Identification of a new series of amides as non-covalent proteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.01.022

Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the β5, β2, and β1 subunits of each heptameric β rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the β5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure.

Full text of DOI:10.1016/j.ejmech.2014.01.022

European Journal of Medicinal Chemistry 76 (2014) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of a new series of amides as non-covalent proteasome
inhibitors
,
b
Kety Scarbaci a, Valeria Troiano a, Nicola Micale a, Roberta Ettari b , Lucia Tamborini ,
Carmen Di Giovanni c, Carmen Cerchia c, Silvana Grasso a, Ettore Novellino c,
Tanja Schirmeister d, Antonio Lavecchia c, Maria Zappalà a
*
a Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
c Dipartimento di Farmacia, “Drug Discovery” Laboratory, Università degli Studi di Napoli Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy
d Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple
myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S
proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-
like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located
Received 17 July 2013
Received in revised form
14 January 2014
Accepted 16 January 2014
Available online 25 January 2014
respectively on the b5, b2, and b1 subunits of each heptameric b rings. Currently a wide number of
covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-
covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all
drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of
amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-
like activity of the b5 proteasome subunit. The non-covalent binding mode was corroborated by docking
simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Amides
Proteasome inhibitors
Non-covalent inhibitors
Docking studies
1. Introduction
counterparts
MECL1 and LMP7, respectively) upon exposure to specific stimuli
such as the inflammatory cytokines IFN- and TNF- [3].
Proteasome is responsible for the turnover of cellular proteins
that regulate cell proliferation and survival pathways, however
defects of its proteolytic activity can lead to anarchic cell prolifer-
ation and as a consequence to tumor development.
b1i, b2i and b5i (>50% of identity; also named LMP2,
The eukaryotic 26S proteasome is a large (1.6e2.4 MDa) multi-
functional particle, composed of a barrel-shaped 20S catalytic core
capped by two 19S regulatory complexes. The 20S proteasome is the
proteolytically active key element of the ubiquitineproteasome
system. It is composed by four heptameric rings stacked in a
g
a
a7b7b7a7 arrangement and contains three proteolytic subunits,
b
1,
It has been demonstrated that inhibition of the chymotrypsin-
like (ChT-L) activity of proteasome represents a valid strategy to
induce antineoplastic effects in hematologic tumors [4]. A growing
number of studies suggest that proteasome inhibitors may become
valuable drugs for the treatment of nontumorous diseases when
used in conditions in which the cellular functions are modulated
without induction of cell death [5]. Diverse beneficial effects are
expected in inflammation [6], neurodegenerative diseases [7],
muscular dystrophies [8] and cachexia [9]. Moreover, they have
therapeutic potential as antiparasitics in malaria [10] and sleeping
syndrome [11] and as antimicrobial agents (tuberculosis) [12].
The majority of 20S proteasome inhibitors, currently reported in
literature [13], are peptide-based compounds, endowed with a C-
terminal electrophilic warhead that forms covalent adducts with
b
2
and 5, which have distinct substrate specificities and
b
are responsible, respectively, for the caspase-, trypsin- and
chymotrypsin-like activities of proteasome [1]. All the three sub-
units possess a catalytic site that employs the nucleophilic g-hy-
droxyl group of the N-terminal Thr to cleave peptide bonds [2]. The
expression of these subunits in normal cells may be replaced
by the synthesis of the immunoproteasome catalytic subunits
Abbreviations: ChT-L, chymotrypsin-like; T-L, trypsin-like; PGPH, post-glutamyl
peptide hydrolyzing; C-L, caspase-like; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide; HOBt, N-hydroxybenzotriazole; DIPEA,
N,N-diisopropylethylamine.
* Corresponding author. Tel.: þ39 02 50319342; fax: þ39 02 50319359.
E-mail address: roberta.ettari@unimi.it (R. Ettari).
the active site Thr1Og (Fig. 1a, for bortezomib). A reversible action
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.022

2
K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
Scheme 2. Reagents and conditions: a) HOBt, EDC$HCl, then suitable amine and
DIPEA, CH2Cl2, ꢀ0 ꢁC / rt, 12 h.
Non-covalent inhibitors, even if less widely investigated, with
respect to covalent inhibitors, provide an alternative mechanism
for proteasome inhibition and, due to the rapid dissociation from
the 20S subunits, may be devoid of all drawbacks related to either
irreversible or slowly reversible enzyme inhibition. Their potential
advantages are improved selectivity, moderate reactivity and
reduced instability, which are often associated with side effects in
therapeutics [17]. Taking into account all these reasons non-
covalent proteasome inhibitors might be a promising alternative
for therapy.
Our research group has been involved in the last years in the
development of peptidomimetic vinyl sulfones [18] and boronates
[19] as irreversible and pseudo-irreversible 20S proteasome in-
hibitors, respectively. During the development of peptidomimetic
boronates, we identified a number of amides 1 (Fig. 1 b), recovered
as by-products of the key step for the introduction of the boronic
ester moiety. After a preliminary screening, some of these mole-
cules were supposed to non-covalently inhibit
b5 proteasome
subunit. Starting from these findings, and on the basis of a docking
model, we synthesized and evaluated as potential 20S proteasome
inhibitors a second series of amides 2 (Fig. 1 b), in which bulkier
substituents directly linked to the amide moiety, in agreement also
to literature data [20], have been introduced in order to extend the
structureeactivity relationship of this class of molecules.
Fig. 1. Structure of representative non-covalent (TMC-95A), covalent (bortezomib)
proteasome inhibitors (a) and amides 1e2 (b).
characterizes aldehydes,
a
-keto-aldehydes,
a-keto-oxadiazoles, b-
lactones, boronates. a0
,b
0-Epoxyketones are characterized by an
2. Results and discussion
irreversible action. Very often this covalent mode of action,
together with high reactivity of compounds, may lead to off-target
interactions.
2.1. Chemistry
In this context, the dipeptide boronate Bortezomib (VelcadeÒ),
the first proteasome inhibitor approved by the FDA for treatment of
multiple myeloma and mantle cell lymphoma, has been demon-
strated to possess low systemic tissue distribution, severe off-target
effects due to lack of specificity [14]. Despite the dipeptide boro-
nates reversibly interact with the target enzyme, the formation of a
long-lasting inhibitor-proteasome adduct is responsible of its un-
favorable pharmacodynamic profile: when it is injected intrave-
nously a big part of administered dose inhibits the chymotrypsin-
like activity of 20S proteasome of non-tumor cells, like hematic
or liver cells. As a consequence while irreversible blockage of an
enzyme should be advantageous for parasitic targets [15], on the
contrary in the case of cancer treatment, non-covalent inhibition
would be desirable. Thus, there is a recent tendency to identify non-
covalent inhibitors, like TMC-95A, (Fig. 1 a) and its linear de-
rivatives [16].
In our multistep approach for the synthesis of peptidomimetic
boronates, carried out with our previously reported procedure [19],
we identified compounds 1 as by-products. More in detail the
synthetic step leading to the introduction of the pharmacophore
portion, realized by coupling the carboxylic acids 3 and pinanediol
leucine boronate 4 in the presence of EDC$HCl, HOBt and DIPEA,
together with the desired product 5, led to the formation, as minor
reaction product, of amides 1 obtained by spontaneous deborona-
tion of boronic esters 5 (Scheme 1).
