Development of novel amides as noncovalent inhibitors of immunoproteasomes

Article abstract of DOI:10.1002/cmdc.201900028

The development of immunoproteasome-selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with K_i values in the low micromolar or sub-micromolar ranges toward the b5i and/or b1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibi-tor with a K_i value of 21 nm against the single b1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.

Full text of DOI:10.1002/cmdc.201900028

Accepted Article
Title: Development of novel amides as non-covalent inhibitors of
immunoproteasome
Authors: Roberta Ettari, Carmen Cerchia, Santina Maiorana, Manuela
Guccione, Ettore Novellino, Alessandra Bitto, Silvana Grasso,
Antonio Lavecchia, and Maria Zappalà
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To be cited as: ChemMedChem 10.1002/cmdc.201900028
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10.1002/cmdc.201900028
ChemMedChem
FULL PAPER
Development of novel amides as non-covalent inhibitors of
immunoproteasome
Roberta Ettari,[a]^ Carmen Cerchia,[b]^ Santina Maiorana,[a] Manuela Guccione,[a] Ettore Novellino,[b]
Alessandra Bitto,[c] Silvana Grasso,[a] Antonio Lavecchia,*[b] Maria Zappalà*
[a]
[a]
[b]
[c]
Prof. R. Ettari 0000-0001-9020-2068, Dr. S. Maiorana, Dr. M. Guccione, Prof. S. Grasso 0000-0002-6368-0045, M. Zappalà 0000-0002-8942-797X
Departmentof Chemical, Biological, Pharmaceutical and Environmental Sciences
University of Messina
Viale Annunziata, 98168 Messina, Italy
E-mail: mzappala@unime.it
Dr. C. Cerchia Prof. E. Novellino 0000-0002-2181-2142, Prof. A. Lavecchia 0000-0002-2181-8026,
Department of Pharmacy, “Drug Discovery” Laboratory
University of NapoliFederico II
Via Domenico Montesano 49, 80131 Napoli, Italy
E-mail: antonio.lavecchia@unina.it
Prof. A. Bitto 0000-0001-9300-7532
Department of Clinical and Experimental Medicine
University of Messina
Via C. Valeria, 98125 Messina, Italy
^These authors contributed equally.
Abstract: The development of immunoproteasome-selective
inhibitors is currently a promising strategy to treat hematologic
malignancies, autoimmune and inflammatory diseases; in this
context we now report the design, synthesis, and biological
subunits are replaced by β1i, β2i, and β5i subunits, respectively.
Overall, substrate preference of β5i and β2i subunits overlaps
with that of their cCP analogues, whereas β1i subunit reduces
its caspase-like activity in favor of a chymotrypsin-like activity.[7]
High levels of immunoproteasome have been detected in a
wide number of inflammatory diseases, such as Crohn’s,
inflammatory bowel and ulcerative diseases or in a panel of
autoimmune diseases like rheumatoid arthritis or systemic lupus
erythematosus.[8] On the other hand, iCPs are overexpressed in
cells of hematopoietic origin, including multiple myeloma (MM)
cells, thus targeting iCPs could be a valuable strategy for the
treatment of this hematologic malignancy.[9-11] and, in this
context, several 5i and/or 1i immunoproteasome selective
inhibitors have been identified.[12-14]
Furthermore, in the last years, there have been some efforts
to generate non-covalent proteasome/immunoproteasome
inhibitors, thus providing important insights into the basic
concepts of non-covalent proteasome inhibitor design.[15-18]
In this context, our research group has been actively
involved in the development of novel 20S proteasome
inhibitors;[19-24] in particular, we identified a series of amides,[21-23]
some of which turned to be active against the ChT-L activity of
20S proteasome with Ki values in the submicromolar range. The
non-covalent binding mode of the most active inhibitors was
corroborated by docking simulations into the yeast 20S
proteasome crystal structure.
With the aim to identify new immunoproteasome inhibitors,
we first screened several amide derivatives, already synthesized
in our laboratories, against the three immune subunits, i.e. β1i,
β2i, β5i. Among the tested compounds, N-benzyl-2-(2-
oxopyridin-1(2H)-yl)acetamide (1) proved to selectively inhibit
β1i subunit with a Ki of 2.23 μM; therefore, it was selected as hit
compound to generate a series of analogues characterized by
structural variations at the N-substituent and at the methylene
linker between the pyridone scaffold and the amide function
(Figure 1). Pyridones are frequently incorporated as
peptidomimetic elements in protease inhibitors, including
cysteine or serine proteases, being a suitable surrogate for a
portion of the peptide or a convenient strategy to lock a defined
conformation of the peptide.[25-27]
evaluation of
immunoproteasome inhibitors. Noteworthy, the designed compounds
act as non-covalent inhibitors, which might be promising
a
new series of amide derivatives as
a
therapeutic option because of the lack of all drawbacks and side-
effects related to irreversible inhibition. Among all the synthesized
compounds, we identified a panel of active inhibitors with Ki values
in the low micromolar or submicromolar range towards β5i and/or β1i
subunits of immunoproteasome. Within the active compounds,
amide 7 proved to be the most potent and selective inhibitor with a Ki
21 nM against the sole β1i subunit. Docking studies allowed us to
clarify the binding mode of the molecules in the catalytic site of
immunoproteasome subunits.
Introduction
The 20S proteasome is the major non-lysosomal proteolytic
system in eukaryotic cells, and it plays a key role in the
degradation of most cellular proteins. Certain tumor cells are
strongly dependent on proteasomal function, which however is
at a normal level, while normal cells can better tolerate
impairment of proteasome function; accordingly, proteasome
inhibition in these cells represents a vulnerability that can be
exploited to selectively kill tumor cells.[1-2] For these reasons,
novel approaches of cancer therapy are based on proteasome
inhibition. [3-5]
The central catalytic core 20S of 26S proteasome shows a
barrel-like structure, with the two outer and the two inner rings
composed of seven different α- and β-subunits, respectively.
The catalytic subunits β1c, β2c and β5c, are responsible for the
caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-
L) activities of the proteasome, respectively.[6]
Besides
the
constitutive
proteasome
(cCP),
immunoproteasome (iCP) is a specialized form of vertebrates’
proteasome, mainly represented in lymphocytes and monocytes.
Under the stimuli of IFN-γ and TNF-α, β1c, β2c, and β5c
1
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10.1002/cmdc.201900028
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In particular, the amide group was functionalized with
hydrophobic aliphatic or aromatic substituents (compounds 2-5),
in agreement to the structural features of the S1 pockets of β1i
and β5i subunits that are large and hydrophobic, whereas the 2-
pyridone at the P3 site was kept unchanged due to the nature of
S3 subsite that is small and polar.[10] The glycine at P2 was
replaced with a β-alanine homologue (compounds 6-11), to
evaluate if a longer distance between the amide portion and the
pyridone scaffold could allow a better accommodation of these
moieties into S1 and S3 pocket, respectively. The glycine
residue at P2 was also replaced with
a phenylalanine
(compounds 12-18), or with the homologous homophenylalanine
(compounds 19-25), in order to explore the size of the S2 pocket
of the catalytic site of the immuno-subunits. With regard to the
stereochemistry, compounds 12-25 have been developed in
homochiral form with S absolute configuration, which appears to
be the preferred one.
Scheme 1. Reagents and conditions: (a) NaH, DMF, 0°C, 1h, N2; ethyl
bromoacetate or methyl 3-bromopropionate, r.t. 12h; (b) LiOH, MeOH, 0°C-r.t.,
6 h ; (c) DCM/DMF, 0°C, HOBt, EDC∙HCl, 10 min, then DIPEA and a suitable
amine, r.t., 12h.
Noteworthy, the designed compounds lack of the
electrophilic warhead and should act as non-covalent
proteasome inhibitors that, with respect to covalent inhibitors,
might be a promising alternative to use in therapy, because of
the lack of all drawbacks and side-effects related to irreversible
inhibition.[28]
The synthesis of the P2 fragments of compounds 12-25 was
achieved starting from the commercially available (R)-2-hydroxy-
3-phenyl propanoic acid methyl ester 31 and (R)-2-hydroxy-3-
phenyl butanoic acid ethyl ester 32, which were activated into
the more reactive methanesulfonates 33-34, by reaction with
mesyl chloride in the presence of triethylamine (Scheme 2).
