Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.12.053

This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the treatment of hematological malignancies. In particular, we focused our efforts on the design of noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of drawbacks and side-effects related to irreversible inhibition. Among all the synthesized compounds, we identified a panel of active inhibitors with K_i values towards one or two chymotrypsin-like activities of proteasome (β5c) and immunoproteasome (β5i and β1i subunits) in the low micromolar range. Docking studies suggested a unique binding mode of the molecules in the catalytic site of immunoproteasome proteolytic subunits.

Full text of DOI:10.1016/j.bmcl.2017.12.053

Accepted Manuscript
Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent
proteasome and immunoproteasome inhibitors
Rosanna Maccari, Roberta Ettari, Ilenia Adornato, Alexandra Naß, Gerhard
Wolber, Alessandra Bitto, Federica Mannino, Federica Aliquò, Giuseppe
Bruno, Francesco Nicolò, Santo Previti, Silvana Grasso, Maria Zappalà, Rosaria
Ottanà
PII:
S0960-894X(17)31227-1
https://doi.org/10.1016/j.bmcl.2017.12.053
BMCL 25509
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
7 November 2017
21 December 2017
23 December 2017
Please cite this article as: Maccari, R., Ettari, R., Adornato, I., Naß, A., Wolber, G., Bitto, A., Mannino, F.,
Aliquò, F., Bruno, G., Nicolò, F., Previti, S., Grasso, S., Zappalà, M., Ottanà, R., Identification of 2-
thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.053
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Bioorganic & Medicinal Chemistry Letters
Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent
proteasome and immunoproteasome inhibitors
Rosanna Maccaria, Roberta Ettaria,*, Ilenia Adornatoa, Alexandra Naßb, Gerhard Wolberb, Alessandra
Bittoc, Federica Manninoc, Federica Aliquòc, Giuseppe Brunod, Francesco Nicolòd, Santo Previtia, Silvana
Grassoa, Maria Zappalàa, Rosaria Ottanàa
aDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Polo Annunziata, Viale SS. Annunziata, 98168
Messina (Italy).
bDepartment of Pharmaceutical and Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, (Germany).
cDepartment of Clinical and Experimental Medicine, University of Messina, Via C. Valeria, 98125 Messina, (Italy).
dDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d'Alcontres, 31, 98166
Messina (Italy).
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-
4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the
treatment of hematological malignancies. In particular, we focused our efforts on the design of
noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of
drawbacks and side-effects related to irreversible inhibition. Among all the synthesized
compounds, we identified a panel of active inhibitors with Ki values towards one or two
chymotrypsin-like activities of proteasome (β5c) and immunoproteasome (β5i and β1i
subunits) in the low micromolar range. Docking studies suggested a unique binding mode of
the molecules in the catalytic site of immunoproteasome proteolytic subunits.
Keywords:
Proteasome
Immunoproteasome
5-Arylidene-2-thioxoimidazolidin-4-ones
Non-covalent inhibitors
Docking studies
2017 Elsevier Ltd. All rights reserved.
The ubiquitin-proteasome system (UPS) is a key pathway
involved in the intracellular protein turnover in eukaryotic cells. ,
In normal cells, proteasome proteolytic activity is responsible for
a regular cell cycle progression; thus, defects in UPS can lead to
uncontrolled cell proliferation and tumor development. 20S
proteasome core shows a barrel-like structure, composed of four
stacked rings, each containing seven subunits, i.e. α1–α7 in the
two outer rings, β1–β7 in the two inner rings.1 The proteolytic
activities are located into β1, β2, and β5 subunits, which are
responsible for the caspase-like (C-L), trypsin-like (T-L), and
chymotrypsin-like (ChT-L) activities, respectively.2
It has been demonstrated that i20S is highly expressed in cells
of hematopoietic origin, including multiple myeloma (MM) cells;
thus, the inhibition of i20S could be a promising strategy to treat
MM.6 In this regard, Parlati et al clearly assessed that selective
inhibition of single β5i or β5c subunit is insufficient to produce
an antitumor response, whereas inhibition of both β5i and β5c is
required to induce an antitumor effect in MM, non-Hodgkin
lymphoma, and leukemia cells, without causing cytotoxicity in
non-transformed cells.7 At present, both non-selective and
selective immunoproteasome inhibition has been validated as
potential strategy for the treatment of MM.8
In addition to the constitutive proteasome (c20S), vertebrates
In the last years, our research group has been widely involved
in the development of novel peptidomimetics as inhibitors of the
ChT-L activity of constitutive proteasome.9-15 In particular, we
focused our efforts on the design of noncovalent inhibitors,
which might be a promising therapeutic option because
potentially devoid of drawbacks and side-effects related to
irreversible inhibition.