The synthesis of amides 2aec, 2eeh and 2j (Scheme 2) has been
carried out by coupling acid 6 or 7, synthesized according to a
previously reported procedure [19], together with the suitable
amines by employing also in this case, HOBt and EDC$HCl as
coupling reagents to get the desired amides in high yields. Different
conditions were adopted for the synthesis of sterically hindered
amides 2d and 2i, in this case acid 6 or 7 was first activated to mixed
ring = 1H-pyridin-2-one or 1,6-naphthyridin-5(6H)-one
Scheme 1. Reagents and conditions: (a) HOBt, CH2Cl2, ꢀ5 ꢁC, 20 min, then EDC$HCl, DIPEA, 7, ꢀ15 ꢁC / rt, 3 h.

K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
3
acids can be heated in 40% aqueous HBr or HI [21], without any
decomposition. However, deboronation has been quite often
observed in basic aqueous solutions [22], thus allowing us to
explain the formation from the boronic ester of the corresponding
amide, probably because of the addition of DIPEA, during the
coupling reaction.
1000
800
600
400
200
0
With the aim to evaluate their inhibitory properties, we
screened amides 1aei at 20 mM against the three 20S proteasome
F
proteolytic activities (ChT-L, T-L, PGPH), in such a way to evaluate
their potential ability to inhibit the target enzyme. For compounds
which passed the initial screening (at least 40% of inhibition),
additional assays were performed. The dissociation constants Ki of
the non covalent complex E$I were obtained from the progress
curves at seven different concentrations of inhibitor, as reported in
Fig. 2 for inhibitor 1f, by fitting the progress curves to the 4-
parameter IC50 equation:
0
200
400
600
ymax ꢀ y
Time (seconds)
y ¼
min þ ymin
ꢀ
ꢁ
s
½Iꢂ
1 þ
Fig. 2. Progress curve of substrate hydrolysis in presence of the inhibitors 1f.
F ¼ fluorescence units. Inhibitor concentrations (from top to bottom): 0, 1.0, 10.0, 20.0,
IC50
40.0, 60.0, 80.0, 100.0 mM.
(with y[dF/min] as the substrate hydrolysis rate, ymax as the
maximum value of the doseeresponse curve that is observed at
very low inhibitor concentrations, ymin as the minimum value that
is obtained at high inhibitor concentrations, and s denotes the Hill
coefficient, and correction to zero substrate concentration from
Ki ¼ IC50/(1 þ [S] Kꢀm1).
anhydride, by employing N-methylmorpholine and isobutyl-
chloroformate, and then coupled with 2-aminobenzophenone.
2.2. Biological activity and docking studies
In this regard compounds 1b and 1f were proven to be the most
Early reports attest the great stability of alkylboronic acids un-
der aqueous acidic solutions, as a matter of fact simple alkylboronic
active compounds, with Ki values of 0.56 and 0.33 mM, respectively
(Table 1).
Table 1
Inhibition of ChT-L proteasome activity by amides 1aei.
Comp
X
R1
Y
R2
ChT-L
Ki (
M)a
n.d.
0.44 ꢃ 0.21
0.56 ꢃ 0.01
0.17 ꢃ 0.01
n.d.
1.34 ꢃ 0.12
10.1 ꢃ 0.9
n.d.
1.62 ꢃ 0.44
0.33 ꢃ 0.05
0.098 ꢃ 0.005
n.d.
m
% inhib. at 20 mM
1a
H
H
Br
Br
H
H
e
e
e
e
e
e
e
e
e
e
e
H
H
H
H
e
e
10
100
56
100
35
100
45
7
84
87
97
28
100
42
100
27
88
100
1a-BAc
1b
e
e
e
e
1b-BAc
1c
e
e
Methyl
Methyl
e
e
1c-BAc
1d
e
e
e
e
e
e
e
e
e
e
e
e
e
H
e
1e
Methyl
Methyl
e
1e-BAc
1f
e
e
e
e
e
e
e
e
e
Phenylamino
Phenylamino
Diethylamino
Diethylamino
Morpholin-4-yl
Morpholin-4-yl
Piperidin-1-yl
Piperidin-1-yl
1f-BAc
1g
1g-BAc
1h
0.80 ꢃ 0.11
7.67 ꢃ 0.23
1.45 ꢃ 0.04
n.d.
1h-BAc
1i
1i-BAc
Bortezomibb
0.30 ꢃ 0.05
0.0098 ꢃ 0.001
n.d. ¼ not determined.
BA ¼ boronic acid.
a
Values represent the mean of three independent determinations.
Values determined in the same experimental conditions of compounds 1.
For comparison, corresponding boronates (1-BA*) [19] and bortezomib are added as reference covalent compounds.
b
c

4
K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
Compound 1b, turned also to be active on the PGPH activity,
with a Ki value of 22.3 M. This outcome is extremely important
taking into consideration that co-inhibition of 5 subunits with
either 1 or 2 subunits exerts the maximal effect that is required
to the other tested derivatives. Specifically, the 8-bromo-naph-
thyridinone C]O of 1b and the 4-phenylamino NH of 1f estab-
m
b
lished an H-bond with the protonated
to the stabilization of both inhibitors.
b6-D114, thus contributing
b
b
to produce an antitumor response.
With this in mind, we developed a series of 4-amino-substituted
pyridone analogs 2fej, with the aim to extend the structureeac-
tivity relationship of this class of molecules. The corresponding
unsubstituted analogs 2aee were also synthesized to assess the
relevance of the phenylamino moiety at position 4 of the pyridone
nucleus. At P1 site, to evaluate the size of the liphophilic pocket that
normally accommodates the isopentyl group, we introduced
bulkier substituents, like aromatic or alicyclic nuclei (e.g. phenyl,
adamantyl).
A comparison between the amides of the present work and the
corresponding boronates (1-BA [19], Table 1) clearly points out the
better inhibitory activity of the compounds bearing the boronic
acid moiety with respect to the ones that are lacking such a
warhead. However, in some cases the inhibitory activity revealed to
be almost comparable (e.g. 1b and 1b-BA with Ki values of 0.56 and
0.17 mM respectively) and this highlights how both inhibitors are
able to establish key interactions with the target enzyme, even if
with a different mode of action.
From a preliminary screening at 20
proven to weakly inhibit the 5 subunit of 20S proteasome, with
the exception of compounds 2e, 2h and 2i, which showed Ki values
of 7.55, 1.48 and 8.03 M respectively (Table 2).
mM amides 2aej were
Inhibitors 1b and 1f were docked into the binding pocket of the
b
b5 subunit (Fig. 3), using the crystal structure of bortezomib bound
to the yeast 20S proteasome (PDB ID: 2F16) [23].
m
Docking simulations clearly displayed 1b and 1f in the proximity
The amide 2h revealed to be the most active compound of this
of the ChT-L active site in subunit
mode of binding. The ligands did not interact with the active-site
5-T1 Og nucleophile, which hitherto has been considered a com-
b
5 and suggested a non-covalent
series of analogs, in agreement with its presumed ability to form an
H-bond with D114 of the b6 subunit, similarly to amide 1f. Overall
b
only the 4-methoxyphenyl substituent directly linked to the amide
function (i.e. 2h) yielded an activity nearly comparable to that of 1f,
bearing an isopentyl group. However, from a general analysis of the
obtained results, the isopentyl substituent, in terms of size, proved
to be optimal for the correct alignment of the inhibitor in the
binding site. Additionally compounds 2e, 2h and 2i were proven to
co-inhibit the PGPH activity of the 20S proteasome (more then 50%
of inhibition at 20 mM) that, as already said, is desirable to enhance
an antitumor response.
