAAP) or small neutral amino
acid preferring (SNAAP) activities.1 These proteolytic activities
lend proteasomes a recycler function for damaged or misfolded
proteins in the cell and a critical role in events in the regulation
of the cell cycle.2 Proteasomes thus offer a new target for
anticancer drugs.3 One proteasome inhibitor, the dipeptidylbo-
ronic acid bortezomib (Velcade, Figure 1),4 is commercialized
for its activity against several hematologic malignancies (par-
ticularly multiple myeloma) and solid tumors. Many other
synthetic inhibitors of the proteasome have been developed.3
These synthetic inhibitors possess a peptide structure necessary
for specific recognition by the proteasome site and an inhibitory
function that interacts with the N-terminal threonine (Thr 1) of
one ꢀ-subunit.5 According to Kisselev et al.,3 peptide aldehyde
inhibitors are an essential family for studies in cell cultures and
tissues. The peptide aldehyde inhibitor MG132 (Figure 1) has
been studied mainly for its significant cytotoxic activity.3 The
peptide aldehyde inhibitor family offers the advantage of
dissociating from the proteasome, conferring rapid reversibility
of action. However, the ubiquitous properties of the proteasome
Results and Discussion
Synthesis of Peptide Inhibitors. The method studied to
synthesize the vector structure precursors 5 and 6 used common
intermediate 3 (Scheme 1). This intermediate, used to introduce
an iodine atom into the structure, was obtained by the Wallach
reaction. Using other methods for introducing an iodine atom
(R ) NH2, NO2, Br, SnBu3), we obtained compounds with two
position isomeric structures when we introduced an N,N-
diethylethylenediamine chain. With the Wallach reaction, we
developed a selective synthesis of precursors 5 and 6 using the
steric hindrance of the triazene group. First, triazene 2 was
prepared by diazotization of dimethyl aminoterephthalate 1 using
sodium nitrite in hydrochloric acid solution. This was followed
* To whom correspondence should be addressed. For M.V.: phone, (33)
4 73 15 08 00; fax, (33) 4 73 15 08 01; e-mail, mag.viv@free.fr. For
J.-C.M.: (33) 4 73 15 08 00; fax, (33) 4 73 15 08 01; e-mail, madelmont@
inserm484.u-clermont1.fr.
†
INSERM, UMR 484, Université Clermont 1, and Centre Jean-Perrin.
OTL Pharma.
§
a Abbreviations: PGPH, post glutamyl peptide hydrolyzing; T-L, trypsin-
like; ChT-L, chymotrypsin-like; BrAAP, branched-chain amino acid prefer-
ring; SNAAP, small neutral amino acid preferring; Cbz, carbobenzyloxy;
DCC, dicyclocarbodiimide; HOBt, hydroxybenzotriazole; tR, retention time;
DMEM, Dulbecco’s modified Eagle’s medium.
10.1021/jm701419g CCC: $40.75
2008 American Chemical Society
Published on Web 02/01/2008

1044 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Brief Articles
(route C) or 8.3% (route B). The pure peptide aldehyde 12 was
isolated as a white solid by flash chromatography and was tested
as its hydrochloride.
Synthesis of Labeled Compound [125I]12. Our first approach
to the carrier-free radioiodinated peptide aldehyde was by the
Wallach triazene reaction. This method involves using a
triazenyl precursor that undergoes nucleophilic radioiodine
substitution when decomposed by acid in the presence of the
radioiodine ion. We used the condition optimized with nonra-
diolabeled 8 to obtain iodine-125 labeled 8 (S4 in Supporting
Information, route A). The highest yield was obtained when
the reaction was carried out with acetonitrile in the presence of
trifluoroacetic acid and left at room temperature for 4 h. On the
basis of this information on the reaction conditions, radioiodi-
nation was undertaken using Na125I. The radiochemical yield
of 72% was detected by AMBIS after TLC and HPLC (tR )
32.4 min for 9 vs 27.2 min for [125I]8). The radiochemical purity
was greater than 99.5%. The reduction of the Weinreb amide
was performed with LiAlH4 at -10 °C for 1 min and quenched
with water. The lithium salts were eliminated by the method of
Mihailovich. The reaction yield was only 7%, and the separation
of [125I]8 and [125I]12 was very difficult, the difference between
the two retention times being only 1 min.
