New aldehyde and vinylsulfone proteasome inhibitors for targeted melanoma therapy

Article abstract of DOI:10.1016/j.ejmech.2011.07.037

The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [ ¹²³I]-N-(2-diethylaminoethyl)benzamide ([¹²³I]BZA) or [¹²⁵I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([ ¹²⁵I]BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the highest cytotoxicity (IC₅₀, 0.71 and 0.64 μM respectively), which persisted in the presence of an MTL derived from N-(dialkylaminoalkylenyl)benzamide residue.

Full text of DOI:10.1016/j.ejmech.2011.07.037

European Journal of Medicinal Chemistry 46 (2011) 5705e5710
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
New aldehyde and vinylsulfone proteasome inhibitors for targeted
melanoma therapy
,
e
,
a
,
,
Magali Vivier a , Maryse Rapp e, Marie-Josephe Galmier a e, Anne-Sophie Jarrousse b,
*
Elisabeth Miot-Noirault a e, Fernand Leal a e, Valérie Weber a e, Jacques Métin c, Jacques Sauzière d,
,
,
,
Jean-Michel Chezal a e, Jean-Claude Madelmont a
,
,e
a Clermont Université, Université d’Auvergne, Imagerie moléculaire et thérapie vectorisée, BP 10448, F-63000, Clermont-Ferrand, France
b Clermont Université, Université d’Auvergne, UMR GReD, CNRS6247 e INSERM U931, 28 place Henri-Dunant, 63000 Clermont-Ferrand, France
c Laboratoire de Chimie Physique et Minérale, 28 place Henri-Dunant, 63000 Clermont-Ferrand, France
d OTL Pharma, 15 rue de Turbigo, Paris F-75002, France
e Inserm, UMR 990, F-63005 Clermont-Ferrand, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause
side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors
of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [123I]-N-
(2-diethylaminoethyl)benzamide ([123I]BZA) or [125I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([125I]
BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the
amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the
Received 13 May 2011
Received in revised form
19 July 2011
Accepted 20 July 2011
Available online 27 July 2011
highest cytotoxicity (IC50, 0.71 and 0.64
derived from N-(dialkylaminoalkylenyl)benzamide residue.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM respectively), which persisted in the presence of an MTL
Keywords:
Proteasome inhibitor
Melanoma
Aldehyde inhibitor
Vinylsulfone inhibitor
MG132
1. Introduction
A class of radiopharmaceuticals has been developed by Moreau
et al. [6] for scintigraphic imaging of melanoma. These radiotracers
e.g. [123I]-N-(2-diethylaminoethyl)benzamide ([123I]BZA, Fig. 1)
share the same N-(dialkylaminoalkylenyl)benzamide residue,
which binds specifically to melanin expressed in more than 92% of
melanoma lesions [8e11]. Also, a lengthening of the alkyl side
chain length of these radiotracers has been correlated with an
enhanced and/or long-lasting tumor uptake: melanotic tumor
The proteasome is a multicatalytic complex that has five
proteolytic activities (chymotrypsin-like, trypsin-like, post-glu-
tamyl peptide hydrolyzing, branched-chain preferring activity and
small neutral amino acids preferring activity) which gives it a crit-
ical role in several events such as inflammatory response, metabolic
pathway regulation..[1e3] Proteasome inhibition is thus a target
of interest in cancer therapeutic strategies. One proteasome
inhibitor, the dipeptidyl boronic acid bortezomib (VelcadeÒ, Fig. 1),
is commercialized for its activity against several hematologic
malignancies (particularly multiple myeloma) and solid tumors
[4,5]. However, the proteasome is ubiquitous and its inhibitors
cause side effects including fatigue, nausea, sensory neuropathy,
diarrhea and thrombocytopenia [6]. To overcome this limitation,
a strategy for targeting cancer tissue would be available [7] (Fig. 2).
retention of
[
125I]-N-(4-dipropylaminobutyl)-4-iodobenzamide
([125I]BZ18, Fig. 1), which has a longer alkyl chain than BZA, was
observed with values of 3.2 ꢀ 0.6% activity/g 72 h in B16 melanoma
after injection in melanoma-bearing mice (versus 0.8 ꢀ 0.3%
activity/g for BZA) [9].
In previous works, we used these melanin-targeting ligands
(MTLs) derived from radiotracer [123I]BZA to target toward mela-
notic tumor proteasome inhibitors, such as peptide aldehyde
inhibitors such as MG132 (Fig. 1), which represent a major
members of this drug class [12]. Aldehyde inhibitors have the
advantage of non-covalent binding with proteasome making its
action rapidly reversible. The substitution of the benzyloxycarboxyl
N-terminal protective group of MG132 by N-(2-diethylaminoethyl)
* Corresponding author. Clermont Université, Université d’Auvergne, Imagerie
moléculaire et thérapie vectorisée, BP 10448, F-63000, Clermont-Ferrand, France.
Tel.: þ33 4 73 15 08 00; fax: þ33 4 73 15 08 01.
E-mail addresses: magali.vivier@u-clermont1.fr, vivier_magali@yahoo.fr (M. Vivier).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.037

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M. Vivier et al. / European Journal of Medicinal Chemistry 46 (2011) 5705e5710
were synthesized according to Schemes 1 and 2. Alkylation of the
-bromobutyr-
dipropylamine with chloroacetonitrile (n ¼ 2) or
g
onitrile (n ¼ 4) followed by the nitrile reduction of compounds 4
and 5 with lithium aluminium hydride (LiAlH4) gave amines 6 and
7
with excellent overall yields (88% and 78%, respectively)
(Scheme 1).