Interestingly, docking of 2h to the ChT-L active site of the yeast
proteasome revealed that it, unlike compounds 1b, 1f and borte-
zomib, did not bind into the S1 region of the ChT-L pocket but in the
mon principle of ChT-L inhibitor binding. The isopentyl group
linked to the amide moiety of compounds 1b and 1f projected into
the S1 pocket, adopting a spatial arrangement similar to that of the
P1-leucine side chain of bortezomib. Both compounds accommo-
dated their aromatic group perfectly in the S3 pocket through a
series of van der Waals interactions, thereby profoundly stabilizing
these moieties in the ChT-L site. In addition, the amide and the
pyridinone C]O oxygens of both ligands are predicted to form H-
bonds with
b
5-A49N,
b
5-A50N and
b
6-D114Og via an intervening
water molecule located crystallographically in the bortezomib
structure and included in our model. It is to note that compound 1f
engaged a further H-bond with
amide C]O group. Interestingly, both compounds are predicted to
establish an H-bond with the side chain of D114 of the 6 subunit,
and this could explain the higher activity of 1b and 1f with respect
b
5-T21 NH backbone through its
S2 and S3 regions between the subunits
addition to forming H-bonds with 5-G47,
D114, 2h accepted a further H-bond from
b
5 and
b
b
6 (Fig. 4a). In
5-A49 and 6-
5-S129 through its 4-
b
b
5-T21,
b
b
b
methoxy oxygen atom, thus stabilizing the position of the 4-
Fig. 3. Binding modes of compounds 1b (a, green), and 1f (c, yellow) into the
Only amino acids located within 4 A of the bound ligand are displayed (white) and labeled. Key H-bonds between the inhibitors and the protein are shown as dashed black lines. An
b
5 (cyan)/
b
6 (white) active site of 20S proteasome represented as a ribbon model (PDB ID: 2F16) [20].
ꢀ
overlay of 1b (b, docked pose), 1f (d, docked pose) with bortezomib (purple, X-ray crystal pose) is shown in the b5/b6 active site of 20S proteasome displayed as Connolly surface.
The defined water molecule forming tight H-bonds to the protein is shown as a red sphere. Specificity pockets S1eS4 are indicated. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
5
Table 2
for a primary in vitro antitumor assay. Each compound was
routinely tested, at 10ꢀ5 M concentration, against 60 human tumor
cell lines, unfortunately within the one dose all compounds turned
out to be essentially inactive.
Inhibition of ChT-L proteasome activity by amides 2aej.
In conclusions with our work we identified a series of amides,
some of which turned to be active against the chymotrypsin-like
activity of 20S proteasome. In particular compounds 1b and 1f
were proven to possess Ki values in the submicromolar range, thus
being reasonable to consider them worthy of further optimization
within the development of non-covalent 20S proteasome inhibitors
for cancer treatment.
Comp
R2
R3
ChT-L
Ki (
M)a
m
% inhib. at 20 mM
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
Benzyl
Phenyl
4-Methoxyphenyl
2-Benzoylphenyl
1-Adamantyl
Benzyl
n.d.
n.d.
n.d.
n.d.
15
23
32
21
86
16
9
3. Experimental protocols
H
7.55 ꢃ 0.86
3.1. Chemistry
Phenylamino
Phenylamino
Phenylamino
Phenylamino
Phenylamino
n.d.
Phenyl
n.d.
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Reactions under
microwave irradiation were performed on a CEM Discover appa-
ratus. Elemental analyses were carried out on a C. Erba Model 1106
(Elemental Analyzer for C, H and N) and the obtained results are
within ꢃ0.4% of the theoretical values. Merck Silica Gel 60 F254
plates were used as analytical TLC; flash column chromatography
was performed on Merck Silica Gel (200e400 mesh). 1H NMR
spectra were recorded on a Varian Gemini 300 MHz spectrometer
using the residual signal of the deuterated solvent as internal
standard. Splitting patterns are described as singlet (s), doublet (d),
doublet of doublet (dd), triplet (t), quartet (q), multiplet (m) and
4-Methoxyphenyl
2-Benzoylphenyl
1-Adamantyl
1.48 ꢃ 0.06 72
8.03 ꢃ 0.25
67
13
2j
n.d.
n.d. ¼ not determined.
a
Values represent the mean of three independent determinations.
methoxyphenyl moiety in the S2 pocket. This explains the high
affinity of 2h to the ChT-L active site in comparison with com-
pounds 2g and 2f.
Structural superposition of 1f and 2h (Fig. 4b) bound to the ChT-
L active site clearly suggests that the decrease in affinity of com-
pounds having a bulkier substituent in R3 (2fej) is due to three
main residues in the S1 site (Fig. 4, Table 2), M45, V31, and A49 of
broad singlet (bs). 1H chemical shifts are expressed in
coupling constants (J) in hertz (Hz).
d (ppm) and
subunit
b5, which create a steric barrier to the P1 R3 moiety.
Similarly, engineered derivatives of omuralide [2] and salinospor-
amide A [24], with a phenyl moiety in P1, have been demonstrated
to exert significantly reduced inhibitory potency toward the ChT-L
activity of yeast proteasome compared to the natural products [25]
These P1 side chain examples highlight the high degree of pro-
teasomal substrate binding channel plasticity, which shows the
complexity of optimizing the binding profile of proteasome
inhibitors.
3.2. General procedure for the synthesis of compounds 1
Compounds 1 have been obtained as by-products of the
coupling reaction between the carboxylic acids 3 and pinanediol
leucine boronate 4, employed for the introduction of boronic ester
moiety, as previously reported [17].
To assess the selectivity for the target enzyme, amides 1e2 were
tested on bovine pancreatic a-chymotrypsin and cathepsin B. None
of them showed no significant inhibition in the selectivity assays,
with the exception of compound 2i, which gave a Ki value of
6.65 mM against bovine pancreatic a-chymotrypsin.
To evaluate the cytotoxic and/or growth inhibitory effects, all
synthesized compounds were submitted for testing at the National
Cancer Institute NCI. Ten amides (1a, 1eeh, 2d, 2gej) were selected
3.2.1. N-Isopentyl-2-(5-oxo-1,6-naphthyridin-6(5H)-yl)acetamide
(1a)
Yield 11%. Rf ¼ 0.38 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3)
d
0.87 (d, 6H, J ¼ 6.6 Hz), 1.37 (q, 2H, J ¼ 7.5 Hz), 1.50e1.62
(m, 1H), 3.22e3.29 (m, 2H), 4.60 (s, 1H), 6.43 (bs, 1H), 6.85 (d, 1H,
J ¼ 7.1 Hz), 7.42 (d, 1H, J ¼ 7.1 Hz), 7.43 (dd, 1H, J ¼ 8.0, 4.9 Hz), 8.66
(dd, 1H, J ¼ 8.0, 1.8 Hz), 8.94 (dd, 1H, 8.0, 1.8 Hz). Anal. Calcd. for
C15H19N3O2: C 65.91, H 7.01, N 15.37. Found: C 65.73, H 7.22, N 15.42.
