Synthesis and biological properties of C-terminal vinyl ketone pseudotripeptides

Article abstract of DOI:10.3109/14756366.2012.657189

The ubiquitin-proteasome pathway responsible for the turnover of many cellular proteins represents an attractive target in the development of new drug therapies: In particular, modulation of the proteasome activity by specific inhibitors may represent a useful tool for the treatment of tumours. Here, we report synthesis and activity of a new series of oligopseudopeptide analogues bearing a vinyl ketone pharmacophoric unit at the C-terminal position. Some derivatives showed inhibition in the M range of the trypsin-like (T-L) active site of the proteasome.

Full text of DOI:10.3109/14756366.2012.657189

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 560–564
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.657189
RESEARCH ARTICLE
Synthesis and biological properties of C-terminal vinyl ketone
pseudotripeptides
Christian Franceschini1, Claudio Trapella1, Fabio Sforza2, Riccardo Gavioli2, and Mauro Marastoni1
1Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, Ferrara, Italy and
2Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy
Abstract
The ubiquitin-proteasome pathway responsible for the turnover of many cellular proteins represents an attractive
target in the development of new drug therapies: In particular, modulation of the proteasome activity by specific
inhibitors may represent a useful tool for the treatment of tumours. Here, we report synthesis and activity of a new
series of oligopseudopeptide analogues bearing a vinyl ketone pharmacophoric unit at the C-terminal position.
Some derivatives showed inhibition in the µM range of the trypsin-like (T-L) active site of the proteasome.
Keywords: vinyl ketone derivatives, inhibitors, proteasome, pseudopeptides
Introduction
e proteasome 26S, a multi-catalytic protease1, is an
essential component of the ubiquitin-proteasome path-
way (UPP) that degrades many proteins in eukaryotic
cells. Fundamental cellular functions are linked to an
ubiquitin- and ATP-dependent degradation of proteins
involved in different pathways such as stress response,
cell cycle control and differentiation, apoptosis and the
regulation of transcription factors generation2. Proteins
destined to degradation are tagged by a covalently linked
polyubiquitin chain in a process involving three enzymes
in a successive action E1 (Ubiquitin-activating enzyme),
E2 (Ubiquitin-conjugating enzyme) and E3 (Ubiquitin-
ligase)3,4. Poliubiquitin chain linked to proteins rep-
resents the signal for degradation by multi-catalytic
complex that contains a central barrel-like core and a
20S proteolytic chamber composed of four stacked rings
capped by two 19S structures5,6. e two outer rings of the
20S are composed of seven α-subunits, whereas the two
inner rings are made up by seven different β-subunits,
and each β-ring contains three different active sites. In
particular, the β1 subunit contains a post-acidic (PGPH)
active site, the β2 subunit expresses trypsin-like (T-L)
activity, and a chymotrypsin-like (ChT-L) proteolytic
function is carried out by the β5 subunit. All the proteo-
lytic cavities utilize the γ-hydroxyl function as a nucleo-
phile and the α-amine as a proton donor-acceptor of the
N-terminal threonine residue in the catalytic cycle7,8
.
A proteasome isoform can be formed in response
to cytokine signalling that induces the expression of
different β-subunits and regulatory cap to constitute
immunoproteasome capable to generate epitopes for
presentation by MHC class I molecules9.
Considering the crucial implication of the proteasome
in various cellular processes, modulation of enzymatic
activities is extremely interesting from a therapeutic
perspective10–12. Natural and synthetic products have
been tested as inhibitors of the different multi-catalytic
complex subunits13–21. In vitro and in vivo studies dem-
onstrated that proteasome inhibitors showed anti-pro-
liferative and pro-apoptotic activities against solid and
haematologic tumours. In particular, the boron deriva-
tive PS341 (Bortezomib) was used in the treatment of
multiple myeloma22,23. Other molecules were evaluated
for their effect on many disease states, including inflam-
mation and cancer, as well as on modulation of immune
responses24.
