Proteasome inhibitors: Synthesis and activity of arecoline oxide tripeptide derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.01.081

We describe the synthesis and biological activities of a series of methyl 3,4-epoxypiperidine-3-carboxylate tripeptide derivatives that inhibit the chymotryptic and tryptic active sites of the 20S proteasome. Of the series, compound 2 which contains 3-hydroxy-2-methylbenzoyl group at its N-terminal position, displayed the greatest inhibitory potency (IC₅₀ <1 μM). All derivatives showed favourable pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2004.01.081

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1965–1968
Proteasome inhibitors:
synthesis and activity of arecoline oxide tripeptide derivatives
Mauro Marastoni,a,* John McDonald,c Anna Baldisserotto,a Alessandro Canella,b
Carmela De Risi,a Gian Piero Pollinia and Roberto Tomatisa
aDepartment of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy
bDepartment of Biochemistry and Molecular Biology, University of Ferrara, I-44100 Ferrara, Italy
cUniversity Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester LEI 5WW, UK
Received 20 October 2003; revised 22 January 2004; accepted 22 January 2004
Abstract—We describe the synthesis and biological activities of a series of methyl 3,4-epoxypiperidine-3-carboxylate tripeptide
derivatives that inhibit the chymotryptic and tryptic active sites of the 20S proteasome. Of the series, compound 2 which contains 3-
hydroxy-2-methylbenzoyl group at its N-terminal position, displayed the greatest inhibitory potency (IC50 <1 mM). All derivatives
showed favourable pharmacokinetic properties.
# 2004 Published by Elsevier Ltd.
The proteasome is a multicatalytic protease complex
and are responsible for the majority of nonlysosomal
proteolysis in prokaryotes and eukaryotes.1 The enzyme
are formed of multiple subunits, with a modular struc-
ture and several activities. The core and proteolytic
chamber is the 20S proteasome formed by four stacked
rings where each of the two inner rings is composed of
seven different b subunits.2 Each b-ring contains three
proteolytic active sites, the b1 subunit has a post-acidic-
like (PGPH) active site, b2 is associated with a trypsin-
like (T-L) activity, while the b5 subunit has a chymo-
trypsin-like (ChT-L) active site.3 All the proteolytic sites
utilize a N-terminal threonine residue of b subunits as
nucleophile with a catalytic mechanism similar to those
of serine proteases.4 The 20S core structure with
attached two 19S regulatory subunits forms 26S pro-
teasome (2,4 MDa) able to recognize and to degrade
ubiquitinated proteins.
agents represents a new approach and now classes of
these substances are in clinical trials or used to study the
role of the ubiquitin–proteasome pathway in various
cellular processes. Typically inhibitors are short pep-
tides linked to a C-terminal pharmacophore which is
responsible for the interaction with catalytic threonine
of the proteasome.6
We recently reported on the identification of a number
of tripeptidic sequences derivatized at the N- and C-
terminal with arecoline derivatives that were able to
efficiently interact with the catalytic subsites of the pro-
teasome 20S.7 Arecoline is the major alkaloid of the
betel nut (the seed of Areca catechu).8 We have chosen
this natural molecule as potential Michael addition
substrate for the N-terminal enzymatic threonine and
because it is easily usable in peptide synthesis. Biological
data have suggested that the arecoline conjugate double
bond in the C-terminal position does not function as
substrate for the catalytic threonine, while the N-term-
inal 1,2,5,6-tetrahydropyridine-3-carbonyl tripeptide
derivatives have shown an interesting inhibition against
tryptic and chymotryptic active sites with an IC50 <1
mM in in vitro enzyme assays.