Intermediates 33-34 were then condensed with the pyridone
scaffold 26 to give the esters 35-36, which were then converted
into the corresponding carboxylic acids 37-38 by alkaline
hydrolysis. Coupling reactions, carried out as reported in
Scheme 1, between the carboxylic acids 37-38 and the suitable
amines gave compounds 12-18 and 19-25 in high yields.
Figure 1.Structure of hit compound 1 and the new designed amide derivatives
2-25.
Results and Discussion
Chemistry
Scheme 2. Reagents and conditions: (a) MsCl, Et3N, dry CH2Cl2, r.t., 2h, N2;
(b) compound 26, NaH, dry DMF, 0°C-r.t., then 33 or 34, 12h, N2; (c) LiOH,
MeOH/H2O/dioxane (1:1:1), 0°C-r.t., 12h; (d) DMF, 0°C, HOBt, EDC∙HCl, 10
min, then DIPEA and a suitable amine, 0°C-r.t., 12h.
The synthesis of amides 2-11 was achieved according to our
previously reported procedure,[21] starting from 2(1H)-pyridone
26 that was N-alkylated with ethyl bromoacetate or methyl 3-
bromopropionate in the presence of NaH, to give esters 27-28.
These latter intermediates were converted into the
corresponding carboxylic acids 29-30 by alkaline hydrolysis with
LiOH. Coupling reactions between the carboxylic acids 29-30
and the suitable amines, in the presence of EDC∙HCl, HOBt, as
coupling reagents, and DIPEA as a base, gave the desired
amides 2-5 and 6-11 in good yields (Scheme 1).
Biological activity and docking studies
All the synthesized compounds were tested for their ability to
inhibit each one of the catalytic subunits of c20S and i20S, by
measuring the rate of hydrolysis of the appropriate fluorogenic
substrate (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). MG-132 (Z-Leu-Leu-Leu-al), a
reversible inhibitor of both proteasome and immunoproteasome,
was used as positive control.
2
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First, compounds underwent a preliminary screening on
each proteolytic subunit at 50 µM. 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.
The β-alanine derivatives 10 and 11, bearing a cyclohexyl
and an n-butyl substituent, on the contrary, targeted both the β1i
and β5i subunits.
When a Phe residue was introduced at P2 site (e.g.
compounds 12 and 13), the activity was switched on both the
constitutive and immuno-core particles (β5i and β5c), with a
strong preference for the constitutive core-particle (see e.g.
compound 12, Ki = 8.81 µM and 45.5 µM for β5c and β5i,
respectively).It is worth noting that, two the compounds bearing
a HomoPhe residue at the P2 site turned to be active on both
the ChT-L activities of i20S (i.e. 19) or against the sole β5i (i.e.
21).
Among all the tested compounds, some of them (i.e. 1, 7,
10-13, 19 and 21) turned to be active on the iCPs. Interestingly,
compounds 1 and 7, which were N-benzyl substituted amides
selectively inhibited the β1i subunit with
a
consistent
improvement of activity observed for the β-alanine derivative
with respect to the glycine derivative (Ki = 0.021 µM vs 2.23 µM).
Table 1. Activity on proteasome and immunoproteasome core-particles of compounds 1-25.
1-5
6-11
12-18
19-25
% of inhibition at 50 µM or Ki (µM)
Comp.
1
R
β1c
32%
24%
32%
36%
36%
23%
37%
24%
22%
23%
19%
15%
14%
10%
15%
5%
β2c
β5c
β1i
β2i
β5i
benzyl
21%
n.i.
n.i.
2.23±0.26
44%
17%
n.i.
n.i.
2
phenylethyl
phenylpropyl
cyclohexyl
n-butyl
n.i.
n.i.
3
n.i.
n.i.
n.i.
11%
10%
5%
11%
25%
n.i.
n.i.
4
n.i.
n.i.
22%
3%
5
n.i.
n.i.
n.i.
n.i.
6
phenyl
n.i.
25%
n.i.
n.i.
25%
7
benzyl
n.i.
0.021±0.002
27%
17%
8
phenylethyl
phenylpropyl
cyclohexyl
n-butyl
n.i.
3%
46%
9
n.i.
18%
12%
4%
37%
n.i.
15.17±0.63
5.74±0.63
14.29±3.0
45.5± 2.6
7.77±1.51
43%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
n.i.
2.92±0.87
3.09±1.06
23%
n.i.
n.i.
n.i.
phenyl
6%
8.81±1.11
3.02±0.29
20.4±2.2
50%
45%
48%
55%
n.i.
n.i.
benzyl
8%
19%
n.i.
phenylethyl
phenylpropyl
cyclohexyl
n-butyl
13%
12%
6%
10%
n.i.
25%
n.i.
45%
16%
n.i.
39%
n.i.
n.i.
30%
9%
n.i.
33%
i-pentyl
8%
n.i.
23%
30%
phenyl
n.i.
23%
26%
31%
33%
30%
31%
30%
5.9± 0.16
21%
n.i.
5.81±0.37
38%
benzyl
n.i.
n.i.
n.i.
phenylethyl
phenylpropyl
cyclohexyl
n-butyl
n.i.
n.i.
48%
14%
4%
6%
n.i.
3.85± 0.46
34%
n.i.
n.i.
36%
n.i
n.i.
40%
42%
n.i.
n.i.
29%
37%
i-pentyl
n.i.
17%
18%
6%
39%
[a] For Ki values, data represent the mean±SD of two independent determinations, each performed in duplicate. n.i.=no inhibition.
…
3
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To help interpret the structure–activity relationship (SAR)
data and elucidate the molecular mechanism of i20S inhibition of
the most active compounds 1, 7, 13 and 21, docking studies
were carried out using the GOLD Suite docking package[29] with
the X-ray crystal structure of the murine i20S in complex with the
epoxyketone inhibitor PR-957 (PDB: 3UNF).[10] This structure
was selected due to the high degree of amino acid sequence
identity (>90%) between mouse and human i20S subunits
around the respective catalytic sites. Moreover, the few non-
identical residues are located at the interface with the
concomitant adjacent subunits and are thus absent from the
catalytic sites. Furthermore, the crystal structure of PR-957
bound to β1i reveals two well-defined water molecules within the
S3 pocket, which coordinate a tight H-bond network between
β1i-A50N, β2i-S118Oγ and the backbone amide nitrogen and
carbonyl oxygen of the P3 alanine residue of PR-957.
Accordingly, the intervening water molecules were included in
the docking experiments.
V20, F31, L45, A49 and A52. The 1 backbone amide in engaged
in H-bonds with the NH group of S21 backbone and the carbonyl
oxygen of G47 backbone, whereas the carbonyl oxygen of the
pyridone ring accepts further H-bonds from A49N, S48Oγ and
A50N, these last two through one of the two intervening water
molecules located crystallographically in the PR-957 structure
and included in our model. Moreover, the second water
molecule forms a H-bond with the first one, while simultaneously
engaging the β2i-S11833Oγ, which is at the bottom of the S3
pocket and contributes to a further stabilization of the ligand into
the active site. Additional hydrophobic interactions occur
between the pyridone ring and the V20 and V27 side chains
located in proximity of S3 pocket of β1i subunit.
The β1i and β1c subunits strongly differ in their amino acid
lining in the unprimed substrate binding channel. The
hydrophobic active site surrounding of subunit β1i is replaced by
a more polar one in subunit β1c. In particular, the amino acid
substitutions V20T, F31T, L45R and A52T increase the polarity
and the size of the β1c S1 pocket. Therefore, the more
hydrophilic S1 pocket of subunit β1c opposes binding of the
hydrophobic P1 substituents. These findings are in agreement
with the poor inhibitory activity displayed by this series of
derivatives towards β1c.
Docking of the amide derivative 1 to the β1i subunit revealed
a binding mode similar to that of PR-957 (Fig. 2a), with an
almost identical positioning of the backbone amides, the P1
phenyl moieties adopting the same spatial arrangement and the
P3 alanine residue of PR-957 nicely overlapped with the
pyridone ring of 1 (Fig. 2b). The P1 benzyl substituent extends
deeply into the S1 pocket, forming hydrophobic contacts with
Figure 2. Binding modes of compounds 1 (A, yellow orange sticks) and 7 (C, lime green sticks) into the β1i (slate)/β2i (light pink) active site of mouse i20S,
represented as a ribbon model. Only amino acids located within 4 Å of the bound ligand are displayed and labelled. H-bonds discussed in the text are depicted as
dashed grey lines. The defined water molecules forming tight H-bonds to the protein are displayed as red spheres. S1-S3 specificity pockets are labeled. An
overlay of 1 (B, docked pose) and 7 (D, docked pose) with PR-957 (white sticks, X-ray crystal pose) is shown in the β1i/β2i active site of mouse i20S.
