Design and NMR conformational analysis in solution of β5i-selective inhibitors of immunoproteasome

Article abstract of DOI:10.1016/j.molstruc.2020.129633

The identification of effective and selective immunoproteasome inhibitors could greatly promote smart treatment of many autoimmune, inflammatory and tumor diseases. This paper explores the activity of some pyridone derivatives which are taking advantage of peptide mimicking moieties and other functional groups whose related effects are taken into consideration. The target compounds were designed and synthesized starting from a lead, next their in vitro biological activity was tested; the following structural and conformational analysis sheds light over the different activity of the tested compounds. Compound 3 bearing a phenylalanine at the P2 site and an allyloxycarbonyl group spanning the P3 region, was found to be a selective inhibitor of β5 subunit of immunoproteasome. It has been demonstrated by Nuclear Magnetic Resonance (NMR) that, in solution, this substrate displays a more rigid conformation respect to the others, especially in apolar media, this feature is likely crucial for the enhancement of the activity towards immunoproteasome. The specific conformational analysis enables insights of the biological activity of new chemicals driving possible advancements based on the suitable structural features to identify novel selective β5 immunoproteasome inhibitor.

Full text of DOI:10.1016/j.molstruc.2020.129633

Journal Pre-proof
Design and NMR conformational analysis in solution of β5i-selective
inhibitors of immunoproteasome
`
Archimede Rotondo , Maria Zappala , Santo Previti ,
Carla Di Chio , Alessandro Allegra , Roberta Ettari
PII:
S0022-2860(20)31946-3
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129633
MOLSTR 129633
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
21 October 2020
9 November 2020
10 November 2020
`
Please cite this article as: Archimede Rotondo , Maria Zappala , Santo Previti , Carla Di Chio ,
Alessandro Allegra , Roberta Ettari , Design and NMR conformational analysis in solution of
β5i-selective inhibitors of immunoproteasome, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.129633
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Highlights
 The study is based on the synthesis of 5i-selective and effective inhibitors of proteasome and
immonoproteasome as effective potential drugs.
 The activity of some pyridone derivates endowed with peptide mimicking moieties is explored
to design drugs.
 Among this group, one allyl and phenylalanine peptide analogue turned out to be more
effective.
 The NMR conformational analysis demonstrated that the most active compound is the most
rigid in the apolar media.
 This conformational behaviour may account for the enhanced activity of the chemical because
of specific biological interactions.

Design and NMR conformational analysis in solution of
β5i-selective inhibitors of immunoproteasome
Archimede Rotondo*a, Maria Zappalàb, Santo Previtib, Carla Di Chiob, Alessandro Allegrac and
Roberta Ettari*b
aDepartment of Biomedical Sciences, Dentistry and Morphological and functional Immages -
BIOMORF, University of Messina, Viale Annunziata, 98168 Messina, Italy. E-mail:
arotondo@unime.it
bDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of
Messina, Viale Annunziata, 98168 Messina, Italy. E-mail: rettari@unime.it
cDepartment of Human Pathology in Adulthood and Childhood, University of Messina, Via C. Valeria,
98125 Messina, Italy.
*Corresponding author: Tel +39-090-6766890, Fax +39-090-393756
e-mail address: arotondo@unime.it; rettari@unime.it
Abstract

The identification of effective and selective immunoproteasome inhibitors could greatly promote smart
treatment of many autoimmune, inflammatory and tumor diseases. This paper explores the activity of
some pyridone derivatives which are taking advantage of peptide mimicking moieties and other
functional groups whose related effects are taken into consideration. The target compounds were
designed and synthesized starting from a lead, next their in vitro biological activity was tested; the
following structural and conformational analysis sheds light over the different activity of the tested
compounds. Compound 3 bearing a phenylalanine at the P2 site and an allyloxycarbonyl group
spanning the P3 region, was found to be a selective inhibitor of 5 subunit of immunoproteasome. It
has been demonstrated by Nuclear Magnetic Resonance (NMR) that, in solution, this substrate
displays a more rigid conformation respect to the others, especially in apolar media, this feature is
likely crucial for the enhancement of the activity towards immunoproteasome. The specific
conformational analysis enables insights of the biological activity of new chemicals driving possible
advancements based on the suitable structural features to identify novel selective 
immunoproteasome inhibitor.
KEYWORDS: Immunoproteasome; Amide derivatives; noncovalent inhibitors; NMR; Conformational
analysis.

