Development of peptidomimetic boronates as proteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.03.032

Proteasome inhibition has emerged over the past decade as an effective therapeutic approach for the treatment of hematologic malignancies. It is a multicatalytic complex, whose proteolytic activity relies in three types of subunits: chymotrypsin-like (β5), trypsin-like (β2) and caspase-like (β1). Most important for the development of effective antitumor agents is the inhibition of the β5 subunits. In this context, the dipeptide boronate bortezomib (Velcade) represents the first proteasome inhibitor approved by the FDA and the lead compound in drug discovery. This paper describes the synthesis and biological evaluation of a series of conformationally constrained pseudopeptide boronates (1-3) structurally related to bortezomib. The synthesized compounds showed a promising inhibitory profile by blocking primarily the chymotrypsin-like activity of the proteasome with K_i values in submicromolar/micromolar range. These compounds also resulted quite selective since no significant inhibition was recorded in the test against bovine pancreatic α-chymotrypsin. The obtained results were rationalized by means of docking experiments based on a model of the crystal structure of bortezomib bound to the yeast 20S proteasome providing essential insights for further optimization of this class of inhibitors.

Full text of DOI:10.1016/j.ejmech.2013.03.032

European Journal of Medicinal Chemistry 64 (2013) 23e34
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of peptidomimetic boronates as proteasome inhibitors
,
b
c
c
Nicola Micale a , Roberta Ettari , Antonio Lavecchia , Carmen Di Giovanni ,
Kety Scarbaci a, Valeria Troiano a, Silvana Grasso a, Ettore Novellino c, Tanja Schirmeister d,
Maria Zappalà a
*
a Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
c Dipartimento di Farmacia, “Drug Discovery” Laboratory, Università degli Studi di Napoli Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy
d Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteasome inhibition has emerged over the past decade as an effective therapeutic approach for the
treatment of hematologic malignancies. It is a multicatalytic complex, whose proteolytic activity relies in
Received 12 December 2012
Received in revised form
1 March 2013
Accepted 20 March 2013
Available online 27 March 2013
three types of subunits: chymotrypsin-like (
b
5), trypsin-like (
b
2) and caspase-like (
b1). Most important
for the development of effective antitumor agents is the inhibition of the
b
5 subunits. In this context, the
dipeptide boronate bortezomib (VelcadeÒ) represents the first proteasome inhibitor approved by the FDA
and the lead compound in drug discovery. This paper describes the synthesis and biological evaluation of
a series of conformationally constrained pseudopeptide boronates (1e3) structurally related to borte-
zomib. The synthesized compounds showed a promising inhibitory profile by blocking primarily the
chymotrypsin-like activity of the proteasome with Ki values in submicromolar/micromolar range. These
compounds also resulted quite selective since no significant inhibition was recorded in the test against
Keywords:
Peptidomimetic boronates
Proteasome inhibitors
Docking studies
bovine pancreatic a-chymotrypsin. The obtained results were rationalized by means of docking exper-
iments based on a model of the crystal structure of bortezomib bound to the yeast 20S proteasome
providing essential insights for further optimization of this class of inhibitors.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
regulate cell proliferation and survival pathways. Thus, defects in
the proteasome activity can lead to anarchic cell proliferation and
The 26S proteasome is a multisubunit, multicatalytic threonine
protease complex involved in different phases of cellular life-cycle.
It is composed of a 20S catalytic core capped by two 19S regulatory
complexes. Its proteolytic activity relies in three types of subunit
tumor development. For these reasons, proteasome inhibition, in
particular of the ChT-L activity, has become a significant strategy for
drug development in cancer treatment [1e3].
In this context, we designed and synthesized conformationally
constrained pseudopeptide boronates (1e3, Fig. 1) using bortezo-
mib as lead compound, the first proteasome inhibitor approved by
FDA in 2003 for the treatment of relapsed and refractory multiple
myeloma [4] and mantle cell lymphoma [5]. These constrained
surrogates should have in principle several advantages comparing
to peptide inhibitors such as stability toward protease degradation,
good cell permeability and target-selectivity by stabilizing a bio-
logically active conformation [6]. This strategy has been accom-
plished by: i) maintaining the Leu-boronic electrophilic warhead at
(
b
1,
b
2 and
b
5) positioned into the barrel-like 20S core. These
5), trypsin-like (T-L)
2), and post-glutamyl peptide hydrolyzing (PGPH) or caspase-like
1) activities, so named for the preferred substrate they hydrolyze.
subunits possess chymotrypsin-like (ChT-L) (
b
(
(
b
b
Targeting of eukaryotic proteins to this proteolytic system requires
their prior marking by polyubiquitin chains. Proteasome is pri-
marily responsible for the turnover of intracellular proteins that
P1 due to its essentiality in binding interaction with the g-OH group
Abbreviations: ChT-L, chymotrypsin-like; T-L, trypsin-like; PGPH, post-glutamyl
peptide hydrolyzing; DMSO, dimethyl sulfoxide; DMF, dimethylformamide;
of the N-terminal catalytic Thr; ii) substituting the Phe residue at P2
with less hindered residues such as Gly or Ala due to the fact that
there is no much specificity assessed for this site; iii) enclosing the
amide moiety at P3 in a 1H-pyridin-2-one ring to reduce the
peptidic character of the compound as well as to reduce its
Pd2(dba)3,
tris(benzylideneacetone)dipalladium;
EDC,
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBt, N-hydroxybenzotriazole; DIPEA, N,N-
diisopropylethylamine.
* Corresponding author. Tel.: þ39 090 6766419; fax: þ39 090 6766402.
E-mail address: nmicale@unime.it (N. Micale).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.032

24
N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
Fig. 1. Structure of bortezomib and compounds 1e3.
conformational freedom; iv) maintaining a basic moiety corre-
sponding to N4 of the pyrazine ring of bortezomib taking into ac-
count that the complex bortezomib-
through an interaction involving this nitrogen and the D114 residue
of the adjacent 6 subunit [7].
Two series of compounds came out from our design. In the first
series (i.e. 1aeg, Scheme 2), the constrained motif is represented by
the sole 1H-pyridin-2-one scaffold bearing secondary or tertiary
amines at C-4 (1beg) with the unsubstituted derivative 1a as
reference compound to validate our hypothesis of the interaction
b5 subunit is stabilized
Scheme 1. Reagents and conditions: (a) BnOH, KH, THF, rt, 18 h, N2, 87e94%; (b)
Pd2(dba)3, DavePhos, amine, t-BuONa, toluene, MW, 120e150 ꢀC, 15 min, N2, 65e88%;
(c) H2, 10% Pd/C, MeOH/EtOAc (2:1), rt, 2 h, 95e99% (9bef) or BBr3, CH2Cl2, ꢁ78 ꢀC, 4 h,
99% (9g).
b
2.2. Biological activity
with b6 subunit. In the second series [i.e. 2aec (Scheme 3) and 3
(Scheme 4)], the constrained motif is represented by a bicyclic 1,6-
naphthyridin-5(6H)-one scaffold.
The newly synthesized peptidomimetic boronates 1e3 have
been tested for their inhibitory properties by using purified 20S
proteasome isolated from human erythrocytes and the appropriate
fluorogenic substrate for each of the proteolytic activities (i.e. Suc-
Leu-Leu-Val-Tyr-AMC for ChT-L; Boc-Leu-Arg-Arg-AMC for T-L; Z-
Leu-Leu-Glu-AMC for PGPH).
2. Results and discussion
2.1. Chemistry
First, compounds 1e3 underwent a preliminary screening for
ChT-L activity at 20 mM using an equivalent volume of dimethyl
The pyridone scaffolds 9aeg (Scheme 1) were synthesized by
means of an initial nucleophilic aromatic substitution reaction of
2-chloropyridines (4e5) with benzyl alcohol in the presence of
potassium hydride followed by debenzylation of the resulting O-
pyridyl benzyl ethers (6e7) under transfer hydrogenation condi-
tions. The 1H-pyridin-2-one 9a was obtained directly by catalytic
debenzylation of 2-benzyloxypyridine 6, whereas, for compounds
9beg, the debenzylation reaction was preceded by a microwave-
sulfoxide (DMSO) as a negative control. The screening showed that
all compounds, with the exception of 1f, inhibited more than 40% of
the enzyme activity.
Continuous assays were then performed (progress curve
method, at seven different concentrations, ranging from those that
minimally inhibited to those that fully inhibited the enzyme, Fig. 2)
to determine the Ki values (Fig. 3), reported in Table 1.
As can be noticed, all compounds showed promising inhibitory
properties toward the ChT-L activity of the proteasome, with Ki
values in the submicromolar/micromolar range.
promoted BuchwaldeHartwig amination at position
4 of 4-
chloro-2-benzyloxypyridine 7 in presence of Pd2(dba)3 as a cata-
lyst, the DavePhos ligand and a suitable amine to give in-
termediates 8beg (Scheme 1), according to a procedure reported
in literature [8]. The obtained 1H-pyridin-2-ones 9aeg were N-
alkylated with ethyl bromoacetate in the presence of NaH to give
esters 10aeg (Scheme 2), which were in turn smoothly converted
into the corresponding acids 11aeg by alkaline hydrolysis with
LiOH. Coupling reactions between acids 11aeg and pinanediol
leucine boronate 12 [9] in the presence of EDC$HCl, HOBt and
DIPEA gave the pinanediol esters 13aeg (Scheme 2), which were
directly used for the successive trans-esterification reaction with
isobutylboronic acid under acid conditions to provide the title
boronic acids 1aeg.
The inhibitory activity toward the other two proteasome activ-
ities, as well as the selectivity of these compounds toward the
target enzyme, by using bovine pancreatic
a-chymotrypsin, was
determined with the same method. None of these pseudopeptide
boronates exhibited any inhibition in the T-L activity assay at
20 mM. Conversely, all of them but 1g inhibited the PGPH activity of
the proteasome, although to a much lesser extent compared with
the ChT-L activity inhibition (generally one or two order of
magnitude), with the exception of 1c which is more potent in PGPH
assay (Table 1). This outcome, per se, is extremely important since
literature data suggest that the inhibition of only
proteasome has a moderate effect on inhibition of protein degra-
dation pathways [12], whereas the co-inhibition of 5 subunits
with either 1 or 2 subunits exerts the maximal effect that is
b5 subunits of the
Synthesis of the boronic acids 2aec and 3 has been conducted
with a similar approach starting from 1,6-naphthyridin-5(6H)-ones
14e15 and 1-methyl-1,2,3,4-tetrahydro-1,6-naphthyridin-5(6H)-
one 18 [10], as depicted in Schemes 3 and 4, respectively.
b
b
b
required to produce an antitumor response [13]. On the other hand,
it is also known that the inhibition of all proteasome subunits is

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
25
Scheme 2. Reagents and conditions: (a) NaH, DMF, 0 ꢀC, 1 h; then ethyl bromoacetate, rt, 2 h, N2, 44e67%; (b) 1 N LiOH, EtOH, 0 ꢀC / rt, 4 h, 75e90%; (c) HOBt, CH2Cl2, ꢁ5 ꢀC,
i
20 min, then EDC$HCl, DIPEA, 12, ꢁ15 ꢀC / rt, 3 h, 83e99%; (d) BuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 30e50%.
generally cytotoxic [14]. The preliminary screening against
a
-
The condensation of a pyridine ring on the 1H-pyridin-2-one
nucleus of 1a does not alter the inhibitory activity. In fact, the
1,6-naphthyridin-5(6H)-one 2a showed Ki values submicromolar
and micromolar toward ChT-L and PGPH activities, respectively. By
replacing the Gly residue at P2 site of 2a with an Ala residue (2b),
both Ki values decreased of one order of magnitude and a compa-
chymotrypsin showed no significant inhibition for all compounds
with the exception of 1e and 2b, which gave Ki values 12.7 ꢂ 7.1 and
9.16 ꢂ 7.53
mM, respectively.