Address for Correspondence: Prof. Mauro Marastoni, Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17–19, 44100, Ferrara,
Italy. Tel: +39-532-291281. Fax: +39-532-291296. E-mail: mru@dns.unife.it
(Received 29 November 2011; revised 09 January 2012; accepted 10 January 2012)
560

C-terminal vinyl ketone pseudotripeptides 561
Our studies report the development of numerous
series of peptide-based proteasome inhibitors contain-
ing a different pharmacophoric moiety as a potential
substrate for the catalytic threonine through a mecha-
nism similar to that of the well-known vinyl sulphone
inhibitors, and as recently reported25, to that the natural
pseudopeptidic compound Syringolin A and, even if in
minor way, the analogue Syringolin B that irreversibly
inhibits the β2 and β5 subunits of the proteasome, using
the same catalytic mechanism.
e column was perfused at a flow rate of 30 mL/min, with
a mobile phase-containing solvent A (10%, v/v, acetonitrile
in 0.1% TFA), and a linear gradient from 0 to 100% of solvent
B (60%, v/v, acetonitrile in 0.1% trifluoroacetic acid (TFA));
30 min was the time adopted for elution of the compounds.
HPLC analysis was performed using a Beckman System
Gold with a Hypersil BDS C18 column (5 μm; 4.6 × 250 mm).
Analytical determination and capacity factor (K′) of the pep-
tides were assayed via HPLC conditions in the above solvent
system (solvents A and B), programmed at flow rates of 1
mL/min, using the following linear gradients: (i) from 0 to
90% B for 25 min and (ii) from 30 to 100% B for 25 min. No
pseudopeptide showed more than 1% impurity when moni-
tored at 220 and 254 nm. e molecular weights of the com-
pounds were determined by electrospray ionisation (ESI)
(MICROMASS ZMD 2000), and the values are expressed as
[MH]+. TLC was performed on pre-coated plates of silica gel
F254 (Merck, Darmstadt, Germany), exploiting the following
solvent systems: (iii) AcOEt/n-hexane (1:1, v/v), (iv) CH2Cl2/
methanol (9.5:0.5, v/v), (v) CH2CL2/methanol (9:1, v/v) and
(vi) 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 apparatus and are uncorrected. Optical rotations
were determined by a Perkin–Elmer 141 polarimeter with
a 10-cm water-jacketed cell. 1H NMR spectroscopy was
obtained using a 400 spectrometer.
Generally, our C-terminal pharmacophoric units
(arecoline, vinyl ester and α,β-unsatured N-acylpyrrole)
present a carbonylic function conjugated to a double
bond. ese electrophilic trap at the C-terminous are
capped with oligopeptides (three residues) variously
functionalized at the N-terminal. e electrophilic part
of the molecules represents a potential substrate for the
Michael addition by catalytic threonine, whereas ami-
noacidic sequences and groups at N-terminous interact
with binding pockets of the enzymatic subunits and
determine active site selectivity26–28
.
Herein, we describe the synthesis and biological
activities of novel vinyl ketone-based peptide deriva-
tives (Figure 1). e aim of our work was to evaluate the
capacity of the new C-terminal vinyl ketone pharma-
cophoric unit to interact with catalytic threonine, and
the influence of the N-derivatized peptide portions on
the inhibition potency and specificity. We synthesized
and tested molecules containing a central tripeptidic
sequence Leu-Leu-Leu (compounds 1–5) or Val-Ser-Leu
(compounds 6–10) carrying 3-Hydroxy-2-methylbenzoyl
(HMB), Z-protected 6-aminohexanoyl or 8-aminooc-
tanoyl groups at the N-terminous, in accordance with the
results obtained in previous series.
Synthesis
Vinyl ketone pseudotripeptides 1–10 were prepared using
a C-terminal stepwise elongation. Following the strategy
reported in Scheme 1, C-terminal dipeptide H-Xaa-
Leu-VK was synthesized starting from leucine acylation
by Boc-protected succinimidyl ester residue (Leu or Ser).
Pharmacophoric unit was introduced by a Wittig reaction
between dipeptide aldheyde29, and the ylide [(methyl-
carbonyl)methylidene]triphenylphosphorane. Boc was
removed by TFA and N-terminal residues were condensed
using water soluble carbodiimide/N-hydroxybenzotri-
azole (WSC/HOBt) to complete the pseudotripeptide
sequence 1,6 that after TFA treatment permitted to obtain
the corresponding free N-terminal analogues. Finally,
the other derivatives were obtained from 2 and 7, respec-
tively by acylation with 3-hydroxy-2-methylbenzoic (3,8),
Z-protected 6-aminohexanoic (4,9) or 8-aminooctanoic
acids (5,10) always with WSC/HOBt as coupling reagent.
