Naphthoquinone amino acid derivatives, synthesis and biological activity as proteasome inhibitors

Article abstract of DOI:10.1080/14756366.2017.1334649

The ubiquitin-proteasome system has been largely investigated for its key role in protein degradation mechanisms that regulate both apoptosis and cell division. Because of their antitumour activity, different classes of proteasome inhibitors have been identified to date. Some of these compounds are currently employed in the clinical treatment of several types of cancer among which multiple myeloma. Here, we describe the design, chemistry, biological activity and modelling studies of a large series of amino acid derivatives linked to a naphthoquinone pharmacophoric group through variable spacers. Some analogues showed interesting inhibitory potency for the β1 and β5 subunits of the proteasome with IC₅₀ values in the sub-μm range.

Full text of DOI:10.1080/14756366.2017.1334649

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Naphthoquinone amino acid derivatives, synthesis
and biological activity as proteasome inhibitors
Mauro Marastoni, Claudio Trapella, Alessandra Scotti, Anna Fantinati,
Valeria Ferretti, Erika Marzola, Gallerani Eleonora, Riccardo Gavioli & Delia
Preti
To cite this article: Mauro Marastoni, Claudio Trapella, Alessandra Scotti, Anna Fantinati, Valeria
Ferretti, Erika Marzola, Gallerani Eleonora, Riccardo Gavioli & Delia Preti (2017) Naphthoquinone
amino acid derivatives, synthesis and biological activity as proteasome inhibitors, Journal of
Enzyme Inhibition and Medicinal Chemistry, 32:1, 865-877, DOI: 10.1080/14756366.2017.1334649
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1334649
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Date: 29 June 2017, At: 20:09

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 865–877
https://doi.org/10.1080/14756366.2017.1334649
ORIGINAL ARTICLE
Naphthoquinone amino acid derivatives, synthesis and biological activity as
proteasome inhibitors
Mauro Marastonia, Claudio Trapellaa, Alessandra Scottia, Anna Fantinatia, Valeria Ferrettia, Erika Marzolaa,
Gallerani Eleonorab, Riccardo Gaviolib and Delia Pretia
b
aDepartment of Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy; Department of Life Sciences and Biotechnology,
University of Ferrara, Ferrara, Italy
ABSTRACT
ARTICLE HISTORY
Received 3 April 2017
Revised 12 May 2017
Accepted 15 May 2017
The ubiquitin-proteasome system has been largely investigated for its key role in protein degradation
mechanisms that regulate both apoptosis and cell division. Because of their antitumour activity, different
classes of proteasome inhibitors have been identified to date. Some of these compounds are currently
employed in the clinical treatment of several types of cancer among which multiple myeloma. Here, we
describe the design, chemistry, biological activity and modelling studies of a large series of amino acid
derivatives linked to a naphthoquinone pharmacophoric group through variable spacers. Some analogues
showed interesting inhibitory potency for the b1 and b5 subunits of the proteasome with IC50 values in
the sub-mm range.
KEYWORDS
Proteasome; naphthoqui-
none; amino acid
derivatives; post-acidic
inhibition
Introduction
synthetic molecules have been studied as 20 S catalytic subunits
inhibitors7–16. The first-generation proteasome inhibitor (PI) borte-
zomib is currently employed as an anti-cancer drug, although its
effectiveness seems to be restricted to a limited number of can-
cers17,18. The FDA-approved carfilzomib19 and ixazomib20 along
with oprozomib21, currently in advanced clinical trials, are exam-
ples of second-generation irreversible PIs with a peptide structure.
In the last years, we developed several classes of peptide-
based PIs having different pharmacophoric units such as
electrophilic groups potentially able to interact with the catalytic
The proteasome enzymatic complex is widely involved in the
cytosolic and nuclear catabolism of most proteins behaving as a
protease with multiple catalytic sites1. The proteolytic action of
proteasome allows the elimination of impaired proteins and
results in the production of short-chain peptides that can be
exposed by the MHC complexes2. The ubiquitin-proteasome
system has been largely investigated for its key role in protein
degradation mechanisms that regulate both apoptosis and cell
division3,4. According to this system, the degradation of proteins is
the result of their conjugation with multiple ubiquitin units that
allow the recognition by the 26 S proteasome. The 26 S prote-
asome is composed of multiple subunits and is characterised by a
central 20 S core and two distal regions (19 S) displaying regulatory
activity. In the 20 S core, two external heptameric rings of a-sub-
units enclose two central heptameric rings of b-subunits. The 20 S
core exerts three typical catalytic activities that are specifically
located in the b1 (the peptidyl glutamyl peptide hydrolysing activ-
ity, PGPH), b2 (the trypsin-like activity, T-L) and b5 (the chymotryp-
sin-like, ChT-L) subunits5. The three catalytic b subunits have a
slightly different substrate specificity with a common mechanism
of proteolysis through an N-terminal threonine-dependent nucleo-
philic attack. Most of the cellular proteins undergo degradation
through this pathway that affects several processes such as cell
division, apoptosis or repair of DNA damages. As a consequence,
any alteration of this system may result in important pathologies,
including cancer.
threonine22–24
of dipeptide-based derivatives bearing at the C-terminal a 2-
chloronaphthoquinone pharmacophoric group (structure in
. We have recently investigated a new series
b
Figure 1)25. Some compounds of this series have been shown
to inhibit the post-acidic-like and the ChT-L active sites of the
proteasome in the mm range. The compound named PI-
083(NSC-45382), bearing the 2-cloronaphthoquinonic unit, and
other non-peptide analogues (general structure a in Figure 1),
are known to express a good inhibition against chymotryptic activ-
ity of the proteasome and the capacity to selectively inhibit
tumour cell proliferation26–28
.
Herein, we describe the synthesis, the in vitro biological evalu-
ation of proteasome inhibition and modelling studies of a new
series of amino acid derivatives linked through the a-carboxylic
function to the 2-chloronaphthoquinone pharmacophoric group
(general structure c in Figure 1). The 2-chloronaphthoquinone
(ClNafQ), a potential electrophilic substrate for the catalytic threo-
nine, is linked to the selected residues by a diamine alkyl spacers.
Studies regarding the non-peptide PI-083 and its analogues, in
addition to our docking analysis with the previous dipeptidic
Proteasome regulation by exogenous molecules able to
manipulate cellular activities has been receiving increasing atten-
tion6. Inhibitors of this multicatalytic complex are potential drugs
suitable in various therapeutic applications, therefore, natural and derivatives, suggest the potential interaction of the c-hydroxyl
CONTACT Delia Preti prtdle@unife.it Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari 46, Ferrara 44121, Italy
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

866
M. MARASTONI ET AL.
exploiting the following solvent systems: (c) AcOEt/n-hexane (1:1,
v/v), (d) CH2Cl2/methanol (9.5:0.5, v/v), (e) CH2CL2/methanol (9:1,
v/v) and (f) CH2CL2/methanol/toluene (17:2:1, v/v/v). Ninhydrin
(1%) or chlorine iodine spray reagents were employed to detect
the peptides. Melting points were determined by a Kofler appar-
atus and are uncorrected. Optical rotations were determined by a
O
Cl
Tailing
linker
(a)
H-bond
acceptor
HN
O
O
1
Perkin–Elmer 141 polarimeter with a 10-cm water-jacketed cell. H
NMR spectroscopy was obtained using a Varian 400 MHz spec-
trometer. All the assayed compounds described in this manuscript
were at least 95% pure as judged by HPLC and NMR.
Cl
O
H
H
N
(b)
R'
N
N
H
N
H
O
R
O
O
Synthesis
The synthesis applied for the preparation of compounds 1–48 is
reported in Scheme 1. This synthetic strategy allowed us to obtain
the compounds with the least possible number of steps. The
entire synthesis was performed in solution wherein the L-amino
acids were suitably protected at the Na and linked to the selected
diamine spacers at the carboxylic group. The alkyldiamine linkers
were protected at one of the amine functionality with the Boc
group. Subsequently, the Fmoc-protected amino acids were con-
densed at the mono-protected diaminic spacers using 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimmide (WSC) and N-hydroxybenzo-
triazole (HOBt) as acylating agents. After treatment with piperidine
to remove the fluorenylmethoxycarbonyl (Fmoc), the Na function-
alisation was carried out by acylation with 3-hydroxy-2-methyl-
benzoic acid, 4-nitrobenzoic acid, benzoic acid or alpha-naphthoic
acid using 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyl-ami-
nium hexafluorophosphate (HATU) as a coupling agent. After the
Boc removal via TFA treatment, we proceeded to the final reaction
with 2,3-dichloro-naphthoquinone that takes place in a solution of
95% EtOH, in presence of N-methyl-morpholine. All products, after
purification by RP-HPLC, were analysed by mass spectrometry and
NMR. The analytical data of the compounds are reported in the
supporting material.