A survey of the results obtained for compounds 1aeg put in
evidence that the 1H-pyridin-2-one nucleus as such is fundamental
in determining the 20S proteasome inhibition. Indeed, 1a is one of
rable activity was also detected toward bovine pancreatic
a-
the most active compounds of this series with a Ki ¼ 0.26
m
M for the
chymotrypsin (Ki ¼ 9.16
m
M). An enhancement of the ChT-L
ChT-L activity and a Ki ¼ 1.38 M for PGPH activity. The introduction
m
inhibitory activity of 2a was achieved by introducing a bromine
atom at C-8 of the 1,6-naphthyridin-5(6H)-one scaffold (i.e. 2c,
of a tertiary amine at C-4 reduces the ChT-L and PGPH inhibition, in
particular, the presence of a piperazine ring is decisively detri-
mental: no PGPH inhibition was detected with 1f and 1g, and only a
fair ChT-L inhibition was recorded for 1g. Out of this trend lies the
4-(1-piperidinyl)-substituted derivative 1d which has an inhibition
profile similar to that of 1a.
Ki ¼ 0.17
m
M). The presence of the bromine atom makes it possible
to discriminate between
b
5 and 1 subunits of the proteasome with
b
a difference of two orders of magnitude in Ki values. Differently, the
N-methyl-tetrahydro derivative 3 showed a similar degree of in-
hibition of ChT-L and PGPH activities, both in a micromolar range.
The most relevant result for this series of compounds was ob-
tained by introducing a secondary amine at C-4. As a matter of fact,
the aniline derivative 1e maintained a micromolar inhibitory pro-
file toward the PGPH activity of the 20S proteasome and reached
nanomolar values of inhibition toward the ChT-L activity
2.3. Docking studies
In order to help interpretation of biological activity data and to
gain a better understanding of how the described peptidomimetic
boronates might bind to the 20S proteasome, we carried out
docking experiments of inhibitors 1aee and 2c into the binding
(Ki ¼ 0.098
m
M). Compound 1e also showed a slight inhibition to-
-chymotrypsin (Ki ¼ 12.7 M).
ward bovine pancreatic
a
m
Scheme 3. Reagents and conditions: (a) NaH, DMF, 0 ꢀC, 1 h, then ethyl bromoacetate or methyl 2(R)-bromopropionate, rt, 2 h, N2, 34e65%; (b) 1 N LiOH, EtOH or MeOH, 0 ꢀC / rt,
i
6 h, 88e98%; (c) HOBt, CH2Cl2, ꢁ5 ꢀC, 20 min, then EDC$HCl, DIPEA, 12, ꢁ15 ꢀC / rt, 3 h, 70e100%; (d) BuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 32e34%.

26
N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
Scheme 4. Reagents and conditions: (a) NaH, DMF, 0 ꢀC, 1 h, then ethyl bromoacetate, rt, 2 h, N2, 78%; (b) 1 N LiOH, EtOH, 0 ꢀC / rt, 6 h, 95%. (c) HOBt, CH2Cl2, ꢁ5 ꢀC, 20 min, then
i
EDC$HCl, DIPEA, 12, ꢁ15 ꢀC / rt, 3 h, 50%; (d) BuB(OH)2, 1 N HCl, MeOH/n-hexane (1:1), rt, 18 h, 28%.
pocket of the
b
5 subunit of the previously reported crystal structure
observed in the crystal structure. H-bonds with
b5-T21, b5-G47, b5-
of bortezomib bound to the yeast 20S proteasome (PDB ID: 2F16)
[7]. Because of the covalent binding of peptide boronic acids with
proteasome, all the covalent docking simulations were performed
by employing GOLD 5.1 [15,16] which has a unique function for
handling covalent docking.
A49, and 6-D114 of proteasome were also reproduced in the
b
docked structure. Thus, this docking protocol was considered to be
suitable enough to be used in the prediction of the binding modes
of 1aee and 2c into the 20S proteasome.
When 1aee and 2c were docked within the b5 subunit binding
The crystal structure of 20S/bortezomib complex revealed one
pocket, a convergent binding mode was largely adopted. All these
proteasome inhibitors were found to be in the same location as
bortezomib in the crystal structure. In particular, compounds 1ae
d and 2c assumed a folded conformation stabilized by an intra-
molecular H-bond involving the pyridone C]O oxygen and one of
the OH groups of the boronic acid fragment. On the contrary, the
most active compound 1e adopted an extended conformation
similar to that observed crystallographically for bortezomib. Fig. 4
depicts the binding mode of the representative compounds 1a, 1e
and 2c into the proteasome binding site and their overlay with
bortezomib.
well-defined water molecule in proximity to
ordinates a tight H-bonding network, interacting with
5-A49 N and 5-A50 N of the protein and with the C]O oxygen of
bortezomib [7]. Moreover, one of the pyrazine nitrogens of borte-
zomib interacted via a direct H-bond with the protonated 6-D114.
It is to note that the pKa of a protonated pyrazine is <1 and thus
presumably 6-D114 is protonated, since in the X-ray structure the
b
6-D114, which co-
b
6-D114 O
g,
b
b
b
b
ꢀ
OeN distance is 2.9 A, indicative of a strong H-bond. Accordingly,
the intervening water molecule and the proper protonation state of
D114 were included in the docking experiments.
The Goldscore-CS docking protocol [17], was adopted in this
study. In this protocol, the poses obtained with the original Gold-
score function are rescored and reranked with the GOLD imple-
mentation of the Chemscore function [17e21].
To test the validity of this protocol for the 20S proteasome
system, the crystal structure of bortezomib was first docked back
As expected, in all docked compounds, the boron atom formed a
covalent interaction with the nucleophilic oxygen of
whereas the two acidic boronate OH groups engaged H-bonds with
the T1 NH2 group and the backbone C]O of 5-Y168. Backbone of
compounds 1aed and 2c formed two H-bonds with residues G47
b5 Og-T1,
b
ꢀ
ꢀ
(3.1 A) and T21 (2.9 A) of
b5 subunit (Fig. 4a,e), whereas compound
ꢀ
into the binding pocket of the b5 subunit. In this docking run, the
1e established H-bonds with residue A49 (2.9 A) and the proton-
ated D114 (3.0 A) in the b6 subunit (Fig. 4c). This latter interaction is
ꢀ
200 poses produced by GOLD resulted in only one prevailing cluster
on the basis of their conformations. The top-ranked pose obtained
after rescoring with Chemscore function closely resembled the
cocrystallized conformation with a heavy atom root-mean-square
crucial to retain the ChT-L inhibitory activity of 1e and further
validates our docking results. In addition, the isopropyl substituent
of all inhibitors was found to project into the S1 pocket adopting
the same spatial arrangement of the P1-leucine side chain of
bortezomib.
ꢀ
deviation (rmsd) of 1.5 A. Orientation of the P1eP3 substituents
of bortezomib in the docked conformation were similar to that
1400
1200
1000
1,8
1,6
1,4
1,2
800
F
1
b
600
400
200
0
0,8
0,6
0,4
0,2
0
0
200
400
600
0
2
4
6
8
10 12 14 16 18 20
I
Time (Seconds)
Fig. 2. Progress curves of substrate hydrolysis in the presence of the inhibitor 2c.
F ¼ fluorescence units. Inhibitor concentrations (from top to bottom): 0, 0.1, 0.5, 1.0,
Fig. 3. The slopes of the progress curve (b) from Fig. 1 were plotted against the in-
hibitor concentrations and fitted to the 4 parameter IC50 equation. Ki was obtained
from the equation Ki ¼ IC50/(1 þ [S] Kꢁm1).
5.0, 10.0, 15.0, 20.0 mM.

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
27
Table 1
As depicted in Fig. 4d, the 4-phenylamino substituent of the
most potent inhibitor 1e projected toward the deep specificity S3
binding pocket and was in close contact with residues of the
Inhibition of ChT-L and PGPH proteasome activities by boron tripeptides 1aeg, 2aec
and 3.
Comp
ChT-L Ki (
m
M)a
PGPH Ki (m
M)a
adjacent b6 subunit. This result is in agreement with biochemical
1a
1b
1c
1d
1e
1f
1g
2a
0.26 ꢂ 0.06
0.80 ꢂ 0.11
1.45 ꢂ 0.04
0.30 ꢂ 0.05
0.098 ꢂ 0.005
30% inhibition at 20
5.90 ꢂ 1.47
0.44 ꢂ 0.21
1.34 ꢂ 0.12
0.17 ꢂ 0.01
1.62 ꢂ 0.44
0.0006b
1.38 ꢂ 0.39
4.42 ꢂ 2.09
0.52 ꢂ 0.36
1.42 ꢂ 0.46
5.14 ꢂ 1.10
n.d.
investigations showing that the size and length of the P3 side chain
is crucial for inhibitor potency [22,23].
The pyridinone ring of 1a (Fig. 4b) and the naphthyridinone
moiety of 2c (Fig. 4f) pointed toward the S2 pocket, a large cavity
able to accept space-demanding substituents, occupying the spatial
position of the P2 phenylalanine side chain of bortezomib. These
mM
n.i.
1.55 ꢂ 0.41
13.6 ꢂ 2.9
17.6 ꢂ 0.8
3.90 ꢂ 0.06
moieties established hydrophobic and
pep stacking interactions
2b
2c
3
with T21, S129 and Y168 side chains. Notably, the 8-Br atom of 2c
was engaged in a halogen bond with the backbone C]O oxygen of
ꢀ
Bortezomib
G23. The distance between the bromine and oxygen atoms is 3.1 A,
which is significantly shorter than the sum of the van der Waals’
radii of the two atoms, consistent with quantum chemical calcu-
lations on the nature of halogen bonds [24]. The halogen-bond
angle (CeBr/O) is 132ꢀ. Although halogen bonds are considered
n.d. ¼ not determined; n.i. ¼ no inhibition.
a
Values represent the mean of three independent determinations.