4
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Elongation of the linker between the pyridone ring and the
amide function from one to two methylene units yielded
compound 7, which showed the highest inhibitory potency and
selectivity towards β1i (Ki = 0.021 μM). As a result of the docking
of 7 into the β1i subunit, we obtained a complex showing an
overall binding mode similar to that of 1 (Fig. 2c,d), with the
ligand forming H-bonds with G47O, S21N and S21Oγ of β1i
through the amide group as well as the same pattern of
hydrophobic interactions within the S1 pocket. These results are
in consonance with the SAR data showing that compounds
bearing a benzyl (7), cyclohexyl (10) or n-butyl (11) residue at
P1 inhibit β1i much stronger than β1c, which confirms that the
S1 pocket of β1i is more hydrophobic than that of β1c.[7] In
addition, neither β1i nor β1c is inhibited by compounds featuring
a bulky phenylethyl (8) or phenylpropyl (9) substituent at P1, an
outcome that supports structural data displaying that β1i/c have
a smaller S1 pocket than β5i/c.[10]
The pyridone moiety of 7 fits into the S3 pocket formed by
S48, S118 and H114, which is beneficial for improving potency
and selectivity for β1i subunit (Fig. 2c). In particular, the pyridone
carbonyl oxygen forms a tight H-bond network with A49N,
S48Oγ and A50N of β1i through one water molecule and with
β2i-S118Oγ via the second water molecule. Additionally, the
aromatic pyridone core makes an edge-to-face π-stacking
interaction with the H114 imidazole ring in the subunit β2i.
Compared to β1c, the S3 pocket of β1i is characterized by the
amino acid replacements T22A, A27V as well as Y114H in the
neighbouring subunit β2i. These differences lead to a more size-
restricted and more hydrophilic S3 pocket in β1i compared to its
counterpart β1c.
A second series of derivatives was designed, in which a
residue of phenylalanine was used to replace the glycine P2
group. Among them, compound 13 was moderately active
against β5i (Ki = 7.77 μM) and displayed a fairly good potency
against β5c (Ki = 3.02 μM). As depicted in Fig. 3a,b, 13 exhibited
a binding mode in β5i similar to that of derivatives 1, 7 and PR-
957. The P1 benzyl substituent fits into the S1 pocket, the
backbone amide is stabilized by H-bonds with G47O, T21N and
T21Oγ of β5i and the pyridone group protrudes into the S3 site
with the carbonyl engaged in H-bonds with A49N and C48S. The
P2 phenylalanine moiety projects into the central core of the 20S
cavity, making little or no contacts with the β5i subunit. Docking
of 13 to the human c20S crystal structure[30] elucidates that the
P1 benzyl and the P3 pyridone ring fit into subunit β5c by
interacting with T21, A27, A49, S129 and D144 of the
neighbouring subunit β6 via an intervening water molecule. This
latter interaction stabilizes the ligand into the S3 pocket and
accounts for the enhanced potency of 13 for β5c.
Figure3. Binding modes of compounds 13 (A, pink sticks) and 21 (C, violet) into the β5i (aquamarine)/β6 (wheat) active site of mouse i20S, represented as a
ribbon model. Only amino acids located within 4 Å of the bound ligand are displayed and labelled. H-bonds discussed in the text are depicted as dashed grey
lines. S1-S3 specificity pockets are labeled. An overlay of 13 (B, docked pose) and 21 (D, docked pose) with PR-957 (white sticks, X-ray crystal pose) is shown in
the β5i/β6 active site of mouse immunoproteasome. Compound 21 overlaps with the X-ray crystal pose of PR-924 (slate sticks) bound to the humanized yeast
i20S (PDB: 5L5H).
5
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Noteworthy, substitution of the glycine residue at P2 with a
homophenylalanine residue yielded a series of derivatives in
which the activity against c20S subunits β1 and β5 was
completely lost. Among them, compound 21 showed selective
inhibitory activity against β5i (Ki = 3.85 μM). Docking of 21 into
the subunit β5i revealed a different binding mode in comparison
to 1 and 7 derivatives, with the P2 substituent protruding into the
S3 subsite instead of pointing toward the initially assumed S2
binding pocket (Fig. 3c). As a consequence, the pyridone
scaffold assumes a folded conformation, engaging a very weak
H-bond with β5i-G23N (distance of 4 Å).[31] The backbone amide
establishes a H-bonding network involving T21O and A49N. The
homophenylalanine residue deeply extends into the S3 subsite
making O-H/π interactions with the hydroxyl groups of S27 from
β5i and S129 from β6. The phenylethyl at P1 perfectly fits into
the spacious S1 pocket of β5i and is stabilized by a cation-
contact with K33, a sulfur–arene interaction with M45[32-33] and
by C-H/ interactions with the side chains of M31, K33, and A49.
Interestingly, the overlay of the docked pose of 21 with the β5i-
specific inhibitors PR-957 (PDB ID: 3UNF) and PR-924 bound to
the chimeric hβ5/hβ6 substrate binding channel (PDB ID:
5L5H)[34] reveals that 21 and PR-924 adopt a similar kinked
binding mode, with an identical positioning of the amide
scaffolds and a similar orientation of the P1 and P3 functions,
whereas PR-957 adopts a linear orientation (Fig. 3d). The
superior β5i-selectivity of 21 compared to ligands that target the
strictly conserved peptide binding sites seems to result from its
ability to exploit subpockets other than the substrate-binding
channels. Moreover, insights into the selectivity of 21 towards
i20S over c20S can be gained when considering amino acid
compositions of β5c and β5i S1 pockets. Although the S1
specificity pockets of both β5c and β5i are formed by the same
residues, the conformation of M45 in β5c is different from that in
β5i, thus resulting in peculiarly sized S1 specificity pockets.[7]
The observed selectivity of 21 can be rationalized by the
impaired accommodation of the phenylethyl moiety into the
smaller β5c S1 pocket because of the closed conformation of
M45.
Experimental Section
Materials & methods
Chemistry.
All reagents and solvents were purchased from commercial suppliers and
used without any further purification. Elemental analyses were performed
on a C. Erba Model 1106 Elemental Analyzer and the results are within
±0.4% of the theoretical values. Merck silica gel 60 F254 plates were
used for analytical TLC; column chromatography was carried out on
Merck silica gel (200–400 mesh). 1H-NMR and 13C-NMR spectra were
recorded on
a Varian 300 MHz NMR spectrometer operating at
frequencies of 300.13 and 75.47 MHz, or on a Varian 500 MHz
spectrometer operating at 499.74 and 125.73 MHz for H-NMR and 13C-
1
NMR spectra, respectively. The residual signal of the deuterated solvent
was used as internal standard. Chemical shifts are given in δ (ppm) and
coupling constants (J) in Hz. Splitting patterns are described as singlet
(s), broad singlet (bs), doublet (d), triplet (t), quartet (q), multiplet (m),
doublet of doublet (dd), or triplet of doublets (td). Compounds 27-30 were
synthesized as previously reported.[20, 23].
General procedures for the synthesis of amides 2-5 and 6-11
The coupling reactions between the carboxylic acid (29 or 30) (1 equiv.)
and the appropriate amines (1.5 equiv.) were carried out by dissolving
compound 29 or 30 in dry DMF, then cooling to 0°C and adding HOBt
(1.5 equiv.) and EDC∙HCl (1.5 equiv.). After 10 minutes, DIPEA (2 equiv.)
and the amine (1.5 equiv.) dissolved in DMF were added dropwise, and
the mixture was stirred at room temperature overnight. The organic layer
was washed with brine, dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude residue were purified by column
chromatography, using CHCl3/MeOH, 95:5 as eluent mixture.
2-(2-Oxopyridin-1(2H)-yl)-N-phenethylacetamide (2): yield: 90%. Rf =
0.32 (CHCl3/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ = 2.78 (t, J = 7.
