Glutamine vinyl ester proteasome inhibitors selective for trypsin-like (β2) subunit

Article abstract of DOI:10.1016/j.ejmech.2006.12.008

Here we report the study of a new series of peptide-based proteasome inhibitors with a vinyl ester moiety at C-terminal. The presence of Tic, a rigid analogue of phenylalanine, in the central portion of some derivatives is not favourable for the activity.

Full text of DOI:10.1016/j.ejmech.2006.12.008

European Journal of Medicinal Chemistry 42 (2007) 586e592
http://www.elsevier.com/locate/ejmech
Original article
Glutamine vinyl ester proteasome inhibitors selective for
trypsin-like (b2) subunit
a
b
Anna Baldisserotto a, Mauro Marastoni a, , Claudio Trapella , Riccardo Gavioli ,
*
Valeria Ferretti c, Loretta Pretto c, Roberto Tomatis a
a Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
b Department of Biochemistry and Molecular Biology, University of Ferrara, I-44100 Ferrara, Italy
c Chemistry Department and Center for Structural Diffractometry, University of Ferrara, I-44100 Ferrara, Italy
Received 16 November 2006; received in revised form 30 November 2006; accepted 5 December 2006
Available online 9 January 2007
Abstract
Here we report the study of a new series of peptide-based proteasome inhibitors with a vinyl ester moiety at C-terminal. The presence of Tic,
a rigid analogue of phenylalanine, in the central portion of some derivatives is not favourable for the activity. The best analogue of the series
shows a potent and selective inhibition for the b2 subunit and good enzymatic stability.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Proteasome inhibitors; b2 selective inhibitors; Constrained residue; Vinyl ester tripeptides
1. Introduction
peptidase activity can be assigned to the specific b subunits:
b1 center cleaves peptide bonds preferentially on the carboxyl
sides of acidic amino acid residues (peptidylglutamyl peptide
hydrolyzing, PGPH), b2 after basic amino acid residues (tryp-
sin-like activity, T-L), and b5 after hydrophobic residues (chy-
motrypsin-like activity, ChT-L) [7e9].
In the last two decades, the ubiquitin-proteasome system
(UPS) has been recognized as an important component in nu-
merous biological processes such as regulation of cell cycle
progression, division, development and differentiation, apo-
ptosis, cell proliferation, stress response, and modulation of
the immune and inflammatory responses. The attractive idea
to target the proteasomal pathway represents a new approach
for the treatment of a range of pathologies such as cancer, in-
flammation, immune diseases and others [10e12].
The 26S proteasome is a multicatalytic and multisubunit
threonine protease complex (2.4 MDa) responsible for the
degradation of normal and abnormal intracellular proteins
and its composed of two subcomplexes, the first one 20S pro-
teasome and the second two 19S regulatory particles, which
cap the ends of the 20S core [1e4]. The substrates, marked
for degradation by the binding of multiple molecules of ubiq-
uitin (Ub), are recognized and bound to the 26S proteasome,
unfolded, deubiquitinated and then transferred and degraded
in the 20S catalytic chamber [5,6]. The 20S proteasome con-
sists of four stacked rings each with seven distinct subunits,
stacked one on top of each other, that are responsible for the
proteolytic activity of the proteasome. The a-rings are outer,
while the two inner b-rings form the central cavity of the cyl-
inder and harbor the proteolytic sites. The proteasome utilizes
in the catalytic site the side chain of an N-terminal threonine
to perform the nucleophilic attack on the target bond. The
Several class of inhibitors have been recognized and used to
study the role of ubiquitin-proteasome pathway in cellular pro-
cesses. In addition, proteasome inhibitors could be utilized as
novel therapeutic agents to arrest tumor cell proliferation and
as modulators of antigen presentation [13e15].
The most common inhibitors are short peptides linked to
a C-terminal pharmacophore, able to interact with enzymatic
* Corresponding author. Tel.: þ39 532 291281; fax: þ39 532 291296.