Data reported in Ref. [11].
b
Fig. 4. Binding modes of compounds 1a (a, green), 1e (c, magenta) and 2c (e, yellow) into the
ID: 2F16) [7]. Only amino acids located within 4 A of the bound ligand are displayed (white) and labeled. Key H-bonds between the inhibitors and the protein are shown as dashed
b
5 (cyan)/
b
6 (white) active site of 20S proteasome represented as a ribbon model (PDB
ꢀ
black lines. An overlay of 1a (b, docked pose), 1e (d, docked pose) and 2c (f, docked pose) with bortezomib (purple, X-ray crystal pose) is shown in the b5/b6 active site of 20S
proteasome displayed as Connolly surface. The defined water molecule forming tight H-bonds to the protein is shown as a red sphere. Specificity pockets S1eS4 are indicated. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

28
N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
weak interactions in comparison to classical H-bonds, they benefit
from a lower desolvation penalty and may very well contribute to
inhibitor specificity [25]. The slight decrease in activity in going
from 2c to 2a is consistent with loss of the halogen bond upon
replacement of bromine with hydrogen.
Similar binding modes were also found for 1bed, where the sub-
stituent at position 4 of the pyridinone ring fitted into the S2 pocket
and formed van der Waals and hydrophobic interactions with T21,
S129 and Y168 residues of proteasome. It was proposed that these
three residues and the P2 moiety of the inhibitors are exposed to the
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Reactions under
microwave irradiation were performed on a CEM Discover appa-
ratus. Elemental analyses were carried out on a C. Erba Model 1106
(Elemental Analyzer for C, H and N) and the obtained results are
within ꢂ0.4% of the theoretical values. Merck Silica Gel 60 F254
plates were used as analytical TLC; flash column chromatography
was performed on Merck Silica Gel (200e400 mesh). 1H and 13C
NMR spectra were recorded on a Varian Gemini 300 MHz spec-
trometer using the residual signal of the deuterated solvent as in-
ternal standard. Splitting patterns are described as singlet (s),
doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet
(m) and broad singlet (bs). 1H and 13C chemical shifts are expressed
water in the central cavity of the
b ring and act as a “gateway” of the
pocket. It is worth noting that the more polar 4-morpholino moiety in
1c does not improve the inhibition of 20S proteasome activity in
comparison with the 4-diethylamine and 4-piperidine moieties in 1b
and 1d, respectively (see Table 1). Besides the hydrophilic differences,
another explanation could be depicted: the electron-withdrawing
effect of the oxygen on the morpholine system tends to decrease the
electron densityat the nitrogen making it less basic. If we compare the
activity and pKa of 4-amino substituents of 1b (diethylamine,
pKa ¼ 10.8), 1c (morpholine pKa ¼ 8.50), and 1d (piperidine
pKa ¼ 11.1), it is observed that piperidine and diethylamine remain
protonated upto physiologic pH. Thiscould result in theformation of a
in
d (ppm) and coupling constants (J) in hertz (Hz). MS analyses
were performed on a Varian 320-MS triple quadrupole mass
spectrometer equipped with an electron spray ionization (ESI)
source.
catione
p
interaction with the phenyl ring of Y168, thus rationalizing
4.1.1. Synthesis of 1H-pyridin-2-ones 9aeg
Compounds 9a [26], 9c, 9d, and 9f [8], were synthesized as re-
ported in the literature.
the difference in potency of 1b and 1d compared to 1c.
The obtained biological data and the consequent docking
studies clearly indicate that the flexibility of these pseudopeptides
at P2 constitutes the main issue on which future research efforts
must be focused. As we noticed, in most cases our compounds
adopted a folded conformation that flip the constrained scaffold to
the S2 pocket instead of the originally assumed S3 pocket. One
possibility to prevent the folding is that of introducing bulky hy-
drophobic residues at P2 that might fit with S2 pocket and obstacle
the intramolecular H-bond formation (C]O/HOeB) at the same
time. In this situation the constrained scaffold should be projected
toward the intended S3 pocket as a consequence. Alternatively, the
Gly residue at P2 of these compounds might be replaced with rigid
synthons that likewise should impede the folding.
4.1.2. 2-Benzyloxy-4-chloro-pyridine (7)
A suspension of KH (1.08 g, 27 mmol) in dry THF (80 mL) at 0 ꢀC
under N2, was treated with benzyl alcohol (1.7 mL, 16.2 mmol) and
stirred for 1 h. Then, a solution of 2,4-dichloropyridine 5 (1.5 mL,
13.5 mmol) in THF (5 mL) was added and the mixture was stirred at
room temperature for 18 h. The reaction was quenched with
saturated NH4Cl (20 mL) and the organic solvent was removed in
vacuo. The product was extracted from the residual aqueous layer
with CH2Cl2 (3 ꢃ 50 mL), and the combined organic phases were
dried over Na2SO4, filtered and concentrated in vacuo. The crude
product was purified by column chromatography (light petroleum/
Et2O 8:2) to yield compound 7 as a pale yellow oil (2.58 g, 87%);
Rf ¼ 0.56 (light petroleum/Et2O 8:2). 1H NMR (300 MHz, CDCl3)
3. Conclusions
d
5.46 (s, 2H), 6.87e6.89 (m, 2H), 7.33e7.52 (m, 5H), 8.09 (dd, 1H,
In conclusion, we have identified two series of conformationally
constrained analogs of bortezomib that efficiently inhibit two out of
three of the proteolytic activities of the proteasome (mainly the
ChT-L activity) and showed a good target-selectivity. In regard to
the first series having a 1H-pyridin-2-one ring as a constrained
motif, the 4-aniline-derivative 1e turned out to be the most active
with a Ki value for ChT-L activity of proteasome in nanomolar range.
With the support of docking studies we found out that 1e adopted
J ¼ 4.7, 1.8 Hz).
4.1.3. 2-Benzyloxy-4-diethylamino-pyridine (8b)
To compound 7 (400 mg, 1.82 mmol) were added in sequence
Pd2(dba)3 (1 mol%), DavePhos ligand (1.5 mol%), diethylamine
(225 mL, 2.18 mmol) and t-BuONa (245 mg, 2.55 mmol). The mixture
was suspended with toluene (4 mL) and degassed with N2 over
5 min. The resulting mixture was heated under microwave irradi-
ation at 120e150 ꢀC for 15 min, then the suspension was taken up
into EtOAc (100 mL), filtered through a short layer of celite and
concentrated in vacuo. The crude product was purified by column
chromatography (light petroleum/EtOAc 7:3) to give compound 8b
(349 mg, 75%) as a yellow oil; Rf ¼ 0.64 (light petroleum/EtOAc 7:3).
an extended conformation within the binding pocket of
(as for bortezomib) with the amine moiety interacting with the
D114 residue of the adjacent 6 subunit, according to our initial
b5 subunit
b
design. This fact may explain its specificity toward the ChT-L ac-
tivity of proteasome, that is two orders of magnitude higher with
respect the PGPH activity. The other compounds of this series (1be
g) as well as compounds of the 1,6-naphthyridin-5(6H)-one series
(2aec and 3), although maintain a good inhibitory profile and
target-selectivity, do not show a high subunit specificity due to the
fact that they adopt a folded conformation within the binding
1H NMR (300 MHz, CDCl3)
d
1.16 (t, 6H, J ¼ 7.0 Hz), 3.32 (q, 4H,
J ¼ 7.0 Hz), 5.33 (s, 2H), 5.94 (d, 1H, J ¼ 2.2 Hz), 6.23 (dd, 1H, J ¼ 6.2,
2.0 Hz), 7.28e7.47 (m, 5H), 7.83 (d, 1H, J ¼ 6.2 Hz).
4.1.4. 2-Benzyloxy-4-phenylamino-pyridine (8e)
pocket of the
b
5 subunit stabilized by an intramolecular H-bond
Compound 7 (400 mg, 1.82 mmol) was reacted with aniline
involving the carbonyl oxygen at C2 of the pyridone ring and one of
the OH groups of the boronic acid warhead. Only the C8eBr de-
rivative 2c showed considerable subunit specificity (two orders of
magnitude) due to additional interactions provided by the bromine
atom. Therefore, 1e and 2c will be taken into account as new lead
structures for further design of proteasome inhibitors.
(197 mL, 2.18 mmol), according to the same procedure described for
compound 8b. Compound 8e was obtained as a yellow oil (356 mg,
71%), after purification by column chromatography (CHCl3);
Rf ¼ 0.16 (CHCl3). 1H NMR (300 MHz, CDCl3)
d 5.35 (s, 2H), 6.07 (bs,
1H), 6.36 (d, 1H, J ¼ 1.3 Hz), 6.47 (dd, 1H, J ¼ 5.8, 1.8 Hz), 7.09e7.46
(m, 10H), 7.92 (d, 1H, J ¼ 5.8 Hz).

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
29
4.1.5. 2-Benzyloxy-4-(4-allylpiperazin-1-yl)pyridine (8g)
Compound 7 (400 mg, 1.82 mmol) was reacted with 1-
allylpiperazine (305 mL, 2.18 mmol), according to the same proce-
(300 MHz, CDCl3)
d
1.18 (t, 6H, J ¼ 7.3 Hz),1.29 (t, 3H, J ¼ 7.3 Hz), 3.31
(q, 4H, J ¼ 7.3 Hz), 4.22 (q, 2H, J ¼ 7.3 Hz), 4.57 (s, 2H), 5.54 (d, 1H,
J ¼ 2.6 Hz), 5.83 (dd, 1H, J ¼ 7.7, 2.9 Hz), 7.01 (d, 1H, J ¼ 7.7 Hz).
dure described for compound 8b. Compound 8g was obtained as a
yellow oil (421 mg, 75%), after purification by column chromatog-
raphy (CHCl3/MeOH 95:5); Rf ¼ 0.27 (CHCl3/MeOH 95:5). 1H NMR
4.1.11. [4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid
ethyl ester (10c)
Pyridone 9c (223 mg, 1.24 mmol) was reacted with ethyl bro-
moacetate (193 mL, 1.74 mmol), by employing the same procedure
(300 MHz, CDCl3)
d
2.52 (t, 4H, J ¼ 4.8 Hz), 3.02 (d, 2H, J ¼ 6.7 Hz),
3.30e3.90 (m, 4H, J ¼ 4.8 Hz), 5.16e5.24 (m, 2H), 5.34 (s, 2H), 5.80e
5.93 (m, 1H), 6.13 (d, 1H, J ¼ 1.9 Hz), 6.40 (dd, 1H, J ¼ 6.2, 1.9 Hz),
7.26e7.46 (m, 5H), 7.89 (d, 1H, J ¼ 6.2 Hz).
described for compound 10a, to provide compound 10c as a yellow
oil (144 mg, 44%); Rf ¼ 0.42 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3)
d
1.29 (t, 3H, J ¼ 7.0 Hz), 3.26 (t, 4H, J ¼ 5.3 Hz), 3.79 (t, 4H,
4.1.6. 4-Diethylamino-1H-pyridin-2-one (9b)
J ¼ 5.3 Hz), 4.23 (q, 2H, J ¼ 7.0 Hz), 4.57 (s, 2H), 5.73 (d, 1H,
To a solution of compound 8b (349 mg, 1.36 mmol) in MeOH/
EtOAc (2:1, 30 mL) at room temperature, 10% Pd/C (10 mol%) was
added and the reaction mixture was fluxed with H2 under stirring
for 2 h. The resulting mixture was filtered off through a short layer
of celite and washed with EtOAc (150 mL). The solvent was
removed in vacuo and the resulting residue was taken up into Et2O
(15 mL) and filtered to provide compound 9b as a white powder
(224 mg, 99%); Rf ¼ 0.48 (CHCl3/MeOH 9:1). 1H NMR (300 MHz,
J ¼ 3.1 Hz), 5.95 (dd, 1H, J ¼ 7.5, 3.1 Hz), 7.05 (d, 1H, J ¼ 7.5 Hz).