O
O
Cl
HMB
NBz
Bz
NH-(CH2)2
NH-(CH2)4
NH-cHx
Leu
Asn
Phe
Ser
(c)
HN
1-NaftCO
Figure 1. (a) Schematic structure of non-peptide inhibitors bearing the 2-cloro-
naphthoquinonic unit. (b) The general structure of dipeptide derivatives with a 2-
chloronaphthoquinone group. (c) The generic structure of the new amino acid
derivatives linked to the 2-chloronaphthoquinone group.
group of catalytic threonine with the 2-chloronaphthoquinone
unit. The L-amino acids (Leu, Asn, Phe, Ser) were selected for their
different physicochemical features. The chloronaphthoquinone
pharmacophore is linked to the carboxylic group of the central
residue by ethylenediamine (compounds 1–16), butylenediamine
(17–32) and cyclohexyldiamine (33–48) spacers having different
length and flexibility (see Table 1 for the detailed structures).
Finally, the a-amino group is functionalised with 2-methyl-3-
hydroxybenzoyl (HMB), p-nitrobenzoyl (NBz), benzoyl (Bz) or
1-naphthoyl (1-NaftCO) aromatic groups having variable electronic
and steric peculiarity.
General synthetic procedures
Methods and materials
a. Condensation with Fmoc-amino acids. The carboxylic compo-
nent (1 mmol) was dissolved DMF (10 ml) and, after cooling at
0 ꢁC, WSC (1.1 mmol), HOBt (1.1 mmol) and the amine compo-
nent (1.1 mmol) were added. The reaction mixture was stirred
for 1 h at 0 ꢁC then overnight at room temperature. The solv-
ent was evaporated to give a residue that was suspended
with EtOAc and washed successively with 10% citric acid
(10 ml), 5% NaHCO3 (10 ml) and again with brine (10 ml). The
organic phase was dried with Na2SO4, filtered and evaporated
to furnish the desired products that were used without fur-
ther purification.
b. Condensation with 2-methyl-3-hydroxybenzoic acid, p-nitroben-
zoic acid, benzoic acid or a-naphthoic acid. The carboxylic
component (1 mmol) was dissolved DMF (6 ml) and HATU
(1mmol) and DIPEA (1 mmol) were added. Then a solution of
the appropriate amine (1 mmol) and TEA (1 mmol) in DMF
(6 ml) was added. The mixture was stirred overnight at room
temperature. The solvent was evaporated to obtain a residue
that was suspended with EtOAc. The organic phase was
washed successively with 10% citric acid (2 ꢀ 5 ml), 5%
NaHCO3 (2 ꢀ 5 ml) and again with brine (2 ꢀ 5 ml). The
organic phase was dried with Na2SO4, filtered and evaporated
to give a solid residue that was crystallised (Et2O) and col-
lected after centrifugation.
Chemistry-general
Amino acids, amino acid derivatives and chemicals were pur-
chased from Bachem, Novabiochem, and Fluka (Switzerland).
Crude products were purified by preparative reversed-phase HPLC
using a Waters Delta Prep 3000 system with a Jupiter column C18
(250 ꢀ 30 mm, 300 Å, 15 m spherical particle size). The column was
perfused at a flow rate of 20 ml/min, with a mobile phase-contain-
ing solvent A (10%, v/v, acetonitrile in 0.1% TFA), and a linear gra-
dient from 0% to 100% of solvent B (60%, v/v, acetonitrile in 0.1%
TFA); 30 min was the time adopted for elution of the compounds.
HPLC analysis was performed using a Beckman System Gold with
a Luna C18 column (4.6 ꢀ 100 mm, 3 m particle size). Analytical
determination and retention time (Tr) of the peptides were
assayed via HPLC conditions in the above solvent system (solvents
A and B), programmed at flow rates of 0.5 ml/min, using the fol-
lowing linear gradients: (a) from 0% to 90% B for 25 min and (b)
from 30% to 100% B for 25 min. No naphthoquinone derivative
showed more than 1% impurity when monitored at 220 and
254 nm. The molecular weights of the compounds were deter-
mined by electrospray ionisation (ESI) (MICROMASS ZMD 2000),
and the values are expressed as [M þ H]þ. TLC was performed on
pre-coated plates of silica gel F254 (Merck, Darmstadt, Germany),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
867
Table 1. Inhibition of the proteasome subunits by the synthesised compounds.
O
Cl
R' Xaa
NH-R-NH
O
Compd
R'
HMB
NBz
Xaa
R
IC50 (lm)a T-L
IC50 (lm)a ChT-L
IC50 (lm)a PGPH
1
2
3
4
5
6
7
8
Leu
Leu
Leu
Leu
Asn
Asn
Asn
Asn
Phe
Phe
Phe
Phe
Ser
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)2ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–(CH2)4ꢂ
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
–cHx-
77.44 6.51
65.32 5.91
58.43 5.13
21.08 1.85
68.35 5.14
17.23 1.09
49.66 3.87
35.23 2.77
>100
35.40 2.91
53.18 6.70
49.75 3.97
>100
63.36 7.01
53.82 4.88
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.21 0.08
2.35 0.21
2.61 0.23
0.91 0.07
6.18 0.55
1.19 0.13
11.23 0.98
10.01 1.27
0.88 0.08
0.24 0.3
1.05 0.09
35.76 4.03
91.52 7.69
78.66 8.14
5.78 4.35
>100
Bz
1-NaftCO
HMB
NBz
0.82 0.08
8.47 0.75
0.92 0.09
7.99 0.67
76.25 6.82
0.85 0.07
0.77 0.06
6.22 0.48
9.14 1.02
>100
Bz
1-NaftCO
HMB
NBz
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
MG132
Bz
1-NaftCO
HMB
NBz
Ser
Ser
Ser
83.47 8.03
24.11 1.98
>100
Bz
1-NaftCO
HMB
NBz
Leu
Leu
Leu
Leu
Asn
Asn
Asn
Asn
Phe
Phe
Phe
Phe
Ser
11.50 1.02
8.40 0.72
10.12 0.70
49.30 5.02
65.38 6.50
28.52 3.05
24.19 2.05
78.34 6.95
17.50 1.07
9.10 0.6
10.12 0.70
9.30 1.02
45.68 2.59
88.74 8.03
84.11 6.08
>100
7.82 0.71
10.54 0.82
2.41 0.14
45.35 4.03
9.52 1.09
>100
Bz
1-NaftCO
HMB
NBz
Bz
>100
>100
>100
>100
>100
>100
1-NaftCO
HMB
NBz
8.82 0.71
10.54 0.82
5.43 0.44
25.65 3.03
61.52 4.99
83.66 8.16
3.55 1.65
83.62 5.48
>100
>100
>100
73.67 1.45
>100
>100
>100
>100
>100
>100
>100
83.37 4.58
>100
5.78 0.62
3.42 0.51
11.71 1.25
>10
Bz
>100
1-NaftCO
HMB
NBz
79.71 6.97
80.24 6.91
83.26 7.21
>100
60.75 4.88
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Ser
Ser
Ser
Bz
1-NaftCO
HMB
NBz
Leu
Leu
Leu
Leu
Asn
Asn
Asn
Asn
Phe
Phe
Phe
Phe
Ser
>100
42.10 5.02
23.12 1.70
59.30 6.42
>100
>100
>100
>100
>100
82.10 6.52
73.31 3.90
>100
Bz
1-NaftCO
HMB
NBz
Bz
1-NaftCO
HMB
NBz
Bz
1-NaftCO
HMB
NBz
Bz
1-NaftCO
>100
>100
>100
>100
Ser
Ser
Ser
>100
>100
>100
1.04 0.092
0.0018 0.00022
aThe values reported are the mean SEM of three independent determinations.
c. Fmoc Removal. The Fmoc protection was removed by treat-
ment at room temperature with a 20% piperidine solution in
DMF for 1 h. The solvent was evaporated and the desired
products were precipitated with ethyl ether, then separated
by centrifugation and collected.
then N-methyl-morpholine (0.3 mmol) and 2,3-dichloro-1,4-
naphthoquinone (0.6 mmol) were added. The mixture was
heated at 115 ꢁC for 3 d under stirring. After evaporation of
the solvent, the residue was triturated with ethyl ether and
separated by centrifugation.
d. Boc Removal. The Boc protection was removed by treatment
with 90% aqueous TFA (1 ml for 1 mmol of the Boc-protected
Preparation of Boc-ethylene/butylene/trans-cyclohexyldiamine
The diamine (10 mmol) was dissolved in a mixture of t-ButOH/H2O
(2:1, 20 ml) then (Boc)2 O (7 mmol) was added and the reaction
was stirred for 2 h at room temperature. Water (20 ml) was added
compound) for 1 h. After evaporation of the solvent, the resi-
due was triturated with ethyl ether and separated by
centrifugation.
e. Condensation with 2,3-dichloro-1,4-naphthoquinone. The amine
component (0.3 mmol) was dissolved in 95% EtOH (15 ml) and the aqueous phase was extracted with n-pentane (2 ꢀ 10 ml).