E-mail address: mru@dns.unife.it (M. Marastoni).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.008

A. Baldisserotto et al. / European Journal of Medicinal Chemistry 42 (2007) 586e592
587
catalytic threonine [16e24]. We have been interested in the
development of peptide-based derivatives with selective activ-
ity towards the three catalytic sites and with good pharmaco-
kinetics properties [25]. We recently report the identification
of a new class of specific proteasome inhibitors selective for
trypsin-like activity (b2 subunit) bearing a C-terminal leucine
vinyl ester as pharmacophore that can function as substrate for
catalytic threonine in Michael addition in the same way that
has been suggested for the well-known peptide vinyl sulfone
inhibitors. HMB-Val-Ser-Leu-VE is the most potent deriva-
tives of the series with an IC50 ¼ 33 nM. These compounds
are non-toxic, and do not affect cell proliferation, but are
able to modulate the generation of immunogenic peptides pre-
sented by MHC class I molecules [26]. Two small libraries of
tripeptidic-based vinyl ester derivatives were synthesized and
tested, starting with the HMB-Val-Gln-Leu-VE prototype. In-
vestigations of P3 and P4 positions with a complete set of
amino acid residues led to the identification of some deriva-
tives that have showed remarkable selective inhibition of the
b2 subunit. The 3-hydroxy-2-methylbenzoyl (HMB) moiety
at the P4 position can be replaced with cyclic N-terminal sub-
stituent with different physicochemical properties or with aro-
matic or hydrophobic amino acid residues [27,28]. Some vinyl
ester tripeptides have undergone X-ray crystallographic stud-
ies, with a conformational study of the molecules in the solid
state, in vacuum and in a polar environment in order to define
the inhibitoreenzyme interaction subsite pockets [29].
Through flexible alignment, our vinyl ester compounds have
shown some conformational similarities with the cyclic inhib-
itors TMC95A (Fig. 1). The opportunity to display the mole-
cules with a rigid conformation and good inhibitory activity
could allow to obtain a lot of structural information and a target
investigation about potency and selectivity.
Fig. 1. Best superposition of HMB-Val-Ser-Leu-VE (light grey) and TMC95A
(black) obtained by the flexible alignment procedure implemented in MOE
(Molecular Operating Environment, Chemical Computing Group Inc., Version
2006.08). The TMC95A molecule was kept fixed in the conformation adopted
in the proteasome binding site. The force field used was AMBER (W.D.
Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz Jr., D.M. Ferguson,
D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A. Kollman, A second generation
force field for the simulation of proteins and nucleic acids, J. Am. Chem.
Soc. 177 (1995) 5179e5197).
On the basis of these evidences we have developed a new
series of peptide-based molecules with the structure reported
in Fig. 2.
Compounds 13e18 bearing a glutamine vinyl ester moiety at
C-terminal were synthesized following the strategy reported in
Scheme 1. The Ng-trityl-glutamine vinyl ester was prepared
from the corresponding aldehyde by reaction with [(ethoxycar-
bonyl)methylidene]triphenylphosphorane without racemiza-
tion [32,33]. WSC/HOBt were employed for the coupling
steps, and Boc and Trt were removed by TFA treatment. Syn-
thesis of compounds 25e28 with leucine vinyl ester at C-ter-
minal follows the procedure describe above. Introduction of
1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (Tic) in P2
or P3 position in compounds 15e18 and 25e28, is possible
without modifying standard acylation protocols. All products
were purified by preparative RP-HPLC, and structural verifica-
tion was achieved by mass spectrometry and NMR spectros-
copy. HPLC capacity factors (KI) and other physicochemical
properties of compounds 13e18 and 25e28 are summarized
in Table 1.
Compounds 13e18 show a C-terminal glutamine vinyl ester
while derivatives 25e28 bear the reference frame Leu-VE as
pharmacophoricunit, thatis, apotentialsubstrateslikeaMichael
acceptor for the catalytic threonine. This residue (data reported
in literature) could confer a higher b2 subunit selectivity [30].
Compounds 13 and 14 have, respectively, the sequence
Leu-Leu and Val-Ser as central dipeptidic portion by analogy
with the most representative inhibitors of the previous series.
We have obtained a major structural rigidity by putting in
the center portion of the sequence the amino acid Tic
(1,2,3,4-tetrahydro-isoquinolinic acid) [31]. This rigid ana-
logue of phenylalanine was inserted in P3 position in the com-
pounds 15, 16, 25 and 26 while in P2 position in the
pseudopeptides 17, 18, 27 and 28. All the derivatives of the
series were functionalized in P4 position with a 2-methyl-
3-hydroxybenzoyl group.