4.1.12. [4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid ethyl
ester (10d)
Pyridone 9d (270 mg, 1.51 mmol) was reacted with ethyl bro-
moacetate (234 mL, 2.11 mmol), by employing the same procedure
described for compound 10a, to provide compound 10d as a yellow
oil (265 mg, 67%); Rf ¼ 0.56 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
DMSO-d6)
d
1.05 (t, 6H, J ¼ 7.3 Hz), 3.26 (q, 4H, J ¼ 7.3 Hz), 5.13 (d,
CDCl3)
d
1.28 (t, 3H, J ¼ 7.0 Hz), 1.60e1.63 (m, 6H), 3.27e3.30 (m,
1H, J ¼ 2.5 Hz), 5.80 (dd, 1H, J ¼ 7.8, 2.5 Hz), 7.05 (d, 1H, J ¼ 7.8 Hz),
4H), 4.21 (q, 2H, J ¼ 7.1 Hz), 4.54 (s, 2H), 5.70 (d, 1H, J ¼ 2.8 Hz), 5.94
(dd, 1H, J ¼ 7.8, 2.8 Hz), 7.00 (d, 1H, J ¼ 7.8 Hz).
10.52 (bs, 1H).
4.1.7. 4-Phenylamino-1H-pyridin-2-one (9e)
4.1.13. (4-Phenylamino-2-oxo-2H-pyridin-1-yl)-acetic acid ethyl
ester (10e)
Pyridone 9e (236 mg, 1.27 mmol) was reacted with ethyl bro-
moacetate (141 mL, 1.27 mmol), by employing the same procedure
Compound 9e was obtained as a white powder (236 mg, 99%)
from 8e (356 mg, 1.29 mmol), by using the same procedure
described for 9b; Rf ¼ 0.33 (CHCl3/MeOH 9:1). 1H NMR (300 MHz,
DMSO-d6)
d
5.62 (d, 1H, J ¼ 1.2 Hz), 5.86 (dd, 1H, J ¼ 7.0, 1.8 Hz), 7.04
described for compound 10a, to provide compound 10e as a yellow
(t, 1H, J ¼ 7.0 Hz), 7.12e7.35 (m, 5H), 8.64 (bs, 1H), 10.66 (bs, 1H).
oil (231 mg, 67%); Rf ¼ 0.42 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
CDCl3)
d
1.27 (t, 3H, J ¼ 7.0 Hz), 4.21 (q, 2H, J ¼ 7.0 Hz), 4.56 (s, 2H),
4.1.8. 4-(4-Allylpiperazin-1-yl)-1H-pyridin-2-one (9g)
5.91 (dd,1H, J ¼ 7.0, 2.3 Hz), 5.99 (d,1H, J ¼ 2.3 Hz), 6.52 (bs,1H), 7.03
(d, 1H, J ¼ 7.0 Hz), 7.09e7.34 (m, 5H), 8.00 (bs, 1H).
To a solution of compound 8g (421 mg, 1.36 mmol) in dry CH2Cl2
(70 mL), at ꢁ78 ꢀC was added dropwise a 1 M solution of BBr3 in
CH2Cl2 (2.7 mL). After 3 h of stirring at ꢁ78 ꢀC the reaction was
quenched with MeOH (20 mL) and the solvent was evaporated in
vacuo to give compound 9g (296 mg, 99%) which was used directly
for the next step without any purification; Rf ¼ 0.11 (CHCl3/MeOH
9:1). 1H NMR (300 MHz, DMSO-d6), 2.50 (t, 4H, J ¼ 5.3 Hz), 3.05 (d,
2H, J ¼ 6.5 Hz), 3.28 (t, 4H, J ¼ 5.2 Hz), 5.16e5.26 (m, 2H), 5.70 (d,
1H, J ¼ 2.6 Hz), 5.80e5.91 (m, 1H), 5.96 (dd, 1H, J ¼ 7.2, 2.6 Hz), 7.04
(d, 1H, J ¼ 7.2 Hz).
4.1.14. [4-(4-Methyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic
acid ethyl ester (10f)
Pyridone 9f (230 mg, 1.19 mmol) was reacted with ethyl bro-
moacetate (185 mL, 1.67 mmol), by employing the same procedure
described for compound 10a, to provide compound 10f as a white
powder (167 mg, 50%); Rf ¼ 0.26 (CHCl3/MeOH 9:1). 1H NMR
(300 MHz, CDCl3)
d 1.25 (m, 3H), 2.27e2.47 (m, 7H), 3.26e3.36 (m,
4H), 4.17e4.21 (m, 2H), 4.54 (s, 2H), 5.71 (d, 1H, J ¼ 2.3 Hz), 5.95 (s,
1H), 7.02 (s, 1H).
4.1.9. (2-Oxo-2H-pyridin-1-yl)-acetic acid ethyl ester (10a)
A suspension of NaH (117 mg, 4.87 mmol) in dry DMF (10 mL) at
0 ꢀC under N2, was treated with a solution of pyridone 9a (386 mg,
4.06 mmol) in dry DMF (20 mL). After 1 h, ethyl bromoacetate
4.1.15. [4-(4-Allyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic
acid ethyl ester (10g)
Pyridone 9g (296 mg, 1.35 mmol) was reacted with ethyl bro-
(623
mL, 5.68 mmol) was added to the mixture and the reaction was
moacetate (269 mL, 2.43 mmol), by employing the same procedure
stirred for a further 2 h at room temperature. The reaction was
quenched with saturated NH4Cl (5 mL) and the product was
extracted with CH2Cl2 (3 ꢃ 70 mL). The combined organic phases
were washed with water (3 ꢃ 100 mL), dried over Na2SO4, filtered
and concentrated in vacuo. The crude was purified by column
chromatography (CHCl3/MeOH 95:5) to give compound 10a as a
yellow oil (457 mg, 62%); Rf ¼ 0.70 (CHCl3/MeOH 9:1). 1H NMR
described for compound 10a, to provide compound 10g as a white
powder (235 mg, 57%); Rf ¼ 0.35 (CHCl3/MeOH 9:1). 1H NMR
(300 MHz CDCl3)
d
1.27 (t, 3H, J ¼ 7.0 Hz), 2.53 (t, 4H, J ¼ 5.3 Hz), 3.03
(d, 2H, J ¼ 6.5 Hz), 3.32 (t, 4H, J ¼ 5.3 Hz), 4.22 (q, 2H, J ¼ 7.0 Hz), 4.55
(s, 2H), 5.17e5.24 (m, 2H), 5.72 (d,1H, J ¼ 2.9 Hz), 5.79e5.90 (m,1H),
5.96 (dd, 1H, J ¼ 7.6, 2.9 Hz), 7.02 (d, 1H, J ¼ 7.6 Hz).
(300 MHz, CDCl3)
d
1.21 (t, 3H, J ¼ 7.3 Hz), 4.16e4.20 (m, 2H), 4.60
4.1.16. (2-Oxo-2H-pyridin-1-yl)-acetic acid (11a)
(s, 2H), 6.15e6.19 (m, 1H), 6.53e6.56 (m, 1H), 7.19e7.33 (m, 2H).
A solution of ester 10a (457 mg, 2.52 mmol) in EtOH/H2O (1:1,
30 mL) at 0 ꢀC was treated with 1 N LiOH (5 mL) and stirred for 4 h
at room temperature. The ethanol fraction was evaporated at
reduced pressure and the residual aqueous solution was treated
with 6 N HCl (pH w 6.8). The solution was concentrated in vacuo
and the residue was purified by column chromatography (3%
HCOOH in CHCl3/MeOH 9:1) to give compound 11a as a white
powder (306 mg, 78%); Rf ¼ 0.27 (3% HCOOH in CHCl3/MeOH 9:1).
4.1.10. (4-Diethylamino-2-oxo-2H-pyridin-1-yl)-acetic acid ethyl
ester (10b)
Pyridone 9b (224 mg, 1.35 mmol) was reacted with ethyl bro-
moacetate (210 mL, 1.89 mmol), by employing the same procedure
described for compound 10a, to provide compound 10b as a pale
yellow oil (204 mg, 60%), after purification by column chromatog-
raphy (CHCl3/MeOH 9:1); Rf ¼ 0.72 (CHCl3/MeOH 9:1). 1H NMR
1H NMR (300 MHz, acetone-d6)
d
4.60 (s, 2H), 6.16 (t, 1H, J ¼ 5.9 Hz),

30
N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
6.55 (dd, 1H, J ¼ 7.4, 2.5 Hz), 7.20 (d, 1H, J ¼ 5.9 Hz), 7.33 (t, 1H,
J ¼ 7.4 Hz).
After 20 min, the reaction mixture was further cooled to ꢁ15 ꢀC and
treated in sequence with EDC$HCl (242 mg,1.56 mmol), a precooled
(0 ꢀC) solution of commercially available pinanediol
L-leucine bor-
4.1.17. (4-Diethylamino-2-oxo-2H-pyridin-1-yl)-acetic acid (11b)
Ester10b (204 mg, 0.81 mmol)was treatedwith 1 N LiOH (1.6mL),
according with the same procedure described for 11a. Title com-
pound 11b was obtained as a white powder (137 mg, 75%); Rf ¼ 0.30
onate trifluoroacetate salt 12 (246 mg, 0.65 mmol) and DIPEA
(136
m
L, 0.78 mmol) in dry CH2Cl2 (5 mL). After stirring at ꢁ15 ꢀC for
1 h and additional 2 h at room temperature, the solutionwas washed
with 0.1 M KHSO4, 5% NaHCO3 and brine, dried over Na2SO4, filtered
and concentrated in vacuo. The residue was taken up into Et2O
(20 mL), filtered and evaporated to give the crude product 13a
(240 mg, 91%) as a pale yellowoil which was directly used in the next
step without purification. Rf ¼ 0.82 (CHCl3/MeOH 95:5).