Starting from these considerations, and assuming as lead
compounds oxathiazolones HT1171 and HT2004 (Fig. 1), which
were reported to irreversibly inhibit both β5i and β5c subunits
with a preference for β5i,16 we planned to assess the inhibitory
activity of a panel of 3-aryl-2-thioxoimidazolidin-4-ones 1-2 and
(5Z)-5-arylidene-3-aryl-2-thioxoimidazolidin-4-ones 3a-3i (Fig.
1) against constitutive proteasome and immunoproteasome.
possess
a
specialized form of proteasome, named
immunoproteasome (i20S), which is predominantly expressed in
lymphocytes and monocytes, and is responsible for the regulation
of major histocompatibility complex (MHC) class I antigen
presentation.3 Under the stimuli of IFN-γ and TNF-α, the
constitutive subunits β1c, β2c and β5c are replaced by the newly
formed immuno counterparts β1i, β2i and β5i.4 β2i and β5i
subunits maintain the same substrate specificity of β2c and β5c
ones; on the contrary, β1i mainly performs a ChT-L activity
whereas its caspase-like activity is reduced to background levels.5
________________
 Corresponding author. Tel.: +39-090-6766554; fax: +39-090-
6766402; e-mail: rettari@unime.it

The selected molecules are endowed with a nonpeptide
structure that gives them a great stability in solution and, in
addition, could be promising as potential immunoproteasome
inhibitors because they possess structural features that make them
suitable for the binding to i20S. In fact, the i20S subunits
responsible for ChT-L activity show a strong preference for
bulky hydrophobic groups able to fit into the S1 pocket, in
particular aromatic for β5i and branched for β1i; conversely, both
ChT-L activities have the same preference for small polar groups
able to establish key interactions with their S3 site.
Fig. 2. ORTEP view of 5-(4-phenoxybenzylidene)-3-phenyl-2-
thioximidazolidin-4-one 3h along the crystallographic b-axis evidencing the
H-interactions and the numbering scheme. Displacement ellipsoids are drawn
at the 50% probability level while hydrogen size is arbitrary.
Compounds 1, 2 and 3a-3i were tested for their inhibitory
properties on 20S immunoproteasome and on 20S proteasome
isolated from human spleen and from human erythrocytes,
respectively, using an appropriate fluorogenic substrate for each
proteolytic activity (Suc-Leu-Leu-Val-Tyr-AMC for β5i and β5c;
Boc-Leu-Arg-Arg-AMC for β2i and β2c; Ac-Pro-Ala-Leu-AMC
for β1i and Z-Leu-Leu-Glu-AMC for β1c). First, compounds
underwent a preliminary screening on each proteolytic subunit at
50 µM. An equivalent volume of DMSO was used as a negative
control, and MG-132, Z-Leu-Leu-Leu-al, a reversible inhibitor of
both proteasome and immunoproteasome, as positive control.
Compounds able to inhibit the enzymatic activity by more
than 60% were characterized in detail: continuous assays were
thus performed (progress curve method, at seven different
concentrations, ranging from those that minimally inhibited to
those that fully inhibited the immunoproteasome or the
proteasome subunit) to determine the Ki values reported in Table
1. All compounds were shown to inhibit immunoproteasome and
proteasome in a reversible manner, as demonstrated by the
analysis of the progress curves at seven different concentrations
(see e.g. 3e tested against β5c subunit, Fig.3). As a matter of fact,
after an incubation time of 10 min (Fig. 3a) and 30 min (Fig. 3b)
the obtained linear progress curves put in evidence a time-
independent inhibition. On the contrary, in time-dependent
inhibition the progress curves follow an exponential equation.19
Reversibility of inhibition was also confirmed by measuring the
recovery of enzymatic activity after dilution of the enzyme–
inhibitor complex with assay buffer (see Supplementary data).
The 3-aryl-2-thioxoimidazolidin-4-ones 1 and 2 did not pass
the initial screening, whereas the insertion of the 5-arylidene
moiety generally resulted in consistent inhibitory effects. In the
series of (5Z)-5-arylidene-3-aryl-2-thioxoimidazolidin-4-ones 3,
we observed that the presence of a methoxy group at the position
3 of the arylidene moiety (compound 3a) led to similar binding
affinity towards both β5 subunits of the constitutive and
immunoproteasomes (Ki =17.5 µM and 17.9 µM for β5i and β5c,
respectively).
Fig. 1. Structures of model and target compounds 1, 2 and 3a-3i.