Reaction of 6 and 7 with dimethyl terephthalate in the presence
of trimethylaluminium afforded benzamides 9 and 10, respectively
with the concomitant formation of the disubstituted derivatives
(not shown), which were easily separated by flash chromatography.
Saponification of methyl esters 9 and 10 with lithium hydroxide
gave compounds 11 and 12 in good yields (87% and 97% respec-
tively, Scheme 2).
The coupling reaction between the lithium carboxylates 11 or 12
and amine 13, which has the same peptidic sequence as MG132,
were undertaken in the presence of DCC and HOBt as coupling
reagents leading to the Weinreb amides 14 and 15 with moderate
yields (34% and 17%, respectively). The Weinreb amides thus
obtained were reduced at ꢁ80 ꢂC with LiAlH4 to give corresponding
aldehydes 16 and 17 (Scheme 3).
Secondly, three vinylsulfone proteasome inhibitors were
developed to enhance proteasome inhibition and cellular uptake.
Vinylsulfones are less reactive than aldehydes and C-terminal
leucine vinyl sulfone appears to be able to interact covalently with
the proteasome catalytic threonine increasing the residence time of
these compounds in cells compared with the aldehyde. A general
synthetic pathway to obtain vinylsulfone peptides is presented in
Scheme 4.
In the first step, commercially available diethyl methyl-
sulfanylmethylphosphate 18 was oxidized in the presence of acetic
acid and hydrogen peroxide to give sulfone 19 in acceptable yield
(59%). The vinylsulfones were obtained by reaction of 19 with the
previously formed aldehydes 20, 22 and 24 [13] in the presence of
NaH at ꢁ70 ꢂC and in anhydrous tetrahydrofuran with yields
ranging from 25% to 42%.
Fig. 1. Compounds discussed in the text.
benzamide residue as MTL does not affect cytotoxicity on human
ocular melanoma IPC227F cells. Unfortunately, the aldehyde
compounds tested are rapidly eliminated in vivo [13,14]. As an
example, the drug biodistribution of [125I]ICF08000 (Fig. 1) in
a subcutaneous melanoma-bearing mice showed a radioactive
accumulation of 2.7% of the injected activity/g in B16 tumors at
15 min, with 1.2% of radioactivity remaining in the tumor at 3 h and
6 h after administration, thus giving a tumor/blood ratio greater
than or equal to 1.
Based on these results and with the aim of enhancing both
tumor uptake and biological stability of these conjugates, structural
modifications of the inhibiting function, peptide composition and
melanin-targeting moieties were undertaken.
3. Pharmacology
The cytotoxicity of these compounds was tested versus MG132
control on human ocular melanoma IPC227F cells. IPC227F cells
were cultured for 24 h on a microplate and then exposed for 48 h to
increasing concentrations of the aldehydes 16, 17, 20, 22 or 24 or
vinylsulfone 21, 23 or 25 derivatives. The effect of these compounds
on cell growth (Table 1) was evaluated by assay with Hoechst dye
33342.
We also report on the synthesis and cytotoxicity assessment for
IPC227F cells of these new proteasome inhibitor-MTL conjugates.
4. Results and discussion
We elected to modify the length of the alkyl side chain of the
vector on trileucine aldehyde inhibitors (same peptidic sequence as
reference MG132) to take advantage (i) of the potential melanotic
tumor retention of these MTLs, and (ii) of the inhibitory function by
vinylsulfone. Peptides possessing a vinylsulfone moiety represent
another class of proteasome inhibitors: they covalently modify the
2. Chemistry
Firstly, we initiated two trileucine aldehyde inhibitors bearing
long alkyl side chain of the vector. The melanin-targeting moieties
catalytic
b-subunits of proteasome, but are less reactive than
aldehydes [12].
Fig. 2. Pharmacomodulated proteasome inhibitors.
Scheme 1. reagents: (i) KI, K2CO3, 80-85 ꢂC, CH3CN; (ii) LiAlH4, -78 ꢂC, ether.

M. Vivier et al. / European Journal of Medicinal Chemistry 46 (2011) 5705e5710
5707
Scheme 4. reagents: (i) H2O2, CH3COOH, reflux, CH2Cl2; (ii) NaH 60% in oil; (iii) R-CHO
(20 or 22 or 24), -70ꢂC, THF.
Scheme 2. reagents: (i) AlMe3, 0ꢂC, CH2Cl2; (ii) reflux; (iii) LiOH, rt, THF/H2O.
Table 1
Concentration inhibiting 50% of cell growth (CI50) in IPC227F cells for compounds
In biological assays, we observed that the cytotoxicity decreased
six-fold (IC50 of 4.18 vs 0.64 for compounds 17 and 22, respectively,
Table 1) when we increased the length of the alkyl chain (from
n ¼ 2 to n ¼ 4). We then evaluated the influence of the inhibitory
function on the cytotoxicity. The replacement of the aldehyde by
the vinylsulfone function resulted in a four- to 45-fold decrease in
the IC50 (Compounds 20 vs 21, 22 vs 23, 24 vs 25; Table 1).
16e25a.