1 Hz, 2H), 3.48 (q, J = 7.1 Hz, 2H), 4.49 (s, 2H), 6.25 (td, J = 6.6, 1.4 Hz,
1H), 6.60 (d, J = 9.1 Hz, 1H), 6.92 (bs, 1H), 7.12 (d, J = 7.1 Hz, 2H),
7.18-7.28 (m, 3H), 7.34-7.44 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 35.45, 40.80, 54.02, 107.02, 120.85, 126.42, 128.54, 128.66, 130.75,
137.98, 140.50, 162.81, 167.25 ppm. Elemental analysis: calcd for
C15H16N2O2: C 70.29, H 6.29, N10.93; found: C 70.11, H5.98, N 11.17.
2-(2-Oxopyridin-1(2H)-yl)-N-(3-phenylpropyl)acetamide (3): yield:
85%. Rf = 0.50 (CHCl3/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ =
1.76-1.86 (m, 2H), 2.59 (t, J = 7.6 Hz, 2H), 3.24 (q, J = 6.9 Hz, 2H), 4.52
(s, 2H), 6.27 (td, J = 6.6, 1.1 Hz, 1H), 6.63 (dd, J = 9.6, 1.1 Hz, 1H), 7.04
(bs, 1H), 7.1-7.31 (m, 5H), 7.38-7.45 (m, 2H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 30.85, 33.02, 39.14, 53.77, 106.99, 120.62, 125.91, 128.34,
138.37, 140.64, 141.30, 162.87, 167.10 ppm. Elemental analysis: calcd
for C16H18N2O2: C 71.09, H 6.71, N 10.36; found: C 71.32, H 6.43, N
10.68.
Conclusions
In conclusions, with our work we identified a series of amides
with Ki values in the low micromolar or submicromalar range
towards one or two chymotrypsin-like activities of
immunoproteasome (β5i and β1i subunits). Amide
7 was
identified as lead compound, due to the selective inhibition of β1i
subunit in the submicromolar range (Ki = 21 nM).
Docking studies allowed us to clarify the binding mode of the
amides in the catalytic site of immunoproteasome proteolytic
subunits, thus explaining the preferential inhibition of
immunoproteasome with respect to proteasome.
Worthy of note, the non-covalent inhibition, characteristic of
our amides, is strongly desirable, because free of drawbacks
and side-effects related to covalent inhibition. Our future efforts
will be devoted to optimize the identified lead compound 7 in
terms of potency and selectivity and to check activity against a
panel of hematological malignancies or against autoimmnune
diseases, because controversial data are currently reported in
literature related to the specific proteasome inhibition in MM
cells and disease models.[35,36]
N-Cyclohexyl-2-(2-oxopyridin-1(2H)-yl)acetamide (4): yield: 65%. Rf =
0.60 (CHCl3/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ = 1.06-1.90 (m,
10H), 3.62-3.74 (m, 1H), 4.49 (s, 2H), 6.25 (td, J = 6.7, 1.3 Hz, 1H), 6.61
(d, J = 8.8 Hz, 1H), 6.81 (bs, 1H), 7.34-7.45 (m, 2H) ppm. 13C NMR (75
MHz, CDCl3): δ = 24.58, 25.40, 32.55, 48.45, 53.45, 106.76, 120.45,
138.55, 140.52, 162.80, 166.05 ppm. Elemental analysis: calcd for
C13H18N2O2: C 66.64, H 7.74, N 11.96; found: C 66.89, H 7.86, N 11.74.
N-Butyl-2-(2-oxopyridin-1(2H)-yl)acetamide (5): yield: 44%. Rf = 0.24
(CHCl3/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ = 0.89 (t, J = 7.2 Hz,
3H), 1.24-1.36 (m, 2H), 1.40-1.52 (m, 2H), 3.21 (q, J = 7.2, 2H), 4.53 (s,
2H), 6.25 (td, J = 6.7, 1.3 Hz, 1H), 6.63 (dd, J = 9.8, 1.3 Hz, 1H), 6.93 (bs,
1H), 7.36-7.45 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 13.67, 19.96,
31.25, 39.35, 53.59, 106.88, 120.56, 138.43, 140.56, 162.84, 166.99
ppm. Elemental analysis: calcd for C11H16N2O2: C 63.44, H 7.74, N13.45;
found: C 63.61, H 7.53, N 13.64.
6
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10.1002/cmdc.201900028
ChemMedChem
FULL PAPER
3-(2-Oxopyridin-1(2H)-yl)-N-phenylpropanamide (6): yield: 62%. Rf=
0.43 (CHCl3/MeOH 95:5). 1H NMR (300 MHz, CDCl3): δ = 2.96 (t, J = 5.8
Hz, 2H), 4.34 (t, J = 5.8 Hz, 2H), 6.19 (td, J = 6.7, 1.2 Hz, 1H), 6.45 (d, J
= 9.0 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 7.24-7.36 (m, 3H), 7.53-7.66 (m,
3H), 9.78 (bs, 1H) ppm.13C NMR (75 MHz, CDCl3): δ = 32.76, 45.21,
111.73, 118.92, 121.65, 124.45, 129.08, 134.52, 138.41, 138.87, 161.71,
173.73 ppm. Elemental analysis: calcd for C14H14N2O2, C 69.41, H 5.82,
N 11.56; found. C 69.28, H 5.91, N 11.46.
oil. Yield: 1.68 g (99%); Rf = 0.73 (light petroleum/EtOAc, 4:6). 1H NMR
(300 MHz, CDCl3): δ = 2.77 (s, 3H), 3.12 (dd, J = 14.4, 8.8 Hz, 1H), 3.31
(dd, J = 14.4, 4.2 Hz, 1H), 3.80 (s, 3H), 5.18 (dd, J = 8.8, 4.2 Hz, 1H),
7.23-7.37 (m, 5H) ppm.
(R)-1-(Ethoxycarbonyl)-3-phenylpropyl
methanesulfonate
(34)
According to the same procedure described for 33, compound 32 (1 g,
4.8 mmol, 0.930 mL) was reacted with methanesulfonyl chloride (1.01 g,
9.6 mmol, 0.74 mL) and Et3N (1.21 g, 12 mmol, 1.67 mL) to give the title
compound 34 as a brown/yellow oil. Yield: 1.4 g (99%); Rf = 0.77 (light
petroleum/EtOAc, 4:6). 1H NMR (300 MHz, CDCl3): δ = 1.29 (t, J = 7.2
Hz, 3H), 2.18-2.30 (m, 2H), 2.75-2.83 (m, 2H) 3.17 (s, 3H), 4.23 (q, J =
7.2 Hz, 2H), 5.02 (dd, J = 7.8, 4.7 Hz, 1H), 7.17-7.35 (m, 5H) ppm.
N-Benzyl-3-(2-oxopyridin-1(2H)-yl)propanamide (7): yield: 46%. Rf =
0.42 (CHCl3/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ = 2.74 (t, J = 6.1
Hz, 2H), 4.21 (t, J = 6.1 Hz, 2H), 4.38 (d, J = 5.8 Hz, 2H), 6.12 (td, J = 6.8,
1.2 Hz, 1H), 6.38 (d, J = 9.1 Hz, 1H), 6.81 (bs, 1H), 7.14 (d, J = 7.1 Hz,
2H), 7.22-7.32 (m, 4H), 7.43 (dd, J = 6.8, 1.6 Hz, 1H) ppm. 13C NMR
(125.73 MHz, CDCl3): δ = 34.99, 43.49, 47.17, 106.13, 120.43, 127.35,
127.62, 128.56, 138.04, 139.10, 139.97, 162.77, 169.93 ppm. Elemental
analysis: calcd for C15H16N2O2: C 70.29, H 6.29, N10.93; found: C 70.56,
H 6.46, N 10.62.
(S)-Methyl 2-(2-oxopyridin-1(2H)-yl)-3-phenylpropanoate (35). To a
suspension of NaH (311.7 mg, 7.8 mmol) in dry DMF, at 0 ºC under N2, a
solution of pyridin-2(1H)-one 26 (741 mg, 7.8 mmol) in dry DMF was
added via syringe. After stirring for 1h, (R)-1-(methoxycarbonyl)-2-
phenylethyl methanesulfonate 33 (1.68 g, 6.5 mmol) was added to the
mixture and the reaction was stirred overnight at room temperature. The
reaction was then quenched with saturated NH4Cl and the product was
extracted with EtOAc. The combined organic layers were washed with
brine, dried over Na2SO4, filtered and evaporated under reduced pressure.