1. Introduction
The 20S proteasome is the most relevant non-lysosomal proteolytic system in eukaryotic cells,
being involved in the degradation of most cellular proteins [1-2]. Tumor cells are strictly dependent on
proteasomal function, thus proteasome inhibition represents a novel therapeutic strategy to kill cancer
cells [3-7]. The 26S proteasome possesses a central 20S catalytic core characterized by a barrel-like
structure, with the two outer and the two inner rings composed of seven different α- and β-subunits,
respectively. β1c, β2c and β5c are the constitutive core particles (cCPs), responsible for the caspase-
like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities, respectively [8]. Another
specialized form of proteasome, mainly expressed in lymphocytes and monocytes is represented by the
immunoproteasome. Under the stimuli of IFN-γ and TNF-α, the cCPs are replaced by the immuno-
core particles (iCPs) β1i, β2i, and β5i subunits, respectively. While β5i and β2i subunits maintain the
same activities of the constitutive counterparts, β1i subunit performs a chymotrypsin-like activity,
while its caspase-like activity is reduced to background levels [9]. An up-regulation of
immunoproteasome has been detected in a large number of autoimmune diseases, like rheumatoid
arthritis or systemic lupus erythematosus and in a panel of inflammatory diseases, such as Crohn’s and
inflammatory bowel or ulcerative diseases, as well [10]. At the same time, iCPs are overexpressed in
hematopoietic cells, including multiple myeloma (MM) cells, thus selectively targeting iCPs could be
an innovative strategy in the drug discovery process of new agents for the treatment of hematologic
malignancy [11-13]. In the last years, many efforts have been made to develop noncovalent
proteasome or immunoproteasome inhibitors, which may lack of several off targets effects with respect
to the irreversible inhibitors of human proteases [14-17]. In this context, our research group has been
actively involved in the development of novel 20S proteasome [18-23] and immunoproteasome [24-
25] inhibitors endowed with Ki values in the submicromolar range. Starting from the lead compound 1
[25], which inhibited β5c subunit more effectively than the β5i counterpart (Ki=8.8 µM and 45.5 µM,
respectively), we decided to extend the P3 site with the introduction of a moiety potentially able to act
as H-bond donor and/or H-bond acceptor, thus we functionalized the amino group at position 3 of the

pyridin-2(1H)-one nucleus into a carbamate function, i.e. compounds 2-3 (Fig. 1). At the P2 site, for
which no specificity is assessed, we first maintained the P2 phenylalanine (Phe) residue, later studying
the impact upon the activity driven by the replacement of this residue with leucine (Leu). Finally, the
isopentyl group was introduced as capping substituent of the amide moiety in order to match the
structural features of the S1 pockets of β1i and β5i subunits, which are large and hydrophobic [12].
Compound 4 was subsequently synthesized as an optimization process of the most active of the two
inhibitors. We now report and discuss the synthesis, biological investigation and conformational
analysis of the three synthesized potential inhibitors of immunoproteasome.
Fig. 1. Molecular representation and labeling scheme of the studied compounds.
2. Results and Discussion
2.1.
Synthesis
The synthesis of novel peptidomimetics 2-4 (Scheme 1) was carried out following a synthetic
strategy widely validated in our laboratories [18]. The commercially available 3-aminopyridin-2(1H)-
one nucleus was first protected at the amino group with allyl or benzyl chloroformate to give
intermediates 5-6; in parallel, Leu and Phe (R)-α-hydroxy ester derivatives were treated with mesyl