Figure 1. Pharmacomodulated proteasome inhibitors: coupling between
BZA and proteasome inhibitors.
Scheme 1. Synthesis of Vector a
Route B was also preferred to develop a radiosynthesis of
12 with a high iodine-125 specific activity. Because of the low
yield (5%) for the introduction of an iodine atom into peptide
aldehyde structure 10 with trifluoroacetic acid, conditions were
optimized to obtain radioiodine compound 12. The highest yield
for the incorporation of 125I (68%) was obtained with 30%
methanesulfonic acid (v/v) in acetonitrile for 3 h. The yield fell
to 5% when we used conditions with 5% methanesulfonic acid
(v/v). The hydrochloride salt was formed by addition of ether/
HCl solution. The mixture could be purified by semipreparative
HPLC (see Experimental Section) because the difference
between the two retention times was 5 min (tR ) 32.5 min for
10 vs 26 min for [125I]12). After purification the radiochemical
yield for [125I]12 by route B was 41% with a radiochemical
purity of g99.3% as determined by HPLC analysis.
a Reagents: (a) (i) NaNO2, H2O/HCl, (ii) pyrrolidine, 1 N KOH; (b)
NH2CH2CH2NEt2, Al(CH3)3, CH2Cl2, reflux; (c) KI, CH3CN, CF3COOH;
(d) LiOH, THF/H2O, room temp.
by immediate trapping with an excess of pyrrolidine in basic
solution (99% from 1, method A in Experimental Section). The
reaction of 2-diethylaminoethylamine with 2 in the presence of
trimethylaluminum gave the monosubstituted derivative 3 (67%
from 2), easily separated by crystallization in ethyl acetate (68%,
method B in Experimental Section). Decomposition of triazene
3 with potassium iodine in trifluoroacetic acid gave the iodine
compound with a yield of 12% (method C in Experimental
Section).8 Esters 3 and 4 were converted into their lithium salts
5 and 6 by saponification at room temperature with lithium
hydroxide (method D in Experimental Section) in quantitative
yield.
General synthetic pathways to obtain peptide structures are
presented in Scheme 2. The final leucine derivative 12 was
prepared by three different routes. Whenever possible, com-
mercially available protected amino acids were used. In the first
step, leucine-derivative-structured peptides 7 were prepared
using standard peptide synthesis procedures, with DCC and
HOBt as catalytic coupling agents9 and a deprotection method.
By route A, the Weinreb amide 7 was coupled to 6 to give 9 in
34% yield (method E in Experimental Section).The Weinreb
amide 9 was reduced with LiAlH4 to give aldehyde 10 (method
F in Experimental Section).10 The reaction must be quenched
before secondary compound 11 is formed. Aldehyde 10 could
be separated from 11 by flash chromatography (14% yield for
10). The final compound 12 was obtained by the Wallach
reaction from 10 (5% from 10; not effective from 11). For the
two other routes, 8 was also obtained by the Wallach reaction
from 9 in 77% yield (route B) or could be obtained by coupling
between 5 and the Weinreb amide 7 in 13% yield (route C).
The Weinreb amide 8 was then reduced by LiAlH4 to give
aldehyde 12 in 48% yield. In summary, the global synthesis
yield of 12 was 0.2% through 6 (route A) and 0.5% through 5
Biological Studies. The studies of benzamide compounds had
been conducted on mice carrying B16 melanoma. We therefore
tested the cytotoxicity of our compounds on this same line. We
also tested them on a human melanic cell line, IPC227F. Human
ocular melanoma IPC227F and murine melanoma B16 cell line
were maintained as monolayers in 75 cm2 culture flasks in
DMEM medium supplemented with 10% fetal calf serum. A
population of 5 × 103 cells were seeded in 96-well plates and
incubated for 24 h at 37 °C in a humidified atmosphere under
5% CO2. The cells were then exposed for 48 h to increasing
concentrations of peptide aldehydes 10, 11, 12, or MG132 and
BZA-CO-LLL-H as controls. The effect of these compounds
on cell outgrowth was evaluated by assay with Hoechst dye
33342. As in previous optimization, we used BZA-CO-LLL-
H7 as control.