All products were purified and isolated by preparative
RP-HPLC, and the homogeneity of the lyophilized prod-
ucts was assessed by HPLC. Analytical characterization
was then achieved by electrospray ionisation (ESI) mass
spectrometry (Table 1) and 1H NMR spectroscopy.
Matherials and methods
Chemistry-general
Amino acids, amino acid derivatives and chemicals were
purchased from Bachem, Novabiochem and Fluka, respec-
tively (Switzerland).Crude products were purified by pre-
parative reversed-phase HPLC using a Water Delta Prep
4000 system with a Waters PrepLC 40 mm Assembly column
C18 (30 × 4 cm, 300 Å, 15 μm spherical particle size column).
General synthetic procedures
TFA deprotection
Boc was removed by treating intermediates with aque-
ous 90% TFA (1:10, w/v) for 30–40 min. After evaporation,
the residue was triturated with Et2O, centrifuged and the
resulting solid was collected and dried.
Figure 1. General structure of the vinyl ketone pseudotripeptides.
© 2013 Informa UK, Ltd.

562 C. Franceschini et al.
(80 mL) and washed consecutively with HCl 0.1 N,
NaHCO3 and brine. e organic phase was dried (MgSO4)
and evaporated to dryness. e residue was treated with
Et2O and the resulting solid separated by centrifugation.
Coupling with WSC/HOBt
e deprotected α-amine intermediate (1 mmol),
N-methylmorpholine (NMM) (2 mmol) WSC (1 mmol)
and HOBt (1 mmol) were added to a solution of carbox-
ylic component (1 mmol) in dimethylformamide (3 mL)
at 0°C. e reaction mixture was stirred for 1 h at 0°C
and 18 h at rt; then the solution was diluted with AcOEt
1H NMR of the selected compounds
1
Boc-Leu-Leu-Leu-VK (1). H NMR (CDCl3): δ 1.01–1.12
(m, 18H); 1.50–1.77 (m, 15H); 1.89-1.97 (m, 3H); 2.41 (s,
3H); 4.11 (m, 1H); 4.37–4.49 (m, 2H); 6.18 (d, J = 16.2, 1H);
6.87 (dd, J = 16.1, 1H); 7.36 (br s, 3H).
Z-NH-(CH2)5-CO-Leu-Leu-Leu-VK (4). 1H NMR
(CDCl3): δ 0.98–1.08 (m, 18H); 1.27 (m, 2H); 1.50–1.65
(m, 6H); 1.81-1.94 (m, 7H); 2.24 (t, 2H); 2.33 (s, 3H); 2.88
(t, 2H); 4.17 (m, 1H); 4.49–4.60 (m, 2H); 5.18 (s, 2H); 6.21
(d, J = 16.4, 1H); 6.85 (dd, J = 16.3, 1H); 7.11–7.23 (m, 5H);
7.58 (br s, 4H).
1
HMB-Val-Ser-Leu-VK (8). H NMR (CDCl3): δ 1.01–
1.12 (m, 12H); 1.47 (m, 2H); 1.85 (m, 1H); 2.25 (br s, 1H);
2.34 (s, 3H); 2.45 (s, 3H); 2.83 (m, 1H); 4.10–4.19 (m, 2H);
4.38 (m, 1H); 4.58-4.70 (m, 2H); 5.08 (br s, 1H); 5.97 (d,
J = 16.0, 1H); 6.72 (dd, J = 16.2, 1H); 7.05–7.21 (m, 3H);
7.76 (br s, 3H).