chloride, allowing us to obtain the corresponding mesylate analogues 7-8; N-alkylation of 5-6 with
mesylates 7-8 gave the dipeptide analogues with inversion of the stereochemical configuration,
according to the SN2 mechanism. Subsequently, their hydrolysis in alkaline condition via LiOH gave
the acids 9-11 [18], which were coupled to the commercially available isopentylamine. Coupling
reactions were performed in dry DCM/DMF (1:1) using HOBt/EDCI as coupling reagents and DIPEA
as a base. The resulting crudes were purified by preparative HPLC providing the desired products 2-4
in good yields (68-77%).
R
O
O
a,b
S
OMe
PG
NH
O
N
H
O
O
PG
5, Alloc
6, Cbz
7, PG= -CH(CH3)2
8, PG= -Ph
O
R
c
PG
N
COOH
R
PG
N
N
N
H
N
H
H
O
O
PG
R
PG
R
9, Alloc -CH(CH3)2
10, Alloc -Ph
2, Alloc -CH(CH3)2
3, Alloc -Ph
4, Cbz -Ph
11, Cbz -Ph
Scheme 1. Reagents and conditions. a) NaH, DMF, 0°C, 1 h, N2; then 7-8, rt, 12h; b) LiOH,
MeOH/H2O (1:1), rt, 5 h; c) HOBt, EDCI, dry DCM/DMF (1:1), 0°C, N2; then isopentylamine,
DIPEA, rt, 12h.
2.2.
Biological activity
All the three 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. First, compounds
underwent a preliminary screening on each proteolytic subunit at 100 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.
Table 1. Activity on proteasome and immunoproteasome core-particles of compounds 1-4
% of inhibition at 100 µM or Ki (µM)a
Comp.
1[23]
β1c
15%
26%
24%
13%
β2c
6%
n.i.
n.i.
n.i.
β5c
8.8±1.1
36%
33%
23%
β1i
23%
7%
2%
n.i.
β2i
n.i.
8%
3%
n.i.
β5i
45.5± 2.6
16%
33.6±0.2
5%
2
3
4
a For Ki values, data represent the mean ± SD of two independent determinations, each performed in
duplicate. n.i.=no inhibition.
Between the two initially synthesized Alloc-derivatives 2-3, only compound 3, bearing a Phe residue
at the P2 site, turned out to be active against immunoproteasome, with a total selectivity against β5i
subunit showed by a Ki value of 33.6 µM, thus suggesting a key role of the P2 Phe in the interaction
with the target enzyme. With respect to the lead compound 1, compound 3 showed both an increase of
the activity against β5i subunit and of the selectivity over the constitutive counterpart β5c. With the
aim to optimize the identified selective immunoproteasome inhibitor 3, also compound 4 bearing a
Cbz N-protecting group was synthesized, in such a way to evaluate if the additional aromatic nucleus
could establish additional interactions with the S3 pocket of immune-core particles, however this
structural modification turned out to be unfruitful.
2.3.
Conformational analysis
In order to better understand the features leading to the sole activity and selectivity of compound 3
against immunoproteasome the three similar compounds 2-4 were totally characterized by homo and
heteronuclear 1H, 13C and 15N NMR and assignments are reported in Tables 2-4; relative spectra with
the structural features are illustrated in Fig. 2 and 3. Beyond the definite structural elucidation, NMR

data enables the conformational analysis of these species in different media (mimicking in vivo drug
behaviour in polar and apolar environments); it accounts for specific biological activities possibly
improved constructs by de novo design [23, 26, 27].
Table 2. Chemical shift (in ppm) recorded for 13C and 15N hetero nuclei (X) and hydrogen nuclei
belonging to the molecule 2 in CDCl3 and in CD3OH. Delta values indicate chemical shift deviation in
ppm respect to expected values calculated on the basis of a wide database of compounds. Remarkable
deviations from expected values are indicated in gray, for compound 3 these values in CDCl3 are
somewhat bigger according to a pretty locked conformation.
Compound 2 in CDCl3
Compound 2 in CD3OH
XHn
X Shift
157.41
X Delta
-0.23
H shift
-
H Delta
-
X Shift
158.90
X Delta
1.26
H shift
-
H Delta
-
2-C
129.04
120.55
107.51
126.02
56.15
n.d
0.48
-3.78
-4.04
-2.54
-0.92
n.d
-
-
129.75
122.52
107.32
128.67
57.19
1.20
-1.82
-4.23
0.10
0.12
2.97
-
-
3-C
8.024
6.314
7.183
5.526
-
0.087
-0.016
-1.018
0.492
-
7.991
6.384
7.397
5.643
-
0.054
0.054
-0.804
0.609
-
4-CH
5-CH
6-CH
7-CH
171.68
8-C
38.13
38.13
38.20
25.80
22.36
153.31
38.83
38.83
24.82
1.49
3.156
3.248
1.337
1.509
0.864
-
-0.118
-0.026
-0.029
-0.048
-0.062
-
10-CH2'
10-CH2"
11-CH2
12-CH
13,14-CH3
16-C
38.80
2.16
3.199
-0.075
1.49
-0.36
-0.72
-0.43
-0.41
-0.59
-0.59
-0.60
39.08
26.62
22.48
155.18
0.52
0.10
-0.31
1.46
1.369
1.565
0.891
-
0.003
0.008
-0.035
-
1.995
1.830
1.430
0.129
-0.036
-0.189
18-CH2'
18-CH2"
19-CH
20-CH3
21-CH3
23-CH2
24-CH
25-CH2c
25-CH2t
1-N
40.95
1.53
1.890
0.024
25.84
21.73
23.11
66.69
133.56
118.13
118.13
Nd
0.42
-0.61
0.77
0.66
1.67
-0.10
-0.10
nd
1.388
0.928
0.944
4.655
5.990
5.232
5.361
-
-0.231
0.049
0.065
0.032
0.116
-0.048
-0.004
-
22.56
0.22
0.931
0.052
66.15
132.59
118.53
118.53
172.58
117.39
96.14
0.12
0.69
0.29
0.29
18.43
7.43
-1.98
4.675
5.959
5.266
5.368
-
0.052
0.085
-0.014
0.003
-
6.296
7.806
-0.477
-0.677
Nd
nd
8.264
8.233
1.491
-0.250
9-NH
Nd
nd
15-NH
Dashes indicate missing resonances, nd in instead used for “not detected” signals probably due to the lack of
sensitivity