First, we demonstrate that the introduction of an iodine atom
in the aromatic ring of the vector structure preserved the
cytotoxic activity of these compounds. As shown in Table 1,
the cytotoxicity of 12 was maintained (IC50 ) 1.79 ( 0.42 µM)
in B16 cells but decreased 5.6-fold in IPC227F cells (IC50
)
3.58 ( 1.9 µM) compared with the BZA-CO-LLL-H control
(IC50 ) 1.3 ( 0.1 µM in B16 cells vs 0.64 ( 0.07 µM in
IPC227F cells). We also investigated the cytotoxicity of inter-
mediates 10 and 11. The cytotoxicity of 10 was increased 3.5-
fold in IPC227F cells (IC50 ) 0.18 ( 0.16 µM) and 65-fold in
B16 cells (IC50 ) 0.02 ( 0.01µM) compared with the BZA-

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 1045
Scheme 2. General Methods for the Preparation of Iodine Leucinal Derivativesa
a Reagents. 7: TFA-Leu-Leu-Leu-NOMeMe. (a) DCC, HOBt, TEA, CH2Cl2/DMF, room temp; (b) AlLiH4, THF, -80 °C; (c) NaI, CH3CN, CH3SO3H;
(d) NaI, CH3CN, CF3COOH; (e) AlLiH4, THF, -80 °C.
Table 1. Concentration Inhibiting 50% of Cell Growth (IC50) in B16
and IPC227F Cellsa
Figure 2. Drug biodistribution of [125I]12.
biodistribution method developed in Supporting Information).
In the tumor, the level of radioactivity was only 2.7% of the
injected dose per unit mass of organs, whereas it was
9.5%/ID in the mice treated with BZA.6 Radioactivity was
distributed in many tissues, especially in the metabolizing and
eliminating organs, i.e., the lung, kidney, and liver (16.3%, 4.9%,
and 8.9%, respectively, as shown in Figure 2). However, the
lungs were also a frequent tissue site for melanoma metastasis,
and the high concentration of the compound [125I]12 in these
a Cbz ) PhCH2OCO-. BZA ) Et2NH(CH2)2NHCOC6H4-. IC50 values
organs could be explained by the presence of σ receptors in tissues
are determined from the survival curves. Each value is the mean ( SD
such as liver, kidney, and lung.11 Compound [125I]12 seems to be
from three independent experiments, each of which was performed in
more fully metabolized than BZA (5.95% in liver for BZA vs
triplicate. All the compounds were tested as hydrochloride salts.
16.3% for [125I]12). No radioactivity was measured in the brain;
[
[
125I]12 did not cross the blood-brain barrier. The elimination of
CO-LLL-H control. Contrary to 10, the reduction of the N-N
bond in the triazene group was correlated with a 3-fold decrease
in the cytotoxicity of BZA-CO-LLL-H in B16 and IPC227F
cells. This increase in toxicity of 10 may be explained by the
alkylating effect of the triazene group, found in dacarbazine,
added to the action of the proteasome inhibitor. The inhibition
of the proteasome associated with its alkylating action may
possibly lead to another type of compound. Generally, the
cytotoxicity of these compounds was higher on the B16 murine
cells than on human IPC227F cells.
125I]12 was mainly fecal, 87% of the radioactivity injected being
found in the feces (2/3) and in urine (1/3) in 72 h. The ratio between
B16 cells and blood was greater than or equal to 1 (see S1 in
Supporting Information). Some selectivity of the benzamide group
toward malignant melanoma tissue was also conserved; the affinity
might well be increased by synthesizing this compound with
another vector structure derived from BZA.
Conclusion
Second, to verify the conservation of BZA affinity for melanic
tissue, we chose the radioiodine compound [125I]12. The
pharmacokinetic study of [125I]12 was carried out in C57Bl/6J
mice bearing murine B16 melanoma. Postinjection points at 15
min, 30 min, 1 h, 3 h, and 6 h were chosen to determine the
distribution of the compound in various organs and tissues (drug
We report a selective synthesis and radiosynthesis of new
leucine iodine peptides linked to the benzamide structure. We
investigated different methods to obtain an iodine aldehyde (12).