2. Chemistry
3. Biological activity
Vinyl ester tripeptides were synthesized by the classical
solution method by means of C-terminal stepwise elongation.
We tested the inhibitory capacity of the new vinyl ester
derivatives on purified 20S proteasome using fluorogenic

588
A. Baldisserotto et al. / European Journal of Medicinal Chemistry 42 (2007) 586e592
I
P4
P3
P2
P1
P1
O
R
O
O
1
H
N
HO
N
H
N
H
O
O
R2
HMB-AA1-AA2-Leu-VE
O
NH2
O
NH2
O
O
O
O
H
N
H
N
HO
HO
O
O
N
N
H
N
H
N
H
H
O
O
O
O
O
OH
13
14
OH
O
O
O
O
O
H
N
H
HO
HO
N
N
H
O
N
H
O
N
N
O
O
O
NH2
O
NH2
15
16
O
O
O
O
H
N
H
N
N
N
HO
HO
N
H
N
H
O
O
O
O
O
O
NH2
O
O
NH2
17
18
OH
O
O
O
O
O
O
H
N
H
HO
HO
N
N
H
N
H
O
O
N
N
O
O
25
26
O
O
O
O
H
H
N
N
N
HO
HO
N
N
H
N
H
O
O
O
O
O
O
27
28
Fig. 2. Structure of the prototype and new vinyl ester pseudotripeptides inhibitors.
substrate, specific for the three major proteolytic activities
of the enzymatic complex [34]. All the compounds were
assayed at different concentrations (from 0.001 to 10 mM)
for their capacity to inhibit the in vitro trypsin-like (T-L)
activity, chymotrypsin-like (ChT-L) and post-acidic
(PGPH) activities of the proteasome purified from lympho-
blastoid cell lines. IC50 values against proteasome subsite
activities obtained after 30 min of incubation are reported
in Table 2. Similar IC50 were obtained after 3 h of
incubation.

A. Baldisserotto et al. / European Journal of Medicinal Chemistry 42 (2007) 586e592
589
Boc-Gln(Trt)-OH + HN(Me)OMe
(a)
half-lives of the derivatives were determined as described in
experimental protocols.
Boc-Gln(Trt)-N(Me)OMe
(b)
1
4. Results and discussion
The data obtained from enzyme inhibition tests underlined
that the glutamine vinyl ester could be a substrate able to in-
teract with catalytic threonine. In comparison to the prototype
sequence bearing the leucine vinyl ester, the pharmacophore
substitution did not yield interesting results in terms of po-
tency and selectivity (Table 2).
Boc-Gln(Trt)-al
(c)
2
3
Boc-Gln(Trt)-VE
Generally, the new sequences confirm the capacity to in-
hibit trypsin activity while only in few cases we have seen
a b5 subunit inhibition at a concentration less than 10 mM;
all the analogues are not active against post-acidic subsite.
P1 substitutions are critical on the catalytic subunits inhibi-
tion. The simple substitution of the Leu-VE with the Gln-VE
in P1 position of the prototype sequence determines an insig-
nificant decrease of the activity on the b2 subunit. Most im-
pressive results are the conformational noise that derives
from the presence of the Tic amino acid in the core of the
structure.
(d)
H-Gln-VE
(e)
H-Leu-VE
(e)
Boc-Xaa-Gln-VE
(f)
4-6
Boc-Xaa-Leu-VE
(f)
19-18
21-24
Boc-Xbb-Xaa-Gln-VE
(g)
7-12
Boc-Xbb-Xaa-Leu-VE
(g)
The most potent and selective inhibitor of the series, com-
pound 14, preserves structural characteristics related to the
prototype. In the new compound the position P1 is substituted
with a propyl amide chain.
HMB-Xbb-Xaa-Gln-VE
13-18
HMB-Xbb-Xaa-Leu-VE
25-28
The presence of the constrained analogue of the phenylala-
nine in the central portion of the sequence is not favourable for
the activity; this conformational constraint is really negative in
compounds that carrying Tic in P2 position compared to P3
position. The pseudotripeptides 17, 18, 27 and 28 have showed
a b2 subunit inhibition less than the other derivatives
(IC50 > 1 mM).