Fig. 4. a) Binding mode of compound 2h (magenta) into the
b
5 (cyan)/
b
6 (white) active site of 20S proteasome represented as a ribbon model (PDB ID: 2F16) [20]. Only amino acids
ꢀ
located within 4 A of the bound ligand are displayed (white) and labeled. Key H-bonds between the inhibitor and the protein are shown as dashed black lines. b) Structural
superposition of 2h (magenta) and 1f (yellow) is shown in the ChT-L active site of 20S proteasome. The defined water molecule forming tight H-bonds to the protein is shown as a
red sphere. Specificity pockets S1eS4 are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

6
K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
3.2.2. 2-(8-Bromo-5-oxo-1,6-naphthyridin-6(5H)-yl)-N-
isopentylacetamide (1b)
3.2.9. N-Isopentyl-2-(20-oxo-3,4,5,6-tetrahydro-2H,20H-[1,40]
bipyridinyl-10-yl)acetamide (1i)
Yield 26%. Rf ¼ 0.58 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
Yield 29%. Rf ¼ 0.54 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3)
d
0.89 (d, 6H, J ¼ 5.9 Hz), 1.40 (q, 2H, J ¼ 6.5 Hz), 1.50e1.64
CDCl3):
d
0.87 (d, 6H, J ¼ 6.5 Hz), 1.33e1.40 (m, 2H), 1.51e1.63 (m,
(m, 1H), 3.28 (q, 2H, J ¼ 6.5 Hz), 4.59 (s, 2H), 7.48e7.57 (m, 1H), 7.78
(s, 1H), 8.70 (d, 1H, J ¼ 7.6 Hz), 9.04e9.12 (m, 1H). Anal. Calcd. for
7H), 3.16e3.23 (m, 2H), 3.30e3.33 (m, 4H), 4.41 (s, 2H), 5.72 (d, 1H,
J ¼ 2.9 Hz), 5.98 (dd, 1H, J ¼ 7.6, 2.9 Hz), 7.16 (d, 1H, J ¼ 7.6), 7.32 (bs,
1H). Anal. Calcd. for C17H27N3O2, C 66.85, H 8.91, N 13.76. Found C
66.87, H 8.84, N 13.88.
C
15H18BrN3O2: C 51.15, H 5.15, N 11.93. Found: C 51.23, H 5.07, N
11.72.
3.3. General procedure for the synthesis of compounds 2aec, 2eeh,
and 2j
3.2.3. N-Isopentyl-2-(5-oxo-1,6-naphthyridin-6(5H)-yl)
propanamide (1c)
20
Yield 7%. Rf ¼ 0.42 (CHCl3/MeOH 95:5). [
a
]
¼ ꢀ47.8 (c 0.2,
D
CHCl3); 1H NMR (300 MHz, CDCl3)
d
ppm 0.85 (d, 6H, J ¼ 4.69 Hz),
The coupling between acids (6e7) and the suitable amines
(1 equiv.) was carried out by employing HOBt (1.2 equiv.), EDCI
(1.2 equiv.), DIPEA (2 equiv.) in dry CH2Cl2 at 0 ꢁC. Then the mixture
was stirred at room temperature for 12 h. The resulting mixture
was washed with KHSO4 0.1 M, NaHCO3 and brine. The crude
product was purified by column chromatography (CHCl3/MeOH
95:5).
1.31e1.45 (m, 2H), 1.46e1.56 (m, 1H), 1.63 (d, 3H, J ¼ 6.5 Hz), 3.13e
3.33 (m, 2H), 5.66 (d, 1H, J ¼ 6.5 Hz), 6.39 (bs, 1H), 6.86 (d, 1H,
J ¼ 7.0 Hz), 7.36e7.46 (m, 1H), 7.56 (d, 1H, J ¼ 7.0 Hz), 8.58e8.70 (m,
1H), 8.86e8.97 (m, 1H). Anal. Calcd. for C16H21N3O2: C 66.88, H 7.37,
N 14.62. Found: C 66.73, H 7.21, N 14.79.
3.2.4. N-Isopentyl-2-(1,2,3,4-tetrahydro-5-oxo-1,6-naphthyridin-
6(5H)-yl)acetamide (1d)
3.3.1. N-Benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (2a)
Yield 86%. Rf ¼ 0.52 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
Yield 13%. Rf ¼ 0.31 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3):
d
4.41 (d, 2H, J ¼ 5.9 Hz), 4.58 (m, 2H), 6.24e6.29 (m, 1H),
CDCl3)
d
ppm 0.88 (d, 6H, J ¼ 6.5 Hz), 1.36 (q, 2H, J ¼ 7.0 Hz), 1.50e
6.59 (dd, 1H, J ¼ 8.8, 1.2 Hz), 7.20e7.43 (m, 8H). Anal. Calcd. for
14H14N2O2, C 69.41, H 5.82, N 11.56. Found C 69.29, H 5.97, N 11.44.
1.64 (m, 1H), 1.85e1.96 (m, 2H), 2.58 (t, 2H, J ¼ 5.9 Hz), 3.20 (q, 2H,
J ¼ 7.0 Hz), 3.25e3.34 (m, 2H), 4.42 (s, 2H), 5.59 (d, 1H, J ¼ 7.6 Hz),
7.03 (d, 1H, J ¼ 7.6 Hz). Anal. Calcd. for C15H23N3O2: C 64.97, H 8.36,
N 15.15. Found: C 64.89, H 8.55, N 15.37.
C
3.3.2. 2-(2-Oxopyridin-1(2H)-yl)-N-phenylacetamide (2b)
Yield 77%. Rf ¼ 0.41 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
DMSO-d6):
d
4.71 (s, 2H), 6.20e6.25 (m, 1H), 6.37 (d, 1H, J ¼ 9.2), 7.0
3.2.5. N-Isopentyl-2-(1,2,3,4-tetrahydro-1-methyl-5-oxo-1,6-
naphthyridin-6(5H)-yl)acetamide (1e)
3 (t, 1H, J ¼ 7.5), 7.26e7.65 (m, 6H). Anal. Calcd. for C13H12N2O2, C
68.41, H 5.30, N 12.27. Found C 68.67, H 5.14, N 12.29.
Yield 40%. Rf ¼ 0.36 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3)
d
0.86 (d, 6H, J ¼ 6.2 Hz), 1.32e1.39 (m, 2H), 1.46e1.62 (m,
3.3.3. N-(4-Methoxyphenyl)-2-(2-oxopyridin-1(2H)-yl)acetamide
(2c)
1H), 1.86e1.96 (m, 2H), 2.56e2.60 (m, 2H), 2.95 (s, 3H), 3.15e3.26
(m, 4H), 4.43 (s, 2H), 5.86 (d, J ¼ 7.6 Hz), 7.10 (d, J ¼ 7.6 Hz), 7.42 (bs,
1H, NH). Anal. Calcd. for C16H25N3O2: C 65.95, H 8.65, N 14.42.
Found: C 65.71, H 8.82, N 14.17.
Yield 79%. Rf ¼ 0.45 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
DMSO-d6):
d 3.69 (s, 3H), 4.67 (s, 2H), 6.21 (m, 1H), 6.37 (m, 1H),
6.36 (d, 1H, J ¼ 9.7 Hz), 6.86 (d, 2H, J ¼ 8.8 Hz) 7.39e7.64 (m, 6H).