Compounds
Cytotoxicity (IC50, mM)
MG132
22
0.077 (ꢀ0.009)
0.64 (ꢀ0.07)
Concomitantly, we evaluated the influence of the amino acid
chain length and nature of the aldehyde and vinylsulfone
compounds. A modification of the peptide chain with a bulkier and
more hydrophobic amino acid such as phenylalanine was per-
formed to increase cytotoxic activities. A hydrophobic substituent
in place of hydrophilic one at the P2 or P3 amino acid positions
could be beneficial for higher cellular activities [15,16]. However, in
biological assays, the presence of the phenylalanine appeared to
decrease the cytotoxicity of the aldehyde and the vinylsulfone
compounds (Compounds 22 vs 24 vs 25; Table 1).
16
0.71 (ꢀ0.34)
17
20
4.18 (ꢀ0.83)
5. Conclusion
In this study, we report the synthesis and pharmacological
characterization of aldehyde and vinylsulfone proteasome inhibi-
tors conjugated to an MTL derived from the benzamide radiotracer
[
123I]BZA or [125I]BZ18. We set out to find the best compromise
20.88 (ꢀ1.96)
between a modification of the inhibition function and a modifica-
tion of the vector moieties with the aim of improving the phar-
macokinetics of these compounds in vivo.
The substitution of the aldehyde function by a vinylsulfone
moiety decreased the cytotoxicity in vitro. Among the compounds
tested, compounds 16 and 22 presented the highest cytotoxicity
21
94.5 (ꢀ3.5)
(IC50, 0.71 and 0.64 mM, respectively). This cytotoxicity persisted in
the presence of an MTL derived from a N-(dialkylaminoalkylenyl)
benzamide residue. With the advantage of the potential melanotic
tumor retention of these MTLs, compound 16 is a good candidate
for further in vivo preclinical assessment on melanoma-bearing
animal models.
23
24
28.7 (ꢀ5.0)
11.92 (ꢀ1.20)
25
120.4 (ꢀ8.4)
a
Values are means of three experiments, standard deviation is given in
Scheme 3. reagents: (i) DCC / HOBt, NEt3, rt, CH2Cl2; (ii) LiAlH4, -80ꢂC, THF.
parentheses.

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M. Vivier et al. / European Journal of Medicinal Chemistry 46 (2011) 5705e5710
6. Experimental
product was purified by flash chromatography (Alumina,
dichloromethane/gradient of methanol from 1 up to 2%) to give
compound 10 as a beige solid (1.44 g, 3.25 mmol, 31%). mp :
60 ꢀ 1 ꢂC; Rf : 0.4 (Alumina, eluent mixture A); 1H NMR (CDCl3,
6.1. Materials and methods
All reagents and solvents were purchased from commercial
suppliers and were used with no further purification except for
tetrahydrofuran (THF), which was distilled over sodium-
benzophenone. Purity was checked by TLC on precoated silica gel
plates (plastic sheet 60 F254, layer thickness 0.25 mm, SDS, Pepin,
France), aluminium oxide plates (60 F254, neutral type E, layer
thickness 0.20 nm, Merk, Darmstadt, Germany) using as eluents:
mixture A: dichloromethane/methanol (95:5, v/v) or mixture B:
cyclohexane/ethyl acetate (95:5, v/v) and visualized with UV light
(254 nm) Melting points weredetermined on a Reichert-Jung Koffler
apparatus. Infrared spectrawere recorded in KBr pellets or in CCl4 on
200 MHz)
d
0.78 (t, 6H, J ¼ 8 Hz, H14), 1.37 (m, 4H, H13), 1.55 (quint,
2H, J ¼ 6 Hz, H9), 2.29 (t, 6H, J ¼ 6 Hz, H12), 2.38 (t, 2H, J ¼ 8 Hz,
H10), 2.41 (m, 2H, H8), 3.39 (q, 2H, J ¼ 8 Hz, H7), 3.87 (s, 3H, OCH3),
7.51 (brs,1H, H6), 7.78 (d, 2H, J ¼ 8 Hz, H3), 7.99 (d, 2H, J ¼ 8 Hz, H2);
13C NMR (CDCl3, 50.3 MHz)
d 11.9 (2C14), 19.4 (2C13), 24.7 (C9), 27.4
(C8), 40.0 (C7), 52.3 (C10), 53.4 (2C12), 55.9 (OCH3), 127.1 (2C3),
129.6 (2C2), 132.9 (C4), 138.9 (C1), 166.3, 166.8 (CO2, C5); IR (KBr)
3337.96, 2953.24, 1718.18, 1640.61, 1279.56, 733.11 cmꢁ1; MS: m/
z ¼ 335.3 [M þ H]þ; Anal. (C19H30N2O3, 0.2H2O) C, H, N.
6.2.3. Lithium 4-((2-(dipropylamino)ethyl)carbamoyl)benzoate
(11)
an FT Vector 22 instrument (n
expressed in cmꢁ1; Bruker, Bremen,
Germany). Proton and carbon nuclear magnetic resonance spectra
(1H and 13C NMR) were recorded in CDCl3 or DMSO-d6 on a Bruker
AM 200 (4.7 T), or Bruker DRX 500 (11.7 T) spectrometer. Chemical
To a solution of lithium hydroxide monohydrate (0.24 g,
5.79 mmol, 1.5 eq) in water (3.5 mL) was added at 0 ꢂC and drop-
wise a solution of 9 (1.18 g, 3.86 mmol) in tetrahydrofuran (3.5 mL).