(3% HCOOH in CHCl3/MeOH 9:1).1H NMR (300 MHz, DMSO-d6)
d 1.05
(t, 6H, J ¼ 7.0 Hz), 3.25 (q, 4H, J ¼ 7.0 Hz), 4.09 (s, 2H), 5.21 (d, 1H,
J ¼ 2.6 Hz), 5.81 (dd, 1H, J ¼ 7.9, 2.9 Hz), 7.19 (d, 1H, J ¼ 7.7 Hz).
4.1.18. [4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid
(11c)
4.1.24. R-1-[2-(4-Diethylamino-2-oxo-2H-pyridin-1-yl)
acetamido]-3-methylbutylboronic acid pinanediol ester (13b)
Compound 11b (100 mg, 0.45 mmol) was reacted with HOBt
Ester 10c (144 mg, 0.54 mmol) was treated with 1 N LiOH (1.1 mL),
according with the same procedure described for 11a. Title com-
pound 11c was obtained as a white powder (104 mg, 87%); Rf ¼ 0.20
(146 mg, 1.08 mmol), EDC$HCl (168 mg, 1.08 mmol), DIPEA (94 mL,
(3% HCOOH in CHCl3/MeOH 9:1).1H NMR (300 MHz, DMSO-d6)
d
3.16
0.54 mmol) and amino boronate 12 (171 mg, 0.45 mmol) according
with the same procedure described for 13a, to yield the crude
product 13b (212 mg, 99%) which was directly used for the next
step without purification. Rf ¼ 0.24 (EtOAc).
(t, 4H, J ¼ 4.8 Hz), 3.65 (t, 4H, J ¼ 4.4 Hz), 4.08 (s, 2H), 5.45 (d, 1H,
J ¼ 3.1 Hz), 6.00 (dd, 1H, J ¼ 7.9, 2.7 Hz), 7.23 (d, 1H, J ¼ 7.9 Hz).
4.1.19. [4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic acid (11d)
Ester 10d (265 mg, 1.0 mmol) was treated with 1 N LiOH (2 mL),
according with the same procedure described for 11a. Title com-
pound 11d was obtained as a white powder (180 mg, 76%); Rf ¼ 0.25
(3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6)
4.1.25. R-1-{2-[4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid pinanediol ester (13c)
Compound 11c (100 mg, 0.42 mmol) was reacted with HOBt
(136 mg, 1.01 mmol), EDC$HCl (157 mg, 1.01 mmol), DIPEA (88 mL,
d
1.48e1.69 (m, 6H), 3.22e3.24 (m, 4H), 4.12 (s, 2H), 5.43 (d, 1H,
0.50 mmol) and amino boronate 12 (159 mg, 0.42 mmol) according
with the same procedure described for 13a, to yield the crude
product 13c (204 mg, 99%) which was directly used for the next
step without purification. Rf ¼ 0.50 (CHCl3/MeOH 9:1).
J ¼ 2.7 Hz), 6.2 (dd, 1H, J ¼ 7.5, 2.7 Hz), 7.21 (d, 1H, J ¼ 7.5 Hz).
4.1.20. (4-Phenylamino-2-oxo-2H-pyridin-1-yl)-acetic acid (11e)
Ester 10e (231 mg, 0.85 mmol) was treated with 1 N LiOH
(1.7 mL), according with the same procedure described for 11a. Title
compound 11e was obtained as a white powder (181 mg, 87%);
Rf ¼ 0.31 (3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz,
4.1.26. R-1-{2-[4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid pinanediol ester (13d)
Compound 11d (100 mg, 0.42 mmol) was reacted with HOBt
DMSO-d6)
d
4.13 (s, 2H), 5.79 (d, 1H, J ¼ 1.8 Hz), 6.08 (dd, 1H, J ¼ 7.0,
(136 mg, 1.01 mmol), EDC$HCl (157 mg, 1.01 mmol), DIPEA (88 mL,
1.8 Hz), 6.98 (t, 1H, J ¼ 7.0 Hz), 7.16e7.31 (m, 5H).
0.50 mmol) and amino boronate 12 (159 mg, 0.42 mmol) according
with the same procedure described for 13a, to yield the crude
product 13d (203 mg, 99%)which was directly used for the next step
without purification. Rf ¼ 0.52 (CHCl3/MeOH 9:1).
4.1.21. [4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic
acid (11f)
Ester 10f (167 mg, 0.60 mmol) was treated with 1 N LiOH
(1.2 mL), according with the same procedure described for 11a,
adjusting the residual aqueous solution to pH w6. Title compound
11f was obtained as a white solid (135 mg, 90%) after purification by
column chromatography (CHCl3/MeOH 9:1 till all impurities came
off, then 100% MeOH to elute the product); Rf ¼ 0.32 (MeOH). 1H
4.1.27. R-1-[2-(4-Phenylamino-2-oxo-2H-pyridin-1-yl)acetamido]-
3-methylbutylboronic acid pinanediol ester (13e)
Compound 11e (100 mg, 0.41 mmol) was reacted with HOBt
(132 mg, 0.98 mmol), EDC$HCl (152 mg, 0.98 mmol), DIPEA (85 mL,
0.49 mmol) and amino boronate 12 (155 mg, 0.41 mmol) according
with the same procedure described for 13a, to yield the crude
product 13e (197 mg, 97%) which was directly used for the next step
without purification. Rf ¼ 0.49 (CHCl3/MeOH 95:5).
NMR (300 MHz, CD3OD)
d
2.34 (s, 3H), 2.54 (t, 4H, J ¼ 5.4 Hz), 3.41
(t, 4H, J ¼ 5.4 Hz), 4.41 (s, 2H), 5.71 (d, 1H, J ¼ 2.9 Hz), 6.27 (dd, 1H,
J ¼ 7.6, 2.9 Hz), 7.37 (d, 1H, J ¼ 7.6 Hz).
4.1.22. [4-(4-Allyl-piperazin-1-yl)-2-oxo-2H-pyridin-1-yl]-acetic
acid (11g)
4.1.28. R-1-{2-[4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid pinanediol ester (13f)
Compound 11f (100 mg, 0.40 mmol) was reacted with HOBt
Ester 10g (235 mg, 0.77 mmol) was treated with 1 N LiOH
(1.5 mL), according with the same procedure described for 11a,
adjusting the residual aqueous solution to pH w7.2. Title com-
pound 11f was obtained as a white solid (189 mg, 88%) after puri-
fication by column chromatography (CHCl3/MeOH 9:1 till all
impurities came off, then 100% MeOH to elute the product);
(130 mg, 0.96 mmol), EDC$HCl (149 mg, 0.96 mmol), DIPEA (83 mL,
0.48 mmol) and amino boronate 12 (152 mg, 0.40 mmol) according
with the same procedure described for 13a, to yield the crude
product 13f (199 mg, 99%) which was directly used for the next step
without purification. Rf ¼ 0.44 (CHCl3/MeOH 9:1).
Rf ¼ 0.30 (MeOH). 1H NMR (300 MHz, CD3OD)
d
2.57 (t, J ¼ 4.7 Hz
4H), 3.07 (d, 2H, J ¼ 7.0 Hz), 3.38 (t, 4H, J ¼ 4.7 Hz), 4.41 (s, 2H),
5.21e5.28 (m, 2H), 5.72 (d, 1H, J ¼ 2.3 Hz), 5.83e5.96 (m, 1H), 6.26
(dd, 1H, J ¼ 7.6, 2.3 Hz), 7.32 (d, 1H, J ¼ 7.6 Hz).
4.1.29. R-1-{2-[4-(4-Allylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid pinanediol ester (13g)
Compound 11g (100 mg, 0.36 mmol) was reacted with HOBt
(116 mg, 0.86 mmol), EDC$HCl (133 mg, 0.86 mmol), DIPEA (75 mL,
4.1.23. R-1-[2-(2-Oxo-2H-pyridin-1-yl)acetamido]-3-
methylbutylboronic acid pinanediol ester (13a)
0.43 mmol) and amino boronate 12 (136 mg, 0.36 mmol) according
with the same procedure described for 13a, to yield the crude
product 13g (157 mg, 83%) which was directly used for the next
step without purification. Rf ¼ 0.46 (CHCl3/MeOH 9:1).
An ice-cold suspension (ꢁ5 ꢀC) of acid 11a (100 mg, 0.65 mmol)
in dry CH2Cl2 (20 mL) was treated with HOBt (211 mg, 1.56 mmol).

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
31
4.1.30. R-1-[2-(2-Oxo-2H-pyridin-1-yl)acetamido]-3-
methylbutylboronic acid (1a)
4.1.34. R-1-[2-(4-Phenylamino-2-oxo-2H-pyridin-1-yl)acetamido]-
3-methylbutylboronic acid (1e)
To a solution of 13a (240 mg, 0.6 mmol) in MeOH/n-hexane (1:1,
28 mL), isobutylboronic acid (306 mg, 3.0 mmol) and 1 N HCl
(1.7 mL) were added. The reaction was stirred for 18 h at room
temperature. The methanolic phase was washed with n-hexane
(3 ꢃ 10 mL) and the n-hexane layer with MeOH (3 ꢃ 10 mL). The
combined methanolic phases were evaporated in vacuo and the
residue was taken up into CH2Cl2 (30 mL) and washed with 5%
NaHCO3 (30 mL). The product was extracted with CHCl3 (3 ꢃ 30 mL)
from the water layer and the combined organic phases were dried
over Na2SO4, filtered and concentrated in vacuo. The crude product
was taken up in Et2O (20 mL), filtered off and concentrated in vacuo
to give the title compound 1a as a white sticky solid (53 mg, 33%);
Rf ¼ 0.29 (CHCl3/MeOH 9:1). MS (ESI) m/z 265.1 [M ꢁ H]ꢁ (100%);
Ester 13e (197 mg, 0.40 mmol) was treated with isobutylboronic
acid (204 mg, 2 mmol) and 1 N HCl (1.2 mL), by employing the same
procedure described for 1a, to give the title compound 1e as a white
sticky solid (64 mg, 45%); Rf ¼ 0.20 (3% HCOOH in CHCl3/MeOH 9:1).
MS (ESI) m/z 356.2 [M ꢁ H]ꢁ (100%); ½a D27
¼ ꢁ11.9 (c 0.3, i-PrOH);
ꢄ
1H NMR (300 MHz, acetone-d6)
d 0.87 (m, 6H), 1.29e1.47 (m, 3H),
3.34 (m, 1 H), 4.05e4.50 (m, 2H), 5.88e6.10 (m, 2H), 7.10e7.43 (m,
6H); 13C NMR (75 MHz, acetone-d6)
d
23.3, 26.4, 32.2, 44.3, 51.9,
88.4, 111.1, 117.1, 118.7, 128.8, 134.6, 144.9, 160.1, 162.3, 170.1; Anal.
Calcd. for C18H24BN3O4: C 60.52; H 6.77; N 11.76. Found: C 60.50; H
6.89; N 11.69.