HMB = 3-hydroxy-2-methylbenzoyl; Xaa = Leu, Ser, Tic; Xbb = Leu, Tic, Val
Scheme 1. Synthesis of the vinyl ester derivatives 13e18 and 25e28.
Reagents: (a) WSC, HOBt, NMM, DMF; (b) LiAlH4, THF; (c) EtO-CO-
CH]PPh3, toluene; (d) TFA; (e) Boc-Xaa-OH, WSC, HOBt, NMM, DMF;
(f) (1) TFA; (2) Boc-Xbb-OH, WSC, HOBt, NMM, DMF; (g) (1) TFA; (2)
HMB-OH, WSC, HOBt, NMM, DMF.
The most active compounds 13, 14, 25 and 26 were evalu-
ated on the basis of their ability to permeate cellular mem-
brane; data obtained confirm the in vitro activity. IC50 values
of inhibition are from 0.06 to 0.2 mM for the trypsin-like
activity while it is >10 mM for the chymotrypsin-like activity
(Table 2). These derivatives have shown the in vivo capacity to
inhibit the enzymatic complex.
The cell membrane permeation of the most representative
compounds 13, 14, 25 and 26 was tested in live cells. After
cell treatment, proteasomes were purified and assayed for pro-
teolytic activity as described above.
The enzymatic stability of inhibitors was determined by
incubation at 37 ꢀC in human plasma [35]. The degradation
Table 1
Analytical data and physicochemical properties of vinyl ester tripeptides
No
Compound
HPLC
m.p. (ꢀC)
[a]2D0a
M þ Hþ
KI(a)
KI(b)
HMB-Val-Ser-Leu-VE
HMB-Leu-Leu-Gln-VE
HMB-Val-Ser-Gln-VE
HMB-Tic-Leu-Gln-VE
HMB-Tic-Ser-Gln-VE
HMB-Val-Tic-Gln-VE
HMB-Leu-Tic-Gln-VE
HMB-Tic-Leu-Leu-VE
HMB-Tic-Ser-Leu-VE
HMB-Leu-Tic-Leu-VE
HMB-Val-Tic-Leu-VE
13
14
15
16
17
18
25
26
27
28
a
7.10
6.15
7.32
6.23
7.15
7.82
8.43
7.11
8.51
8.22
5.68
4.98
5.95
5.02
5.77
6.45
6.98
5.66
7.13
6.48
178e181
202e207
183e186
198e200
156e159
177e180
154e158
191e195
170e172
173e176
ꢁ25.5
ꢁ15.9
ꢁ18.8
ꢁ30.4
ꢁ23.7
ꢁ20.9
ꢁ36.4
ꢁ37.2
ꢁ16.9
ꢁ25.1
560.7
520.6
607.5
581.4
593.4
607.5
592.5
566.4
592.5
578.4
c ¼ 1, MeOH.

590
A. Baldisserotto et al. / European Journal of Medicinal Chemistry 42 (2007) 586e592
Table 2
Subsites of proteasome inhibition and enzymatic stability of new vinyl ester derivatives
No
Compound
IC50 (mM)a
Isolated enzyme
T-L
Half-life of plasma (min)
In vivo inhibition
T-L
ChT-L
PGHP
ChT-L
HMB-Val-Ser-Leu-VE
HMB-Leu-Leu-Gln-VE
HMB-Val-Ser-Gln-VE
HMB-Tic-Leu-Gln-VE
HMB-Tic-Ser-Gln-VE
HMB-Val-Tic-Gln-VE
HMB-Leu-Tic-Gln-VE
HMB-Tic-Leu-Leu-VE
HMB-Tic-Ser-Leu-VE
HMB-Leu-Tic-Leu-VE
HMB-Val-Tic-Leu-VE
0.033
0.145
0.056
0.435
0.349
2.340
5.890
0.157
0.098
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.050
0.180
0.067
>10
>10
>10
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
>360
13
14
15
16
17
18
25
26
27
28
a
8.410
>10
>10
>10
>10
>10
6.890
7.360
>10
>10
0.196
0.119
>10
>10
8.510
The values reported are the average of two independent determinations.