The mixture was allowed to reach at room temperature and then
stirred for 1 h. The reaction mixture was evaporated under reduced
pressure and the crude precipitate was taken up with acetone
(7 mL) and filtered to give compound 11 as a yellow solid (1.00 g,
3.35 mmol, 87%). Dec : 200 ꢀ 1 ꢂC; 1H NMR (DMSO-d6, 200 MHz)
shifts (d) are reported in parts per million relative to the internal
standard (CH3)3Si or relative to solvent signals (CDCl3,
d
¼ 7.26 ppm
for 1H NMR and
for 1H NMR and
d
¼ 77.1 ppm for 13C NMR or DMSO-d6,
d
¼ 2.50 ppm
d
¼ 39.5 ppm for 13C NMR). Electrospray ionization
mass spectra (ESI-MS) were obtained on an ESQUIRE-LC spectrom-
eter in positive mode (solvent: CH3CN or CH3CN/H2O 1:1; Bruker,
Bremen, Germany). Main fragmentations of [M þ H]þ ions from
electrospray of synthesized derivatives were determined. Micro-
analyses were performed by the Analytical Laboratory of the CNRS
(Vernaison, France) for the elements indicated and were within 0.4%
of the theoritical value unless indicated. The peptidic precursor 13
was synthesized by standart peptide chemistry methods [17]. The
aldehyde inhibitors 20, 22 and 24 were obtained according to the
procedure described in a previous report [13].
d
0.81 (t, 6H, J ¼ 7 Hz, H12), 1.39 (m, 4H, H11), 2.35 (t, 4H, J ¼ 6 Hz,
H10), 2.50 (m, 2H, H8), 3.29 (m, 2H, H7), 7.70 (d, 2H, J ¼ 8 Hz, H2),
7.89 (d, 2H, J ¼ 8 Hz, H3), 8.39 (brs, 1H, H6); 13C NMR (DMSO-d6,
50.3 MHz)
d 11.3 (2C12), 19.6 (2C11), 37.0 (C7), 52.9 (C8), 55.4
(2C10), 125.6 (C3), 128.4 (C2), 134.3 (C1, C4), 165.7, 168.0 (C]O, C5);
IR (KBr) 3323.36, 2958.59, 1596.72, 1411.78, 744.10 cmꢁ1; MS: m/
z ¼ 293.15 [M þ H ꢁ Li]þ.
6.2.4. Lithium 4-((4-(dipropylamino)butyl)carbamoyl)benzoate
(12)
6.2. Chemical synthesis
Compound 12 was synthesized according to the procedure
described for 11 starting from 10 (2.07 g, 6.19 mmol, 1 eq) in THF
(20 mL). The crude product was taken up with acetone (15 mL),
filtered and then washed with diethyl ether (20 mL) to give
compound 12 (1.95 g, 6.0 mmol, 97%). dec : 200 ꢀ 1 ꢂC; 1H NMR
6.2.1. Methyl 4-((2-(dipropylamino)ethyl)carbamoyl)benzoate (9)
To a solution of 6 (2.05 g,14.2 mmol,1 eq) in dry dichloromethane
(50 mL) was added dropwise at 0 ꢂC under a nitrogen atmosphere
a solution of trimethylaluminium 2 M in hexane (7.8 mL,15.6 mmol).
The reaction mixture was stirredat 0e5 ꢂC for 2 h beforethe addition
of a solution of dimethyl terephthalate (8.28 g, 42.7 mmol, 3 eq) in
dry dichloromethane (60 mL). The reaction mixture was refluxed for
77 h, cooled down to room temperature and then quenched with
water (40 mL). The white precipitate obtained was filtered. The
filtrate was decanted and the aqueous layer was extracted with
dichloromethane (4 ꢃ 100 mL). The combined organic layers were
washed with brine (1 x 100 mL), dried over magnesium sulfate and
concentrated under vacuum. The crude product was purified by
flash chromatography (alumina, dichloromethane/gradient of
methanol from 0 up to 2%) to give compound 9 as an orange solid
(1.47 g, 4.82 mmol, 34%). mp : 53 ꢀ 1 ꢂC; Rf : 0.5 (Alumina, eluent
(CDCl3, 200 MHz)
d
0.78 (t, 6H, J ¼ 8 Hz, H14), 1.31 (m, 6H, H13, H9),
2.29 (m, 6H, H10, H12), 3.21 (m, 2H, H8), 3.54 (m, 2H, H7), 7.72 (d,
2H, J ¼ 8 Hz, H3), 7.90 (d, 2H, J ¼ 8 Hz, H2), 8.46 (t, 1H, J ¼ 4 Hz, H6);
13C NMR (CDCl3, 50.3 MHz)
d 11.4 (2C14),19.5 (2C13), 23.9 (C9), 26.7
(C8), 40.3 (C7), 52.9 (C10), 55.1 (2C12), 125.7 (2C3), 128.3 (2C2),
134.5 (C4), 142.2 (C1), 165.8, 168.2 (CO2, C5); IR (KBr) 3303.58,
2960.55, 1593.37, 1401.34, 741.42 cmꢁ1; MS: m/z ¼ 321.31 [M þ H]þ.