Column chromatography (light petroleum/EtOAc, 2:8) of the crude
afforded compound 35 as white crystals. Yield: 869 mg (52%); Rf = 0.62
3-(2-Oxopyridin-1(2H)-yl)-N-phenethylpropanamide (8): yield: 33%. Rf
1
= 0.34 (CHCl3/MeOH, 95:5). H NMR (500 MHz, CDCl3): δ = 2.66 (t, J =
6.4 Hz, 2H), 2.74 (t, J = 7.0 Hz, 2H), 3.47 (q, J = 7.0 Hz, 2H), 4.22 (t, J =
6.4 Hz, 2H), 5.81 (bs, 1H), 6.16 (t, J = 6.4 Hz, 1H), 6.55 (d, J =9.1 Hz,
1H), 7.11 (d, J = 7.1 Hz, 2H), 7.19-7.38 (m, 4H), 7.44 (dd, J = 6.4, 1.6 Hz,
1H) ppm. 13C NMR (125.73 MHz, CDCl3): δ = 35.14, 35.55, 40.73, 46.94,
105.98, 120.61, 126.50, 128.64, 128.61, 138.62, 139.01, 139.93, 162.75,
169.92 ppm. Elemental analysis: calcd for C16H18N2O2: C 71.09; H 6.71;
N 10.36; found: C 71.39, H 6.56, N 6.96.
1
(light petroleum/EtOAc, 2:8 ). H NMR (300 MHz, CDCl3): δ = 3.33 (dd, J
= 14.3, 10.0 Hz, 1H), 3.50 (dd, J = 14.3, 5.3 Hz, 1H), 3.73 (s, 3H), 5.29
(dd, J = 10.0, 5.3 Hz, 1H), 6.00 (td, J = 6.7, 1.3 Hz, 1H), 6.49 (dd, J = 9.4,
1.3 Hz, 1H), 6.95 (dd, J = 7.0, 1.8 Hz, 1H), 7.03-7.08 (m, 2H), 7.15-7.27
(m, 4H) ppm.
3-(2-Oxopyridin-1(2H)-yl)-N-(3-phenylpropyl)propanamide (9): yield:
33%. Rf =0.34 (CHCl3/MeOH, 95:5). 1H NMR (500 MHz, CDCl3): δ =
1.72-1.78 (m, 2H,), 2.55 (t, J = 7.7 Hz, 2H), 2.67 (t, J = 6.1 Hz, 2H), 3.22
(q, J = 6.8 Hz, 2H), 4.21 (t, J = 6.1 Hz, 2H), 6.12 (td, J = 6.7, 0.8 Hz, 1H),
6.45 (bs, 1H), 6.49 (d, J = 9.1 Hz, 1H), 7.1-7.26 (m. 5H), 7.30 (ddd, J =
9.1, 6.7, 1.9 Hz, 1H), 7.43 (dd, J = 6.7, 1.5 Hz, 1H) ppm. 13C NMR
(125.73 MHz, CDCl3): δ = 30.98, 33.14, 35.14, 39.18, 47.05, 106.11,
120.44, 125.94, 128.30, 128.39, 139.09, 140.05, 141.36, 162.79, 169.95
ppm. Elemental analysis: calcd for C17H20N2O2: C 71.81; H 7.09; N 9.85;
found: C 71.45, H 7.21, N 9.73.
(S)-Ethyl 2-(2-oxopyridin-1(2H)-yl)-4-phenylbutanoate (36). The
synthesis of ester 36 was carried out in agreement with the procedure
described for compound 35, by reacting pyridin-2(1H)-one 26 (799 mg,
8.4 mmol), NaH (336 mg, 8.4 mmol), and (R)-1-(ethoxycarbonyl)-3-
phenylpropyl methanesulfonate 34 (2 g, 7 mmol). Consistency: yellow oil.
Yield: 1.2 g (50%); Rf = 0.61 (light petroleum/EtOAc, 3:7). 1H NMR (300
MHz, CDCl3): δ = 1.24 (t, J = 7.1 Hz, 3H), 2.19-2.34 (m, 1H), 2.45-2.72
(m, 3H), 4.19 (q, J = 7.1 Hz, 2H), 5.50 (dd, J = 9.9, 4.5 Hz, 1H), 6.22 (td,
J = 6.3, 1.3 Hz, 1H), 6.59 (dd, J = 9.1, 1.3 Hz, 1H), 7.10-7.39 (m, 7H)
ppm.
N-Cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propanamide (10): yield: 36%.
Rf = 0.41 (CHCl3/MeOH, 95:5). 1H NMR (500 MHz, CDCl3): δ = 0.98-1.83
(m, 10H), 2.67 (t, J = 6.1 Hz, 2H), 3.61-3.72 (m, 1H), 4.22 (t, J = 6.1 Hz,
2H), 6.07 (bs, 1H), 6.15 (td, J = 6.7, 1.2 Hz, 1H), 6.52 (d, J = 9.1 Hz, 1H),
7.34 (ddd, J = 9.1, 6.7, 2.0 Hz, 1H), 7.43 (dd, J = 6.7, 1.6 Hz, 1H) ppm.
13C NMR (125.73 MHz, CDCl3): δ = 24.78, 25.41, 32.89, 35.37, 47.19,
48.28, 105.97, 120.48, 139.08, 139.93, 162.78, 168.93 ppm. Elemental
analysis: calcd for C14H20N2O2: C, 67.71; H, 8.12; N, 11.28; found: C
67.94, H 7.93, N 11.55.
(S)-2-(2-Oxopyridin-1(2H)-yl)-3-phenylpropanoic acid (37). A solution
of ester 35 (275 mg, 1.07 mmol) in MeOH/H2O/dioxane (1:1:1) at 0°C
was treated with LiOH (128 mg, 5.34 mmol) and stirred for 12h. MeOH
and dioxane was evaporated in vacuo and the aqueous solution was
treated with 5% citric acid and extracted with EtOAc. The organic layer
was dried over Na2SO4, filtered and concentrated to afford the acid 37 as
20
a white powder. Yield: 240 mg (92%). []D = -33.1 (c = 0.6, DMSO).
1H NMR (300 MHz, CH3OH-d4): δ = 3.45 (dd, J = 14.2, 9.9 Hz, 1H), 3.59
(dd, J = 14.2, 5.2 Hz, 1H), 5.37 (dd, J = 9.9, 5.2 Hz, 1H), 6.18 (td, J = 6.6,
1.3 Hz, 1H), 6.45 (dd, J = 9.1, 1.3 Hz, 1H), 7.05-7.20 (m, 5H), 7.29-7.33
(m, 1H), 7.37-7.44 (m, 1H) ppm.
N-Butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (11): yield: 57%. Rf =
0.37 (CHCl3/MeOH, 95:5). 1H NMR (500 MHz, CDCl3): δ = 0.87 (t, J = 7.3
Hz, 3H), 1.21-1.30 (m, 2H), 1.37-1.44 (m, 2H), 2.70 (t, J = 6.1 Hz, 2H),
3.19 (q, J = 7.0, 2H), 4.23 (t, J = 6.1 Hz, 2H), 6.16 (td, J = 6.7, 1.1 Hz,
1H), 6.40 (bs, 1H), 6.50 (d, J = 9.1 Hz, 1H), 7.34 (ddd, J = 9.1, 6.7, 2.0
Hz, 1H), 7.45 (dd, J = 6.7, 1.6 Hz, 1H) ppm. 13C NMR (125.73 MHz,
CDCl3): δ = 13.66, 19.93, 31.45, 35.14, 39.26, 47.10, 106.04, 120.43,
139.10, 139.97, 162.78, 169.87 ppm. Elemental analysis: calcd for
C12H18N2O2: C 64.84; H 8.16; N 12.60; found: C 64.55, H 7.78, N 12.77.
(S)-2-(2-Oxopyridin-1(2H)-yl)-4-phenylbutanoic
acid
(38).
The
synthesis of acid 38 was carried out in agreement with the procedure
described for compound 37, by reacting ester 36 (435 mg, 1.52 mmol)
and LiOH (182 mg, 7.62 mmol). Consistency: white powder. Yield: 360
20
mg (89%). []D = +27.4 (c = 0.6, DMSO). 1H NMR (300 MHz, CH3OH-
d4): δ = 2.35-2.48 (m, 1H), 2.49-2.64 (m, 3H), 5.30 (dd, J = 10.2, 4.1 Hz,
1H), 6.42 (td, J = 6.8, 1.3 Hz, 1H), 6.55 (dd, J = 8.7, 1.3 Hz, 1H), 7.11-
7.29 (m, 5H), 7.53 (ddd, J = 8.9, 6.7, 2.0 Hz, 1H), 7.61 (dd, J = 6.8, 1.9
Hz, 1H) ppm.