13
15
Table 3. Chemical shift (in ppm) recorded for C and N hetero nuclei (X) and hydrogen nuclei
belonging to the molecule 3 in CDCl3 and in CD3OH. Delta values indicate chemical shift deviation
in ppm respect to expected resonances calculated on the basis of a wide database of compounds.
Remarkable deltas are indicated in gray, for compound 3 these values in CDCl3 are somewhat
bigger according to a pretty locked conformation
compound 3 in CDCl3
compound 3 in CD3OH
XHn
2-C
X Shift
157.23
128.92
120.24
107.17
125.87
59.27
X Delta
-0.24
-0.39
-3.59
-4.04
-4.15
-1.85
-0.66
H Shift
-
H Delta
-
X Shift
158.21
130.22
122.58
107.31
129.25
60.65
X Delta
0.74
H Shift
-
H Delta
-
3-C
-
-
0.91
-
-
4-CH
8.000
6.300
7.296
5.671
-
0.048
0.146
-1.208
0.627
-
-1.24
-3.90
-0.77
-0.47
1.81
7.930
6.322
7.476
5.746
-
-0.022
0.168
-1.028
0.702
-
5-CH
6-CH
7-CH
8-C
167.98
170.45
10-CH2''
10-CH2'
11-CH2
12-CH
13/14-CH3
16-C
3.220
3.117
1.244
1.387
0.820
-
-0.087
-0.190
-0.049
-0.170
-0.106
-
3.129
3.179
1.295
1.443
0.854
-
-0.178
-0.128
0.002
-0.114
-0.072
-
37.90
1.71
38.63
2.45
38.00
25.63
22.37
153.22
-0.40
-0.88
-0.42
-0.49
38.76
26.50
22.45
154.75
0.36
-0.02
-0.33
1.04
18-CH2"
18-CH2'
19-C
3.541
3.111
-
0.307
-0.123
-
3.192
3.434
-
-0.042
0.2
36.40
0.15
37.81
1.56
135.85
129.05
128.70
127.02
65.89
-1.40
-0.73
0.41
0.83
-0.14
0.33
137.49
130.66
129.84
127.84
66.70
0.24
0.87
1.54
1.65
0.67
1.86
-
20-CH
21-CH
22-CH
23-CH2
24-CH
25-CH2c
25-CH2t
9-NH
7.195
7.260
7.217
4.662
5.951
5.262
5.360
6.111
7.751
-0.027
-0.050
0.130
0.039
0.077
-0.018
-0.005
-0.884
-0.846
7.195
7.223
7.169
4.636
5.976
5.228
5.347
8.167
8.189
-0.027
-0.087
0.082
0.013
0.102
-0.052
-0.018
1.172
-0.408
132.22
133.75
118.27
0.03
118.14
-0.10
119.66
96.26
9.50
n.d
n.d
15-NH
-1.91
n.d.
n.d.
Dashes indicate missing resonances, nd is instead used for “not detected” signals probably due to the lack of
sensitivity