6.2.5. N1-((5S,8S,11S)-5,8-Diisobutyl-3,13-dimethyl-4,7,10-trioxo-
2-oxa-3,6,9-triazatetradecan-11-yl)-N4-(2-(dipropylamino)ethyl)
terephthalamide (14)
To a solution of acid salt 11 (1.00 g 3.35 mmol, 1.1 eq) in dry
dichloromethane at 0 ꢂC and under argon atmosphere were added
successively hydroxybenzotriazole (0.10 g, 0.90 mmol, 0.3 eq), N,N0-
dicyclohexylcarbodiimide (0.72 g, 3.49 mmol, 1.15 eq) and trie-
thylamine (1 mL, 7.6 mmol, 2.5 eq). The mixture was stirred at 0 ꢂC
for 1 h. A solution of compound 13 (1.47 g, 3.04 mmol, 1 eq) in dry
dichloromethane was added dropwise at 0 ꢂC and the reaction
mixture was allowed to reach at room temperature and then stirred
overnight. The white precipitate of dicyclohexylurea (DCU) was
eliminated by filtration and the filtrate was washed with saturated
aqueous sodium bicarbonate solution (100 mL) and brine (100 mL),
dried over magnesium sulfate, filtered and concentrated under
vacuum. The crude product was purified by flash chromatography
(silica gel, dichloromethane/gradient of methanol from 0 up to 4%)
to give 14 as a beige solid (319 mg 0.5 mmol, 16%). mp: 132 ꢀ 1 ꢂC;
mixture A); 1H NMR (CDCl3, 200 MHz)
d
0.80 (t, 6H, J ¼ 7.5 Hz, H12),
1.23 (sex, 4H, J ¼ 7.5 Hz, H11), 2.36 (t, 4H, J ¼ 7.5 Hz, H10), 2.58 (t, 2H,
J ¼ 6 Hz, H8), 3.41 (q, 2H, J ¼ 6 Hz, H7), 3.84(s, 3H, OCH3), 7.18 (brs,1H,
H6), 7.77 (d, 2H, J ¼ 8.5 Hz, H2), 8.00 (d, 2H, J ¼ 8.5 Hz, H3); 13C NMR
(CDCl3, 50.3 MHz) d 11.7 (2C12), 20.0 (2C11), 37.2 (C7), 52.1 (OCH3),
52.2 (C8), 55.6 (2C10),126.2 (2C3),129.6 (2C2),132.3 (C1),138.4 (C4),
166.1 (CO2), 166.1 (C5); IR (KBr) 3338.31, 2958.68, 2872.95, 1720.19,
1638.61, 1279.96, 735.37 cmꢁ1 MS: m/z ¼ 307.16 [M þ H]þ; Anal.
(C17H26N2O3, 0.1H2O) C, H, N.
6.2.2. Methyl 4-((2-(dipropylamino)butyl)carbamoyl)benzoate (10)
Compound 10 was synthesized according to the procedure
described for 9 starting from 7 (2.37 g, 13.7 mmol, 1 eq). The crude

M. Vivier et al. / European Journal of Medicinal Chemistry 46 (2011) 5705e5710
5709
Rf : 0.5 (alumina, eluent mixture A); 1H NMR (CDCl3, 200 MHz)
0.91 (m, 24H, H120, H ), 1.66 (m, 13H, H110, H
, H ), 3.03 (m, 4H,
(C
g
), 24.9 (C
g
), 33.9 (C70), 36.9 (C
b
), 37.3 (C
b
), 40.7 (C
b
), 51.9 (C2),
d
d
b
g
52.7 (C80, C5), 55.7 (2C100), 57.3 (C8), 127.2 (C30), 127.7 (C20), 134.3
(C10), 137.2 (C40), 157.1 (C]O), 166.5 (C]O), 166.9 (C]O), 172.4
(C]O), 199.9 (C1); IR (KBr) 3320.82, 2930.35, 1626.72,
1539.25 cmꢁ1; MS: m/z ¼ 616.43 [M þ H]þ; Anal. (C34H57N5O5,
2H2O, 2 HCl) C, H, N.
H100), 3.19 (s, 3H, NCH3), 3.31 (m, 2H, H80), 3.78 (s, 3H, OCH3), 3.89
(m, 2H, H70), 4.57 (m,1H, H8), 4.71 (q,1H, J ¼ 7.5 Hz, H5), 5.05 (q,1H,
J ¼ 6.5 Hz, H2), 6.75 (d, 1H, J ¼ 7.5 Hz, H9), 7.10 (brs, 1H, H3), 7.29
(brs, 1H, H6), 7.88 (d, 2H, J ¼ 8 Hz, H20), 8.12 (d, 2H, J ¼ 8 Hz, H30),
9.12 (brs, 1H, H60); 3C NMR (CDCl3, 50.3 MHz)
d
11.3 (2C120), 16.0
), 23.4 (C ), 23.5 (C ),
), 33.6 (NCH3), 35.0 (C70), 41.2 (C
), 41.3
b), 49.2 (C2), 53.1 (C8’or C5), 53.2 (C8’or C5), 53.6 (C8),
(2C110), 22.0 (C
25.0 (C ), 25.1 (C
(C ), 41.7 (C
d), 22.5 (C
d), 22.6 (C
d), 23.2 (C
d
d
d
6.2.8. N1-(4-(Dipropylamino)butyl)-N4-((S)-4-methyl-1-((S)-4-
methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-
ylamino)-1-oxopentan-2-yl)terephthalamide, hydrochloride salt
(17)
g
g), 25.3 (C
g
b
b
56.1 (2C100), 61.2 (OCH3),127.3 (2C30),127.6 (2C20),135.3 (C10),136.9
(C40), 166.9 (2C]O), 167.6 (C]O), 171.5 (C]O), 172.4 (C]O); IR
(KBr) 3285.48, 2956.42, 1636.96, 1540.79 cmꢁ1; MS: m/z ¼ 675.56
[M þ H]þ.