The different iodine compounds were obtained by the Wallach
triazene reaction and the aldehyde by Weinreb amide reduction.
The radiosynthesis of [125I]12 gave the highest radiochemical

1046 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Brief Articles
(3.5 mL) at 0 °C. The mixture was allowed to reach room
temperature and then stirred for 1 h. The mixture was evaporated
under reduced pressure and the crude product was washed with
acetone and dried to give 842 mg of 5 (yield, 100%): white solid;
mp >200 ( 1 °C, TLC Rf ) 0.67 (RP18; H2O/CH3CN/TFA, 40:
60:0.1%).
yield by route B (41% with a radiochemical purity of g99.3%
as determined by HPLC analysis). Cytotoxic assay showed that
10 had the highest cytotoxicity (0.18 ( 0.16 µM) probably
because of the presence of the triazene group. The drug
biodistribution of [125I]12 was also studied in mice bearing a
subcutaneous implantation of malignant melanoma cells. These
experiments showed an accumulation of 2.7% 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/blood ratio greater than or equal to 1. Also, some
selectivity of the benzamide group toward malignant melanoma
tissue was conserved. The affinity of the compounds might be
increased by using another vector structure derived from BZA and
N4-(2-(Diethylamino)ethyl)-2-iodo-N1-(1-(1-(1-(methoxy(me-
thyl)amino)-4-methyl-1-oxopentan-2-ylamino)-4-methyl-1-oxo-
pentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)terephthala-
mide (8). The iodination of Weinreb amide 9 (1 equiv, 0.51 mmol,
380 mg) was achieved using method C.
The crude product was purified by flash chromatography
(aluminum oxide, CH2Cl2/gradient of MeOH from 0 up to 2%) to
give 290 mg of 11 (yield, 73%): beige solid; mp 120 ( 1 °C; TLC
Rf ) 0.4 (alumina, CH2Cl2/MeOH, 95:5). Anal. (C34H57IN6O6) C,
H, N.
might also offer a better selectivity for melanoma cells.6
A
derivative of 10 bearing a triazene group will be tested in in vitro
and in vivo experiments on tumor-bearing animals with radiola-
beled compounds to determine whether this new compound has a
greater selectivity for malignant melanoma and metastases.
N4-(2-(Diethylamino)ethyl)-2-iodo-N1-(4-methyl-1-(4-methyl-
1-(4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-ylamino)-1-
oxopentan-2-yl)terephthalamide (12). A solution of lithium
aluminum hydride in ether (1 M/THF, 1.1 equiv, 0.2 mmol) was
added dropwise at -90 °C under a nitrogen atmosphere to a solution
of 8 (1 equiv, 0.17 mmol, 130 mg) in anhydrous THF (5 mL). The
mixture was stirred for 2 h and 30 min and then allowed to warm
to 0 °C. The reaction was quenched by adding water (5 mL), and
excess lithium aluminum hydride was eliminated by the Mihailovic
method. The resulting crude mixture was extracted with CH2Cl2
(2 × 50 mL). The combined organic layers were dried over MgSO4,
concentrated under vacuum, and purified by flash chromatography
(aluminum oxide, CH2Cl2/gradient of MeOH from 2% to 3%) to
give 59.1 mg of 12 (yield, 48%): yellow solid; mp >200 °C; TLC
Rf ) 0.4 (alumina, CH2Cl2/MeOH, 95:5). Anal. (C32H52IN5O5 ·
HCl·1H2O) C, H, N.
Experimental Section
General procedures and synthesis of 6, 9-11, and [125I]8 are
described in Supporting Information.