Table 4. Chemical shift (in ppm) recorded for 13C and 15N hetero nuclei (X) and hydrogen nuclei
belonging to the molecule 4 in CDCl3 and in CD3OH. Delta values indicate chemical shift deviation
in ppm respect to expected resonances calculated on the basis of a wide database of compounds.
Remarkable deltas are indicated in gray, for compound 4 these values in CDCl3 are somewhat more
respect to compound 2 but less pronounced respect to the records concerning compound 3.
compound 4 in CDCl3
compound 4 in CD3OH
Atom
type and
label
X Shift
X Delta
H Shift
H Delta
X Shift
X Delta
H Shift
H Delta
2-C
3-C
157.20
128.94
120.32
107.19
126.00
59.26
-0.19
-0.10
-3.13
-4.02
-4.02
-1.87
-0.69
1.82
-
-
158.35
129.75
122.46
107.17
129.09
60.75
0.96
0.72
-0.99
-4.04
-0.92
-0.38
1.53
2.51
2.51
0.36
-0.15
-
-
-
-
-
-
4-CH
8.016
6.298
7.303
5.664
-
0.149
0.144
-1.201
0.620
-
7.940
6.319
7.470
5.735
0.073
0.165
-1.034
0.691
0.000
-0.184
-0.124
-0.007
-0.117
5-CH
6-CH
7-CH
8-C
167.95
38.01
170.17
38.70
10-CH2'
10-CH2"
11-CH2
12-CH
13-CH3
14-CH3
16-C
3.206
3.110
1.246
1.384
-0.101
-0.197
-0.047
-0.173
3.123
3.183
1.286
1.440
38.01
1.82
38.70
38.03
-0.37
-0.88
-0.47
-0.43
0.33
38.76
25.64
26.37
22.32
0.811
-0.115
22.46
-0.33
0.846
-0.080
22.36
153.29
36.45
-
-
154.86
37.74
1.90
1.49
1.49
-0.13
0.12
0.97
1.53
0.95
1.01
1.28
0.73
1.34
24.09
11.58
-6.72
-
-
18-CH2'
18-CH2"
19-C
0.20
3.095
3.530
-
-0.139
0.296
-
3.194
3.425
-
-0.040
0.191
-
36.45
0.20
37.74
135.85
129.12
128.72
127.14
67.16
-1.40
-0.66
0.43
137.12
129.90
129.26
127.72
67.74
20-CH
21-CH
22-CH
23-CH2
24-C
7.190
7.250
7.216
5.198
-
-0.032
-0.060
0.129
0.069
-
7.193
7.211
7.166
5.177
-
-0.029
-0.099
0.079
0.048
-
0.95
0.37
135.91
128.65
128.23
128.41
172.63
119.75
96.66
-0.52
0.12
137.43
128.84
129.26
128.96
172.60
121.75
95.76
25-CH
26-CH
27-CH
1-N
7.380
7.389
7.349
-
0.059
-0.023
0.155
-
7.396
7.361
7.325
-
-0.016
0.040
0.131
-
0.67
0.80
24.13
9.59
9-NH
6.158
7.780
-0.837
-0.958
8.154
8.211
1.159
-0.527
15-NH
-5.82
Dashes indicate missing resonances, nd in instead used for “not detected” signals probably due to the lack of
sensitivity
As demonstrated in other similar cases, compounds 2, 3 and 4 in polar and protic solvents (CD3OH)
display a greater conformational freedom, whereas, in apolar environments (CDCl3) data confirm
the permanent presence of intramolecular interactions [26] eliciting limited molecular

arrangements. Any information about conformational features [28, 29] and kinetic freedom [30] is
crucial to understand the possible efficacy of drugs, since it provides the descriptors shared as the
basement for any leads [31]. In this case, all the NMR data (see below and supporting information)
are consistent with the presence of a prevailing conformation in CDCl3 stabilized by intramolecular
dipolar interactions; this conformation is very close to the minimized models obtained through
optimization calculations. The detected stiffness concerns the N1-C7-C8-N9 and C3-N15-C16
frameworks connected to the pyridone core ring, however, only for compounds 2 and 4, some space
contacts (NOE NMR) reveal the allowed rotation through the N1-C7 dihedral angle. It is not the
case of compound 3 which also present more defined elements featuring the prevalent conformation
represented in Fig. 3 and reported in details into the supplementary text. On the other hand, all the
three molecules display some dynamic freedom concerning the peripheral chains attached to the 10-
CH2 and 23-CH2 knots, this fluxional behavior is enhanced in CD3OH and involves also the core
structure by the weakening of intramolecular interactions replaced by the intermolecular
interactions (solvation). On the basis of the mentioned basic knowledge about drugs, the less
pronounced conformational freedom of 3 in apolar solvents accounts for its enhanced activity
towards the biological substrate, whereas the conformational changes in polar media will guarantee
the necessary drug ability to kinetically fit in to the bioactive site.