868
M. MARASTONI ET AL.
H-Asn-NH-(CH2)2-NH-Boc. White solid, yield 67%; 1H NMR,
(CDCl3) d: 7.82 (bs, 1H), 5.11 (s, 2H), 3.79–3.76 (m, 1H), 3.58–3.54
(m, 2H), 3.47–3.42 (m, 2H), 3.12–2.90 (m, 1H), 2.85–2.72 (m, 1H),
1.45 (s, 9H);MS (M þ Hþ) 275.32.
H2N-R-NH2
(Boc)2O
H2N-R-NH-Boc
1) Fmoc-Xaa-OH, WSC, HOBt
H-Phe-NH-(CH2)2-NH-Boc. White solid, yield 88%; 1H NMR,
(CDCl3) d: 7.76 (bs, 1H), 7.44–7.42 (m, 2H), 7.32–7.29 (m, 3H), 5.13
(s, 2H), 3.91–3.88 (m, 1H), 3.56–3.52 (m, 2H), 3.46–3.45 (m, 2H),
3.44–3.42 (m, 1H), 3.20–3.18 (m, 1H), 1.44 (s, 9H);MS (M þ Hþ)
308.30.
2) Piperidine in DMF (20%)
1
H-Ser-NH-(CH2)2-NH-Boc. White solid, yield 67%; H NMR, (CDCl3)
NH-R-NH-Boc
H Xaa
d: 7.74 (bs, 1H), 5.13 (bs, 2H), 4.18–4.16 (m, 1H), 3.92–3.90 (m, 1H),
3.66–3.64 (m, 1H), 3.53–3.50 (m, 2H), 3.48–3.46 (m, 2H), 1.38 (s,
9H);MS (M þ Hþ) 248.28.
1) R'-OH, HATU, DIPEA
2) TFA
H-Leu-NH-(CH2)4-NH-Boc. White solid, yield 77%; 1H NMR,
(CDCl3) d: 8.00 (bs, 1H), 5.40 (bs, 2H), 3.49–3.41 (m, 5H), 1.78–1.76
(m, 1H), 1.55–1.54 (m, 2H), 1.51–1.49 (m, 2H), 1.43 (s, 9H),
1.34–1.33 (m, 2H), 0.93 (d, 3H, J ¼ 7.4 Hz), 0.90 (d, 3H,
J ¼ 7.4 Hz);MS (M þ Hþ) 302.24.
NH-R-NH2
R' Xaa
H-Asn-NH-(CH2)4-NH-Boc. White solid, yield 87%; 1H NMR,
(CDCl3) d: 7.79 (bs, 1H), 5.33 (bs, 2H), 3.79–3.77 (m, 1H), 3.55–3.53
(m, 2H), 3.44–3.42 (m, 2H), 2.91–2.89 (m, 1H), 2.87–2.86 (m, 1H),
1.54–1.53 (m, 2H), 1.50–1.49 (m, 2H), 1.43 (s, 9H);MS (M þ Hþ)
303.27.
2,3-dichloro-1,4-naphthoquinone
EtOH (95%), Δ
O
Cl
H-Phe-NH-(CH2)4-NH-Boc. White solid, yield 64%;1H NMR, (CDCl3)
d: 7.99 (bs, 1H), 7.44–7.42 (m, 2H), 7.31–7.29 (m, 3H), 5.22 (bs, 2H),
3.89–3.87 (m, 1H), 3.56–3.53 (m, 1H), 3.22–3.18 (m, 5H), 1.52–1.50
(m, 2H), 1.49–1.47 (m, 2H), 1.42 (s, 9H);MS (M þ Hþ) 336.18.
R' Xaa NH-R-NH
O
R = -(CH2)2-, -(CH2)4-, cHx;
Xaa = Leu, Asn, Phe, Ser;
R' = Hmb, NBz, Bz, 1-NaftCO
1
H-Ser-NH-(CH2)4-NH-Boc. White solid, yield 77%; H NMR, (CDCl3)
d: 8.03 (bs, 1H), 5.20 (bs, 2H), 4.17–4.15 (m, 1H), 3.90–3.89 (m, 1H),
3.64–3.62 (m, 1H), 3.50–3.49 (m, 2H), 3.48–3.47 (m, 2H), 1.54–1.53
(m, 2H), 1.51–1.49 (m, 2H), 1.43 (s, 9H);MS (M þ Hþ) 276.29.
H-Leu-NH-cHx-NH-Boc. White solid, yield 77%; 1H NMR, (CDCl3)
d: 7.64 (bs, 1H), 5.44 (bs, 2H), 3.55–3.54 (m, 1H), 3.52–3.51 (m, 1H),
3.35–3.33 (m, 1H), 1.77–1.75 (m, 1H), 1.70–1.69 (m, 4H), 1.54–1.53
(m, 4H), 1.39 (s, 9H), 1.34–1.32 (m, 2H), 0.94 (d, 3H, J ¼ 7.4 Hz), 0.89
(d, 3H, J ¼ 7.4 Hz); MS (M þ Hþ) 328.22.
Scheme 1. Synthesis of naphthoquinone amino acid derivatives.
After separation, the aqueous phase was further extracted with
EtOAc (2 ꢀ 50 ml) and the latter organic phase was dried with
anhydrous Na2SO4 and evaporated to yield the desired com-
pounds that were employed without further purification.
Boc-ethylenediamine. Colourless oil, yield 85%.1H NMR (CDCl3)
d5.98 (bs, 1H), 3.08 (m, 2H), 2.69 (m, 2H), 1.75 (bs, 2H), 1.39 (s, 9H);
MS (M þ Hþ) 161.20; HPLC (Tr) 6.54 min. Spectroscopic data are
consistent with those previously reported25.
H-Asn-NH-cHx-NH-Boc. White solid, yield 87%; 1H NMR, (CDCl3)
d: 8.26 (bs, 1H), 7.24 (bs, 2H), 5.54 (bs, 2H), 3.78–3.76 (m, 1H),
3.55–3.53 (m, 2H), 2.90–2.88 (m, 1H), 2.85–2.83 (m, 1H), 1.77–1.73
(m, 4H), 1.50–1.47 (m, 4H), 1.38 (s, 9H);MS (M þ Hþ) 329.32.
H-Phe-NH-cHx-NH-Boc. White solid, yield 56%; 1H NMR, (CDCl3)
d: 8.44 (bs, 1H), 7.42–7.40 (m, 2H), 7.31–7.29 (m, 3H), 5.44 (bs, 2H),
3.89–3.87 (m, 1H), 3.58–3.56 (m, 1H), 3.54–3.53 (m, 2H), 3.19–3.18
(m, 1H), 1.78–1.73 (m, 4H), 1.49–1.44 (m, 4H), 1.38 (s, 9H);MS
(M þ Hþ) 362.15.
Boc-butylenediamine. Colourless oil, yield 75%. 1H NMR (CDCl3)
d 4.70 (bs, 1H), 3.14 (m, 2H), 2.68 (t, 2H, J ¼ 6.7), 1.68 (bs, 2H),
1.50–1.45 (m, 4H), 1.48 (s, 9H). MS (M þ Hþ) 189.22; HPLC (Tr)
7.24 min. Spectroscopic data are consistent with those previously
reported29.
Boc-trans-cyclohexyldiamine. White solid, yield 96%. 1H NMR
(CDCl3): d 4.91–5.12 (bs, 1H), 3.31–3.41 (bs, 1H), 2.57–2.68 (m, 1H),
1.90–2.03 (bs, 2H), 1.87–1.97 (m, 4H), 1.44 (s, 9H), 1.10–1.24 (m,
4H). MS (M þ Hþ) 214.26; HPLC (Tr) 7.56 min. Spectroscopic data
are consistent with those previously reported30.
1
H-Ser-NH-cHx-NH-Boc. White solid, yield 57%; H NMR, (CDCl3) d:
8.15 (bs, 1H), 5.44 (bs, 2H), 4.16–4.13 (m, 1H), 3.97–3.96 (m, 1H),
3.64–3.62 (m, 1H), 3.58–3.56 (m, 1H), 3.54–3.52 (m, 1H), 1.77–1.73
(m, 4H), 1.54–1.51 (m, 4H), 1.38 (s, 9H);MS (M þ Hþ) 302.28.