Synthesis of carboxylic acids 37-38
(R)-1-(Methoxycarbonyl)-2-phenylethyl methanesulfonate (33)
A
solution of 31 (1.2 g, 6.6 mmol) in dry DCM was reacted with
methanesulfonyl chloride (1.5 g, 13.2 mmol, 1.02 mL) and Et3N (1.67 g,
16.5 mmol, 2.29 mL). The mixture was stirred at room temperature under
nitrogen for 2 hours, then washed with water and dried (Na2SO4), filtered
and evaporated under reduced pressure to afford 33 as a brown/yellow
General procedures for the synthesis of amides 12-18 and 19-25 The
coupling reactions between the carboxylic acids 37-38 (1 equiv.) and the
suitable amines (1.5 equiv.) were carried out as above described for
7
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10.1002/cmdc.201900028
ChemMedChem
FULL PAPER
compounds 2-11. The final compounds were purified by column
chromatography, using EtOAc/light petroleum, 8:2 as eluent mixture.
139.62, 162.60, 168.38 ppm. Elemental analysis: calcd for C18H22N2O2: C
72.46; H 7.43; N 9.39; found: C 72.06, H 7.54, N 9.31.
(S)-2-(2-Oxopyridin-1(2H)-yl)-N,3-diphenylpropanamide (12): yield:
44%. Rf = 0.55 (EtOAc/light petroleum, 8:2). H NMR (300 MHz, CDCl3):
(S)-N-Isopentyl-2-(2-oxopyridin-1(2H)-yl)-3-phenylpropanamide (18):
yield: 69%. Rf = 0.57 (EtOAc/light petroleum, 9:1). 1H NMR (300 MHz,
CDCl3): δ = 0.80 (d, J = 6.5 Hz, 6H), 1.17-1.42 (m, 3H), 2.99-3.28 (m, 3H),
3.51 (dd, J = 13.6, 8.9 Hz, 1H), 5.78 (t, J = 7.9 Hz, 1H), 6.26 (td, J = 6.9,
1.2 Hz, 1H), 6.53 (dd, J = 9.1, 1.2 Hz, 1H), 6.78 (bs, 1H), 7.14-7.38 (m,
6H), 7.73 (dd, J = 6.9, 1.8 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3): δ =
22.34, 25.50, 36.85, 37.86, 39.37, 51.45, 106.87, 120.02, 126.98, 128.58,
129.13, 134.63, 135.85, 139.69, 162.60, 168.34 ppm. Elemental
analysis: calcd for C19H24N2O2: C 73.05; H 7.74; N 8.97; found: C 73.25,
H 7.85, N 8.70.
1
δ = 3.19 (dd, J = 13.8, 7.5 Hz, 1H), 3.65 (dd, J = 13.8, 8.3 Hz, 1H), 6.04 (t,
J = 7.8 Hz, 1H), 6.31 (td, J = 7.0, 1.3 Hz, 1H), 6.57 (dd, J = 9.1, 1.3 Hz,
1H), 7.02-7.50 (m, 11H), 7.71 (dd, J = 7.0, 1.5 Hz, 1H), 9.24 (bs, 1H)
ppm. 13C NMR: (75 MHz, CDCl3): δ = 36.45, 63.76, 107.26, 119.95,
120.19, 124.45, 127.09, 128.69, 128.87, 129.12, 135.66, 137.63, 140.00,
142.78, 164.81, 166.72 ppm. Elemental analysis: calcd for C20H18N2O2: C
75.45; H 5.70; N 8.80; found: C 75.56, H 5.65, N 8.95.
(S)-N-Benzyl-2-(2-oxopyridin-1(2H)-yl)-3-phenylpropanamide
(13):
yield: 48%. Rf = 0.68 (EtOAc/light petroleum, 9:1). 1H NMR (300 MHz,
CDCl3): δ = 3.10 (dd, J = 13.5, 6.9 Hz, 1H), 3.52 (dd, J = 13.5, 9.1 Hz,
1H), 4.16 (dd, J = 15.0, 5.1 Hz, 1H), 4.48 (dd, J = 15.0, 6.6 Hz, 1H), 5.91
(t, J = 7.9 Hz, 1H), 6.25 (td, J = 7.0, 1.3 Hz, 1H), 6.33 (dd, J = 9.2, 1.3 Hz,
1H), 6.91-7.01 (m, 2H), 7.16-7.31 (m, 9H), 7.42 (bs, 1H), 7.76 (dd, J =
7.0, 1.3 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3): δ = 37.03, 43.43,
53.79, 106.78, 120.02, 127.01, 127.26, 127.38, 128.51, 128.64, 129.24,
134.64, 137.53, 139.62, 141.70, 162.54, 168.51 ppm. Elemental
analysis: calcd for C21H20N2O2: C 75.88; H 6.06; N 8.43; found: C 75.99,
H 6.17, N 8.12.
(S)-2-(2-Oxopyridin-1(2H)-yl)-N,4-diphenylbutanamide (19): yield:
50%. Rf = 0.72 (EtOAc/light petroleum, 8:2). H NMR (300 MHz, CDCl3):
1
δ = 2.14-2.30 (m, 1H), 2.57-2.77 (m, 3H), 5.61 (t, J = 7.4 Hz, 1H), 6.31
(td, J = 6.8, 1.4 Hz, 1H), 6.66 (d, J = 9.2 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H),
7.13-7.24 (m, 3H), 7.25-7.35 (m, 4H), 7.39 (ddd, J = 8.9, 6.6, 2.0 Hz, 1H),
7.50-7.58 (m, 3H), 9.11 (bs, 1H) ppm. 13C NMR: (75 MHz, CDCl3): δ =
32.07, 44.13, 47.01, 101.50, 110.00, 119.90, 120.45, 121.81, 128.40,
128.63, 128.96, 136.31, 139.95, 140.07, 142.35, 165.12, 166.54 ppm.
Elemental analysis: calcd for C21H20N2O2: C 75.88; H 6.06; N 8.43;
found: C 75.56, H 6.016, N 8.58.
(S)-2-(2-Oxopyridin-1(2H)-yl)-N-phenethyl-3-phenylpropanamide
(14): yield: 92%. Rf = 0.63 (EtOAc/light petroleum, 9:1). 1H NMR (300
MHz, CDCl3): δ = 2.54-2.73 (m, 2H), 3.06 (dd, J = 13.7, 7.2 Hz, 1H),
3.24-3.37 (m, 1H), 3.39-3.53 (m, 2H), 5.80 (t, J = 7.9 Hz, 1H), 6.22 (td, J
= 7.0, 1.4 Hz, 1H), 6.39 (dd, J = 9.1, 0.8 Hz, 1H), 6.93-7.01 (m, 2H), 7.10
(bs, 1H), 7.13-7.35 (m, 9H), 7.69 (dd, J = 7.0, 1.8 Hz, 1H) ppm. 13C NMR:
(75 MHz, CDCl3): δ = 35.37, 36.99, 40.92, 57.71, 106.68, 120.04, 126.34,
127.00, 128.48, 128.60, 128.64, 129.22, 134.67, 135.98, 138.66, 139.59,
162.45, 168.54 ppm. Elemental analysis: calcd for C22H22N2O2: C 76.28;
H 6.40; N 8.09; found: C 76.20, H 6.12, N 8.29.
(S)-N-Benzyl-2-(2-oxopyridin-1(2H)-yl)-4-phenylbutanamide
(20):
yield: 95%. Rf = 0.63 (EtOAc/light petroleum, 8:2). 1H NMR (300 MHz,
CDCl3): δ = 2.07-2.24 (m, 1H), 2.43-2.71 (m, 3H), 4.30 (dd, J = 14.8, 5.3
Hz, 1H), 4.51 (dd, J = 14.8, 6.0 Hz, 1H), 5.60 (t, J = 7.7 Hz, 1H), 6.26 (td,
J = 6.8, 1.4 Hz, 1H), 6.48 (d, J = 9.2 Hz, 1H), 7.08-7.36 (m, 11H), 7.43
(bs, 1H), 7.59 (dd, J = 6.8, 1.8 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3):
δ = 32.04, 32.42, 43.58, 56.29, 107.00, 120.14, 126.32, 127.42, 127.55,
128.35, 128.55, 128.64, 134.31, 137.77, 139.68, 140.24, 162.77, 168.92
ppm. Elemental analysis: calcd for C22H22N2O2: C 76.28; H 6.40; N 8.09;
found: C 76.01, H 6.35, N 8.19.