Fig. 2. 1H-NMR profiles of the compounds 2, 3 and 4 in CDCl3 and CD3OH. The main differences
are highlighted.
Fig. 3. Schematic conformational representation of 3, whose conformational freedom results limited
in CDCl3 and is enhanced in polar protic solvents like CD3OH. Darker rotation arrows indicate
more dynamic freedom.

3. Material and Methods
3.1. 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. Semipreparative HPLC was performed on a Waters 1525 binary
HPLC pump system equipped with a Rheodyne model 3725i injector (2 mL sample loop). Reverse-
phase chromatography was carried out on a Merck Chromolith SemiPrep RP-18e column (100x10
mm i.d.), at a temperature of 20°C, with a mobile phase of water/acetonitrile (90:10) and a flow rate
of 5 mL min-1. A Waters 2489 UV/Vis dual-wavelength absorbance detector was used, and
chromatograms were processed using Waters HPLC Breeze 2 software. The detector wavelength
was set at 266 and 310 nm. All solutions employed in the analysis were filtered through 25 mm x
0.45 mm polypropylene (PP) membrane. 1H-NMR and 13C-NMR spectra were recorded on a Varian
1
13
500 MHz spectrometer operating at 499.74 and 125.73 MHz for H-NMR and C-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).
(S)-allyl
(1-(1-(isopentylamino)-4-methyl-1-oxopentan-2-yl)-2-oxo-1,2-dihydropyridin-3-
yl)carbamate 2. In a round bottom flask at 0°C and under nitrogen atmosphere, acid 9 (1.0 eq.) was
solubilized in DCM/DMF (1:1) and HOBt (1.5 eq) and EDCI (1.5 eq.) were added. After 10 min,
DIPEA (2 eq.) and isopentylamine (1.2 eq.) were added and the reaction was stirred at rt overnight.
Subsequently, solvents were removed in vacuo and the resulting residue was dissolved in EtOAc
and washed with 1 M HCl (x3), saturated NaHCO3 solution (x3) and brine (x3), dried over MgSO4,
filtered and concentrated under reduced pressure following preliminary purification by flash column
chromatography (EtOAc/light petroleum 9/1) and subsequent semipreparative RP-HPLC.