Stability to enzymatic hydrolysis of the vinyl ester deriva-
(5 mm; 4.6 ꢂ 250 mm). Analytical determination and capacity
factor (KI) of the peptides were determined using HPLC con-
ditions in the above solvent system (solvents A and B) pro-
grammed at flow rates of 1 mL/min using the following
linear gradients: (a) from 0 to 100% B for 25 min and (b)
from 30 to 90% B for 25 min. All pseudopeptides showed
less than 1% impurity when monitored at 220 and 254 nm.
The molecular weight of the compounds was determined by
electrospray ionization (ESI) (MICROMASS ZMD 2000) and
the values are expressed as M þ Hþ. TLC was performed in
precoated plates of silica gel F254 (Merck, Darmstadt,
Germany) using 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), (f) CH2Cl2/methanol/toluene
(17:2:1, v/v/v). Ninhydrin (1%) or chlorine iodine spray re-
agents were employed to detect the peptides. Melting points
were determined by a Kofler apparatus and are uncorrected.
Optical rotations were determined by a PerkineElmer 141 po-
larimeter with a 10-cm water-jacketed cell. 1H NMR spectros-
copy was obtained on a Bruker AC 200 spectrometer.
tives was evaluated towards human plasma. All the com-
pounds of the series have underlined a high resistance to the
proteases with an half-lives upper to 6 h (Table 2).
5. Conclusions
We designed, synthesized and tested as proteasome inhibi-
tors new tripeptidic-based derivatives bearing a vinyl ester
C-terminal pharmacophoric unit as substrate of the catalytic
g-hydroxy threonine side chain in Michael addition. With the
aim to obtain rigid molecules useful for close structural stud-
ies, we have inserted the Tic residue in the prototype sequence.
Biological results of the new constrained compounds are not
so interesting in terms of inhibition of the trypsin-like activity
of the proteasome. The compounds that differ from the proto-
type only for the residue bearing the vinyl ester function in the
C-terminal position are the most active and selective.
Considering that these molecules show good cell membrane
permeation and resistance to proteolysis, we can use this new
series of compounds as the base for the development of rigid
structures able to show both potency and selectivity of the cat-
alytic subunits of the proteasome.
6.2. Chemistry
6.2.1. Boc-Gln(Trt)-VE (3)
6. Experimental protocols
To a solution of the Boc-Gln(Trt)-al (3 mmol) in toluene
(15 mL) was added [(ethoxycarbonyl)methylidene]triphenyl-
phosphorane (5 mmol). The reaction mixture was stirred for
12 h at room temperature. The residue obtained after evapora-
tion of the solvent was purified by flash chromatography (sil-
ica gel, CH2Cl2/MeOH 20:1). [a]2D0 ¼ ꢁ41.5 (c ¼ 1, MeOH).
1H NMR (CDCl3): d 1.08 (t, 3H), 1.33 (s, 9H), 1.82 (q, 2H),
2.18 (t, 2H), 4.08 (q, 2H), 4.29 (m, 1H), 5.85 (d, J ¼ 16.3,
1H), 6.95 (dd, J ¼ 16.4, 1H), 7.06e7.20 (m, 15H), 8.02 (s,
2H). HPLC: KI(a) ¼ 5.90. MS: M þ Hþ ¼ 543.7. Anal. Calcd.
for C33H38N2O5: C, 73.04; H, 7.06; N, 5.16; O, 14.74. Found:
C, 73.28; H, 7.11; N, 5.23.
6.1. General
Amino acids, amino acid derivatives and chemicals were pur-
chased from Bachem, Novabiochem and Fluka (Switzerland).
Crude vinyl ester tripeptides were purified by preparative
reversed-phase HPLC using a Water Delta Prep 4000 system
with a Waters PrepLC 40 mm Assembly column C18
(30 ꢂ 4 cm, 300 A, 15 mm spherical particle size column).
The column was perfused at a flow rate of 40 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% TFA), 30 min was adopted
for the elution of compounds. HPLC analysis was performed
by a Beckman System Gold with a Hypersil BDS C18 column
6.2.2. H-Leu-VE
Leucine vinyl ester was prepared as described in Ref. [26].