Anal. Calcd. for C14H14N2O3, C 65.11, H 5.46, N 10.85. Found C 65.22,
H 5.31, N 10.77.
3.2.6. N-Isopentyl-2-(2-oxo-4-phenylamino-2H-pyridin-1-yl)
acetamide (1f)
Yield 37%. Rf ¼ 0.43 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
3.3.4. N-Adamant-1-yl-2-(2-oxo-2H-pyridin-1-yl)acetamide (2e)
Yield 71%. Rf ¼ 0.41 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
DMSO-d6):
d
0.88 (d, 6H, J ¼ 6.6 Hz) 1.33e1.41 (m, 3H),
d 4.53 (s, 2H),
CHCl3): d 1.63 (s, 6H),1.88e2.02 (m,10H), 4.43 (s, 2H), 6.21e6.26 (m,
5.90e5.93 (m, 1H), 6.15 (s, 1H), 7.09e7.72 (m, 11H), 8.55 (bs, 1H).
Anal. Calcd. for C18H23N3O2, C 68.98, H 7.40, N 13.41. Found C 68.74,
H 7.44, N 13.33.
1H), 6.61 (d, 1H, J ¼ 9.7 Hz), 7.36e7.41 (m, 2H). Anal. Calcd. for
C17H22N2O2, C 71.30, H 7.74, N 9.78. Found 71.43, H 7.48, N 9.83.
3.3.5. N-Benzyl-2-(2-oxo-4-phenylamino-pyridin-1(2H)-yl)
acetamide (2f)
3.2.7. 2-(4-Diethylamino-2-oxo-2H-pyridin-1-yl)-N-isopentyl
acetamide (1g)
Yield 57%. Rf ¼ 0.46 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
Yield 30%. Rf ¼ 0.57 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
DMSO-d6):
d
4.27 (d, 2H, J ¼ 6.2 Hz), 4.43 (s, 2H), 5.70 (d, 1H,
CDCl3):
d
0.88 (d, 6H, J ¼ 6.6 Hz),1.18 (t, 6H, J ¼ 7.1 Hz),1.33e1.41 (m,
J ¼ 2.6 Hz) 5.92 (dd, 1H, J ¼ 7.5, 2.6 Hz), 7.02e7.39 (m, 10H), 8.56 (t,
1H, J ¼ 6.2 Hz). Anal. Calcd. for C20H19N3O2, C 72.05, H 5.74, N 12.60.
Found C 72.35, H 5.49, N 12.99.
2H), 1.53e1.64 (m, 1H), 3.17e3.24 (m, 2H), 3.28e3.35 (q,
4H, J ¼ 7.1 Hz), 4.42 (s, 2H), 5.57 (d, 1H, J ¼ 1.8 Hz), 5.85 (dd, 1H,
J ¼ 7.5, 1.8 Hz), 7.16 (dd, 1H, J ¼ 1.3, 7.5 Hz), 7.39 (bs, 1H). Anal. Calcd.
for C16H27N3O2, C 65.50, H 9.28, N 14.32. Found C 65.66, H 9.13, N
14.43.
3.3.6. 2-(2-Oxo-4-phenylamino-pyridin-1-(2H)-yl)-N-phenyl
acetamide (2g)
Yield 63%. Rf ¼ 0.31 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
3.2.8. 2-(4-Morpholin-4-yl-2-oxo-2H-pyridin-1-yl)-N-isopentyl
acetamide (1h)
DMSO): d 4.55 (s, 2H), 5.90e5.93 (m, 1H), 6.15 (s, 1H), 7.09e7.72 (m,
11H), 8.55 (bs, 1H). Anal. Calcd. for C19H17N3O2, C 71.46, H 5.37, N
13.16. Found C 71.43, H 5.57, N 13.11.
Yield 33%. Rf ¼ 0.49 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3):
d
0.87 (d, 6H, J ¼ 5.7 Hz), 1.32e1.39 (m, 2H), 1.52e1.61
(m, 1H), 3.16e3.26 (m, 6H), 3.78 (s, 4H), 4.43 (s, 2H), 5.73 (s, 1H),
5.97 (d, 1H, J ¼ 7.9 Hz), 7.22 (d, 1H, J ¼ 7.5). Anal. Calcd. for
3.3.7. N-(4-Methoxyphenyl)-2-(2-oxo-4-phenylamino-pyridin-1-
(2H)-yl)acetamide (2h)
C
16H25N3O3, C 62.52, H 8.20, N 13.67. Found C 62.31, H 8.44, N
Yield 67%. Rf ¼ 0.35 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
13.51.
DMSO-d6): d 3.70 (s, 3H), 4.52 (s, 2H), 5.80e5.91 (m, 1H), 6.17 (s,

K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
7
1H), 6.45e7.32 (m, 10H), 8.57 (bs, 1H). Anal. Calcd. (%) for
20H19N3O3, C 68.75, H 5.48, N 12.03. Found C 68.69, H 5.88, N 12.01.
supported by the fact that in the X-ray structure the OeN distance
is 2.9 A, indicative of a strong H-bond. Accordingly, the intervening
ꢀ
C
water molecule and the proper protonation state of D114 were
included in the docking experiments. The protonation and flip
states of the imidazole rings of the histidine residues were adjusted
together with the side chain amides of glutamine and asparagine
residues in an H-bonding network optimization process. Succes-
sively, the protein hydrogens only were minimized using the
Impref module of Impact with the OPLS_2005 force field. The initial
structures of 1b, 1f and 2h were created, modified and energy-
minimized with Schrödinger’s Maestro [26].
3.3.8. N-Adamant-1-yl-2-(2-oxo-4-phenylamino-2H-pyridin-1-yl)
acetamide (2j)
Yield 61%. Rf ¼ 0.22 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CD3OH):
d
1.71 (s, 6H), 1.99e2.09 (m, 10H), 5.94 (d, 1H, J ¼ 2.3), 6.11
(dd, 1H, J ¼ 7.6, 2.3 Hz), 6.11 (dd, 1H, J ¼ 7.6, 2.3 Hz), 8.60 (bs, 1H).
Anal. Calcd. for C23H27N3O2, C 73.18, H 7.21, N 11.13. Found C 73.28,
H 7.11, N 11.55.
3.4. General procedure for the synthesis of compounds 2d and 2i
3.5.2. Docking studies
For the synthesis of amides 2d and 2i, a suspension of acids 6
and 7 in dry CH2Cl2 was treated with N-methylmorpholine
(1.1 equiv.) and isobutylchloroformate (1.1 equiv.) at 0 ꢁC. After
30 min, a solution of 2-aminobenzophenone in dry CH2Cl2 was
added to the refluxing mixture and the resulting solution was
stirred at room temperature for 12 h. The organic layer was washed
with brine:HCl 1 N (3:1). The crude product was purified by column
chromatography (CHCl3/MeOH 95:5).