Preparation of R'-Xaa-NH-R-NH2
Preparation of H-Xaa-NH-R-NH-boc
The compounds with general structure R'-Xaa-NH-R-NH2 were pre-
pared through a first HATU-mediated coupling of intermediates H-
Xaa-NH-R-NH-Boc with 2-methyl-3-hydroxybenzoic acid, p-nitro-
benzoic acid, benzoic acid or a-naphthoic acid following the gen-
eral procedure (b). The resulting Boc-protected derivatives were
then treated according to the general method for Boc removal (d)
to give the target compounds as trifluoroacetate salts. Analytical
data and 1H-NMR spectra of representative compounds are listed
below. NMR spectra of the whole series can be found in the
Supplementary Material.
The intermediates with general structure Fmoc-Xaa-NH-R-NH-Boc
were first prepared by acylation of the appropriate Boc-protected
diamine with Fmoc-Xaa-OH following the general procedure (a).
Fmoc-protected derivatives were then treated according to the
general procedure for Fmoc removal (c) to give the desired H-Xaa-
NH-R-NH-Boc intermediates.
H-Leu-NH-(CH2)2-NH-Boc. White solid, yield 77%; 1H NMR,
(CDCl3) d: 3.49–3.41 (m, 2H), 3.18–3.14 (m, 1H), 3.05–3.00 (m, 1H),
2.90–2.88 (m, 1H), 1.78–1.76 (m, 1H), 1.39 (s, 9H), 1.36–1.32 (m,
2H), 0.94 (d, 3H, J ¼ 7.4 Hz), 0.89 (d, 3H, J ¼ 7.4 Hz); MS (M þ Hþ)
274.31.
HMB-Leu-NH-(CH2)2-NH2. White solid, yield 54%; 1H NMR
(400 MHz, CDCl3) d 7.30 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.21–7.04 (m, 1H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
869
6.90 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.55 (s, 1H), 5.19 (s, 3H), 5.02 (s, 1H), 4.31–4.06 (m, 2H), 4.03–3.88 (m, 1H), 3.87–3.66 (m, 1H), 3.61–3.32
4.41 (t, J ¼ 7.6 Hz, 1H), 3.95 (t, J ¼ 7.6 Hz, 2H), 3.55 (s, 1H), 3.03 (t, (m, 2H), 2.29 (s, 3H), 2.23–2.10 (m, 2H), 2.05–1.79 (m, 4H),
J ¼ 7.6 Hz, 2H), 2.31 (s, 3H), 1.98–1.66 (m, 2H), 1.58 (t, J ¼ 7.5 Hz, 1.67–1.45 (m, 2H); MS (M þ Hþ) 336.52.
1H), 1.23–0.86 (m, 6H);MS (M þ Hþ) 308.21.
HMB-Asn-NH-(CH2)2-NH2. Pale yellow solid, yield 79%; 1H NMR
Preparation of R'-Xaa-NH-R-NH-ClNafQ
(400 MHz, CDCl3) d 7.30 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.22–7.06 (m, 1H),
The final compounds 1–48 were obtained by the reaction of inter-
6.91 (dd, J ¼ 7.5, 1.4 Hz, 1H), 6.55 (s, 1H), 5.58 (s, 3H), 5.19 (s, 1H),
mediates R'-Xaa-NH-R-NH2 with 2,3-dichloro-1,4-naphthoquinone
following the general procedure (e). All the compounds were puri-
fied by RP-HPLC. Analytical data and 1H-NMR spectra of represen-
tative compounds (1–16) are listed below. NMR spectra of the
whole series can be found in the Supplementary Material.
5.07 (s, 2H), 4.83–3.91 (m, 2H), 3.61 (s, 1H), 3.14–2.96 (m, 3H),
2.87–2.83 (m, 1H), 2.19 (s, 3H);MS(M þ Hþ) 309.17.
HMB-Phe-NH-(CH2)2-NH2. White solid, yield 81%; 1H NMR
(400 MHz, CDCl3) d 7.36–7.33 (m, 1H), 7.30–7.03 (m, 6H), 6.91 (dd,
J ¼ 7.5, 1.4 Hz, 1H), 6.80 (s, 1H), 5.07 (s, 3H), 4.69–4.67 (m, 1H), 4.30
(s, 1H), 3.97–3.90 (m, 2H), 3.56 (s, 1H), 3.30–3.28 (m, 1H), 3.03–2.78
(m, 3H), 2.31 (s, 3H); MS(M þ Hþ) 342.18.
HMB-Leu-NH-(CH2)2-NH-ClNafQ (1). White solid; yield 58%;mp
¼111–114 ꢁC; [a]D20–30.2 (c ¼ 1, MeOH). H NMR (400 MHz, CDCl3)d
1
HMB-Ser-NH-(CH2)2-NH2. White solid, yield 77%; 1H NMR
(400 MHz, CDCl3)d 7.18–7.01 (m, 2H), 6.97–6.87 (m, 1H), 5.50 (bs,
1H), 5.21 (s, 3H), 4.99 (s, 1H), 4.75–4.73 (m, 1H), 4.29–4.28 (m, 1H),
4.02–3.75 (m, 3H), 3.54 (s, 1H), 3.05–3.02 (m, 2H), 2.29 (s, 3H); MS
(M þ Hþ) 282.27.
8.29–8.07 (m, 2H), 8.05 (bs, 1H), 7.78–7.66 (m, 2H), 7.23–6.98 (m,
2H), 6.95–6.78 (m, 1H), 5.79 (bs, 1H), 5.71 (bs, 1H), 4.72–4.66 (m,
1H), 3.56–3.45 (m, 4H), 2.29 (s, 3H), 1.85–1.69 (m, 2H), 1.56 (t,
J ¼ 6.6 Hz, 1H), 0.97–0.87 (m,6H); MS (ESI): [MH]þ ¼ 498.12.HPLC (Tr)
16.76 min.
HMB-Leu-NH-(CH2)4-NH2. Pale yellow solid, yield 57%; 1H NMR
(400 MHz, CDCl3)d 7.30–7.26 (m, 1H), 7.14–7.11 (m, 1H), 6.91–6.89
(m, 1H), 6.03 (s, 3H), 5.26 (s, 1H), 5.23–5.11 (m, 2H), 3.74 (s, 1H),
3.50–3.48 (m, 1H), 3.19–3.17 (m, 1H), 2.82–2.75 (m, 2H), 2.24 (s,
3H), 2.02–1.89 (m, 2H), 1.83–1.58 (m, 4H), 1.54–1.52 (m, 1H),
1.04–0.93 (m, 6H); MS (M þ Hþ) 336.31.
NBz-Leu-NH-(CH2)2-NH-ClNafQ (2). White solid; yield 66%;mp
1
¼134–137 ꢁC; [a]D20–26.4 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
8.22–8.13 (m, 4H), 8.08 (d, J ¼ 7.4 Hz, 2H), 7.80–7.69 (m, 2H), 7.41
(bs, 1H), 6.80 (bs, 1H), 5.65 (bs, 1H), 3.93–3.86 (m, 1H), 3.89–3.41
(m, 4H), 1.86–1.57 (m, 2H), 1.45–1.43 (m, 1H), 0.97–0.87 (m, 6H);
MS (ESI): [MH]þ ¼ 513.11; HPLC (Tr) 18.40 min.
HMB-Asn-NH-(CH2)4-NH2. White solid, yield 73%; 1H NMR
(400 MHz, CDCl3)d 7.70 (s, 1H), 7.61 (s, 1H), 7.32–7.28 (m, 1H), 7.15
(t, J ¼ 7.5 Hz, 1H), 6.91–6.94 (m, 1H), 5.26 (s, 2H), 4.94 (s, 3H),
4.63–4.58 (m, 1H), 3.31–3.17 (m, 2H), 3.05–3.02 (m, 1H), 2.82–2.65
(m, 3H), 2.32 (s, 3H), 1.86–1.79 (m, 2H), 1.55–1.50 (m, 2H); MS
(M þ Hþ) 337.32.
Bz-Leu-NH-(CH2)2-NH-ClNafQ (3). White solid; yield 89%;mp
20
¼128–130 ꢁC; [a]D
–22.4 (c ¼ 1, MeOH); 1H NMR (400 MHz,
CDCl3)d 8.25–8.10 (m, 2H), 7.89–7.83 (m, 2H), 7.79–7.68 (m, 2H),
7.53–7.358 (m, 3H), 6.54 (s, 1H), 5.45 (s, 1H), 5.31 (s, 1H), 4.23–4.18
(m, 1H), 3.58–3.44 (m, 3H), 3.40–3.36 (m, 1H), 1.68–1.42 (m, 3H),
1.06 (d, J ¼ 6.1 Hz, 6H); MS (ESI): [MH]þ ¼ 468.03; HPLC (Tr)
17.71 min.