4.1.35. R-1-{2-[4-(4-Methylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid (1f)
½
a 2D7
ꢄ
¼ ꢁ4.0 (c 0.1, i-PrOH); 1H NMR (300 MHz, acetone-d6)
d 0.87 (t,
6H, J ¼ 6.6 Hz), 1.29e1.72 (m, 3H), 3.39e3.44 (m, 1H), 4.06e4.92 (m,
2H), 6.25 (s, 1H), 6.39e6.47 (m, 1H), 7.45 (s, 1H), 7.60e7.64 (m,
Ester 13f (199 mg, 0.4 mmol) was treated with isobutylboronic
acid (204 mg, 2 mmol) and 1 N HCl (1.7 mL), by employing the same
procedure described for 1a, to give the title compound 1f as a white
sticky solid (41 mg, 30%); Rf ¼ 0.36 (3% HCOOH in CHCl3/MeOH 9:1).
1H);13C NMR (75 MHz, acetone-d6)
d 23.4, 26.5, 32.0, 44.3, 51.8,
111.5, 118.8, 134.3, 138.7, 162.4, 170.4; Anal. Calcd. for C12H19BN2O4:
C 54.16; H 7.20; N 10.53. Found: C 54.07; H 7.17; N 10.66.
MS (ESI) m/z 363.2 [M ꢁ H]ꢁ (100%); ½a D26
ꢄ
¼ ꢁ10.7 (c 0.4, MeOH); 1H
NMR: (300 MHz)
d
1.16 (t, 6H, J ¼ 5.4 Hz), 1.30e1.57 (m, 3H), 2.37 (s,
4.1.31. R-1-[2-(4-Diethylamino-2-oxo-2H-pyridin-1-yl)acetamido]-
3-methylbutylboronic acid (1b)
3H), 2.56 (t, 4H, J ¼ 5.3 Hz), 3.34e3.44 (m, 5H), 4.51 (s, 2H), 6.16 (d,
1H, J ¼ 2.9 Hz), 6.44 (dd, 1H, J ¼ 6.7, 2.9 Hz), 7.36 (d, 1H, J ¼ 6.7 Hz);
Ester 13b (212 mg, 0.45 mmol) was treated with isobutylboronic
acid (229 mg, 2.25 mmol) and 1 N HCl (1 mL), by employing the
same procedure described for 1a, to give the title compound 1b as a
white sticky solid (61 mg, 40%); Rf ¼ 0.17 (CHCl3/MeOH 9:1). MS
13C NMR (75 MHz, CD3OD)
d 23.3, 26.4, 32.2, 43.6, 44.3, 50.5, 51.9,
54.0, 86.2, 111.1, 134.6, 162.3, 166.3, 170.7; Anal. Calcd. for
17H29BN4O4: C 50.06; H 8.02; N 15.38. Found: C 50.01; H 8.09; N
C
15.42.
(ESI) m/z 335.8 [M ꢁ H]ꢁ (100%); ½a D24
ꢄ
¼ ꢁ5.8 (c 0.4, MeOH); 1H
NMR (300 MHz, acetone-d6)
d
0.84 (t, 6H, J ¼ 5.6 Hz), 1.16 (t, 6H,
4.1.36. R-1-{2-[4-(4-Allylpiperazin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid (1g)
J ¼ 6.2 Hz), 1.35e1.69 (m, 3H), 3.37e3.39 (m, 5H), 4.28e4.78 (m,
2H), 5.38 (d, 1H, J ¼ 2.6 Hz), 5.97 (dd, 1H, J ¼ 7.9, 2.9 Hz), 7.38 (d, 1H,
Ester 13g (157 mg, 0.3 mmol) was treated with isobutylboronic
acid (153 mg, 1.5 mmol) and 1 N HCl (1 mL), by employing the same
procedure described for 1a, to give the title compound 1g as a white
sticky solid (35 mg, 30%); Rf ¼ 0.47 (3% HCOOH in CHCl3/MeOH 9:1).
J ¼ 7.7 Hz), 8.71 (bs, 1H); 13C NMR (75 MHz, acetone-d6)
d 14.0, 23.4,
26.5, 32.0, 44.3, 44.6, 51.9, 86.2,111.2,134.3,162.4,166.1,169.8; Anal.
Calcd. for C16H28BN3O4: C 56.99; H 8.37; N 12.46. Found: C 56.91; H
8.47; N 12.44.
MS (ESI) m/z 389.2 [M ꢁ H]ꢁ (100%); ½a D26
ꢄ
¼ ꢁ12.3 (c 0.3, MeOH); 1H
NMR (300 MHz, CD3OD)
d
1.18 (t, 6H, J ¼ 6.2 Hz), 1.25e1.57 (m, 3H),
4.1.32. R-1-{2-[4-(Morpholin-4-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid (1c)
2.44 (t, 4H, J ¼ 4.7 Hz), 3.11 (d, 2H, J ¼ 7.0 Hz), 3.23e3.44 (m, 5H),
4.54 (s, 2H), 5.20e5.26 (m, 2H), 6.22 (d, 1H, J ¼ 2.3 Hz), 6.64e6.73
(m, 1H), 6.89 (dd, 1H, J ¼ 7.2, 2.3 Hz), 7.22 (d, 1H, J ¼ 7.2 Hz); 13C
To a solution of 13c (204 mg, 0.42 mmol) was treated with
isobutylboronic acid (214 mg, 2.10 mmol) and HCl 1 N (1 mL), by
employing the same procedure described for 1a, to give the title
compound 1c as a white sticky solid (60 mg, 40%); Rf ¼ 0.28 (3%
HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 350.2 [M ꢁ H]ꢁ (100%);
NMR (75 MHz, CD3OD) d 23.3, 26.4, 32.2, 43.6, 44.3, 50.5, 54.0, 51.2,
52.2, 53.8, 57.4, 86.2, 111.1, 116.1, 133.2, 134.6, 162.5, 166.4, 170.9;
Anal. Calcd. for C19H31BN4O4: C 58.47; H 8.01; N 14.36. Found: C
58.34; H 8.05; N 14.28.
½
a 2D5
ꢄ
¼ ꢁ6.8 (c 0.4, MeOH); 1H NMR (300 MHz, acetone-d6)
d 0.84 (t,
6H, J ¼ 5.6 Hz), 3.24e3.30 (m, 4H), 3.59e3.78 (m, 5H); 4.26e4.78
4.1.37. (5-Oxo-5H-1,6-naphthyridin-6-yl)acetic acid (16a)
(m, 2H), 5.60 (s, 1H), 6.14 (s, 1H), 7.42 (s, 1H); 13C NMR (75 MHz
Step 1. Commercially available 1,6-naphthyridin-5(6H)-one 14
(200 mg, 1.37 mmol) and NaH (39 mg, 1.64 mmol) were suspended
in dry DMF (10 mL) at 0 ꢀC and the reaction mixture was stirred for
CDCl3) d 14.0, 23.4, 26.5, 32.0, 44.3, 49.4, 60.0, 67.9, 86.2,111.0,134.3,
162.4, 166.2, 170.7; Anal. Calcd. for C16H26BN3O5: C 54.72; H 7.46; N
11.96. Found: C 54.71; H 7.53; N 11.84.
1 h. After this time, a solution of ethyl 2-bromoacetate (212 mL,
1.92 mmol) in dry DMF (3 mL) was added and the reaction mixture
was stirred for 2 h, then was quenched with saturated NH4Cl so-
lution (5 mL) and extracted with CHCl3 (3 ꢃ 15 mL) and the organic
phase was washed with saturated NaHCO3 solution (2 ꢃ 20 mL) and
distilled water (2 ꢃ 20 mL). The organic layer was dried over
Na2SO4, filtered and evaporated in vacuo. The crude was purified by
column chromatography (CHCl3/MeOH 95:5) to give (5-oxo-5H-
1,6-naphthyridin-6-yl)acetic acid ethyl ester as yellow crystals
(190 mg, 60%); Rf ¼ 0.69 (CHCl3/MeOH 95:5). 1H NMR (300 MHz,
4.1.33. R-1-{2-[4-(Piperidin-1-yl)-2-oxo-2H-pyridin-1-yl]
acetamido}-3-methylbutylboronic acid (1d)
Ester 13d (203 mg, 0.42 mmol) was treated with iso-
butylboronic acid (214 mg, 2.1 mmol) and 1 N HCl (1 mL), by
employing the same procedure described for 1a, to give the title
compound 1d as a white sticky solid (73 mg, 50%), Rf ¼ 0.32 (3%
HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 348.1 [M ꢁ H]ꢁ (100%);
½
a 2D6
ꢄ
¼ ꢁ4.5 (c 0.5, MeOH); 1H NMR (300 MHz, acetone-d6)
d 0.84
(t, 6H, J ¼ 7.0 Hz), 1.29e1.62 (m, 9H), 3.34e3.44 (m, 5H), 4.30e4.77
CDCl3)
d
1.30 (t, 3H, J ¼ 7.3 Hz), 4.26 (q, 2H, J ¼ 7.3 Hz), 4.72 (s, 2H),
(m, 2H), 5.55e5.59 (m, 1H), 6.12 (t, 1H, J ¼ 7.5 Hz), 7.39 (d, 1H,
6.81 (d, 1H, J ¼ 7.7 Hz), 7.25 (d, 1H, J ¼ 7.7 Hz), 7.42 (dd, 1H, J ¼ 8.1,
4.4 Hz), 8.67 (dd, 1H, J ¼ 8.1, 1.8 Hz), 8.92 (dd, 1H, J ¼ 4.4, 1.8 Hz).
Step 2. To a cooled solution (0 ꢀC) of ester intermediate (190 mg,
0.82 mmol) in EtOH (10 mL), 1 N LiOH (1.6 mL) was added, and the
mixture was stirred at 25 ꢀC for 6 h, until TLC showed the complete
J ¼ 7.5 Hz); 13C NMR (75 MHz, acetone-d6)
d 23.3, 26.0, 26.2, 26.5,
32.0, 44.3, 48.4, 51.8, 86.3, 111.0, 134.3, 162.4, 166.2, 170.7; Anal.
Calcd. for C17H28BN3O4: C 58.47; H 8.08; N 12.03. Found: C 58.52;
H 8.06; N 12.09.

32
N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
hydrolysis of the starting material. Upon concentration in vacuo
and pH regulation to 6.8, the aqueous layer was completely evap-
orated in vacuo and the resulting crude was purified by column
chromatography (3% HCOOH in CHCl3/MeOH 9:1) to obtain the title
compound 16a as a pale yellow solid (151 mg, 90%); Rf ¼ 0.27 (3%
(89 mg, 0.41 mmol), HOBt (132 mg, 0.98 mmol), EDC$HCl (188 mg,
0.98 mmol), DIPEA (85 L, 0.49 mmol), pinanediol -leucine boro-
nate 12 (155 mg, 0.41 mmol). After work-up, the crude product
(191 mg, 100%) was directly used in the next reaction without
further purification; Rf ¼ 0.60 (CHCl3/MeOH 9:1).
m
L
HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6)
d 4.57
(s, 2H), 6.67 (d, 1H, J ¼ 7.3 Hz), 7.50 (dd, 1H, J ¼ 7.9, 4.4 Hz), 7.68 (d,
1H, J ¼ 7.3 Hz), 8.24 (bs, 1H), 8.51 (dd, 1H, J ¼ 7.9, 1.8 Hz), 8.92 (dd,
1H, J ¼ 4.4, 1.8 Hz).