General Method A: (E)-Dimethyl 2-(Pyrrolidin-1-yldiazenyl)-
terephthalate (2). To a solution of dimethyl aminoterephthalate
(1) (1 equiv, 61.8 mmol, 12.90 g) in water (100 mL) was added a
solution of 37% HCl to obtain a red suspension, and the mixture
was vigorously stirred for 30 min at 0 °C under a nitrogen
atmosphere. To the reaction mixture was added dropwise a solution
of sodium nitrite (1.3 equiv, 81.2 mmol, 5.56 g) in 20 mL of water
at 0 °C. The mixture was stirred for 45 min, and 8 mL of pyrrolidine
(1.5 equiv, 92.7 mmol) and 12 mL of 1 N potassium hydroxide at
0 °C were then added. The mixture was stirred for 3 h at room
temperature, made alkaline to pH 12 with NaOH, and extracted
with CH2Cl2 (3 × 100 mL). The organic layer was dried over
MgSO4 and concentrated in part under vacuum to obtain 17.79 g
of 2 (yield 99.0%): mp 83 ( 1 °C; TLC Rf ) 0.8 (alumina, CH2Cl2).
Anal (C14H17N3O4) C, H, N.
General Method B: (E)-Methyl 4-((2-(Diethylamino)ethyl)-
carbamoyl)-2-(pyrrolidin-1-yldiazenyl)benzoate (3). To a solution
of N,N-diethylethylenediamine (1 equiv, 1.72 mmol, 0.24 mL) in
20 mL of CH2Cl2 was added dropwise a solution of 2 M
trimethylaluminum in hexane (50 mL, 100 mmol) at 0 °C under a
nitrogen atmosphere. The mixture was stirred at 0–5 °C for 2 h
and was added to a solution of ester 2 (3 equiv, 5.1 mmol, 1.51 g)
in 60 mL of dichloromethane. The mixture was refluxed for 77 h
and then quenched with water (40 mL). The white precipitate
obtained was filtered and extracted with CH2Cl2 (4 × 100 mL).
The combined organic layers were washed with brine, dried over
MgSO4, and concentrated in part under vacuum. The pure 3 was
obtained by recrystallization in ethyl acetate (yield, 68%): beige
solid; mp 100 ( 1 °C; TLC Rf ) 0.4 (alumina, CH2Cl2). Anal.
(C19H29N5O3) C, H, N.
General Method C: Methyl 4-((2-(Diethylamino)ethyl)car-
bamoyl)-2-iodobenzoate (4). To a solution of 3 (1 equiv, 2.66
mmol, 1.00 g), potassium iodide (2 equiv, 5.33 mmol, 884 mg) in
acetonitrile (CH3CN, 1.5 mL) at -10 °C was added a solution of
trifluoroacetic acid (2 equiv, 5.33 mmol, 3.9 mL). The mixture was
stirred at room temperature under a nitrogen atmosphere. The crude
mixture in 10 mL of water was extracted with CH2Cl2 (3 × 15
mL). The combined organic layers were washed with brine (50
mL), dried over MgSO4, and concentrated under vacuum. The crude
product was purified by flash chromatography (aluminum oxide,
CH2Cl2/gradient of MeOH from 0 to 3%) to give 126.6 mg of 4
(yield, 12.0%): yellow solid; mp >200 ( 1 °C; TLC Rf ) 0.9
(alumina, CH2Cl2/MeOH, 95:5).
N4-(2-(Diethylamino)ethyl)-2-iodo-N1-(4-methyl-1-(4-methyl-
1-(4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-ylamino)-1-
oxopentan-2-yl)terephthalamide ([125I]12). To a solution of 10
(8 µmol, 5.31 mg) in acetonitrile (CH3CN, 50 µL) at -10 °C was
added 2.1 mCi of Na125I in 0.1 N NaOH, followed by a solution
of methanesulfonic acid (37 µL). The mixture was stirred at room
temperature and made alkaline with 400 µL of a saturated solution
of NaHCO3 to give [125I]12 (radiochemical yield, 68%). The crude
product was purified with HPLC to give 860 µCi of [125I]12 (yield,
41%): TLC Rf ) 0.4 (alumina, CH2Cl2/MeOH, 95:5); HPLC tR )
32.5 min.
Supporting Information Available: Experimental details of the
synthesis of the compounds described here, spectroscopic data,
elemental analysis results, details of all biological methods, ratio
of the injected dose of [125I]12 in B16 tumor and in blood, main
fragmentations of [M + H]+ ions from electrospray of aldehyde
12, and the synthesis scheme for radiolabeled [125I]12. This material
is available free of charge via the Internet at http://pubs.acs.org.
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