1590 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Brief Articles
(2) Coux, O.; Tanaka, K.; Golberg, A. L. Structure and function of
the 20S and 26S proteasomes. Annu. Rev. Biochem. 1996, 65,
801-847.
(3) Matthews, W.; Dricoll, J .; Tanaka, K.; Ichihara, A.; Golberg, A.
L. Involvment of the proteasome in various degradative pro-
cesses in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 1989,
86, 2597-2601.
(4) Tanaka, K.; Tanahashi, N.; Tsurumi, C.; Yokata, K. Y.; Shim-
bara, N. Proteasomes and antigen processing. Adv. Immunol.
1997, 64, 1-38.
(5) Rock, K. L.; Golberg, A. L. Degradation of cell proteins and the
generation of MHC class I-presented peptides. Annu. Rev.
Immunol. 1999, 17, 739-779.
(6) Orlowski, R. Z. The role of the ubiquitin-proteasome pathway
in apoptosis. Cell Death Differ. 1999, 6, 303-313.
(7) Kisselev, A. F.; Golberg, A. L. Proteasome inhibitors: from
research tools to drugs candidates. Chem. Biol. 2001, 8, 739-
758.
(8) Iqbal, M.; Chatterjee, S.; Kauer, J . C.; Das, M.; Messina, P. A.;
Freed, B.; Biazzo, W.; Siman, R. Potent inhibitors of proteasome.
J . Med. Chem. 1995, 38, 2276-2277.
(9) Iqbal, M.; Chatterjee, S.; Kauer, J . C.; Mallamo, J . P.; Messina,
P. A.; Reiboldt, A.; Siman, R. Potent R-ketocarbonil and boronic
ester derived inhibitors of proteasome. Bioorg. Med. Chem. Lett.
1996, 6, 287-290.
(10) Bogyo, M.; McMaster, J . S.; Gaczynska, M.; Tortorella, D.;
Goldberg, A. L.; Ploegh, H. Covalent modification of the active
site Thr of proteasome beta-subunits and the E. coli homologue
HslV by a new class of inhibitors. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 6629-6634.
(11) Craiu, A.; Gaczynska, M.; Akopian, T.; Gramm, C. F.; Fenteany,
G.; Golberg, A. L.; Rock, K. L. Lactacystin and clasto-lactacystin
-lactone modify multiple proteasome -subunits and inhibit
intracellular protein degradation and major histocompatibility
complex class I antigen presentation. J . Biol. Chem. 1997, 272,
13437-13445.
(12) Meng, L.; Mohan, R.; Kwok, B. H.; Elofsson, M.; Sin, N.; Crews,
C. M. Epoxomicin, a potent and selective proteasome inhibitor,
exhibits in vivo antiinflammatory activity. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 10403-10408.
(13) Loidl, G.; Groll, M.; Musiol, H. J .; Ditzel, L.; Huber, R.; Moroder,
L. Bifunctional inhibitors of the trypsin-like activity of eukaryotic
proteasomes. Chemistry & Biology. 1999, 6, 197-203.
(14) Chatterjee, S.; Dunn, D.; Mallya, S.; Ator, M. A. P′-extended
R-ketoamide inhibitors of proteasome. Bioorg. Med. Chem. Lett.
1999, 9, 2603-2606.
(15) Olofson, R. A.; Martz, J . T.; Senet, J .-P.; Piteau, M.; Malfroot,
T. A. New reagent for the selective, high-yield N-dealkylation
of tertiary amines: improved syntheses of naltrexone and
nalbuphine. J . Org. Chem. 1984. 49, 2081-2082.
(16) J akobsen, P.; Lundbeck, J . M.; Kristiansen, M.; Breinholt, J .;
Demuth, H.; Pawlas, J .; Torres Candela, M. P.; Andersen, B.;
Westergaard, N.; Lundgren, K.; Asano, N. Iminosugar: potential
inhibitor of liver glycigen phosphorylase. Bioorg. Med. Chem.
2001, 9, 733-744.
(17) Hendil, K. B.; Uerkvitz, W. The human multicatalytic protein-
ase: affinity purification using a monoclonal antibody. J . Bio-
chem. Biophys. Methods. 1991, 22, 159-165.
(18) Elofsson, M.; Splittgerber, U.; Myung, J .; Mohan, R.; M Crews,
C. Towards subunit-specific proteasome inhibitors: synthesis
and evaluation of peptide R′,â′-epoxyketones. Chem. Biol. 1999,
6, 811-822.