3-Aryl-2-thioxoimidazolidin-4-ones 1 and 2 were synthesized
by reacting glycine with phenyl- or 1-naphthyl-isothiocyanate.
The subsequent Knoevenagel condensation of compounds 1 and
2 with the appropriate arylaldehyde, in the presence of piperidine
as a base, afforded (5Z)-3-aryl-5-arylidene-2-thioxoimidazolidin-
4-ones 3a-3i in high yields (Scheme 1).
The structures of compounds 1-3 were confirmed by
analytical and spectroscopic data (1H and 13C NMR) and by X-
ray diffraction study (see Supplementary data).
Scheme 1. Reagents and conditions: i, EtOH/H2O, Δ. ii, arylaldehyde,
piperidine, EtOH, Δ.
Conversely, the presence of a methoxy group at the position 4
of the arylidene portion allowed us to obtain a moderately
selective β5i inhibitor (i.e. 3b, Ki =10.9 µM); the presence of a
methylthio group in the same position (i.e. compound 3f) led to a
loss of potency towards the β5i subunit while the inhibition of
subunits β2i and β2c was favoured (Table 1).
X-ray crystallographic studies of 5-(4-phenoxybenzylidene)-
3-phenyl-2-thioximidazolidin-4-one 3h (CCDC # 1811484),
selected as representative, unambiguously attributed the Z
configuration at the chiral axis of derivatives 3 (Figure 2). This is
1
consistent with the presence of only one set of signals in H
NMR spectra, which indicated that, in the reported experimental
conditions, compounds 3 were obtained as the most stable Z
geometric isomers, similarly to previously reported 5-arylidene
substituted 2,4-thiazolidindiones17 and 2-phenylimino-4-
thiazolidinones.18

Table 1.
Activity on proteasome and immunoproteasome core-particles of 2-thioxoimidazolidin-4-ones 1, 2 and 3a-3i.
% of inhibition at 50 µM or Ki (µM)
R
R’
β1c
β2c
β5c
β1i
β2i
β5i
Comp.
-
-
32%
47%
52%
56%
54%
28%
58%
59%
n.i.
n.i.
47%
n.i.
n.i.
53%
1
-
-
38%
36%
35%
32%
37%
29%
20.0±1.1
n.i.
n.i.
6%
n.i.
n.i.
2
OCH3
H
H
17.9±0.3
45%
34%
18%
17.5±3.2
10.9±0.9
11.5±0.2
2.35±1.16
2.01±0.17
50%
3a
OCH3
OH
OCH3
OCH3
SCH3
H
57%
29%
3b
OCH3
OH
OCH3
H
23.6±1.6
45%
5%
6.71±0.58
45%
3c
6.53±0.91
2.64±0.63
n.i.
3d
13.6±0.9
28%
50%
3e
4.38±0.37
32%
3f
OPh
H
39%
17%
56%
3g
OPh
OPh
-
n.i.
5%
13%
n.i.
53%
58%
3h
-
9.64±0.98
22.1±3.3
57%
5.52±0.23
20%
n.i.
5.57±1.23
0.001±0.0002
56%
3i
--
0.004±0.001
0.13±0.01
0.07±0.01
MG-132
a)
b)
18000
2600
2400
2200
2000
1800
1600
1400
1200
1000
16000
14000
12000
10000
8000
F
F
6000
4000
0
200
400
time (seconds)
600
0
200 400 600 800 10001200140016001800
time (seconds)
Fig. 3. Progress curves of substrate hydrolysis in the presence of the inhibitor 3e tested against β5c subunit after an incubation time of 10 min (Fig. 3a) and 30
min (Fig. 3b). F = fluorescence units. Inhibitor concentrations (from top to bottom): 0, 0.1, 0.5, 1, 5, 10, 25, 50 µM.
Overall, compounds bearing two polar groups on the arylidene
moiety were active against two or three chymotrypsin-like
activities of immunoproteasome and proteasome, but with a
different trend of selectivity. 4-Hydroxy-3-methoxybenzylidene
substituted compound 3c, was proven to be active on both β5i
and β5c, with a 2-fold higher activity against β5i. Its 3-hydroxy-
4-methoxybenzylidene isomer 3d showed a preferential binding
(~20-fold) towards subunit β5i over β5c, whereas 3,4-
dimethoxybenzylidene analogue 3e targeted β5i, β1i and β5c,
with a ratio Ki β5c/ Ki β5i=~7 (Table 1).