4.1.42. R-1-[(8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl)
acetamido]-3-methylbutylboronic acid pinanediol ester (17c)
The synthesis of compound 17c was performed following the
same procedure employed for compound 17a using carboxylic acid
16c (150 mg, 0.53 mmol), HOBt (243 mg, 1.27 mmol), EDC$HCl
4.1.38. (S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl)propionic acid
(16b)
(1.27 mmol, 171 mg), DIPEA (112
mL, 0.64 mmol), pinanediol L-
Step 1. Compound 16b was obtained in the same way as for 16a
using 14 (200 mg, 1.37 mmol), methyl 2(R)-bromopropionate [27]
(321 mg, 1.92 mmol) and NaH (39 mg, 1.64 mmol). Purification by
column chromatography (EtOAc) provided the ester intermediate
leucine boronate 12 (201 mg, 0.53 mmol). After work-up, the crude
product (196 mg, 70%) was directly used in the next reaction
without further purification; Rf ¼ 0.59 (CHCl3/MeOH 9:1).
(110 mg, 34%); Rf ¼ 0.51 (EtOAc). 1H NMR (300 MHz, CDCl3)
d
1.70
4.1.43. R-1-[(5-Oxo-5H-1,6-naphthyridin-6-yl)acetamido]-3-
methylbutylboronic acid (2a)
(d, 3H, J ¼ 7.5 Hz), 3.76 (s, 3H), 5.61 (q, 1H, J ¼ 7.5 Hz), 6.83 (d, 1H,
J ¼ 7.5 Hz), 7.34 (d, 1H, J ¼ 7.5 Hz), 7.40 (dd, 1H, J ¼ 8.0, 4.4 Hz), 8.66
(dd, 1H, J ¼ 8.0, 1.8 Hz), 8.90 (dd, 1H, J ¼ 4.4, 1.8 Hz). Step 2. The
hydrolysis was realized with the same procedure of 16a using the
ester intermediate (110 mg, 0.47 mmol), 1 N LiOH (0.9 mL) and
MeOH (10 mL) as solvent, to give the title compound 16b as a pale
yellow solid (89 mg, 88%); Rf ¼ 0.24 (3% HCOOH in CHCl3/MeOH
A solution of 17a (334 mg, 0.74 mmol) and isobutylboronic acid
(377 mg, 3.70 mmol) in MeOH/n-hexane (1:1, 12 mL) was treated
with 1 N HCl (1.8 mL), and the mixture was stirred at room tem-
perature for 18 h. The methanolic phase was washed with n-hexane
(3 ꢃ 10 mL) and the combined hexanic phases were re-extracted
with MeOH (3 ꢃ 15 mL). The resulting methanolic layer was
concentrated in vacuo and the residue was dissolved in CHCl3
(15 mL) and washed with 5% NaHCO3. The aqueous phase was
extracted with CHCl3 (3 ꢃ 15 mL) and the organic layer was evap-
orated in vacuo to give a solid which was purified by trituration
with Et2O to give title product 2a (75 mg, 32%); Rf ¼ 0.21 (3%
HCOOH in CHCl3/MeOH 9:1). MS (ESI) m/z 318.3 [M þ H]þ (100%);
9:1); 1H NMR (300 MHz, DMSO-d6)
d
1.59 (d, 3H, J ¼ 6.9 Hz), 5.31 (q,
1H, J ¼ 6.9 Hz), 6.72 (d, 1H, J ¼ 7.4 Hz), 7.51 (dd, 1H, J ¼ 7.7, 4.4 Hz),
7.75 (d, 1H, J ¼ 7.4 Hz), 8.52 (dd, 1H, J ¼ 7.7, 1.8 Hz), 8.93 (dd, 1H,
J ¼ 4.4, 1.8 Hz).
4.1.39. (8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl)acetic acid (16c)
Step 1. Compound 16c was obtained in the same way as for 16a
½
a 2D6
ꢄ
¼ ꢁ20.8 (c 0.2, i-PrOH); 1H NMR (300 MHz, acetone-d6)
d 0.87
using 15 [10] (187 mg, 0.83 mmol), ethyl bromoacetate (129
mL,
(d, 6H, J ¼ 6.7 Hz), 1.37 (q, 2H, J ¼ 7.5 Hz), 1.50e1.64 (m, 1H), 3.22e
3.29 (m, 1H), 4.63 (s, 2H), 6.57 (d, 1H, J ¼ 5.9 Hz), 6.82 (d, 1H,
J ¼ 7.6 Hz), 7.40 (d, 1H, J ¼ 7.6 Hz), 7.42 (dd, 1H, J ¼ 8.2, 4.7 Hz), 8.66
(dd, 1H, J ¼ 8.2, 1.8 Hz), 8.92 (dd, 1H, J ¼ 4.7, 1.8 Hz); 13C NMR
1.16 mmol) and NaH (24 mg, 1.00 mmol). Purification by column
chromatography (CHCl3/MeOH 95:5) provided the ester interme-
diate (168 mg, 65%); Rf ¼ 0.67 (CHCl3/MeOH 95:5). 1H NMR
(300 MHz, CDCl3)
d
1.32 (t, 3H, J ¼ 7.0 Hz), 4.28 (q, 2H, J ¼ 7.0 Hz),
(75 MHz, acetone-d6) d 23.3, 26.1, 32.0, 44.5, 52.0,105.4,124.0,124.1,
4.72 (s, 2H), 7.47e7.55 (m, 1H), 7.62 (s, 1H), 8.70 (d, 1H, J ¼ 7.5 Hz),
9.06 (s, 1H). Step 2. The successive hydrolysis was realized with the
same procedure employed for 16a using the ester intermediate
(168 mg, 0.54 mmol), 1 N LiOH (1.1 mL) and ethanol as solvent
(10 mL), to give 16c as a pale pink solid (150 mg, 98%); Rf ¼ 0.14 (3%
129.4, 138.0, 147.4, 156.4, 157.6, 170.8; Anal. Calcd. for C15H20BN3O4:
C 56.81, H 6.36, N 13.25. Found: C 56.61, H 6.51, N 13.15.
4.1.44. R-1-[(S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl)
propanamido]-3-methylbutylboronic acid (2b)
HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6)
d
4.64
Compound 2b was obtained following the same procedure
employed for compound 2a using 17b (191 mg, 0.41 mmol), 2-
methylpropylboronic acid (209 mg, 2.05 mmol), 1 N HCl (1 mL)
and a mixture of MeOH/n-hexane (1:1, 7 mL) as solvent. After pu-
rification, the reaction gave the title compound 2b (30 mg, 30%);
Rf ¼ 0.40 (CHCl3/MeOH 9:1). MS (ESI) m/z 330.2 [M ꢁ H]ꢁ (100%);
(s, 2H), 7.63 (dd, 1H, J ¼ 7.9, 4.7 Hz), 8.22 (s, 1H), 8.57 (dd, 1H, J ¼ 7.9,
1.0 Hz), 9.04 (dd, 1H, J ¼ 4.7, 1.0 Hz).
4.1.40. R-1-[(5-Oxo-5H-1,6-naphthyridin-6-yl)acetamido]-3-
methylbutylboronic acid pinanediol ester (17a)
To an ice-cold suspension (ꢁ5 ꢀC) of [(6H)-5-oxo-1,6-
naphthyridinyl]acetic acid 16a (151 mg, 0.74 mmol) in dry CH2Cl2
(15 mL), was added HOBt (240 mg, 1.78 mmol). After 20 min, the
reaction system was further cooled (ꢁ15 ꢀC) and was treated with
EDC$HCl (341 mg, 1.78 mmol). After that, a cold solution of pina-
½
a 2D7
ꢄ
¼ ꢁ41.6 (c 0.1, i-PrOH); 1H NMR (300 MHz, acetone-d6)
d 0.76
(d, 6H, J ¼ 7.6 Hz), 1.24e1.33 (m, 2H), 1.37e1.45 (m, 1H), 1.66 (d, 3H,
J ¼ 7.0 Hz), 3.34e3.46 (m, 1H), 5.77 (q, 1H, J ¼ 7.0 Hz), 6.75 (d, 1H,
J ¼ 7.0 Hz), 7.46 (d, 1H, J ¼ 7.0 Hz), 7.71 (m, 1H), 8.56 (m, 1H), 8.89
(m, 1H); 13C NMR (75 MHz, (CD3)2CO)
d 15.1, 23.3, 25.2, 30.2, 54.0,
nediol
(155
L
-leucine boronate 12 (281 mg, 0.74 mmol) and DIPEA
72.1, 105.4, 124.0, 124.1, 129.4, 138.0, 147.4, 156.1, 157.6, 171.6; Anal.
Calcd. for C16H22BN3O4: C 58.03, H 6.70, N 12.69. Found: C 58.28, H
6.89, N 12.47.
m
L, 0.89 mmol) in dry CH2Cl2 (5 mL) was added and the re-
action mixture was stirred at ꢁ15 ꢀC for 1 h and then at room
temperature for 2 h. Finally, the organic phase was washed with
0.1 M KHSO4, 5% NaHCO3 and brine, dried over Na2SO4 and filtered.
The solution was evaporated to give a crude (334 mg, 100%) which
was used directly in the next reaction without purification;
Rf ¼ 0.42 (EtOAc/acetone/MeOH 80:15:5).