(19) Groll, M.; Nazif, T.; Huber, R.; Bogyo, M. Probing structural
determinants distal to the site of hydrolysis that control
substrate specificity of the 20S proteasome. Biol. Chem. 2002,
9, 655-662.
linear regression analysis of a plot of the logarithmic inhibitor
concentration versus time, using a minimum of five points.
In h ibitor s P u r ifica tion a n d An a lytica l Deter m in a -
tion s. Crude compounds were purified by preparative reversed-
phase HPLC using a Water Delta Prep 4000 system with a
Waters PrepLC 40 mm Assembly column C18 (30 × 4 cm, 300
A, 15 µm spherical particle size column). HPLC analysis was
performed by a Beckman System Gold with a Beckman
ultrasphere ODS column (5 µm; 4.6 × 250 mm). Molecular
weight of compounds was determined by a MALDI-TOF
(Matrix Assisted Laser Desorption Ionization Time-of-Flight)
analysis using a Hewlett-Packard G2025A LD-TOF system
mass spectrometer and R-cyano-4-hydroxycinnamic acid as a
matrix. The values are expressed as MH+. Elemental analyses
were carried out on a TermoQuest CE instruments EA 1110-
CHNS-O elemental analyzer. Optical rotations were deter-
mined by a Perkin-Elmer 241 polarimeter with a 10 cm cell
using methanol as solvent and at an analyte concentration of
1%. 1H NMR spectroscopy was obtained on a Bruker spec-
trometer (Bruker WM 500 MHz).
Ch em istr y. Gen er a l P r oced u r es. Cou p lin g w ith WSC/
HOBt. To a solution of the carboxy component (1 mmol) in
DMF (10 mL) were added the amino component (1 mmol),
NMM (N-methylmorpholine, 1 mmol), HOBt (1.1 mmol), and
WSC (1-ethyl-3-(3I-dimethylaminopropyl)carbodiimide, 1.1
mmol) in this order at 0 °C. The reaction mixture was stirred
for 1 h at 0 °C and 18 h at room temperature; then the solution
was diluted with EtOAc (100 mL) and washed consecutively
with 0.1 N HCl, brine, NaHCO3, and brine. The organic phase
was dried (MgSO4), filtered, and evaporated to dryness. The
residue was treated with Et2O and resulting solid separated
by centrifugation.
Hyd r ogen a tion Dep r otection . The Z (benzyloxycarbonyl)
protecting group was removed by treating the protected
intermediates in methanol (5 mL) with hydrogen (120 min)
in the presence of 10% Pd/C. The reaction mixture was filtered,
the solvent was evaporated, the resulting crude product was
treated with Et2O, and resulting solid was separated by
centrifugation.
In tr od u ction of F m oc. To a solution of tripeptide benzy-
lamide (0.5 mmol) in DMF (5 mL) was added Fmoc-OSu
(fluorenylmethyloxycarbonyl-N-hydroxysuccinimide, 0.5 mmol)
at 0 °C. The mixture was stirred for 1 h at 0 °C and 15 h at
room temperature and then evaporated. The resulting target
compounds were purified by preparative HPLC.
TF A Dep r otection . tert-Butyloxycarbonyl (Boc) protection
was removed from N-arecoline tripeptide derivatives by treat-
ing intermediates with aqueous 90% TFA (trifluoroacetic acid
1:10, w/v) for 30-40 min. After evaporation, the residue was
treated with Et2O and resulting solid separated by centrifuga-
tion.
Ack n ow led gm en t. Financial support of this work
by University of Ferrara, by Ministero dell’Universita`
e della Ricerca Scientifica e Tecnologica (MURST),
Associazione Italiana per la Ricerca sul Cancro (AIRC),
and Istituto Superiore di Sanita` (progetto AIDS).
(20) Gavioli, R.; Vertuani, S.; Masucci, M. G. Proteasome inhibitors
reconstitute the presentation of cytotoxic T-cell epitopes in
Epstein-Barr virus-associated tumors. Int. J . Cancer 2002, 101,
532-538.
(21) Abbreviations: Fmoc, fluorenylmethoxycarbonyl; Boc, tert-bu-
toxycarbonyl; Fmoc-OSu, 9-fluorenylmethoxycarbonyl-N-hydroxy
succinimide; HOBt, N-hydroxybenzotriazole; Suc, succinyl; TFA,
trifluoroacetic acid; WSC (water soluble carbodiimide), 1-ethyl-
3-(3I-dimethylaminopropyl)carbodiimide; Z, benzyloxycarbonyl.
Su p p or tin g In for m a tion Ava ila ble: Analytical data of
arecoline inhibitors are available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces
(1) Ciechanover, A. The ubiquitin-proteasome proteolytic pathway.
Cell 1994, 79, 13-21.
J M0309102