(S)-2-(2-Oxopyridin-1(2H)-yl)-3-phenyl-N-(3-
(S)-2-(2-Oxopyridin-1(2H)-yl)-N-phenethyl-4-phenylbutanamide (21):
yield: 92%. Rf = 0.56 (EtOAc/light petroleum, 8:2). 1H NMR (300 MHz,
CDCl3): δ = 1.98-2.15 (m, 1H), 2.39-2.66 (m, 3H), 2.68-2.85 (m, 2H),
3.38-3.56 (m, 2H), 5.43 (t, J = 7.8 Hz, 1H), 6.31 (td, J = 6.8, 1.4 Hz, 1H),
6.55 (dd, J = 9.2, 1.4 Hz, 1H), 6.84 (bs, 1H), 7.02-7.38 (m, 11H), 7.50 (dd,
J = 6.8, 1.4 Hz, 1H) ppm.13C NMR: (75 MHz, CDCl3): δ = 32.00, 32.09,
35.48, 40.77, 56.63, 106.96, 120.27, 126.31, 126.45, 127.5, 128.34,
128.54, 128.64, 134.09, 138.52, 139.57, 140.27, 162.69, 168.81 ppm.
Elemental analysis: calcd for C23H24N2O2: C 76.64; H 6.71; N 7.77; trov:
C 76.34, H 6.81, N 7.50.
phenylpropyl)propanamide (15): yield: 84%. Rf = 0.64 (EtOAc/light
1
petroleum, 9:1). H NMR (300 MHz, CDCl3): δ = 1.61-1.73 (m, 2H), 2.44
(t, J = 8.4 Hz, 2H), 3.02-3.14 (m, 2H), 3.17-3.29 (m, 1H), 3.50 (dd, J =
13.6, 8.8 Hz, 1H), 5.84 (dd, J = 8.4, 7.5 Hz, 1H), 6.24 (td, J = 7.0, 1.4 Hz,
1H), 6.47 (dd, J = 9.1, 0.8 Hz, 1H), 6.99-7.36 (m, 12H), 7.73 (dd, J = 7.0,
1.8 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3): δ = 30.76, 32.88, 36.88,
39.06, 57.80, 106.76, 120.02, 125.90, 127.01, 128.35, 128.60, 129.16,
134,69, 135.92, 139.69, 141.30, 143.72, 162.57, 168.55 ppm. Elemental
analysis: calcd for C23H24N2O2: C 76.64; H 6.71; N 7.77; found: C 76.50,
H 6.83, N 7.40.
(S)-2-(2-Oxopyridin-1(2H)-yl)-4-phenyl-N-(3-
(S)-N-Cyclohexyl-2-(2-oxopyridin-1(2H)-yl)-3-phenylpropanamide
(16): yield: 65%. Rf = 0.64 (EtOAc/light petroleum, 9:1). 1H NMR (300
MHz, CDCl3): δ = 0.90-1.79 (m, 10H), 3.09 (dd, J = 13.6, 7.0 Hz, 1H),
3.48 (dd, J = 13.6, 8.9 Hz, 1H), 3.55-3.71 (m, 1H), 5.78 (dd, J = 8.9, 7.0
Hz, 1H), 6.24 (td, J = 7.0, 1.4 Hz, 1H), 6.50 (dd, J = 9.1, 1.4 Hz, 1H), 6.66
(bs, 1H), 7.14-7.36 (m, 6H), 7.75 (dd, J = 7.0, 1.7 Hz, 1H) ppm. 13C NMR:
(75 MHz, CDCl3): δ = 24.57, 25.36, 32.38, 32.52, 37.14, 48.40, 106.55,
119.98, 126.94, 128.55, 129.18, 134.73, 135.95, 139.54, 162.48, 167.54
ppm. Elemental analysis: calcd for C20H24N2O2: C 74.04; H 7.46; N 8.64;
found: C 74.25, H 7.30, N 8.75.
phenylpropyl)butanamide (22): yield: 67%. Rf = 0.53 (EtOAc/light
petroleum, 8:2). 1H NMR (300 MHz, CDCl3): δ = 1.68-1.88 (m, 2H), 2.06-
2.22 (m, 1H), 2.41-2.74 (m, 5H), 3.10-3.33 (m, 2H), 5.48 (t, J = 7.7 Hz,
1H), 6.26 (td, J = 6.8, 1.4 Hz, 1H), 6.58 (dd, J = 9.2, 1.4 Hz, 1H), 6.93 (bs,
1H), 7.03-7.41 (m, 11H), 7.55 (dd, J = 6.8, 2.1 Hz, 1H) ppm. 13C NMR:
(75 MHz, CDCl3): δ = 31.98, 30.99, 32.04, 33.04, 39.12, 56.45, 107.08,
120.24, 125.97, 126.36, 128.37, 128.43, 128.57, 130.81, 134.14, 139.72,
140.26, 140.91, 165.48, 168.86 ppm. Elemental analysis: calcd for
C24H26N2O2: C, 76.98; H, 7.00; N, 7.48; found: C 76.68, H 7.32, N 7.28.
(S)-N-Cyclohexyl-2-(2-oxopyridin-1(2H)-yl)-4-phenylbutanamide (23):
yield: 89%. Rf = 0.56 (EtOAc/light petroleum, 8:2). 1H NMR (300 MHz,
CDCl3): δ = 0.97-1.80 (m, 10H), 2.03-2.24 (m, 1H), 2.38-2.72 (m, 3H),
3.58-3.78 (m, 1H), 5.47 (t, J = 7.4 Hz, 1H), 6.24 (td, J = 6.8, 1.4 Hz, 1H),
6.58 (dd, J = 9.2, 1.4 Hz, 1H), 6.65 (bs, 1H), 7.09-7.41 (m, 6H), 7.57 (dd,
J = 6.8, 1.8 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3): δ = 24.70, 25.40,
32.06, 32.56, 32.81, 48.48, 56.35, 106.84, 120.07, 126.28, 128.34,
128.52, 134.38, 139.60, 140.36, 162.69, 167.91 ppm. Elemental
analysis: calcd for C21H26N2O2: C 74.52; H 7.74; N 8.28; found: C 74.59,
H 7.60, N 8.39.
(S)-N-Butyl-2-(2-oxopyridin-1(2H)-yl)-3-phenylpropanamide
(17):
yield: 47%. Rf = 0.60 (EtOAc/light petroleum, 9:1). 1H NMR (300 MHz,
CDCl3): δ = 0.82 (t, J = 7.3 Hz, 3H), 1.06-1.21 (m, 2H), 1.22-1.38 (m, 2H),
2.98-3.13 (m, 2H), 3.14-3.26 (m, 1H), 3.52 (dd, J = 13.6, 8.9 Hz, 1H),
5.78 (t, J = 7.9 Hz, 1H), 6.25 (td, J = 7.0, 1.2 Hz, 1H), 6.52 (dd, J = 9.1,
1.2 Hz, 1H), 6.67 (bs, 1H), 7.14-7.35 (m, 6H), 7.69 (dd, J = 7.0, 1.8 Hz,
1H) ppm. 13C NMR: (75 MHz, CDCl3): δ = 13.64, 19.77, 31.13, 36.70,
39.26, 57.83, 106.73, 120.07, 126.98, 128.60, 129.13, 134.54, 135.91,
8
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(S)-N-Butyl-2-(2-oxopyridin-1(2H)-yl)-4-phenylbutanamide (24): yield:
51%. Rf = 0.57 (EtOAc/light petroleum, 8:2). H NMR (300 MHz, CDCl3):
ID: 3UNF)[7] for docking studies. Murine and human 20S subunits share a
sequence identity of more than 90%, and the few non-identical residues
are external to the active sites.