The reduction of compound 15 (1 eq, 0.54 mmol, 380 mg) was
realized according to the procedure developed for 16 to give
200 mg of compound 17 (0.31 mmol, 58%). The chlorohydrate of
compound 17 was obtained by addition of a solution of ether/HCl
2N as a white solid (211 mg; 0.31 mmol). dec : 200 ꢀ 1 ꢂC; 1H NMR
6.2.6. N1-((5S,8S,11S)-5,8-Diisobutyl-3,13-dimethyl-4,7,10-trioxo-
2-oxa-3,6,9-triazatetradecan-11-yl)-N4-(4-(dipropylamino)butyl)
terephthalamide (15)
(CDCl3. 200 MHz)
(m, 13H, H , H
d
0.93 (m, 24H, H13, H
d
), 1.24 (m, 4H, H130), 1.74
b
d
, H80, H100), 2.95 (m, 4H, H120), 3.48 (m, 2H, H90),
Compound 15 was obtained according to the procedure devel-
oped for 14 from compounds 4 (1.95 g, 6 mmol, 1.1 eq) and 13
(2.95 g, 5.74 mmol, 1 eq) in dry dichloromethane (25 mL)/dry
dimethylformamide (15 mL). The crude product was purified by
flash chromatography (alumina, dichloromethane/gradient of
methanol from 0 up to 4%) to give 15 as a beige solid (400 mg,
0.9 mmol, 17%). Rf : 0.6 (alumina, eluent mixture A); 1H NMR
4.17 (m, 1H, H2), 4.47 (m, 1H, H5), 4.64 (m, 2H, H70), 4.66 (m, 1H,
H8), 6.96 (brs, 1H, H9), 7.19 (brs, 1H, H3), 7.78e7.89 (m, 4H, H20,
H30), 7.82 (brs, 1H, H60), 8.10 (brs, 1H, H6), 9.38 (s, 1H, H1); 13C NMR
(CDCl3, 50.3 MHz)
3C
d
11.1 (2C140), 16.7 (2C130), 21.1e25.6 (C90, 6C
), 40.8 (C ), 41.1 (C ), 49.1 (C2),
d,
g
), 29.6 (C80), 33.9 (C70), 40.7 (C
b
b
b
52.7 (C5, C100), 54.0 (C8, 2C120), 127.4 (2C20, 2C30), 136.0 (C1’or C40),
138.5 (C1’or C40), 167.9 (C50 or C10), 168.0.0 (C50 or, C10), 173.0 (C4,
C7), 200.2 (C1); MS: m/z ¼ 646.55 [M þ H]þ; Anal. (C36H61N5O5,
0.5H2O, 0.5 HCl) C, H, N.
(CDCl3, 200 MHz)
d
0.79 (t, 6H, J ¼ 8 Hz, H140), 0.93 (m, 18H, H
d),
1.63 (m, 4H, H130), 1.71 (m, 9H, H ), 1.75 (m, 4H, H80, H100), 2.71
b, Hg
(m, 4H, H120), 2.79 (m, 2H, H90), 3.17 (s, 3H, NCH3), 3.53 (m, 2H, H70),
3.77 (s, 3H, OCH3), 4.58 (m, 1H, H8), 4.71 (m, 1H, H5), 5.02 (m, 1H,
H2), 6.95 (brs,1H, H9), 7.25 (d,1H, J ¼ 8 Hz, H3), 7.56 (d,1H, J ¼ 8 Hz,
H6), 7.82 (brs, 1H, H60), 7.90 (m, 4H, H20, H30); 13C NMR (CDCl3,
6.2.9. N1-(2-(Diethylamino)ethyl)-N4-((S)-1-((S,E)-5-methyl-1-
(methylsulfonyl)hex-1-en-3-ylamino)-1-oxo-3-phenylpropan-2-yl)
terephthalamide (21)
50.3 MHz)
d
11.2 (2C140), 16.7 (2C130), 21.2e25.6 (C90, 6C
d
, 3C
g
),
A solution of 19 (482 mg, 2.09 mmol, 1.5 eq) in dry tetrahy-
drofuran (10 mL) was added at ꢁ70 ꢂC under nitrogen to a solution
of sodium hydride 60% in mineral oil (83.6 mg, 2.09 mmol,1.5 eq) in
dry tetrahydrofuran (10 mL). After 30 min at ꢁ70 ꢂC, a solution of
20 (710 mg, 1.39 mmol, 1 eq) in dry tetrahydrofuran (10 mL) was
added to the mixture. After return back to room temperature, the
reaction was quenched with methanol (4 mL). The reaction mixture
was evaporated under vacuum, then diluted with water (50 mL)
and extracted with dichloromethane (3 x 50 mL). The organic layer
was washed with aqueous saturated sodium bicarbonate solution
(100 mL), dried with magnesium sulphate, filtered and evaporated
under vacuum. The crude product was purified by flash chroma-
tography (alumina, ethyl acetate/gradient of methanol from 0 up to
4%) to give compound 21 as an oil (200 mg, 0.34 mmol, 25%). Rf :
0.40 (alumina, eluent mixture A); 1H NMR (DMSO-d6, 200 MHz)
29.7 (C80), 33.3 (NCH3), 38.3 (C70), 40.8 (C
b), 4.09 (Cb), 41.0(Cb), 49.1
(C2), 52.7 (C5, C100), 53.9 (C8), 54.0 (2C120), 63.0 (OCH3), 127.5
(2C20, 2C30), 135.0 (C1’or C40), 137.5 (C1’or C40), 167.5 (C50, C10),
172.0 (C1, C4, C7); IR (KBr) 3288.01, 2933.23, 1634.97, 1540.31 cmꢁ1
;
MS: m/z ¼ 703.54 [M þ H]þ; Anal. (C38H66N6O6, 2H2O) C, H, N.