Consistency: white powder; yield: 68%. Rf=0.47 Elemental analysis for C20H31N3O4: calculated: C
1
63.64, H 8.28, N 11.13; C 63.79, H 8.52, N 10.84. H NMR (500 MHz, chloroform-d) δ ppm 0.86
(dd, J=6.71, 2.44 Hz, 6 H) 0.90 - 0.96 (m, 6 H) 1.40 - 1.47 (m, 2 H) 1.47 - 1.54 (m, 2 H) 1.83 (ddd,
J=14.12, 8.32, 5.95 Hz, 1 H) 1.95 - 2.08 (m, 2 H) 3.11 - 3.20 (m, 1 H) 3.21 - 3.35 (m, 2 H) 4.67 (br
d, J=5.49 Hz, 1 H) 5.27 (br dd, J=10.53, 1.07 Hz, 1 H) 5.37 (br dd, J=17.24, 1.37 Hz, 1 H) 5.53 (br
t, J=7.78 Hz, 1 H) 5.91 - 6.01 (m, 1 H) 6.28 - 6.31 (m, 1 H) 6.31 (t, J=7.32 Hz, 2 H) 7.18 (dd,
J=7.02, 1.53 Hz, 1 H) 7.81 (br s, 1 H) 7.96 - 8.09 (m, 1 H). Extensive spectral data are reported in
the supportive file.
(S)-allyl
(1-(1-(isopentylamino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-
yl)carbamate 3. Synthesis of 3 was carried out following the same procedure used for 2, using the
intermediate 10 as acid. Consistency: white powder; Yield: 73%. Rf= 0.50 (EtOAc/light petroleum
9/1) Elemental analysis for C23H29N3O4: calculated: C 67.13, H 7.10, N 10.21; found: C 66.94, H
1
7.34, N 10.14. H NMR (500 MHz, chloroform-d) δ ppm 0.82 (dd, J=6.60, 1.32 Hz, 6 H) 1.21 -
1.27 (m, 2 H) 1.38 (br dd, J=13.43, 6.68 Hz, 1 H) 3.11 (dd, J=13.72, 6.82 Hz, 1 H) 3.15 (br s, 1 H)
3.19 - 3.25 (m, 1 H) 3.54 (dd, J=13.72, 8.58 Hz, 1 H) 4.66 (d, J=5.58 Hz, 1 H) 5.26 (dd, J=10.42,
1.03 Hz, 1 H) 5.36 (br dd, J=17.24, 1.39 Hz, 1 H) 5.67 (br t, J=7.92 Hz, 1 H) 5.86 - 6.02 (m, 2 H)
6.11 (br s, 1 H) 6.30 (t, J=7.26 Hz, 1 H) 7.16 - 7.21 (m, 1 H) 7.22 (br d, J=7.04 Hz, 1 H) 7.24 - 7.28
(m, 9 H) 7.30 (dd, J=7.04, 1.32 Hz, 1 H) 7.75 (s, 1 H) 8.00 (br d, J=6.60 Hz, 1 H) Extensive
spectral data are reported in the supportive file.
(S)-benzyl
(1-(1-(isopentylamino)-1-oxo-3-phenylpropan-2-yl)-2-oxo-1,2-dihydropyridin-3-
yl)carbamate 4. Synthesis of 4 was carried out following the same procedure used for 2, using the
intermediate 11 as acid. Consistency: white powder; yield: 77%; Rf = 0.53 (EtOAc/light petroleum
9/1). Elemental analysis for C27H31N3O4: calculated: C 70.26, H 6.77, N 9.10; C 69.97, H 6.72, N
1
9.29. H NMR (500 MHz, chloroform-d) δ ppm 0.81 (dd, J=6.72, 1.34 Hz, 6 H) 1.20 - 1.28 (m, 2
H) 1.38 (dq, J=13.40, 6.62 Hz, 1 H) 3.08 (s, 1 H) 3.09 - 3.14 (m, 2 H) 3.21 (td, J=13.53, 7.23 Hz, 1
H) 3.53 (dd, J=13.61, 8.57 Hz, 1 H) 5.20 (s, 1 H) 5.66 (br t, J=7.90 Hz, 2 H) 6.16 (br s, 1 H) 6.30 (t,

J=7.23 Hz, 1 H) 7.16 - 7.20 (m, 2 H) 7.22 (br d, J=1.34 Hz, 1 H) 7.23 - 7.26 (m, 1 H) 7.30 (dd,
J=7.06, 1.34 Hz, 1 H) 7.34 - 7.36 (m, 1 H) 7.38 (s, 1 H) 7.38 - 7.41 (m, 2 H) 7.78 (s, 1 H) 7.97 -
8.09 (m, 1 H).
3.2.
Conformational analyses
Small amounts of pure compounds 2, 3 and 4 (0.6, 1.2 and 2.4 mg respectively) were first
dissolved in CDCl3 (600 µL) and poured in the 5 mm test tubes in order to run the thorough NMR
analysis for the total assignment. After these analyses the same tubes were freeze-dyed under
vacuum to eliminate the solvent and re-dissolve the same matter in CD3OH (650 µL) clearly known
as polar and protic solvent mimicking polar media in biological conditions. Again, the complete
NMR analyses were accomplished to elicit the elements supporting similar and different features of
the conformational behaviour of such species.
13
15
1H, C{1H} and N{1H} NMR spectra of 2, 3 and 4 Varian 500 MHz spectrometer equipped
with a ONE_NMR probe and operating at 499.74, 125.73, 50.65 MHz respectively. For CDCl3
solutions calibration was set according to signals of the added standard TMS (tetra-methyl-silane;
H= 0.00 ppm;C= 0.00 ppm); for CD3OH calibration is fixed using the residual proton signals of
CD2HOH (H= 3.30 ppm) and the solvent septuplet 13C resonance (C= 39.52ppm). 15N calibration
was referred to the CH3NO2 as external standard (90% CH3NO2 in CD3OH  = 380.5 ppm).
Complete and unambiguous assignment was pursued by several 1D and 2D homo- and hetero-
13
13
nuclear NMR experiments such as 2D-TOCSY, 2D-NOESY or ROESY, C-HSQC, C-HMBC,
15
15N-HSQC and N-HMBC (Tables 2-4). In order to summarize all the data related to molecular
structures we have used the ACD/lab 2012 free version. Extended material is reported into the
supplementary data. In order to support the detected conformation of compound 3 we have used
GAUSSIAN03 software package for modeling and DFT B3LYP method with G6-31 basis sets for
energy optimization. Two stable conformations changing dihedral angle around the N1-C7 bond
were explored. As expected, the conformation close to the main conformation detected in CDCl3 in