HMB-Phe-NH-(CH2)4-NH2. White solid, yield 77%; 1H NMR
(400 MHz, CDCl3)d 7.40–7.36 (m, 1H), 7.34–7.08 (m, 7H), 6.95–6.91
(m, 1H), 5.16 (s, 3H), 4.73–4.70 (m, 1H), 4.39 (s, 1H), 3.41 (s, 1H),
3.28–3.17 (m, 3H), 2.92–2.89 (m, 1H), 2.78–2.75 (m, 2H), 2.31 (s,
3H), 2.02–1.77 (m, 2H), 1.65–1.55 (m, 2H); MS (M þ Hþ) 370.22.
HMB-Ser-NH-(CH2)4-NH2. Pale yellow solid, yield 44%; 1H NMR
(400 MHz, CDCl3)d 7.18–7.13 (m, 1H), 7.10–7.05 (m, 1H), 6.88–6.97
(m, 1H), 5.32 (s, 2H), 4.96 (s, 3H), 4.67–4.58 (m, 1H), 4.14–4.10 (m,
1H), 3.90–3.85 (m, 1H), 3.56 (s, 1H), 3.32–3.18 (m, 2H), 2.76–7.68
(m, 2H), 2.32 (s, 3H), 1.99–1.85 (m, 2H), 1.76–1.62 (m, 2H); MS
(M þ Hþ) 310.17.
1-NaftCO-Leu-NH-(CH2)2-NH-ClNafQ (4). White solid; yield
74%;mp ¼145–148 ꢁC; [a]D20–33.2 (c ¼ 1, MeOH); 1H NMR
(400 MHz, CDCl3)d 8.52–8.47 (m, 1H), 8.18–8.02 (m, 2H), 7.99–7.93
(m, 2H), 7.82–7.78 (m, 1H), 7.74–7.64 (m, 2H), 7.58–7.46 (m, 3H),
7.21 (s, 1H), 7.05 (s, 1H), 5.75 (s, 1H), 4.52–4.47 (m, 1H), 3.64–3.39
(m, 4H), 1.99–1.86 (m, 1H), 1.73–1.34 (m, 2H), 1.12–0.93 (m, 6H);
MS (ESI): [MH]þ ¼ 518.15; HPLC (Tr) 19.11 min.
HMB-Asn-NH-(CH2)2-NH-ClNafQ (5). White solid; yield 66%;mp
1
¼128–130 ꢁC; [a]D20–42.1 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
8.57 (s, 1H), 8.34–8.23 (m, 1H), 7.95–7.86 (m, 1H), 7.67–7.58 (m,
2H), 7.32–7.29 (m, 1H), 7.14–7.10 (m, 1H), 6.89–6.81 (m, 1H), 5.50
(s, 1H), 5.35 (s, 1H), 5.23–5.18 (m, 1H), 4.73 (s, 2H), 3.62–3.54 (m,
3H), 3.46–3.40 (m, 1H), 3.03–2.98 (m, 1H), 2.51–2.46 (m, 1H), 2.33
(s, 3H); MS(ESI): [MH]þ ¼ 499.24; HPLC (Tr) 13.21 min.
HMB-Leu-NH-cHx-NH2. Pale yellow solid, yield 81%; 1H NMR
(400 MHz, CDCl3)d 7.40 (s, 1H), 7.30 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.14 (t,
J ¼ 7.5 Hz, 1H), 6.91 (dd, J ¼ 7.5, 1.4 Hz, 1H), 5.70 (s, 3H), 4.96 (s,
1H), 4.72–4.70 (m, 1H), 3.67–3.63 (m, 1H), 3.59–3.45 (m, 1H),
2.36–2.29 (m, 3H), 2.17–2.05 (m, 2H), 1.84–1.55 (m, 7H), 1.07–0.96
(m, 6H); MS (M þ Hþ) 362.14.
NBz-Asn-NH-(CH2)2-NH-ClNafQ (6). White solid; yield 52%;mp
¼168–170 ꢁC; [a]D20–38.8 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
1
HMB-Asn-NH-cHx-NH2. Pale yellow solid, yield 72%; 1H NMR
(400 MHz, CDCl3)d 7.50 (s, 1H), 7.36 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.17 (t,
J ¼ 7.5 Hz, 1H), 6.91 (dd, J ¼ 7.5, 1.4 Hz, 1H), 5.27 (s, 2H), 5.16 (s,
3H), 4.49–4.45 (m, 1H), 4.32–4.28 (m, 1H), 3.56 (s, 1H), 3.36–3.31
(m, 1H), 3.07–3.03 (m, 1H), 2.68–2.64 (m, 1H), 2.55–2.49 (m, 2H),
2.35–2.22 (m, 5H), 2.03–1.96 (m, 2H), 1.74–1.68 (m, 2H); MS
(M þ Hþ) 363.32.
8.74 (s, 1H), 8.20–8.05 (m, 6H), 7.79–7.68 (m, 2H), 5.69–5.66 (m,
3H), 5.30 (s, 1H), 4.72–4.66 (m, 1H), 3.70–3.56 (m, 1H), 3.55–3.32
(m, 3H), 2.96–2.91 (m, 1H), 2.50–2.45 (m, 1H); MS (ESI):
[MH]þ ¼ 514.08; HPLC (Tr) 13.66 min.
Bz-Asn-NH-(CH2)2-NH-ClNafQ (7). White solid; yield 48%;mp
20
¼165–167 ꢁC; [a]D
–26.6 (c ¼ 1, MeOH); 1H NMR (400 MHz,
CDCl3)d 8.27–8.08 (m, 2H), 7.87 (dd, J ¼ 7.4, 1.3 Hz, 2H), 7.74–7.65
(m, 2H), 7.41–7.21 (m, 3H), 7.24 (s, 1H), 6.47 (s, 1H), 6.00 (s, 1H),
5.02 (s, 2H), 4.87–4.82 (m, 1H), 3.60–3.37 (m, 4H), 3.13–3.08 (m,
1H), 2.86–7.82 (m, 1H);MS (ESI): [MH]þ ¼ 469.12; HPLC (Tr)
1
HMB-Phe-NH-cHx-NH2. White solid, yield 68%; H NMR (400 MHz,
CDCl3)d 7.41 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.34–7.24 (m, 4H), 7.24–7.08
(m, 2H), 7.02–6.85 (m, 2H), 5.27 (s, 1H), 5.18 (s, 3H), 4.90 (t,
J ¼ 3.6 Hz, 1H), 4.19–4.15 (m, 1H), 3.56–3.34 (m, 2H), 3.00–2.97 (m, 14.30 min.
1H), 2.40–2.22 (m, 5H), 2.10–2.05 (m, 2H), 1.81–1.76 (m, 2H),
1-NaftCO-Asn-NH-(CH2)2-NH-ClNafQ (8). White solid; yield
1.48–1.36 (m, 2H); MS (M þ Hþ) 396.33.
87%;mp ¼178–180 ꢁC; [a]D20–42.8 (c ¼ 1, MeOH); 1H NMR
1
HMB-Ser-NH-cHx-NH2. Pale yellow, yield 69%; H NMR (400 MHz, (400 MHz, CDCl3) d 8.57–8.41 (m, 2H), 8.20–8.04 (m, 2H), 8.00–7.87
CDCl3)d 7.32 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.18 (t, J ¼ 7.5 Hz, 1H), 6.91 (m, 2H), 7.84–7.80 (m, 1H), 7.76–7.60 (m, 2H), 7.61–7.44 (m, 3H),
(dd, J ¼ 7.5, 1.4 Hz, 1H), 5.04 (s, 1H), 4.75 (s, 1H), 4.68 (s, 3H), 5.66 (s, 1H), 5.38 (s, 2H), 4.59–4.45 (m, 1H), 4.17 (s, 1H), 3.60–3.51

870
M. MARASTONI ET AL.
(m, 1H), 3.45–3.34 (m, 4H), 2.91–2.89 (m, 1H);MS (ESI): to determine chymotrypsin-like, trypsin-like and post-acidic prote-
[MH]þ ¼ 519.14; HPLC (Tr) 15.43 min.
asome activities, respectively. Substrates were incubated at 37 ꢁC
for 30 min with proteasomes, untreated or pretreated with
0.1–100 mm of test compounds 1–48 and reference inhibitor
MG132, in activity buffer. Fluorescence was determined by a
fluorimeter (Spectrafluor plus, Tecan, Salzburg, Austria), using an
excitation of 360 nm and emission of 465 nm. Activity was eval-
uated in fluorescence units and the inhibitory capacity of the
compounds is expressed as IC50.