4.1.45. R-1-[(8-Bromo-5-oxo-5H-1,6-naphthyridin-6-yl)
acetamido]-3-methylbutylboronic acid (2c)
Compound 2c was obtained following the same procedure
employed for compound 2a using 17c (196 mg, 0.37 mmol), iso-
butylboronic acid (188 mg, 1.85 mmol), 1 N HCl (0.9 mL) and a
mixture of MeOH/n-hexane (1:1, 6 mL) as solvent. After purifica-
tion, the reaction gave of the title compound 2c (50 mg, 34%);
Rf ¼ 0.31 (CHCl3/MeOH 9:1). MS (ESI) m/z 395.05 [M ꢁ H]ꢁ (100%);
4.1.41. R-1-[(S)-2-(5-Oxo-5H-1,6-naphthyridin-6-yl)
propanamido]-3-methylbutylboronic acid pinanediol ester (17b)
Synthesis of compound 17b was performed following the same
procedure employed for compound 17a using carboxylic acid 16b
½
a 2D7
ꢄ
¼ ꢁ8.9 (c 0.3, i-PrOH); 1H NMR (300 MHz, acetone-d6)
d 0.89

N. Micale et al. / European Journal of Medicinal Chemistry 64 (2013) 23e34
33
(d, 6H, J ¼ 5.9 Hz), 1.37e1.44 (m, 2H), 1.55e1.66 (m, 1H), 3.22e3.29
(m, 1H), 4.59 (s, 2H), 7.46e7.59 (m, 1H), 7.78 (s, 1H), 8.69 (d, 1H,
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. Since the X-ray
structure of the proteasome/bortezomib complex is consistent with
J ¼ 7.6 Hz), 9.02e9.14 (m,1H); 13C NMR (75 MHz, acetone-d6)
d 23.3,
25.2, 30.2, 46.8, 71.8, 103.9, 124.0, 124.1, 125.1, 138.0, 147.4, 156.4,
158.4, 169.6; Anal. Calcd. for C15H19BBrN3O4: C 45.49, H 4.84, N
10.61. Found: C 45.23, H 4.99, N 10.50.
a protonated b6-D114, it was treated as protonated during docking
simulations. 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 hydrogens
only were minimized using the Impref module of Impact with the
OPLS_2005 force field. The initial structure of bortezomib was
derived from the crystal complex coordinates, and the original
structures of 1aee and 2c were constructed on the basis of the
bortezomib crystal structure conformation followed by energy
minimization with the PolakeRibiere conjugated gradient (PRCG)
4.1.46. (1-Methyl-1,2,3,4-tetrahydro-5-oxo-5H-1,6-naphthyridin-
6-yl)acetic acid (19)
Step 1. Compound 19 was obtained in the same way as for 16a
using 18 [10] (159 mg, 0.97 mmol), ethyl bromoacetate (151 mL,
1.36 mmol) and NaH (28 mg, 1.16 mmol). Purification by column
chromatography (CHCl3/MeOH 95:5) provided the ester interme-
diate (190 mg, 78%); Rf ¼ 0.54 (CHCl3/MeOH 95:5). 1H NMR
(300 MHz, CDCl3)
d
1.28 (t, 3H, J ¼ 7.3 Hz),1.85e1.96 (m, 2H), 2.58 (t,
2H, J ¼ 6.0 Hz), 2.95 (s, 3H), 3.22 (t, 2H, J ¼ 5.6 Hz), 4.22 (q, 2H,
J ¼ 7.3 Hz), 4.56 (s, 2H), 5.85 (d, 1H, J ¼ 7.7 Hz), 6.95 (d, 1H,
J ¼ 7.7 Hz). Step 2. The successive hydrolysis was performed in the
same way as for 16a using the ester intermediate (190 mg,
0.76 mmol), 1 N LiOH (1.5 mL), and ethanol (10 mL) as solvent to
give the title compound 19 as white solid (160 mg, 95%); Rf ¼ 0.30
(3% HCOOH in CHCl3/MeOH 9:1). 1H NMR (300 MHz, DMSO-d6)
ꢀ
method with a convergence gradient of 0.001 kJ/mol A.
4.2.2. Docking studies
Compounds 1aee and 2c were covalently docked to the binding
ꢀ
pocket of
from the
b
b
5 subunit using GOLD, version 5.1 [15,16]. A radius of 20 A
5 catalytic N-terminal threonine was used to direct site
d
1.71e1.83 (m, 2H), 2.31 (t, 2H, J ¼ 5.9 Hz), 2.88 (s, 3H), 3.15 (t, 2H,
location. For each of the genetic algorithm runs, a maximum of
100,000 operations were performed on a population of 100 in-
dividuals with a selection pressure of 1.1. Operator weights for
crossover, mutation, and migration were set to 95, 95, and 10,
respectively, as recommended by the authors of the software. The
J ¼ 4.7 Hz), 4.41 (s, 2H), 5.91 (d, 1H, J ¼ 7.6 Hz), 7.27 (d, 1H,
J ¼ 7.6 Hz).
4.1.47. R-1-[(1-Methyl-1,2,3,4-tetrahydro-5-oxo-5H-1,6-
ꢀ
naphthyridin-6-yl)-acetamido]-3-methylbutylboronic acid (3)
Step 1. Synthesis of compound 3 was performed following the
same procedure employed for compound 17a using acid 19
(160 mg, 0.72 mmol), HOBt (234 mg, 1.73 mmol), EDC$HCl (332 mg,
distance for H-bonding was set to 2.5 A, and the cutoff value for van
ꢀ
der Waals calculation was set to 4 A. Covalent docking was applied,
and the terminal boron atoms of all the ligands were bonded to the
hydroxyl oxygen of b5-T1. The water molecule near b6-D114 (crys-
1.73 mmol), DIPEA (150
m
L, 0.86 mmol), pinanediol
L
-leucine bor-
tallographically determined for the proteasome/bortezomib com-
plex) was specified in GOLD by switching state settings to ‘toggle’
and orientation mode to ‘spin’. The Goldscore-CS docking protocol
[17], was adopted in this study. In this protocol, the poses obtained
with the original GoldScore function are rescored and reranked with
the GOLD implementation of the ChemScore function [17e21]. To
perform thorough and unbiased search of the conformation space,
each docking run was allowed to produce 200 poses without the
option of early termination, using standard default settings. The top
solution obtained after re-ranking of the poses with ChemScore was
selected to generate the proteasome/ligand complexes.
onate 12 (273 mg, 0.72 mmol). After work-up, the crude product
was directly used in the next reaction without further purification
(169 mg, 50%); Rf ¼ 0.57 (CHCl3/MeOH 9:1). Step 2. Compound 3
was obtained following the same procedure of compound 2a but
using the crude pinanediol ester intermediate (169 mg, 0.36 mmol),
isobutylboronic acid (183 mg, 1.80 mmol), 1 N HCl (0.9 mL) and a
mixture of MeOH/n-hexane (1:1, 6 mL) as solvent. After purifica-
tion, the reaction gave the title compound 3 as white solid (35 mg,
28%); Rf ¼ 0.25 (CHCl3/MeOH 9:1). MS (ESI) m/z 334.1 [M ꢁ H]ꢁ
(100%); ½a 2D7
ꢄ
¼ ꢁ27.7 (c 0.1, i-PrOH); 1H NMR (300 MHz, acetone-d6)
d
0.86 (t, 6H, J ¼ 6.2 Hz), 1.36 (q, 2H, J ¼ 7.1 Hz), 1.49e1.62 (m, 1H),
1.87e1.97 (m, 2H), 2.58 (t, 2H, J ¼ 6.2 Hz), 2.95 (s, 3H), 3.14e3.28 (m,
3H), 4.28 (d, 1H, J ¼ 14.3 Hz), 4.46 (d, 1H, J ¼ 14.3 Hz), 5.98 (d, 1H,
J ¼ 7.6 Hz), 7.30 (d, 1H, J ¼ 7.6 Hz); 13C NMR (75 MHz, acetone-d6)
4.3. Pharmacology
4.3.1. In vitro 20S proteasome inhibition assays
d
20.0, 23.3, 23.5, 26.1, 32.0, 38.5, 44.5, 47.8, 52.2, 106.5, 111.1, 134.5,
Human 20S proteasome was obtained from Biomol GmbH,
Hamburg, Germany. The three distinct proteolytic activities of the
20S proteasome were measured by monitoring the hydrolysis of
the peptidyl 7-amino-4-methyl coumarin substrates (all obtained
from Bachem) Suc-Leu-Leu-Val-Tyr-AMC, Boc-Leu-Arg-Arg-AMC,
and Cbz-Leu-Leu-Glu-AMC for ChT-L, T-L and PGPH activity of the
enzyme, respectively. Fluorescence of the product AMC of the
substrates’ hydrolyzes was measured using an Infinite 200 PRO
microplate reader (Tecan, Männedorf, Switzerland) at 30 ꢀC with a
380 nm excitation filter and a 460 nm emission filter. The pre-
liminary screening for the inhibition of the three proteolytic ac-
157.7, 157.9, 170.8; Anal. Calcd. for C16H26BN3O4: C 57.33, H 7.82, N
12.54. Found: C 57.15, H 7.72, N 12.77.
4.2. Computational chemistry
Molecular modeling and graphics manipulations were per-
formed using Maestro 9.2 (Schrödinger) [28] and UCSF-CHIMERA
software packages [29], running on a E4 Computer Engineering
E1080 workstation provided of an Intel Core i7-930 Quad-Core
processor. GOLD 5.1 [15,16] was used for all docking calculations.
Figures were generated using Pymol 1.0 [30].
tivities of the 20S proteasome was performed at 20
concentrations using an equivalent amount of DMSO as a negative
control. Compounds showing at least 40% inhibition at 20 M were
mM inhibitor
4.2.1. Protein and ligands preparation
m
Coordinates for the chymotrypsin-like
b
5 subunit derived from
subjected to detailed assays. The dissociation constants Ki of the
non covalent complex E$I were obtained from progress curves
(10 min) at various concentrations of inhibitor by fitting the prog-
ress curves to a 4 parameter IC50 equation, and correction to zero
substrate concentration from Ki ¼ IC50/(1 þ [S] Kꢁm1). The Km values
the X-ray crystal structure of the yeast 20S proteasome determined
ꢀ
at 2.8 A resolution (PDB ID: 2F16) were employed for the automated
docking studies [7]. The protein setup was carried out by Protein
Preparation Wizard in Maestro. Hydrogen atoms were added to the

34
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Leu-Leu-Val-Tyr-AMC 13 M, and PGPH activity with Cbz-Leu-Leu-
Glu-AMC 53 M.
m
m
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Product release from substrate hydrolysis (75
continuously over a period of 10 min.
mM) was monitored
4.3.3. Assaying the tryptic activity of the 20S proteasome
Human 20S proteasome was incubated at 30 ꢀC at a final con-
centration of 0.0025 mg mLꢁ1 with test compound present at
20
mM. The reaction buffer consisted of 50 mM Tris buffer pH 7.4,
50 mM NaCl, 0.5 mM EDTA, 0.03% SDS, and 7.5% DMSO. Product
release from substrate hydrolysis (85
uously over a period of 10 min.
mM) was monitored contin-
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centration of 0.004 mg mLꢁ1 with the test compound present at
variable concentrations. The reaction buffer consisted of 50 mM
Tris buffer pH 7.5 containing 25 mM KCl, 10 mM NaCl, 1 mM MgCl2,
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m
g mLꢁ1) was incubated at 20 ꢀC with test
compound. The reaction buffer consisted of 50 mM Tris buffer pH
8.0 containing 100 mM NaCl and 5 mM EDTA and 7.5% DMSO.
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Acknowledgments
This work was financially supported by the Ministero dell’Is-
truzione, dell’Università e della Ricerca Scientifica e Tecnologica
(MIUR-PRIN2010-2011). We would like to thank the Deutscher
Akademischer Austausch Dienst (DAAD) (Vigoni project 2011/12)
for partial support of this work. TS thanks the Deutsche For-
schungsgemeinschaft (DFG) for financial support. R.E. acknowl-
edges the support of her postdoctoral fellowship from “Dote
Ricerca”: FSE, Regione Lombardia.
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