Compounds 1b, 1f and 2h were docked to the binding pocket of
5 subunit using GOLD, version 5.2 [28,29]. A radius of 20 A from
the b5 catalytic N-terminal threonine was used to direct site loca-
ꢀ
b
tion. For each of the genetic algorithm runs, a maximum of 100,000
operations were performed on a population of 100 individuals with
a selection pressure of 1.1. Operator weights for crossover, muta-
tion, and migration were set to 95, 95, and 10, respectively, as
recommended by the authors of the software. The distance for H-
ꢀ
bonding was set to 2.5 A, and the cutoff value for van der Waals
ꢀ
3.4.1. N-(2-Benzoylphenyl)-2-(2-oxo-2H-pyridin-1-yl)acetamide
(2d)
calculation was set to 4 A. The crystallographically determined
water molecule near b6-D114 was specified in GOLD by switching
Yield 55%. Rf ¼ 0.32 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
state settings to ‘toggle’ and orientation mode to ‘spin’. The
Goldscore-CS docking protocol [31] was adopted in this study. In
this protocol, the poses obtained with the original GoldScore
function are rescored and reranked with the GOLD implementation
of the ChemScore function [19,31e35]. To perform thorough and
unbiased search of the conformation space, each docking run was
allowed to produce 200 poses without the option of early termi-
nation, using standard default settings. The top solution obtained
after re-ranking of the poses with ChemScore was selected to
generate the proteasome/ligand complexes.
DMSO-d6):
d
5.12 (s, 2H), 6.19e6.24 (m, 1H), 6.38 (d, 1H, J ¼ 8.3 Hz),
7.41e7.78 (m, 8H), 8.03e8.09 (m, 1H), 9.58e9.61 (m, 1H). Anal.
Calcd. (%) for C20H16N2O3, C 72.28, H 4.85, N 8.43. Found C 72.48, H
4.56, N 8.74.
3.4.2. N-(2-Benzoylphenyl)-2-(2-oxo-4-phenylamino-2H-pyridin-
1-yl)acetamide (2i)
Yield 53%. Rf ¼ 0.58 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CD3OH):
d
4.55 (s, 2H), 5.91e5.93 (m, 1H), 6.15 (dd, 1H, J ¼ 7.5,
2.2 Hz), 7.09e7.72 (m, 14H), 8.12 (d, 1H, J ¼ 7.9 Hz), 8.69 (bs, 1H).
Anal. Calcd. (%) for C26H21N3O3, C 73.74, H 5.00, N 9.92. Found C
73.94, H 4.88, N 9.99.
3.6. Pharmacology
3.6.1. In vitro 20S proteasome inhibition assays
3.5. Computational chemistry
Human 20S proteasome was obtained from Biomol GmbH,
Hamburg, Germany. The three distinct proteolytic activities of the
20S proteasome were measured by monitoring the hydrolysis of
the peptidyl 7-amino-4-methyl coumarin substrates (all obtained
from Bachem) Suc-Leu-Leu-Val-Tyr-AMC, Boc-Leu-Arg-Arg-AMC,
and Cbz-Leu-Leu-Glu-AMC for ChT-L, T-L and PGPH activity of the
enzyme, respectively. Fluorescence of the product AMC of the
substrates’ hydrolyses was measured using an Infinite 200 PRO
microplate reader (Tecan, Männedorf, Switzerland) at 30 ꢁC with a
380 nm excitation filter and a 460 nm emission filter. The pre-
liminary screening for the inhibition of the three proteolytic ac-
Molecular modeling and graphics manipulations were per-
formed using Maestro 9.2 (Schrödinger) [26] and UCSF-CHIMERA
software packages [27], running on an E4 Computer Engineering
E1080 workstation provided of an Intel Core i7-930 Quad-Core
processor. GOLD 5.2 [28,29] was used for all docking calculations.
Figures were generated using Pymol 1.0 [30].
3.5.1. Protein and ligands preparation
Coordinates for the chymotrypsin-like b5 subunit derived from
the X-ray crystal structure of the yeast 20S proteasome determined
at 2.8 A resolution (PDB ID: 2F16) were employed for the automated
tivities of the 20S proteasome was performed at 20
concentrations using an equivalent amount of DMSO as a negative
control. Compounds showing at least 40% inhibition at 20 M were
subjected to detailed assays. The dissociation constants Ki of the
non covalent complex E$I were obtained from progress curves
(10 min) at various concentrations of inhibitor by fitting the slopes
of the progress curves to a 4 parameter IC50 equation, and correc-
tion to zero substrate concentration from Ki ¼ IC50/(1 þ [S] Kꢀm1).
The Km values were determined in separate experiments: ChT-L
mM inhibitor
ꢀ
docking studies [23]. The protein setup was carried out by Protein
Preparation Wizard in Maestro. Hydrogen atoms were added to the
protein consistent with the neutral physiologic pH. Arginine and
lysine side chains were considered as cationic at the guanidine and
ammonium groups, and the aspartic and glutamic residues were
considered as anionic at the carboxylate groups. The crystal struc-
ture of 20S/bortezomib complex revealed one well-defined water
molecule in proximity to D114Og, which coordinates a tight H-
m
activity with Suc-Leu-Leu-Val-Tyr-AMC 13
with Cbz-Leu-Leu-Glu-AMC 53 M.
mM, and PGPH activity
bonding network, interacting with
b
6-D114Og,
b
5-A49N and
b5-
m
A50N of the protein and with the C]O oxygen of bortezomib
[23]. Moreover, one of the pyrazine nitrogens of bortezomib was
3.6.2. Assaying the chymotryptic activity of the 20S proteasome
Human 20S proteasome was incubated at 30 ꢁC at a final con-
centration of 0.004 mg mLꢀ1 with test compound present at vari-
able concentrations. The reaction buffer consisted of 50 mM Tris pH
found to interact via a direct H-bond with the protonated
In fact, it has been observed that the pKa of pyrazine is approxi-
mately 1.0 and thus 6-D114 is most likely protonated. This is
b6-D114.
b

8
K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
[8] a) G. Bonuccelli, F. Sotgia, F. Capozza, E. Gazzerro, C. Minetti, M.P. Lisanti,
Localized treatment with a novel FDA-approved proteasome inhibitor blocks
the degradation of dystrophin and dystrophin-associated proteins in mdx
mice, Cell Cycle 6 (2007) 1242e1248;
7.5, 10 mM NaCl, 25 mM KCl, 1 mM MgCl2, 0.03% SDS, and 5% DMSO.
Product release from substrate hydrolysis (75
continuously over a period of 10 min.
mM) was monitored
b) B.A. Azakir, S. Di Fulvio, J. Kinter, M. Sinnreich, Proteasomal inhibition re-
stores biological function of mis-sense mutated dysferlin in patient-derived
muscle cells, J. Biol. Chem. 287 (2012) 10344e10354.
3.6.3. Assaying the tryptic activity of the 20S proteasome
Human 20S proteasome was incubated at 30 ꢁC at a final con-
[9] R.T. Jagoe, A.L. Goldberg, What do we really know about the ubiquitinepro-
teasome pathway in muscle atrophy? Curr. Opin. Clin. Nutr. Metab. Care 4
(2001) 183e190.
centration of 0.0025 mg mLꢀ1 with test compound present at
20
mM. The reaction buffer consisted of 50 mM Tris buffer pH 7.4,
[10] a) C. Lindenthal, N. Weich, Y.S. Chia, V. Heussler, M.Q. Klinkert, The protea-
some inhibitor MLN-273 blocks exoerythrocytic and erythrocytic develop-
ment of Plasmodium parasites, Parasitology 131 (Part 1) (2005) 37e44;
b) H. Li, E.L. Ponder, M. Verdoes, K.H. Asbjornsdottir, E. Deu, L.E. Edgington,
J.T. Lee, C.J. Kirk, S.D. Demo, K.C. Williamson, M. Bogyo, Validation of the
proteasome as a therapeutic target in Plasmodium using an epoxyketone
inhibitor with parasite-specific toxicity, Chem. Biol. 19 (2012) 1535e1545.