Z-NH-(CH2)7-CO-Val-Ser-Leu-VK (10). 1H NMR
(CDCl3): δ 1.04–1.13 (m, 12H); 1.31-1.42 (m, 6H); 1.55-
1.64 (m, 6H); 1.87 (m, 1H); 2.15 (br s, 1H); 2.24 (t, 2H);
2.39 (s, 3H); 2.73 (m, 1H); 3.01 (t, 2H); 4.11 (m, 2H); 4.28
(m, 1H); 4.50-4.59 (m, 2H); 5.37 (s, 2H); 6.07 (d, J = 16.3,
1H); 6.92 (dd, J = 16.1, 1H); 7.09–7.23 (m, 5H); 7.83 (br s,
4H).
Biological investigation
Proteasome purification
Proteasomes were isolated from lymphoblastoid cell
lines (LCL) as previously described30.
Proteasome subunit inhibition assays
Suc-LLVY-AMC, Boc-LRR-AMC and Z-LLE-AMC (Sigma)
were used to measure chymotrypsin-like, trypsin-like
and post-acidic proteasome activities, respectively.
Substrates were incubated at 37°C for 30 min with pro-
teasomes, untreated or pre-treated with 0.001–10 μM
of test compounds, in activity buffer. Fluorescence was
determined by a fluorimeter (Spectrafluor plus, Tecan,
Scheme 1. Synthesis of new vinyl ketone derivatives 1–10.
Table 1. Analytical data and physicochemical properties of the novel pseudotripeptides 1–10.
HPLC
KI (a)
7.01
6.31
5.80
7.59
7.89
6.30
5.70
5.47
7.07
7.22
KI (b)
5.47
4.72
4.61
6.63
6.95
5.21
4.43
3.97
6.32
6.54
a[α]D
m.p. (°C)
130–133
138–140
128–130
118–121
140–142
145–148
141–143
135–137
123–125
119–124
M + H+
482.35
382.55
516.34
629.42
657.45
442.29
342.23
476.27
589.36
617.39
20
No.
1
2
3
4
5
6
7
8
Compound
Boc-Leu-Leu-Leu-VK
−10.9
−14.9
−9.2
H-Leu-Leu-Leu-VK
HMB-Leu-Leu-Leu-VK
Z-NH-(CH2)5-CO-Leu-Leu-Leu-VK
Z-NH-(CH2)7-CO-Leu-Leu-Leu-VK
Boc-Val-Ser-Leu-VK
−8.5
−8.7
−22.3
−11.4
−12.9
−10.67
−9.98
H-Val-Ser-Leu-VK
HMB-Val-Ser-Leu-VK
9
10
Z-NH-(CH2)5-CO-Val-Ser-Leu-VK
Z-NH-(CH2)7-CO-Val-Ser-Leu-VK
ac = 1, MeOH.
Journal of Enzyme Inhibition and Medicinal Chemistry

C-terminal vinyl ketone pseudotripeptides 563
Table 2. Inhibition of proteasome subunits and metabolic stability of vinyl ketone derivatives.
Isolated enzyme LCL aIC50 (µM)
Half-life (min)
human plasma
N
1
Compound
ChT-L
>10
T-L
PGHP
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Boc-Leu-Leu-Leu-VK
>10
>10
8.53
4.22
3.81
>10
>10
>10
8.02
6.65
2
H-Leu-Leu-Leu-VK
>10
3
4
5
6
HMB-Leu-Leu-Leu-VK
Z-NH-(CH2)5-CO-Leu-Leu-Leu-VK
Z-NH-(CH2)7-CO-Leu-Leu-Leu-VK
Boc-Val-Ser-Leu-VK
>10
>360
>360
>360
9.13
7.95
>10
7
H-Val-Ser-Leu-VK
>10
8
HMB-Val-Ser-Leu-VK
>10
9
10
Z-NH-(CH2)5-CO-Val-Ser-Leu-VK
Z-NH-(CH2)7-CO-Val-Ser-Leu-VK
8.54
>360
>360
6.41
ae values reported are the average of three independent determinations.
ChT-L, chymotrypsin-like; LCL, lymphoblastoid cell lines; T-L, trypsin-like.
Salzburg, Austria), using an excitation of 360 nm and
emission of 465 nm. Activity was evaluated in fluores-
cence units and the inhibitory activity of the compounds
is expressed as IC50. e data were plotted as percentage
control (the ratio of percentage conversion in the pres-
ence and absence of inhibitor) versus inhibitor concen-
tration, and fitted with the equation Y = 100/1 + (X/IC50)A,
where IC50 is the inhibitor concentration at 50% inhibi-
tion and A is the slope of the inhibition curve.
evaluated in fluorescence units, and the inhibitory activ-
ity of the compounds is expressed here as IC50.