Since the proteasome hydrolyze various cell cycle reg-
ulators, transcription factors and antigenic proteins, it is
a promising target for the development of drug for the
treatment of a range of pathologies such as cancer,
inflammation, immune diseases and others.5 The devel-
opment of proteasome inhibitors into novel therapeutic
Here, we describe the synthesis and biological activities
of a new series of arecoline tripeptide derivatives with
a methyl 3,4-epoxypiperidine-3-carboxylate (guvacine
oxide methyl ester) moiety representing the C-terminal
* Corresponding author. Tel.: +39-0532-291281; fax: +39-0532-
291296; e-mail: mru@dns.unife.it
0960-894X/$ - see front matter # 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.01.081

1966
M. Marastoni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1965–1968
Scheme 1. Synthesis of the arecoline oxide tripeptides 1, 6. Reagents and conditions: (a) ClCOOCHClMe, CH2Cl2; (b) MeOH, ꢀ; (c) Z-OSu, DMF;
(d) MCPBA, CH2Cl2; (e) H2, C/Pd, MeOH; (f) Z-Xaa-OSu, DMF; (g) Z-Xbb-OSu, DMF; (h) WSC, HOBt, DMF; (i) Hmba, WSC, HOBt, DMF;
(l) Boc-Guv, HATU, DIPEA, DMF; (m) TFA.
pharmacophore (Scheme 1). We have chosen this are-
coline oxide derivative as a potential substrate for N-
terminal nucleophile threonine of the catalytic b sub-
units, in analogy with the chemical properties of the
natural epoxyketone inhibitors, such as the potent and
selective epoxomycin and eponemycin.9 Tripeptidic
sequences Val-Ser-Leu (1–3) and Leu-Leu-Leu (4–6) are
functionalized at N-terminal position with benzyloxy-
carbonyl (1,4), 3-hydroxy-2-methylbenzoyl (2,5) or
1,2,5,6-tetrahydropyridine-3-carbonyl (3,6) groups. The
potential sites of interaction between the proteasome
catalytic subunits and the arecoline oxide tripeptide
derivative, compound 2, are shown in Figure 1. The
hydrophobic residues leucine and valine interact with S1
and S3enzymatic pockets and the N-terminal six mem-
bered ring located in P4 position is favourable for
interaction with the S4 region. Serine was inserted in the
core structure to improve pharmacokinetic properties
such as water solubility.
Arecoline oxide tripeptide derivatives 1–6 were synthe-
sized by the classical solution method by means of C-
terminal stepwise elongation (Scheme 1).10 Arecoline
derivatives with the C-terminal pharmacophore methyl
(3RS,4RS)-3,4-epoxypiperidine-3-carboxylate and N-
terminal substituent 1,2,5,6-tetrahydropyridine-3-car-
bonyl (Guv) were prepared according to procedures
reported in literature.11 Benzyloxycarbonyl (Z) protect-
ing groups and N-protected amino acids were coupled
as succinimidyl esters. WSC/HOBt were employed to
introduce N-terminal 3-hydroxy-2-methylbenzoyl (Hmb)
and C-terminal methyl 3,4-epoxypiperidine-3-carboxylate
(AE) substituents in tripeptidic sequences. The coupling
agent HATU was used to condense N-protected guva-
cine (Boc-Guv) in derivatives 3 and 6. Z Protecting
groups were removed by catalytic hydrogenation while
Boc was removed by treating with aqueous TFA. After
purification by preparative reversed-phase HPLC,
purity of all target compounds were determined by
Figure 1. Arecoline oxide tripeptide derivative 2: potential interactions
with proteasome 20S catalytic subsites.

M. Marastoni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1965–1968
Table 1. Metabolic stability and inhibitory activities of compounds 1–6
1967
Compd
Half-life (min)
Proteasome inhibition IC50 (mM)
Culture medium
Human plasma
Chymotr.
Tryptic
Post acid.
1 Z-Val-Ser-Leu-AE
>360
>360
>360
>360
>360
>360
148
>360
295
193
>360
>360
2.14
0.73
0.86
0.98
0.17
0.77
1.13
0.14
0.31
13.20
3.41
4.55
>20
>20
>20
>20
>20
>20
2 Hmb-Val-Ser-Leu-AE
3 Guv-Val-Ser-Leu-AE
4 Z-Leu-Leu-Leu-AE
5 Hmb-Leu-Leu-Leu-AE
6 Guv-Leu-Leu-Leu-AE
HPLC and elemental analysis; structure verification was
achieved by mass spectrometry and NMR spectroscopy.12
capacities.14 All compounds showed proteasome inhibi-
tion similar to that observed in the in vitro assays (data
not shown). The ability of the tripeptide derivatives to
permeate cell membranes is an essential pharmacoki-
netic property of these molecules regarding their inhibit
intracellular proteolytic complexes.