[11] a) Z. Li, C.B. Zou, Y. Yao, M.A. Hoyt, S. McDonough, Z.B. Mackey, P. Coffino,
C.C. Wang, An easily dissociated 26S proteasome catalyzes an essential
ubiquitin-mediated protein degradation pathway in Trypanosoma brucei,
J. Biol. Chem. 277 (2002) 15486e15498;
50 mM NaCl, 0.5 mM EDTA, 0.03% SDS, and 7.5% DMSO. Product
release from substrate hydrolysis (85
uously over a period of 10 min.
mM) was monitored contin-
3.6.4. Assaying the post glutamyl-peptide hydrolyzing activity of
the 20S proteasome
Human 20S proteasome was incubated at 30 ꢁC at a final con-
centration of 0.004 mg mLꢀ1 with the test compound present at
variable concentrations. The reaction buffer consisted of 50 mM
Tris buffer pH 7.5 containing 25 mM KCl, 10 mM NaCl, 1 mM MgCl2,
0.03% SDS, 5% DMSO. Product release from substrate hydrolysis
b) D. Steverding, The proteasome as a potential target for chemotherapy of
African trypanosomiasis, Drug Dev. Res. 68 (2007) 205e212;
c) D. Steverding, X. Wang, B.C. Potts, M.A. Palladino, Trypanocidal activity of
beta-lactone-gamma-lactam proteasome inhibitors, Planta Med. 78 (2012)
131e134.
(80 mM) was monitored continuously over a period of 10 min.
[12] G. Lin, D. Li, L.P. de Carvalho, H. Deng, H. Tao, G. Vogt, K. Wu, J. Schneider,
T. Chidawanyika, J.D. Warren, H. Li, C. Nathan, Inhibitors selective for myco-
bacterial versus human proteasomes, Nature 461 (2009) 621e626.
[13] P. Beck, C. Dubiella, M. Groll, Covalent and non-covalent reversible protea-
some inhibition, Biol. Chem. 393 (2012) 1101e1120.
[14] S. Arastu-Kapur, J.L. Anderl, M. Kraus, F. Parlati, K.D. Shenk, S.J. Lee,
T. Muchamuel, M.K. Bennett, C. Driessen, A.J. Ball, et al., Nonproteasomal
targets of the proteasome inhibitors bortezomib and carfilzomib: a link to
clinical adverse events, Clin. Cancer Res. 17 (2011) 2734e2743.
[15] (a) R. Ettari, L. Tamborini, I.C. Angelo, N. Micale, A. Pinto, C. De Micheli,
P. Conti, Inhibition of rhodesain as a novel therapeutic modality for human
African trypanosomiasis, J. Med. Chem. 56 (2013) 5637e5658;
3.6.5. Assays for bovine pancreatic
a-chymotrypsin inhibition
The enzyme (250
m
g mLꢀ1) was incubated at 20 ꢁC with test
compound. The reaction buffer consisted of 50 mM Tris buffer pH
8.0 containing 100 mM NaCl and 5 mM EDTA and 7.5% DMSO.
Product release from substrate hydrolysis (75 mM final concentra-
tion, Suc-Leu-Leu-Val-Tyr-AMC from Bachem) was determined
over a period of 10 min.
(b) R. Ettari, M. Zappalà, N. Micale, G. Grazioso, S. Giofrè, T. Schirmeister,
S. Grasso, Peptidomimetics containing a vinyl ketone warhead as falcipain-2
inhibitors, Eur. J. Med. Chem. 46 (2011) 2058e2065;
3.6.6. Assays for cathepsin B inhibition
Assays with cathepsins B were performed as described previ-
(c) F. Bova, R. Ettari, N. Micale, C. Carnovale, T. Schirmeister, C. Gelhaus,
M. Leippe, S. Grasso, M. Zappalà, Constrained peptidomimetics as anti-
plasmodial falcipain-2 inhibitors, Bioorg. Med. Chem. 18 (2010) 4928e4938;
(d) N. Micale, R. Ettari, T. Schirmeister, A. Evers, C. Gelhaus, M. Leippe,
M. Zappalà, S. Grasso, Novel 2H-isoquinolin-3-ones as antiplasmodial
falcipain-2 inhibitors, Bioorg. Med. Chem. 17 (2009) 6505e6511;
(e) R. Ettari, M. Zappalà, N. Micale, T. Schirmeister, C. Gelhaus, M. Leippe,
A. Evers, S. Grasso, Synthesis of novel peptidomimetics as inhibitors of pro-
tozoan cysteine proteases falcipain-2 and rhodesain, Eur. J. Med. Chem. 45
(2010) 3228e3233.
ously [36]. Cbz-Phe-Arg-AMC was used as substrate (80
Km value used to calculate Ki values from IC50 values was 150
m
M). The
mM.
Acknowledgments
This work was financially supported by the Ministero dell’Is-
truzione, dell’Università e della Ricerca Scientifica e Tecnologica
[MIUR-PRIN2010-2011, grant Nꢁ 2010W7YRLZ_003 (A.L.) and
2010W7YRLZ_004 (S.G.)]. We would like to thank the Deutscher
Akademischer Austausch Dienst (DAAD) (Vigoni project 2011/12)
for partial support of this work. T.S. thanks the Deutsche For-
schungsgemeinschaft (DFG) for financial support. R.E. acknowl-
edges the support of her postdoctoral fellowship from “Dote
Ricerca”: FSE, Regione Lombardia.
[16] (a) M. Groll, N. Gallastegui, X. Maréchal, V. Le Ravalec, N. Basse, N. Richy,
E. Genin, R. Huber, L. Moroder, J. Vidal, M. Reboud-Ravaux, 20S proteasome
inhibition: designing noncovalent linear peptide mimics of the natural
product TMC-95A, ChemMedChem 5 (2010) 1701e1705;
(b) E. Genin, M. Reboud-Ravaux, J. Vidal, Proteasome inhibitors: recent ad-
vances and new perspectives in medicinal chemistry, Curr. Top. Med. Chem.
10 (2010) 232e256.
[17] J. Kaffy, G. Bernadat, S. Ongeri, Non-covalent proteasome inhibitors, Curr.
Pharm. Des. 19 (2013) 4115e4130.
[18] R. Ettari, C. Bonaccorso, N. Micale, C. Heindl, T. Schirmeister, M.L. Calabrò,
S. Grasso, M. Zappalà, Development of novel peptidomimetics containing a
References
vinyl sulfone moiety as proteasome inhibitors, ChemMedChem
6 (2011)
1228e1237.
[1] M. Unno, T. Mizushima, Y. Morimoto, Y. Tomisugi, K. Tanaka, N. Yasuoka,
ꢀ
[19] N. Micale, R. Ettari, A. Lavecchia, C. Di Giovanni, K. Scarbaci, V. Troiano,
S. Grasso, E. Novellino, T. Schirmeister, M. Zappalà, Development of pepti-
domimetic boronates as proteasome inhibitors, Eur. J. Med. Chem. 64 (2013)
23e34.
T. Tsukihara, The structure of the mammalian 20S proteasome at 2.75 A res-
olution, Structure 10 (2002) 609e618.