6.2.7. N1-(2-(Dipropylamino)ethyl)-N4-((S)-4-methyl-1-((S)-4-
methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-
ylamino)-1-oxopentan-2-yl)terephthalimide, hydrochloride salt
(16)
A solution of lithium aluminum hydride 1M in dry ether (1.1
eq, 0.42 mmol) was added dropwise at e 80 ꢂC under a nitrogen
atmosphere to a solution of 14 (1 eq, 0.38 mmol) in anhydrous
tetrahydrofuran (5 mL). The mixture was stirred for 90 min
at ꢁ80 ꢂC and was then allowed to warm to 0 ꢂC. The reaction was
quenched by the addition of water (10 mL), and excess lithium
aluminum hydride was eliminated by the Mihailovic method [18].
The resulting crude reaction mixture was filtered and the filtrate
was extracted with dichloromethane (3 ꢃ 20 mL). The combined
organic layers were dried over magnesium sulfate, filtered and
concentrated under vacuum. The crude product was diluted
under argon in dry dichloromethane (2 mL) before addition of
a solution of dry ether/hydrochloride acid 2N (0.25 mL). The
reaction mixture was stirred at room temperature for 30 min and
filtered to give 16 as a white solid (0.86 g, 0.14 mmol, 36.5%). dec :
d
0.84 (m, 6H, H3
g
d
), 1.22 (t, 6H, J ¼ 8 Hz, H110), 1.66 (m, 3H, H3
b,
H3
), 2.61 (s, 3H, SCH3), 3.10 (q, 4H, J ¼ 8 Hz, H100), 3.18 (t, 2H,
J ¼ 8 Hz, H80), 3.64 (m, 2H, H70), 4.08 (m, 2H, H6
b), 4.70 (m, 1H, H3),
4.92 (m, 1H, H6), 5.90 (d, 1H, J ¼ 16 Hz, H1), 6.05 (d, 1H, J ¼ 16 Hz,
H2), 7.20 (m, 5H), 7.57 (d, 2H, J ¼ 8 Hz, H20), 8.15 (d, 2H, J ¼ 8 Hz,
H30), 8.60 (brs, 1H, H4), 8.01 (brs, 1H, H60), 8.80 (d, 1H, J ¼ 8.5 Hz,
H7); 13C NMR (DMSO-d6, 50.3 MHz)
d
9.5 (2C110), 20.3, 22.6, 24.9
(2Cd, Cg), 35.9 (Cb b
), 36.0 (C70), 37.5 (C ), 39.3 (2C100), 46.4 (SO2CH3),
51.9 (C80), 54.9 (C6), 62.1 (C3), 116.0 (C1), 126.1e130.0 (2C20, 2C30,
5CPhe), 136.2 (C10 or C40, CPhe), 136.5 (C10 or C40), 140.5 (C2), 165.9,
166.0, 171.5 (C5, C8, C50); IR (KBr) 3269.80, 2956.78, 1644.72,
1540.40, 1300.11, 1133.47 cmꢁ1; MS: m/z ¼ 585.23 [M þ H]þ, Anal.
(C31H44N4O5S, 10H2O) C, N.