1
δ = 0.87 (t, J = 7.2 Hz, 3H), 1.18-1.35 (m, 2H), 1.37-1.54 (m, 2H), 2.03-
2.21 (m, 1H), 2.40-2.72 (m, 3H), 3.05-3.34 (m, 2H), 5.53 (t, J = 7.6 Hz,
1H), 6.26 (td, J = 6.7, 1.4 Hz, 1H), 6.57 (dd, J = 9.2, 1.4 Hz, 1H), 6.95 (bs,
1H), 7.07-7.41 (m, 6H), 7.59 (dd, J = 6.7, 1.8 Hz, 1H) ppm. 13C NMR: (75
MHz, CDCl3): δ =13.70, 19.97, 31.30, 32.06, 32.65, 39.34, 56.25, 106.88,
119.99, 126.26, 128.32, 128.51, 134.50, 139.68, 140.35, 162.72, 168.87
ppm. Elemental analysis: calcd for C19H24N2O2: C 73.05; H 7.74; N 8.97;
found: C 72.95, H 7.85, N 8.81.
For docking purpose, we selected the catalytic subunits β1i (LMP2,
PSMB9)/β2i (MECL-1, PSMB10) and β5i (LMP7, PSMB8)/β6i. The
crystal structure of PR-957 bound to β1i revealed two well-defined water
molecules within the S3 pocket, which coordinate a tight H-bond network
between β1i-A50N, β2i-S118Oγ and the carbonyl oxygen and the amide
nitrogen of PR-957 P3 alanine, respectively. Accordingly, the intervening
water molecules were included in the docking experiments. The protein
setup was carried out using the Protein Preparation Wizard implemented
in Maestro 11.3 (Schrödinger, LLC, New York, NY, 2018). 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
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 a H-bonding network optimization process.
Successively, the protein hydrogen atoms only were minimized using the
Impref module of Impact with the OPLS_2005 force field.
(S)-N-Isopentyl-2-(2-oxopyridin-1(2H)-yl)-4-phenylbutanamide (25):
yield: 37%. Rf = 0.55 (EtOAc/light petroleum, 8:2). 1H NMR (300 MHz,
CDCl3): δ = 0.86 (d, J = 6.6 Hz, 6H), 1.16-1.46 (m, 3H), 2.04-2.25 (m, 1H),
2.40-2.73 (m, 3H), 3.09-3.35 (m, 2H), 5.44 (t, J = 7.6 Hz, 1H), 6.25 (td, J
= 6.7, 1.3 Hz, 1H), 6.59 (dd, J = 9.2, 1.4 Hz, 1H), 6.76 (bs, 1H), 7.06-7.43
(m, 6H), 7.59 (dd, J = 6.7, 1.8 Hz, 1H) ppm. 13C NMR: (75 MHz, CDCl3):
δ =22.35, 22.40, 25.77, 32.03, 32.27, 37.98, 38.04, 56.34, 107.08,
120.13, 126.31, 128.34, 128.54, 134.25, 139.72, 140.27, 162.80, 168.72
ppm. Elemental analysis: calcd for C20H26N2O2: C 73.59; H 8.03; N 8.58;
found: C 73.79, H 8.24, N 8.41.
In vitro 20S immunoproteasome/proteasome inhibition assays
Initial coordinates of compounds 1, 7, 13 and 21 were constructed by
using the Molecular Builder module in Maestro. The structures were
energy-minimized using Macromodel 10.8 (Schrödinger, LLC, New York,
NY, 2018) using the MMFF force field with the steepest descent (1000
steps) followed by truncated Newton conjugate gradient (500 steps)
methods. Partial atomic charges were computed using the OPLS-AA
force field.
Human 20S immunoproteasome, obtained from human spleen, and
human 20S proteasome, isolated from human erythrocytes, were
purchased from Enzo Life Science. The hydrolysis of the appropriate
peptidyl 7-amino-4-methyl-coumarin substrate was monitored to measure
the different proteolytic activities of both proteasome and
immunoproteasome.
The
substrates
Suc-Leu-Leu-Val-Tyr-AMC
(Bachem) for β5c-β5i, Boc-Leu-Arg-Arg-AMC (Bachem) for β2c-β2i, Z-
Leu-Leu-Glu-AMC (Adipogen) for β1c and Ac-Pro-Ala-Leu-AMC (Biomol
GmbH) for β1i subunits were employed at 50 µM, with the exception of Z-
Leu-Leu-Glu-AMC (80 µM). Fluorescence of the product AMC of the
substrate hydrolyses was measured at 30°C with a 380 nm excitation
filter and a 460 nm emission filter, using an Infinite 200 PRO microplate
reader (Tecan, Männedorf, Switzerland). A preliminary screening at 50
µM inhibitor concentrations was carried out on the three proteolytic
activities of proteasome and immunoproteasome; an equivalent amount
of DMSO as a negative control and MG-132 (a reversible inhibitor of
immunoproteasome) as positive control were employed. Compounds
showing at least 60% inhibition at the screening concentration were then
progressed into detailed assays. Continuous assays were performed at
seven different concentrations ranging from those that minimally inhibited
to those that fully inhibited each proteolytic activity to calculate the
dissociation constants Ki of the enzyme-inhibitor complex by means of
the Cheng-Prusoff equation Ki = IC50/(1 + [S] Km-1). Inhibitor solutions
were prepared from stocks in DMSO. Each independent assay was
performed in duplicate in 96-well-plates in a total volume of 200 µL. For
the assay on β5i, β1i, β1c and β5c subunits, human 20S
immunoproteasome or human 20S proteasome was incubated at 30°C
obtaining a final concentration of 0.004 mg/mL with the inhibitor at seven
different concentrations. The reaction buffer comprised: 50 mM Tris HCl,
pH 7.4, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.03% SDS. AMC
released from substrate hydrolysis was monitored in kinetic cycle over a
period of 10 min. For the assay on β2i and β2c subunits, final
concentration of immunoproteasome or proteasome was of 0.0025
mg/mL. The reaction buffer comprised: 50 mM Tris HCl, pH 7.4, 0.5 mM
EDTA III, 50 mM NaCl, 0.03% SDS.
Molecular Docking
Docking of compounds 1, 7, 13 and 21into the active site of β1i or β5i
was performed with the genetic algorithm implemented in GOLD 5.5
(CCDC Software Limited: Cambridge, U.K., 2008).[29] The coordinates of
the cocrystallized ligand PR-957, in β1i and β5i respectively, were
chosen as active-site origin. The active-site radius was set equal to 10 Å.
Explicit water molecules were allowed to toggle on or off during the
individual docking runs (i.e., these waters were not automatically present
in the binding site, but were included if their presence strengthened the
interaction of the ligand with the receptor, as determined by the scoring
function).[37] Orientation mode of water hydrogen atoms was set to “spin”.
We adopted all the program default parameters: for each molecule tested
the number of islands was set to 5, population size to 100, number of
operations was 100,000 with a selective pressure of 1.1. For these
experiments, the number of GA runs was set to 200 without the option of
early termination, and scoring of the docked poses was performed with
the original ChemPLP scoring function rescoring with ChemScore.[38] The
final receptor-ligand complex for each ligand was chosen interactively by
selecting the highest scoring pose that was consistent with the
experimentally derived information about the binding mode of the ligand.
Figures were prepared using Pymol (Schrödinger, LLC, New York, NY,
2018). All computations were performed on an E4 Computer Engineering
E1080 workstation provided with an Intel Xeon processor.
Acknowledgements
Computational details & methodology
S.M. acknowledges the support of “Programma Operativo Fondo
Sociale Europeo (FSE) Regione Siciliana 2014–2020 – Asse 3
Ob. 10.5″ for her PhD fellowship. A.L. thanks “Combattere la
resistenza tumorale: piattaforma integrata multidisciplinare per
un approccio tecnologico innovativo alle oncoterapie-
CAMPANIA ONCO TERAPIE” of Regione Campania (Italy) for
partial support of this work.
Protein and ligand preparation
Since the only available crystal structures of the human i20S are yeast
chimera that incorporate key parts of human β5i and the neighboring β6
subunit but are devoid of the β1i subunit,[34] we employed the crystal
structure of the murine i20S in complex with the inhibitor PR-957 (PDB
9
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2003, 46, 4572-4585.
Keywords: Immunoproteasome •Amide derivatives
covalent inhibitors • Docking studies•
• Non-
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Entry for the Table of Contents
S2
O
N
O
N
H
S3
S1
Immunoproteasome inhibitors: the aim of this research was the identification of new immunoproteasome inhibitors. Among the
synthesized derivatives, we identified a lead compound able to selectively inhibit β1i subunit of immunoproteasome. Docking studies
clarified the binding mode in the catalytic site of immunoproteasome subunits, thus explaining the preferential inhibition of
immunoproteasome with respect to proteasome.
11
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