[2] M. Groll, L. Ditzel, J. Lowe, M. Bochtler, H.D. Bartunik, R. Huber, Structure of
ꢀ
20S proteasome from yeast at 2.4 A resolution, Nature 386 (1997) 463e471.
[20] (a) R.T. Lum, M.G. Nelson, A. Joly, A.G. Horsma, G. Lee, S.M. Meyer, M.M. Wick,
S.R. Schow, Selective inhibition of the chymotrypsin-like activity of the 20S
proteasome by 5-methoxy-1-indanone dipeptide benzamides, Bioorg. Med.
Chem. Lett. 8 (1998) 209e214;
[3] P.M. Kloetzel, Antigen processing by the proteasome, Nat. Rev. Mol. Cell Biol. 2
(2001) 179e187.
[4] A. Sacco, M. Aujay, B. Morgan, A.K. Azab, P. Mais, Y. Liu, Y. Zhang, F. Azab,
H.T. Ngo, G.C. Issa, P. Quang, A.M. Roccaro, I.M. Ghobria, Chymotrypsin-like
activity of the proteasome leads to antitumor carfilzomib-dependent selective
inhibition of the activity in Waldenstrom’s macroglobulinemia, Clin. Cancer
Res. 17 (2011) 1753e1764.
(b) C. Garcìa-Echeverrìa, P. Imbach, D. France, P. Fürst, M. Lang, M. Noorani,
D. Scholz, J. Zimmermann, P. Furet, A new structural class of selective and
non-covalent inhibitors of the chymotrypsin-like activity of the 20S protea-
some, Bioorg. Med. Chem. Lett. 11 (2001) 1317e1319.
[5] (a) S. Meiners, A. Ludwig, V. Stangl, K. Stangl, Proteasome inhibitors: poisons
and remedies, Med. Res. Rev. 28 (2008) 309e327;
[21] H.R. Snyder, J.A. Kuck, J.R. Johnson, Organoboron compounds, and the study of
reaction mechanisms. Primary aliphatic boronic acids, J. Am. Chem. Soc. 60
(1938) 105e111.
[22] J.R. Johnson, M.G. Van Campen Jr., O. Grummit, Organoboron compounds. II.
The reducing action of some organoboronic acids, J. Am. Chem. Soc. 60 (1938)
111e115.
[23] M. Groll, C.R. Berkers, H.L. Ploegh, H. Ovaa, Crystal structure of the boronic
acid-based proteasome inhibitor bortezomib in complex with the yeast 20S
proteasome, Structure 14 (2006) 451e456.
b) E.M. Huber, M. Groll, Inhibitors for the immuno- and constitutive protea-
some: current and future trends in drug development, Angew. Chem. Int. Ed.
51 (2012) 8708e8720.
[6] J. Wang, M.A. Maldonado, The ubiquitineproteasome system and its role in
inflammatory and autoimmune diseases, Cell. Mol. Immunol. 3 (2006) 255e
261.
[7] B. Skaug, X. Jiang, Z.J. Chen, The role of ubiquitin in NFkappaB regulatory
pathways, Annu. Rev. Biochem. 78 (2009) 769e796.

K. Scarbaci et al. / European Journal of Medicinal Chemistry 76 (2014) 1e9
9
[24] M. Groll, R. Huber, B.C. Potts, Crystal structures of salinosporamide A (NPI-
0052) and B (NPI-0047) in complex with the 20S proteasome reveal important
F. Loiodice, New 2-aryloxy-3-phenyl-propanoic acids as PPAR
nists with improved potency and reduced adverse effects on skeletal muscle
function, J. Med. Chem. 52 (2009) 6382e6393.
a
/
g
dual ago-
consequences of
b-lactam ring opening and a mechanism for irreversible
binding, J. Am. Chem. Soc. 128 (2006) 5136e5141.
[33] C. La Motta, S. Sartini, L. Mugnaini, S. Salerno, F. Simorini, S. Taliani,
A.M. Marini, F. Da Settimo, A. Lavecchia, E. Novellino, L. Antonioli, M. Fornai,
C. Blandizzi, M. Del Tacca, Exploiting the pyrazolo[3,4-d]pyrimidin-4-one ring
system as a useful template to obtain potent adenosine deaminase inhibitors,
J. Med. Chem. 52 (2009) 1681e1692.
[34] L. Porcelli, F. Gilardi, A. Laghezza, L. Piemontese, N. Mitro, A. Azzariti, F. Altieri,
L. Cervoni, G. Fracchiolla, M. Giudici, U. Guerrini, A. Lavecchia, R. Montanari,
C. Di Giovanni, A. Paradiso, G. Pochetti, G.M. Simone, P. Tortorella, M. Crestani,
F. Loiodice, Synthesis, characterization and biological evaluation of
ureidofibrate-like derivatives endowed with peroxisome proliferator-
activated receptor activity, J. Med. Chem. 55 (2012) 37e54.
[35] M.G. Perrone, P. Vitale, P. Malerba, A. Altomare, A. Lavecchia, C. Di Giovanni,
E. Novellino, A. Scilimati, Core ring nature in diarylheterocycle COX-1 in-
hibitors, ChemMedChem 7 (2012) 629e641.
[36] (a) R. Vicik, M. Busemann, C. Gelhaus, N. Stiefl, J. Scheiber, W. Schmitz,
F. Schulz, M. Mladenovic, B. Engels, M. Leippe, K. Baumann, T. Schirmeister,
Aziridide-based inhibitors of cathepsin L: synthesis, inhibition activity, and
docking studies, ChemMedChem 1 (2006) 1126e1141;
[25] (a) E.J. Corey, W.D. Li, Total synthesis and biological activity of lactacystin,
omuralide and analogs, Chem. Pharm. Bull. 47 (1999) 1e10;
b) M. Nett, T.A. Gulder, A.J. Kale, C.C. Hughes, B.S. Moore, Function-oriented
biosynthesis of beta-lactone proteasome inhibitors in Salinispora tropica,
J. Med. Chem. 52 (2009) 6163e6167.
[26] Maestro, Version 9.2, Schrödinger, LLC, New York, NY, 2011.
[27] C.C. Huang, G.S. Couch, E.F. Pettersen, T.E. Ferrin, Chimera: an extensible
molecular modelling application constructed using standard components, Pac.
Symp. Biocomput. 1 (1996) 724. http://www.cgl.ucsf.edu/chimera.
[28] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997) 727e
748.
[29] GOLD, Version 5.2, The Cambridge Crystallographic Data Centre, Cambridge,
U.K., 2012.
[30] W.L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific LLC,
San Carlos, CA, USA, http://www.pymol.org/.
[31] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Improved
protein-ligand docking using GOLD, Proteins Struct. Funct. Genet. 52 (2003)
609e623.
[32] G. Fracchiolla, A. Laghezza, L. Piemontese, P. Tortorella, F. Mazza, R. Montanari,
G. Pochetti, A. Lavecchia, E. Novellino, S. Pierno, D. Conte Camerino,
(b) R. Ettari, E. Nizi, M.E. Di Francesco, M.A. Dude, G. Pradel, R. Vicik,
T. Schirmeister, N. Micale, S. Grasso, M. Zappalà, Development of peptido-
mimetics with a vinyl sulfone warhead as irreversible falcipain-2 inhibitors,
J. Med. Chem. 51 (2008) 988e996.