200 ꢀ 1 ꢂC; 1H NMR (DMSO-d6, 200 MHz)
d
0.89 (m, 18H, H
d
d
), 1.04
(m, 6H, H120), 1.44 (m, 6H, H2
b
, H2
d
, H5
b
, H5
)), 1.67 (m, 7H, H110,
H8
b
, H8
d
), 3.07 (t, 4H, J ¼ 4.5 Hz, H100), 3.24 (m, 2H, H80), 3.66 (m,
2H, H70), 4.09 (m, 1H, H2), 4.32 (m, 1H, H5), 4.49 (m, 1H, H8, 7.97
(m, 4H, H20, H30), 8.13 (brs, 1H, H6), 8.29 (brs, 1H, H3), 8.66 (brs,
1H, H9), 9.14 (brs, 1H, H60), 9.35 (s, 1H, H1), 10.52 (brs, 1H, H90);
6.2.10. N1-(2-(Diethylamino)ethyl)-N4-((S)-4-methyl-1-((S)-4-
methyl-1-((S,E)-5-methyl-1-(methylsulfonyl)hex-1-en-3-ylamino)-
1-oxopentan-2-ylamino)-1-oxopentan-2-yl)terephthalamide (23)
Compound 23 was obtained according to the procedure
described for compound 21 starting from 22 (530 mg, 0.9 mmol, 1
13C NMR (CDCl3, 50.3 MHz)
22.1 (C ), 22.5 (C ), 22.7 (C
d
11.7 (2C120), 19.6 (2C110), 21.7 (C
), 22.8 (C ), 23.0 (C ), 24.7 (C ), 24.8
d),
d
d
d
d
d
g

5710
M. Vivier et al. / European Journal of Medicinal Chemistry 46 (2011) 5705e5710
eq) and sulfone 19 (311 mg, 1.35 mmol, 1.5 eq) to give compound 23
as an beige powder (250 mg, 0.38 mmol, 42%). mp: 102 ꢀ 1 ꢂC; Rf:
Hoechst dye 33342 solution was added to each sample (final
concentration 15 g/mL), and plates were incubated for 1 h at room
m
0.40 (alumina, eluent mixture A); 1H NMR (CDCl3, 200 MHz)
d
0.89
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
(m. 18H, H
d
), 1.39 (t, 6H, J ¼ 6 Hz, H110), 1.65 (m, 9H, H
b, Hg), 2.88 (s,
3H, SCH3), 3.22 (q, 4H, J ¼ 6 Hz, H100), 3.33 (t, 2H, J ¼ 6 Hz, H80), 3.88
(m, 2H, H70), 4.45 (m, 1H, H3), 4.66 (m, 1H, H6), 4.70 (m, 1H, H9),
6.65 (d, 1H, J ¼ 16 Hz, H1), 6.82 (d, 1H, J ¼ 16 Hz, H2), 7.50 (m, 1H,
H10), 7.68 (m, 1H, H4), 7.93 (m, 1H, H7), 7.88 (d, 2H, J ¼ 8 Hz, H20),
7.95 (d, 2H, J ¼ 8 Hz, H30), 9.03 (brs, 1H, H60); 13C NMR (CDCl3,
expressed as the concentration inhibiting cell growth by 50% (IC50
calculated from the survival curves.
)
50.3 MHz)
22.9 (C ), 23.6 (C
(C ), 40.1 (C ), 40.9 (Cb
d
10.3 (2C110), 21.32 (C
), 24.1 (C ), 24.2 (C
), 42.7 (SO2CH3), 47.4 (2C100), 47.9 (C3), 52.0
d), 21,7 (C
d), 21.9 (C
d), 22.7 (Cd),
d
d
g
g), 24.8 (C
g
), 36.3 (C70), 39.7
Acknowledgments
b
b
(C80), 52.5, 53,4 (2C), 112.5 (C1), 129.2 and 129.3 (2C20, 2C30), 135.9,
137.3 (2C), 147.8 (C2), 166.6, 167.3, 171.6, 172.5 (4 C]O); IR (CCl4)
3200e3550, 2870e2958, 1542e1642, 1167, 967 cmꢁ1; MS: m/
z ¼ 664.5 [M þ H]þ; Anal. (C34H57N5O6S, 2H2O) C, H, N.
We thank Dr J. Helfenbein and Dr M.-F. Moreau for many helpful
discussions throughout the course of this work.
Appendix. Supplementary data
6.2.11. N1-(2-(Diethylamino)ethyl)-N4-((S)-1-((S)-4-methyl-1-
((S,E)-5-methyl-1-(methylsulfonyl)hex-1-en-3-ylamino)-1-
oxopentan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.07.037.
terephthalamide (25)
Compound 25 was obtained according to the procedure
described for compound 21 starting from 24 (770 mg, 1.24 mmol, 1
eq) and sulfone 19 (428 mg, 1.86 mmol,1.5 eq) to give compound 25
as an oil (230 mg, 0.32 mmol, 27%). Rf : 0.50 (alumina, eluent
References
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(m, 6H, H110), 1.40 (m, 6H, H3
, H6 , H
2H, H9
), 3.08 (m, 4H, H100), 3.12 (m, 2H, H80), 3.8 (m, 2H, H70), 4.75
d
0.87 (m, 12H, H
d), 1.24
b
b
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b
[5] R. Schots, Transfus. Apher. Sci. 44 (2011) 223e229.
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H1), 5.30 (d, 1H, J ¼ 12 Hz, H2), 7.03e7.80 (m, 3H, H4, H7, H10), 7.27
(m, 5H), 7.80 (m, 4H, H20, H30); 13C NMR (DMSO-d6, 50.3 MHz)
d
9.3
(2C110), 20.9e24.7 (4C
d,2Cg), 35.9 (Cb), 37.0 (Cb), 39.6 (Cb), 44.2
(SO2CH3), 47.7 (C70), 52.1 (C80), 54.9 (C6), 56.3 (C9), 61.9 (C3), 114.0
(C1), 127.0e129.7 (9C), 136.2, 136.5 (2C), 139.0 (C2), 166.7, 166.9,
171.5, 171.9 (C5, C8, C11, C50); IR (KBr) 2922.86, 1636.73, 1540.71,
1292.51, 112.69 cmꢁ1; MS: m/z ¼ 698.4 [M þ H]þ.
6.3. Cytotoxicity assay
Attached human ocular melanoma IPC227F cells were seeded in
96 well plates and incubated for 24 h at 37 ꢂC 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 ꢂC. After thawing at room temperature, cells were incubated
for 1 h at room temperature with a 0.01% SDS solution, frozen again
at ꢁ80 ꢂC, and then thawed again at room temperature. The
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