HMB-Phe-NH-(CH2)2-NH-ClNafQ (9). White solid; yield 72%;mp
¼121–124 ꢁC; [a]D20–31.4 (c ¼ 1, MeOH); 1H NMR (400 MHz, CDCl3)
d 8.17–8.02 (m, 2H), 7.72–7.66 (m, 2H), 7.35–7.04 (m, 8H), 6.90 (dd,
J ¼ 7.5, 1.4 Hz, 1H), 6.20 (s, 1H), 5.05 (s, 1H), 4.77–4.68 (m, 1H),
3.64–3.40 (m, 4H), 3.42–3.22 (m, 2H), 2.95–2.92 (m, 1H), 2.28 (s,
3H); MS (ESI): [MH]þ ¼ 532.02; HPLC (Tr) 16.91 min.
NBz-Phe-NH-(CH2)2-NH-ClNafQ (10). White solid; yield 59%;mp
1
¼141–144 ꢁC; [a]D20–28.9 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
8.83 (s, 1H), 8.25–8.15 (m, 3H), 8.09 (dd, J ¼ 7.5, 1.6 Hz, 1H), 8.03 (d,
J ¼ 7.5 Hz, 2H), 7.84–7.80 (m, 1H), 7.70–7.66 (m, 1H), 7.23–7.20 (m,
2H), 7.16–7.04 (m, 3H), 6.23 (s, 1H), 4.69 (s, 1H), 4.03–3.98 (m, 1H),
3.68–3.50 (m, 2H), 3.50–3.42 (m, 1H), 3.45–3.32 (m, 1H), 3.25–3.15
(m, 1H), 2.98–2.96 (m, 1H); MS (ESI): [MH]þ ¼ 547.30; HPLC (Tr)
18.73 min.
Growth inhibition assays
Cell growth inhibition assays were carried out using the breast
cancer cell line MDA and ovarian cancer cell line A2780. Cells were
obtained from ATCC (Manassas, VA) and maintained in DMEM and
RPMI respectively, supplemented with 10% foetal bovine serum,
penicillin (100 U mlꢂ1), streptomycin (100 U mlꢂ1) and glutamine
(2 mm); incubation was performed at 37 ꢁC in a 5% CO2 atmos-
phere. Cells were routinely passaged every 3 d at 70% confluence;
0.05% trypsin-EDTA was used. The antiproliferative activity of new
molecules was tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells were seeded in
triplicate in 96-well plates at a density of 15 ꢀ 103 in 50 ml of com-
plete medium. Stock solutions (10 mm) of selected analogues were
made in DMSO and diluted in complete medium to give final con-
Bz-Phe-NH-(CH2)2-NH-ClNafQ (11). White solid; yield 86%;mp
1
¼122–124 ꢁC; [a]D20–23.4 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
8.23–8.13 (m, 1H), 8.05–7.83 (m, 3H), 7.77–7.65 (m, 3H), 7.52–7.48
(m, 3H), 7.31–7.19 (m, 2H), 7.19–7.11 (m, 3H), 4.87 (s, 1H), 4.55 (s,
1H), 4.10–4.02 (m, 1H), 3.57–3.41 (m, 4H), 3.30–3.27 (m, 1H),
2.98–2.96 (m, 1H);MS (ESI): [MH]þ ¼ 502.15; HPLC (Tr) 18.05 min.
1-NaftCO-Phe-NH-(CH2)2-NH-ClNafQ (12). White solid; yield
66%;mp ¼130–134 ꢁC; [a]D20–33.2 (c ¼ 1, MeOH); 1H NMR
(400 MHz, CDCl3)d 8.51 (dd, J ¼ 7.4, 1.5 Hz, 1H), 8.39 (s, 1H),
8.23–8.03 (m, 2H), 7.95–7.94 (m, 1H), 7.84–7.81 (m, 1H), 7.74–7.60 centrations of 10 and 100 mm. MG-132 was employed as a control.
Untreated cells were placed in every plate as a negative control.
The cells were exposed to the compounds, in 100 ml total volume,
for 72 h, and then 25 ml of a 12 mm solution of MTT was added.
After 2 h of incubation, 100 ml of lysing buffer (50% DMF þ20%
SDS, pH 4.7) was added to convert the MTT solution into a violet-
coloured formazan. After an additional 18 h the solution absorb-
ance, proportional to the number of live cells, was measured by
spectrophotometer at 570 nm and converted into % of growth
inhibition.
(m, 2H), 7.58–7.56 (m, 2H), 7.49–7.48 (m, 1H), 7.28–7.18 (m, 3H),
7.10–7.01 (m, 3H), 6.67 (s, 1H), 5.56 (s, 1H), 4.81–4.76 (m, 1H),
3.59–3.51 (m, 1H), 3.51–3.39 (m, 3H), 3.33–3.30 (m, 1H), 3.03–3.00
(m, 1H); MS (ESI): [MH]þ ¼ 552.16; HPLC (Tr) 19.65 min.
HMB-Ser-NH-(CH2)2-NH-ClNafQ (13). White solid; yield 76%;mp
¼117–120 ꢁC; [a]D20–45.1 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
1
8.11–7.99 (m, 2H), 7.70–7.64 (m, 2H), 7.36–7.32 (m, 1H), 7.10–7.07
(m, 2H), 6.91–6.89 (m, 1H), 6.08 (s, 1H), 5.06 (s, 1H), 4.28–4.17 (m,
1H), 4.19–4.17 (m, 1H), 3.78–3.76 (m, 1H), 3.56–3.40 (m, 4H), 2.32
(s, 3H); MS (ESI): [MH]þ ¼ 472.08; HPLC (Tr) 12.84 min.
NBz-Ser-NH-(CH2)2-NH-ClNafQ (14). White solid; yield 56%;mp
1
¼142–146 ꢁC; [a]D20–39.8 (c ¼ 1, MeOH); H NMR (400 MHz, CDCl3)d
Docking
8.21–8.10 (m, 3H), 8.08–8.00 (m, 3H), 7.91 (s, 1H), 7.79–7.62 (m,
2H), 5.56 (s, 1H), 5.21 (s, 1H), 4.26–4.08 (m, 2H), 3.99–3.96 (m, 1H),
3.68–3.50 (m, 2H), 3.50–3.41 (m, 1H), 3.41–3.27 (m, 1H);MS (ESI):
[MH]þ ¼ 487.09; HPLC (Tr) 13.22 min.
The equilibrium geometry of selected molecules was obtained
using semi-empirical PM3 calculations; the molecules were subse-
quently docked to both b1 and b5 binding sites of 20 S prote-
asome. The simulation was performed utilising the crystal
structures of PDB codes 1G6532 and 3E4733 for b1 and b5 bind-
ing sites, respectively. All molecules were placed in b1 and b5
binding sites using a pharmacophore query derived from the
bound inhibitors epoxomicin (structure 1G65) and homobelacto-
sin C (structure 3E47), respectively, as a filter for docking place-
ment. Epoxomicin and homobelactosin C inhibitors have been
chosen because they share size/shape similarities with the pre-
sent molecules. Before the simulation, hydrogen atoms were
added to the inner part of the enzyme and the energy of the
structure was minimised keeping fixed the atoms of the main
frame and using the MMFF94 molecular mechanics force field34.
Out of 50 unique poses obtained, the 10 having the highest
score on the base of the value assumed by the enthalpic contri-
bution to the free energy of binding were retained. These poses
were in turn rescored considering the estimation of the free
energy of binding of the ligand, i.e. the sum of the electrostatic
and dispersive interaction energy between the ligand and the
target as well as the intramolecular energy of the ligand due to
changes in its conformation. All the calculations were performed
Bz-Ser-NH-(CH2)2-NH-ClNafQ (15). White solid; yield 69%;mp
20
¼155–157 ꢁC; [a]D
–30.6 (c ¼ 1, MeOH); 1H NMR (400 MHz,
CDCl3)d 8.25–8.19 (m, 1H), 8.10–8.05 (m, 1H), 7.74–7.70 (m, 4H),
7.66–7.64 (m, 1H), 7.48–7.41 (m, 1H), 7.36 (t, J ¼ 7.4 Hz, 2H), 5.71 (s,
1H), 5.54 (s, 1H), 4.74–4.65 (m, 1H), 4.24–4.20 (m, 1H), 3.84–3.80
(m, 1H), 3.62–3.32 (m, 4H); MS (ESI): [MH]þ ¼ 442.11; HPLC (Tr)
13.97 min.