General analysis of the activity profile shows that the
new compounds have a low capacity to inhibit the pro-
teasome activities suggesting that the C-terminal new
pharmacophoric group is not a good substrate for the
catalytic threonine. Indeed, all compounds were less
active compared to previously described inhibitors26–28
.
Chymotrypsin-like activity was in a µM range for ana-
logues presenting N-terminal linear amino acids with
a long Z-protected chain on the terminal amine group.
Likewise, inhibition of the trypsin-like was relatively
pronounced, with IC50 values in the order of 3–10 µM
for compounds 4, 5, 9 and 10. Furthermore, 3-Hydroxy-
2-methylbenzoyl N-functionalized derivative showed
mild inhibitory capacity of the trypsin-like activity of
the proteasome. Generally, the biological response was
independent from the central tripeptide sequence but it
correlated to N-terminal substituents; in particular, com-
pounds 5 and 10 with the more bulky groups resulted the
best analogues of the series. All compounds were unable
to inhibit post-acidic (PGPH) activity.
Enzymatic stability assays
e stability of the vinyl ketones under proteases degra-
dation was studied in human plasma. Test compounds
were incubated with plasma (0.6 mL) in a total volume
of 1.5 mL of 10 mM Tris-HCl buffer, pH 7.5. Incubation
was performed at 37° C for 360 min. e incubation was
terminated by addition of ethanol (0.2 mL), the mixture
poured at 21°C, and, after centrifugation (5000 rpm for
10 min) aliquots (20 µL) of the clear supernatant were
injected into an RP-HPLC column. HPLC was performed
as described in analytical determinations. e degrada-
tion half-life (T1/2) was obtained by a least-squares linear
regression analysis of a plot of the logarithmic inhibi-
tor concentration versus time, using a minimum of five
points.
e susceptibility of five selected vinyl ketone deriva-
tives to enzymatic hydrolysis was determined by incu-
bation at 37°C in human plasma. e pseudopeptides,
according to terminal modifications of the peptide chain,
showed great stability to plasma proteases (Table 2).
Results and discussion
Vinyl ketone pseudotripeptides 1–10 were synthesized
following the strategy reported in Scheme 1.
Conclusions
e UPP plays an important role in many cellular pro-
cesses. Considering the high therapeutic potential of
inhibitors selective and specific for the catalytic subunits
of the 20S proteasome, we synthesized and tested new
peptide-based compounds with new C-terminal phar-
macophoric units. Clinical trials indicate that inhibitors
exhibit toxic effects during prolonged drug treatment,
therefore the availability of new potent and selective
molecules without side-effects is ever required. In this
optic, we prepared a new series of peptide-based com-
pounds containing a vinyl ketone pharmacophoric unit
Inhibition of β1, β2 and β5 active sites of the 20S
proteasome, previously purified from lymphoblastoid
cell lines, was determined using fluorogenic substrates
specific for the three main proteolytic activities of the
enzymatic complex. Suc-LLVY-AMC, Boc-LRR-AMC
and Z-LLE-AMC were used to measure chymotrypsin-
like, trypsin-like and caspase-like proteasome activi-
ties, respectively. Substrates were incubated, at 37°C for
30 min, with the proteasome, pre-treated with incre-
mented concentrations (from 0.001 to 10 µM) of the new
vinyl ketone derivatives in activity buffer. Activity was
© 2013 Informa UK, Ltd.

564 C. Franceschini et al.
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γ-hydroxy threonine side-chain in Michael addition.
Generally, inhibition of active subsites of the proteasome
is detectable in a µM range only for some derivatives. e
biological response is function of the N-terminal substit-
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of the central tripeptidic sequence. Finally, vinyl ketone
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Declaration of interest
Financial support of this work was provided by the
University of Ferrara, the Ministero dell’Università e della
Ricerca Scientifica e Tecnologica (MURST).
20. Kaiser M, Groll M, Renner C, Huber R, Moroder L. e core
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