Compounds 3d and 3e were the most active inhibitors,
endowed with Ki values in the low micromolar range. 3,4-
Dimethoxybenzylidene-substituted derivative 3e was shown to be
the most active compound of the series with Ki values of 2.01 µM
and 2.64 µM towards β5i and β1i, respectively.
naphthyl)-substituted derivative 3i showed appreciable inhibitory
effects against subunits β1c and β2i with Ki values in the low
micromolar range (Table 1). Overall, these inhibition results
suggest that the presence of small polar groups could strongly
contribute to stabilize the potential interactions with the S3
pocket of immuno subunits. However, docking studies reported
below indicated a different binding mode of the molecule in the
catalytic site.
The most interesting proteasome/immunoproteasome
inhibitors 3a-3e were selected to evaluate their growth inhibitory
activity on human multiple myeloma cells (MM.1R). These cells
are dexamethasone-resistant and show a poor response to
chemotherapy being representative of patients in the later stages
of the disease. Compounds 3a-3e and MG132 were tested in
triplicate at different doses (1-10-20-40-80-100 µM) for 24 h and
the test was repeated 4 times. The cytotoxic effect of the
compounds was determined by MTT assay; untreated cells were
used as reference and assumed as the 100% of vital cells. MG132
was used as a positive control and showed an IC50 value of
14.4±1.0 µM. The IC50 values of compounds 3a-3e (Table 2)
The replacement of small polar substituents such as methoxy
or hydroxyl groups with a phenoxy moiety (compounds 3g-3i)
resulted in a general marked decrease of inhibitory potency. 3-
Phenoxy- and 4-phenoxy-substituted analogues 3g and 3h
showed poor or moderate inhibitory capability, whereas 3-(1-

ranged from 19.4±1.1 μM (compound 3b) to 40.7±1.2 μM
(compound 3d). The lack of a perfect correlation between
enzyme inhibition and cellular activity suggests that these
inhibitors could have additional targets at cellular level.
However, a partial and non-homogeneous cellular penetration
could not be ruled out.
poses with the arylidene moiety and its substituents faced
towards the catalytic Thr1 and the 3-aryl-2-thioxoimidazolidin-4-
one moiety stretched out along the line of the backbone of known
peptide inhibitors (see Figure 6 for orientation). The N-
substituent is located in the extension of the S3 pocket.
Figure 4A shows interactions of the selected binding pose of
compound 3a with the catalytic cavity of subunit β5c. The pose is
characterized by very good steric complementarity of the ligand
to the catalytic cavity as well as several hydrogen bonding and
hydrophobic interactions: the carbonyl group interacts with the
side chain of Ser129 of the β6 chain, while the imidazolidinone
NH shows an interaction with the backbone of Thr21.
Additionally, the 3-methoxy group on the arylidene moiety shows
a hydrogen bond to the side chain of the catalytic Thr1. Ala20,
Ala27 and Ala49 of the β5c chain are in proximity of
hydrophobic parts of the ligand and positively contribute to the
binding. The slightly higher activity of 3,4-dimethoxy-substituted
derivative 3e can be explained by the favorable electrostatic
complementarity of the additional 4-methoxy group with the side
chain of Thr21, thus expanding the good sterical
complementarity of 3a.
Table 2.
IC50values of tested compounds against MM.1R cells.
Comp.
IC50 (M)
25.9 ± 1.2
19.4± 1.1
20.2 ± 1.1
40.7 ± 1.2
36.7 ± 1.1
14.4± 1.0
3a
3b
3c
3d
3e
MG-132
Docking experiments in the active site cavity of β5c (Figure 4)
and β5i (Figure 5) subunits were carried out for compounds 3a,
3d and 3e. Overall, the investigated compounds showed binding
A
B
C
D
E
F
Fig. 4. Docking poses of compounds 3a, 3d and 3e in the β5 subunit of the human constitutive proteasome. Electrostatic interactions: hydrogen bonds depicted as
yellow lines (A, compound 3a; B, compound 3d; C, compound 3e). Shape complementarity: surface coloring by hydrophilicity (cyan)/ lipophilicity (sand) (D,
compound 3a; E, compound 3d; F, compound 3e).

A hydroxyl group at 3-position of the arylidene moiety,
however, leads to reduced steric complementarity of compound
3d as depicted in Figure 4B/E, as a consequence of the hydrogen
bonding interaction with Thr1. This also leads to the loss of the
favourable electrostatic complementarity of the 4-methoxy group
of the arylidene moiety. The combination of both effects could
explain the reduced activity of this compound compared to the
closely related compounds 3a and 3e.