1-NaftCO-Ser-NH-(CH2)2-NH-ClNafQ (16). White solid; yield
65%;mp ¼163–166 ꢁC; [a]D20–41.5 (c ¼ 1, MeOH);1H NMR (400 MHz,
CDCl3) d 8.78 (dd, J ¼ 7.4, 1.5 Hz, 1H), 8.15–7.99 (m, 2H), 7.95–7.94
(m, 1H), 7.79–7.77 (m, 1H), 7.72–7.62 (m, 4H), 7.56–7.41 (m, 3H),
5.68 (s, 1H), 5.08–5.05 (m, 2H), 4.20–4.17 (m, 1H), 3.96–3.94 (m,
1H), 3.67–3.61 (m, 1H), 3.49–3.47 (m, 3H); MS (ESI): [MH]þ ¼ 492.12;
HPLC (Tr) 14.86 min.
Biological assay
Proteasome purification and subunit inhibition
Proteasomes were isolated and purified from lymphoblastoid
cell lines (LCL) as previously described31. Suc-LLVY-AMC, Boc- using MOE-Dock integrated into the MOE system of programs
LRR-AMC and Z-LLE-AMC (Sigma-Aldrich, Milano, Italy) were used [MOE, Chemical Computing Group, release 2016.08].

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
871
inhibitor of the whole series. A similar structure-activity relation-
ship profile is evident within the naphthoquinone amino acid
derivatives bearing the butylendiaminic linker (17–32). Also in
this subset, the inhibition is mainly directed to the catalytic cav-
ity b1 and b5, while the answer against the tryptic activity is
irrelevant. Generally, it can be speculated that the increase of
the distance between the pharmacophoric unit and the aminoa-
cidic residue at the P1 position is negative for the interaction
with the catalytic pocket of the enzyme complex. Finally, ana-
logues 33–48 are the least active of the whole series with only
a mild inhibition of the b5 subsite for derivatives with leucine
(i.e. 34–36) and of the post-acidic activity (b1) for products hav-
ing serine as the central residue (i.e. 46–48). Clearly, the length
and molecular rigidity of the cyclohexyldiamine bridge are not
favourable for the interaction with the catalytic proteasome
pockets.
Considering the fundamental role of the proteasome for cell via-
bility and proliferation, we investigated the antiproliferative activ-
ities of five selected compounds (4, 6 and 9–11) on MDA and
A2780 tumour cells compared to the reference aldehydic inhibitor
MG13235. The choice of analogues has been carried out in view of
their ability to inhibit the chymotryptic subunit of the proteasome,
directly related to the anti-cancer activity. The cells were treated
with 10 or 100 mm concentrations of the selected naphthoquinone
derivatives and MG132. After 3 d, cell proliferation was evaluated
and, as shown in Figure 2(a and b), compounds 4, 6 and 9 were
shown to inhibit cell proliferation at levels slightly lower than the
Results and discussion
The synthesised compounds were evaluated for their potency in
inhibiting the b1, b2 and b5 catalytic activities of the 20 S prote-
asome isolated from LCLs31. Specific fluorogenic substrates for
each active site were employed: Suc-LLVY-AMC (for the ChT-L),
Boc-LRR-AMC (for the T-L) and Z-LLE-AMC (for the PGPH). The
proteasome was pretreated with increasing concentrations
(0.1–100 mm) of the new naphthoquinone amino acid derivatives
and MG132 as
a reference inhibitor in an activity buffer.
Substrates were then incubated with the proteasome at 37 ꢁC for
30–180 min. Substrate degradation was evaluated in fluorescence
units. The inhibitory activity of all compounds is expressed here as
IC50. From the data reported in Table 1, after 30 min of incubation,
it can be observed that some of the naphthoquinone analogues
present a significant biological response. The inhibition of the
PGPH and ChT-L activities is remarkable for some of the deriva-
tives. In particular, the inhibition of the b1 and b5 subunits is
interesting for compounds 4, 6, 9 and 10 with IC50 values lower
than 1 mm. Generally, the molecules bearing the ethylenediamine
linker (1–16) displayed a quite good activity against post-acidic
and chymotryptic sites of the proteasome and retain a detectable
(IC50< 100 mm) inhibition of the tryptic-like activity in the b2 sub-
unit. The most effective b1 inhibitors of the subseries with the
ethylenediamine spacer present leucine or phenylalanine, while for
the b5 inhibition are favourable asparagine and phenylalanine.
Regarding the Na substituents, the NBz group is slightly pre-
ferred as indicated by the analogue 10 which is the most potent reference pseudotripeptide MG132 in both tumoural cell lines at
Figure 2. Effect of compounds 4, 6, 9–11 on cell proliferation. (a) MDA and (b) A2780 tumour cells cultured for 3 d in the presence or absence of the indicated con-
centrations of compounds. The means of three independent experiments performed in duplicate are shown.

872
M. MARASTONI ET AL.
100 mm. No significant antiproliferative activity was observed at influence the capacity of inhibition of the new compounds. It can
10 mm. reasonably be assumed that the inhibition is reversible by analy-
A general analysis of the structure-activity relationship of this sing the data obtained with the docking experiments that exclude
new series of amino acidic derivatives suggests that the naphtho- a covalent bond between the inhibitor and the enzyme (see
quinone pharmacophoric unit can function as potential electro- below). In addition, the enzymatic inhibition assays of catalytic
philic substrate for the proteasome N-terminal catalytic threonine. subsites of the isolated enzyme confirm that the IC50 values for
The strength of the interaction with the enzyme subsites depends the naphthoquinone amino acid derivatives, progressively
on the molecular components that bind the naphthoquinonic decrease during the time of incubation.
group. The ethylenediamine binder between the amino acid resi-
A computational docking study was performed for selected
due and the pharmacophore was shown to be the most effective compounds (10, 16, 26 and 42) on the basis of their different
since increasing the alkyl chain with the insertion of the butylene- molecular characteristics and biological profile. The results of the
diamine and making it more rigid with the cyclohexyldiamine, a docking study of the most active molecule 10 to both b1 and b5
progressive reduction of activity was observed. The biological binding sites of 20 S proteasome seem to support the previous
response is more evident for phenylalanyl and asparagyl deriva- considerations. In the docking simulation to the b1 site, five poses
tives that display a significant preference for the chymotryptic and (out of 10) having the highest docking scores are characterised by
post-acidic proteasome subunits. In addition, the different Na-aryl the presence of the naphthoquinonic moiety located ca. 3.5 Å
groups, with variable physicochemical characteristics, poorly away from the nucleophilic threonine residue. In view of the fact
Figure 3. (a) Molecule 10 in the b1 active site (best pose). (b) Schematic view of the interactions between the receptor and the docked molecule.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
873
Figure 4. (a) Molecule 10 in the b5 active site. (b) Schematic view of the interactions between the receptor and the docked molecule.
that the protein frame is kept rigid during the simulation, such a
The same considerations can be drawn from the molecule
distance is compatible with an H-bonding interaction. Molecule 10 10 docked to the b5 site. Indeed, the interactions with the
docked to the enzyme and a scheme of the ligand-enzyme inter- binding-site residues (Figure 4) are even more significant: in this
actions are depicted in Figure 3(a and b), respectively. The ligand case, for instance, a direct tight interaction with the active
10-binding pocket presents a Y shape, that is appropriate to well threonine has been found, with a ligand-residue distance of
fit the molecule conformation. Moreover, it is formed by a number some 2.8 Å. A number of additional short contacts can be
of amino acid residues, i.e. Thr21, Ala20, Gli47, Lys33, Asp114, observed between the functional groups of the ligand and the
which are found to be involved in epoxomicin-enzyme interactions amino acids constituting the homobelactosin receptor, e.g.
as well. Additionally, the aromatic rings seem to play an important Ser129, Gly47, Lys33 and, just like the b1 case, C-H … p inter-
role in “anchoring” the molecule inside the binding pocket actions involving the ligand aromatic parts are established
through C-H … p short contacts (Figure 3(a)).
(Figure 4(b)).

874
M. MARASTONI ET AL.
Figure 5. Molecule 16 (a), 26 (b) and 42 (c) docked into the β1 binding site. Molecule 10 docked in the same binding site is reported for comparison.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
875
Figure 6. Molecule 16 (a), 26 (b) and 42 (c) docked into the b5 binding site. Molecule 10 docked in the same binding site is reported for comparison.

876
M. MARASTONI ET AL.
The different biological behaviour of molecules 16, 26 and 42
8. Nazif T, Bogyo M. Global analysis of proteasomal substrate
specificity using positional-scanning libraries of covalent
inhibitors. Proc Natl Acad Sci USA 2001;98:2967–72.