We tested the inhibitory capacity of compounds 1–6
against the isolated 20S proteasome using specific
fluorogenic substrates. Proteasomes were isolated from
lymphoblastoid cell lines and purified on affinity col-
umns containing a derivatized matrix (specific Ab for a
subunit of proteasome, Affinity).13 The fluorogenic
substrates Suc-LLVY-AMC, Boc-LRR-AMC and Ac-
YVAD-AMC were used to measure chymotripsyn-like,
trypsin-like and postacidic-like proteasome activities,
respectively. Peptide substrates were incubated at 37 ꢀC
for 30 min with purified proteasomes, untreated or pre-
treated with 0.01–20 mM of test compounds and finally
fluorescence was determined using an excitation of 360
nm and emission at 465 nm. Proteasome activity was
evaluated in fluorescence units and the inhibitory activ-
ity of the compounds are expressed as IC50 (Table 1).
Biological data show, if compared to natural epoxy-
ketones, that the insertion of a potential pharmaco-
phore at the C-terminal is not beneficial with regard to
inhibitory activity. This suggests that the C-terminal
arecoline oxide does not function as a substrate for the
hydroxyl group in the catalytic threonine side-chain.
3-Hydroxy-2-methyl benzoyl (Hmb) N-terminal con-
jugates 2 and 5 are the more potent inhibitors of the
series, suggesting that the substituted aromatic ring is
oriented so that in may favour interactions with cataly-
tic S4 pockets. A trileucyl sequence confers to com-
pounds 4–6 a degree of selectivity for chymotrypsin-like
subunit, derivatives 1–3 exhibit a slight preference for
tryptic inhibition. Compound 2 shows an IC50<1 mM
for both b2 and b5 subunits, while analogue 5 has a
good selectivity for the chymotryptic active site. All
members of the series were unable to inhibit the post-
acidic-like b1 subunit. IC50 reported in Table 1 are
relative to the effect of the inhibitors on the 1 h degra-
dation of peptide substrates used to evaluate the differ-
ent activities of the proteasome. However, we observed
that the inhibitory activities of the tested compounds
decreased after 2 h and was almost undetectable at 8 h,
suggesting a reversible inhibition.
In order to evaluate the susceptibility to enzymatic
hydrolysis, compounds 1–6 were incubated at 37 ꢀC in
culture medium (RPMI) in the presence of 10% fetal
calf serum (RPMI+10% FCS) or in human plasma for
different periods of time and kinetics were studies by
HPLC.15 The degradation half-life (T1/2) was obtained
by a least-squares linear regression analysis of a plot of
the logarithmic inhibitor concentration versus time,
using a minimum of five points (Table 1). All arecoline
oxide derivatives show a great stability in cell culture
medium and have good enzymatic resistance against
human plasma proteases; in particular Hmb N-terminal
functionalized inhibitors 2 and 5 displayed a half-live of
over 6 h.
In this work, we identified some tripeptidic derivatives
able to efficiently interact with the catalytic subsites of
the proteasome 20S. The C-terminal arecoline oxide
pharmacophore is not particularly favourable for the
primary interaction with catalytic threonine. 3-
Hydroxy-2-methyl benzoyl groups situated at the N-
terminal position, give remarkable inhibitory capacity
against chymotryptic and tryptic activities. In summary
we have developed new small peptide derivatives able to
produce powerful inhibition of the proteasome having
favourable pharmacokinetic properties and will prove
useful tools for studying the biological role of the
ubiquitin–proteasome pathway.
Acknowledgements
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).
The cell membrane permeation and proteasome inhibi-
tory capacities of the compounds 1–6 were tested in live
cells. Lymphoblastoid cell lines were treated over 12 h
with 0.01–20 mM of test compounds, followed by pro-
teasome isolation using an affinity column and activity
measured against specific fluorogenic substrates for
chymotripsyn-like, trypsin-like hydrolytic proteasome
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