Figure 5A shows interactions of compound 3a with the
immunoproteasome active site formed by the subunits β5i and
β6. The position in the binding site is similar to that found in the
corresponding constitutive proteasome pocket; however, in the
immunoproteasome, the exchange of Thr21 to Ser21 leads to
higher flexibility of the hydroxyl group at the protein side,
favouring a position closer to Thr1. This leads to a weakening of
the hydrogen bond formed in the constitutive proteasome with
Ser129 (Ser130 in the immunoproteasome) of chain β6 in favour
of the newly formed one between the hydroxyl group of Ser21
and the 3-methoxy group of the arylidene moiety of compound
3a. The similar steric fit and position in the binding site explain
the comparable activity of compound 3a against the β5 subunits
of both the constitutive and immunoproteasome.
pockets explored by our docking studies. Again, changes in the
amino acid sequence from β5c to β5i, namely Thr21Ser, and the
different position of the interacting Ala28 in the S3 pocket favour
the binding affinity of compound 3e towards immunoproteasome.
This is also due to the additional hydrogen bonding interaction
between the 4-carbonyl group and Ser130, as depicted in Figure
5C.
In the docking experiment, compound 3d again showed an
only slightly different binding pose characterized by several
interactions depicted in Figure 5B. Again, hydrogen bonding
interactions are formed with the backbone of Ser21 and the
sidechain of Thr1; moreover, the 4-methoxy and 3-hydroxyl
groups of the arylidene moiety establish additional hydrogen
bond interactions with the sidechain of Ser21 and the backbone
of Tyr169, respectively. The preferential binding of this
compound to the immunoproteasome is therefore mainly
introduced by the good fit of the arylidene substituents in close
proximity to the catalytic Thr1, which is not possible in the
constitutive proteasome due to reduced flexibility of Thr21
compared to Ser21.
Interestingly, the reported docking poses are able to explain
the obtained SAR, also disclosing novel interactions with respect
to those commonly reported in literature.3
Compound 3e also shows similar binding pose in both binding
A
B
C
D
E
F
Fig. 5. Docking poses of compounds 3a, 3d and 3e in the β5 subunit of the human immunoproteasome. Electrostatic interactions: hydrogen bonds depicted as
yellow lines (A, compound 3a; B, compound 3d; C, compound 3e). Shape complementarity: surface coloring by hydrophilicity (cyan)/ lipophilicity (sand) (D,
compound 3a; E, compound 3d; F, compound 3e).

Figure 6. Crystal structure of the β5 subunit of the immunoproteasome in complex with a humanized version of subunit β6 and the covalent inhibitor
carfilzomib; surface coloring by hydrophobicity (cyan)/ lipophilicity (sand).
In conclusion, in this work we tested a series of 3-aryl-5-arylidene-2-thioxoimidazolidin-4-one derivatives targeting human
proteasome and immunoproteasome, as potential targets for the treatment of hematological malignancies. We identified several
active inhibitors with Ki values in the low micromolar range towards one or two chymotrypsin-like activities of proteasome and
immunoproteasome.
With respect to conventional proteasome inhibitors, which normally give rise to an irreversible inhibition of the target enzyme,
(5Z)-3-aryl-5-arylidene-2-thioxoimidazolidin-4-ones 3 behaved as noncovalent inhibitors and might be devoid of all drawbacks and
side-effects related to the irreversible inhibition. Thus, also considering their drug-like profile (see Supplementary data), they could
be considered as a starting point for further optimization in order to identify more effective proteasome and immunoproteasome
inhibitors for the treatment of hematological malignancies.
Acknowledgements
This work was financially supported by the “Research & Mobility Program 2015”of the University of Messina.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/XXX/j.bmcl.XXXX
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Identification of 2-thioxoimidazolidin-4-one
derivatives as novel noncovalent
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proteasome and immunoproteasome inhibitors
Rosanna Maccaria , Roberta Ettaria,*, Ilenia Adornatoa, Alexandra Naßb, Gerhard Wolberb, Alessandra Bittoc,
Federica Manninoc, Federica Aliquòc, Giuseppe Brunod, Francesco Nicolòd, Santo Previtia, Silvana Grassoa,
Maria Zappalàa, Rosaria Ottanàa
aDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
Polo Annunziata, Viale SS. Annunziata,98168 Messina (Italy).
bDepartment of Pharmaceutical and Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str. 2+4,
14195 Berlin, (Germany).
cDepartment of Clinical and Experimental Medicine, University of Messina, Via C. Valeria, 98125 Messina,
(Italy).
dDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
Viale Ferdinando Stagno d'Alcontres, 31, 98166 Messina (Italy).
R
N
S
R
N
S
O
HN
O
HN
Ar
3a-h
R=C6H5 or 1-naphthyl
1, R = C6H5,
2, R = 1-naphthyl
20.