9. Kisselev AF, van der Linden WA, Overkleeft HS. Proteasome
inhibitors: an expanding army attacking a unique target.
Chem Biol 2012;19:99–115.
10. Groll M, Huber R, Moroder L. The persisting challenge of
selective and specific proteasome inhibition. J Pept Sci
2009;15:58–66.
11. Genin E, Reboud-Ravaux M, Vidal J. Proteasome inhibitors:
recent advances and new perspectives in medicinal chemis-
try. Curr Top Med Chem 2010;10:232–56.
12. Micale N, Scarbaci K, Troiano V, et al. Peptide-based prote-
asome inhibitors in anticancer drug design. Med Res Rev
2014;34:1001–69.
can be rationalised by directly comparing their best-docked poses
with 10 (Figures 5 and 6). The inactive molecule 16 does not fit in
both the b1 and the b5 sites, the terminal naphthyl group being
located in an area outside the binding pocket (Figures 5(a) and
6(a)). As for molecules 26 and 42, they share with 10 the terminal
parts (ClNafQ and NBz-Phe) but with linkers different in length
(molecule 26) or in flexibility (molecule 42). As a consequence, 26
can achieve a not very strained conformation to accommodate
itself into the receptor in a position close to 10, due to the flexi-
bility of the alkyl chain (Figures 5(b) and 6(b)); conversely, in 42
the presence of a rigid cyclohexane linker restricts the conform-
ational adjustments adoptable by the molecule.
13. Dorsey BD, Iqbal M, Chatterjee S, et al. Discovery of a
potent, selective, and orally active proteasome inhibitor for
the treatment of cancer. J Med Chem 2008;51:1068–72.
14. Mroczkiewicz M, Winkler K, Nowis D, et al. Studies of the
synthesis of all stereoisomers of MG-132 proteasome inhibi-
tors in the tumor targeting approach. J Med Chem 2010;53:
1509–18.
15. Bazzaro M, Anchoori RK, Mudiam MK, et al. a,b-unsaturated
carbonyl system of chalcone-based derivatives is responsible
for broad inhibition of proteasomal activity and preferential
killing of human papilloma virus (HPV) positive cervical can-
cer cells. J Med Chem 2011;54:449–56.
Conclusion
In summary, this work reports the design, synthesis and molecular
modelling study of 48 amino acid derivatives assayed as inhibitors
of three major catalytic activities of the proteasome. These com-
pounds have at the Ca position of the aminoacidic residues a 2-
chloronaftoquinone group pharmacophoric unit primary respon-
sible for the interaction with the active subunits of the enzymatic
complex. The same scaffold is present in non-peptidic molecules
and pseudodipeptide derivatives previously reported as inhibitors
of the 20 S proteasome. Data on the biological response of some
analogues of this new series showed an interesting inhibition of
the proteasome. Derivatives 4, 6, 9 and 10 were the most active
against the b1 and b5 subunits, with a biological profile that
makes them potentially capable of operating such as antineoplas-
tic agents. The molecular structure of the new inhibitors allows
further studies on the structure-activity relationships.
ꢀ
16. Desvergne A, Genin E, Marechal X, et al. Dimerized linear
mimics of a natural cyclopeptide (TMC-95A) are potent non-
covalent inhibitors of the eukaryotic 20S proteasome. J Med
Chem 2013;56:3367–78.
17. Moreau P, Richardson PG, Cavo M, et al. Proteasome inhibi-
tors in multiple myeloma: 10 years later. Blood 2012;120:
947–59.
18. Adams J. The development of proteasome inhibitors as anti-
cancer drugs. Cancer Cell 2004;5:417–21.
Disclosure statement
The authors declare no competing financial interest.
19. Demo SD, Kirk CJ, Aujay MA, et al. Antitumor activity of
PR-171, a novel irreversible inhibitor of the proteasome.
Cancer Res 2007;67:6383–91.
ORCID
20. Richardson PG, Baz R, Wang M, et al. Phase 1 study of twi-
ce-weekly ixazomib, an oral proteasome inhibitor, in
re-lapsed/refractory multiple myeloma patients. Blood
2014;124:1038–46.
Delia Preti
http://orcid.org/0000-0002-1075-3781
21. Zhou HJ, Aujay MA, Bennett MK, et al. Design and
synthesis of an orally bioavailable and selective peptide
epoxyketone proteasome inhibitor (PR-047). J Med Chem
2009;52:3028–38.
22. Marastoni M, Baldisserotto A, Canella A, et al. Arecoline tri-
peptide inhibitors of proteasome. J Med Chem 2004;47:
1587–90.
23. Baldisserotto A, Ferretti V, Destro F, et al. Alpha, beta-unsat-
urated N-acylpyrrole peptidyl derivatives: new proteasome
inhibitors. J Med Chem 2010;53:6511–15.
References
1. Ciechanover A. Proteolysis: from the lysosome to ubiquitin
and the proteasome. Nat Rev Mol Cell Biol 2005;6:79–87.
2. Inobe T, Matouschek A. Paradigms of protein degradation
by the proteasome. Curr Opin Struct Biol 2014;24:156–64.
3. Finley D. Recognition and processing of ubiquitin-protein
conjugates by the proteasome. Annu Rev Biochem 2009;78:
477–513.
4. Ciechanover A. The ubiquitin-proteasome pathway: on pro-
tein death and cell life. EMBO J 1998;17:7151–60.
24. Franceschini C, Trapella C, Calia R, et al. C-terminal trans,
trans-muconic acid ethyl ester partial retro-inverso pseudo-
peptides as proteasome inhibitors. J Enzyme Inhib Med
Chem 2013;28:1034–9.
25. Scotti A, Trapella C, Ferretti V, et al. Studies of C-terminal
naphthoquinone dipeptides as 20S proteasome inhibitors.
J Enzyme Inhib Med Chem 2016;3:456–63.
26. Lawrence HR, Kazi A, Luo Y, et al. Synthesis and biological
evaluation of naphthoquinone analogs as a novel class of
proteasome inhibitors. Bioorg Med Chem 2010;18:5576–92.
€
5. Groll M, Heinemeyer W, Jager S, et al. The catalytic sites of
20S proteasomes and their role in subunit maturation: a
mutational and crystallographic study. Proc Natl Acad Sci
USA 1999;96:10976–83.
6. Borissenko L, Groll M. 20S proteasome and its inhibitors:
crystallographic knowledge for drug development. Chem
Rev 2007;107:687–717.
7. Kisselev AF, Goldberg AL. Proteasome inhibitors: from
research tools to drug candidates. Chem Biol 2001;8:739–58.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
877
27. Xu K, Xiao Z, Tang YB, et al. Design and synthesis of
naphthoquinone derivatives as antiproliferative agents and
Epstein-Barr virus-associated tumors. Int J Cancer 2002;
101:532–8.
20S proteasome inhibitors. Bioorg Med Chem Lett 2012;22: 32. Groll M, Kim KB, Kairies N, et al. Crystal structure of epoxo-
2772–4.
micin: 20S proteasome reveals a molecular basis for selectiv-
ity of a, b-epoxyketone proteasome inhibitor. J Am Chem
Soc 2000;122:1237–8.
28. Ge Y, Kazi A, Marsilio F, et al. Discovery and synthesis
of hydronaphthoquinones as novel proteasome inhibitors.
J Med Chem 2012;55:1978–98.
29. Chadwick J, Jones M, Mercer AE, et al. Design, synthesis and
antimalarial/anticancer evaluation of spermidine linked arte-
misinin conjugates designed to exploit polyamine transport-
33. Groll M, Larionov OV, Huber R, de Meijere A. Inhibitor-bind-
ing mode of homobelactosin C to proteasomes: new insights
into class I MHC ligand generation. Proc Natl Acad Sci USA
2006;103:4576–9.
ers in Plasmodium falciparum and HL-60 cancer cell lines. 34. Halgren TA. Merck molecular force field. I. Basis, form, scope,
Bioorg Med Chem 2010;18:2586–97.
parameterization, and performance of MMFF94. J Comput
30. Yang H, Xiang J, Wang N, et al. Converse conformational
Chem 1996;17:490–519.
control of smoothened activity by structurally related small 35. Hansen MB, Nielsen SE, Berg K. Re-examination and
molecules. J Biol Chem 2009;284:20876–84.
further development of a precise and rapid dye method for
31. Gavioli R, Vertuani S, Masucci MG. Proteasome inhibitors
reconstitute the presentation of cytotoxic T-cell epitopes in
measuring cell growth/cell kill.
1989;119:203–10.
J
Immunol Methods