Fig. (3) was about 18.43 kJ/mol, in the gas phase, more stable than the best found alternative (the
two minimized structures reported in Section 4 show an energy gap of 0.00702 Hartrees equal to
18.43 kJ/mol). It is supporting the overall discussion concerning the conformational analysis.
3.3.
In vitro 20S immunoproteasome/proteasome inhibition assays
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
-1
means of the Cheng-Prusoff equation Ki = IC50/(1 + [S] Km ). 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.
4. Conclusions
This study is based on novel derivatives generated from a lead compound with the purpose to
design an active inhibitor against immunoproteasome with specific selectivity toward the important
5i subunit, i.e compound 3. In this context three compounds with different substitution pattern
were synthesized and tested in vitro for their biological activity. In order to find out the rationale of
the obtained results we achieved an extensive hetero-nuclear NMR analysis taking advantage of a
careful global evaluation of many data processed in paragraph 2 (see also supporting information).
As expected, given a thermodynamically favoured conformation, all of the three molecules present
a certain dynamic freedom in CD3OH which is a protic solvent mimicking biological polar media.
On the other hand, in the apolar solvent CDCl3 the conformational freedom is greatly reduced by
the clear detection of dipolar intramolecular interactions; interestingly this trend is definitely more
pronounced for the more active and selective compound. The conformational stiffness in apolar
media accounts for the thermodynamic advantage of this construct to enter the macromolecular
apolar binding site, still keeping the necessary kinetic mobility coming from the polar (water-like)
biological media. This strategy can be adopted to study also many other biologically active
molecules.
List of Abbreviations
CPs = constitutive core particles
C-L = caspase-like
T-L = trypsin-like

ChT-L = chymotrypsin-like
iCPs = immuno-core particles
MM = multiple myeloma
Phe = phenylalanine
Leu = leucine
DCM= dichoromethane
DMF = dimethylformamide
HOBt= Hydroxybenzotriazole
EDCI=N-Ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride
DIPEA= N,N-diisopropylethylamine
NMR = Nuclear Magnetic Resonance
NOESY = nuclear Overhauser effect spectroscopy
2D = two-dimensional
ROESY = rotating-frame Overhauser effect spectroscopy
HSQC = hetero-nuclear single quantum coherence spectroscopy
HMBC = hetero-nuclear multi-bond correlation spectroscopy
TMS = Tetra-Methyl-Sylane
EDTA = Ethylene-diamino-tetra-acetic acid
SDS = sodium dodecyl sulphate
Acknowledgements
This work was financially supported by the Ministero dell’Istruzione, dell’Universita‘ e della
Ricerca Scientifica e Tecnologica [MIUR-PRIN2010-2011, grant 2010W7YRLZ_004 (M.Z.)].
Declaration of interests
x The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
CREDIT AUTHOR STATEMENT
Archimede Rotondo: Conceptualization, Methodology, Software, Writing,Original draft
preparation, final editing. Maria Zappalà: Supervision, coordination. Santo Previti: Data
extraction, writing collaboration. Carla Di Chio: Data analysis (pharmacological). Alessandro
Allegra: Data analysis (pharmacological): Roberta Ettari: Co-Writing, Co-Editing, Co
Supervising
Supplementary Material Content
Section 1. Detailed NMR analysis.
Section 2. Extended spectra and data concerning compound 2 in CDCl3 and in CD3OD
Section 3. Extended spectra and data concerning compound 3 in CDCl3 and in CD3OD
Section 4. Extended spectra and data concerning compound 4 in CDCl3 and in CD3OD
Section 5. Gaussian optimization (DFT B3LYP methods with 6/3/1 G basis sets) for compound
3 in two different conformations
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GRAPHICAL ABSTRACT