A. Baldisserotto et al. / European Journal of Medicinal Chemistry 42 (2007) 586e592
591
6.2.3. General synthetic procedures
and post-acidic proteasome activities, respectively. Substrates
were incubated at 37 ꢀC for 30 min with proteasomes, un-
treated or pretreated with 0.001e10 mM of test compounds,
in activity buffer. Fluorescence was determined by a fluorime-
ter (Spectrafluor plus, Tecan, Salzburg, Austria) using an exci-
tation of 360 nm and emission of 465 nm. Activity was
evaluated in fluorescence units and the inhibitory activity of
the compounds expressed as IC50. The data were plotted as
percentage control (the ratio of percentage conversion in the
presence and absence of inhibitor) vs. inhibitor concentration,
and fitted with the equation Y ¼ 100/(1 þ (X/IC50))A, where
IC50 is the inhibitor concentration at 50% inhibition and A is
the slope of the inhibition curve.
6.2.3.1. TFA deprotection. Boc was removed by treating vinyl
ester intermediates with aqueous 90% TFA (1:10, w/v) for
30e40 min. After evaporation, the residue was worked up as
described, triturated with Et2O, centrifugated and the resulting
solid was collected and dried.
6.2.3.2. Coupling with WSC/HOBt. The deprotected a-amine
intermediate (1 mmol), NMM (1 mmol), WSC (1 mmol) and
HOBt (1 mmol) were added to a solution of carboxylic com-
ponent (1 mmol) in DMF (3 mL) at 0 ꢀC. The reaction mixture
was stirred for 1 h at 0 ꢀC and 18 h at rt; then the solution was
diluted with AcOEt (80 mL) and washed consecutively with
0.1 N HCl, brine, NaHCO3 and brine. The organic phase
was dried (MgSO4), filtered and evaporated to dryness. The
residue was treated with Et2O and the resulting solid separated
by centrifugation.
6.5. Enzymatic stability assays
The degradation kinetics of new vinyl ester tripeptides 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
TriseHCl buffer, pH 7.5. Incubation was performed at 37 ꢀC
for up to 360 min. The 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 mL) of the
clear supernatant were injected into RP-HPLC column.
HPLC was performed as described in analytical determina-
tions. The degradation half-life (T1/2) was obtained by
a least-squares linear regression analysis of a plot of the loga-
rithmic inhibitor concentration versus time, using a minimum
of five points.
1
6.2.4. H NMR data of compounds 13, 14, 25 and 26
1
HMB-Leu-Leu-Gln-VE (13). H NMR (CDCl3): d 1.02 (s,
12H), 1.27 (t, 3H), 1.75e1.84 (m, 8H), 2.15 (t, 2H), 4.15 (q,
2H), 4.35e4.51 (m, 3H), 5.07 (s, 1H), 5.92 (d, J ¼ 16.2,
1H), 6.09 (s, 2H), 6.88 (dd, J ¼ 16.4, 1H), 7.02e7.24 (m,
3H), 7.89 (s, 3H).
1
HMB-Val-Ser-Gln-VE (14). H NMR (CDCl3): d 1.07 (d,
6H), 1.29 (t, 3H), 1.72 (m, 2H), 2.09 (s, 1H), 2.18 (t, 2H), 2.37
(s, 3H), 2.55 (m, 1H), 4.11 (q, 2H), 4.19e4.25 (m, 3H), 4.39e
4.50 (m, 2H), 5.04 (s, 2H), 5.82 (d, J ¼ 16.0, 1H), 6.06 (s,
2H), 6.84 (dd, J ¼ 16.2, 1H), 7.11e7.33 (m, 3H), 8.02 (s, 3H).
1
HMB-Tic-Leu-Leu-VE (25). H NMR (CDCl3): d 0.99 (s,
Acknowledgements
12H), 1.25 (t, 3H), 1.56e1.82 (m, 6H), 2.30 (s, 3H), 2.9e
3.2 (m, 2H), 4.03 (q, 2H), 4.21 (m, 1H), 4.42e4.55 (m, 3H),
4.89 (m, 1H), 5.02 (s, 1H), 5.95 (d, J ¼ 16.3, 1H), 6.89 (dd,
J ¼ 16.1, 1H), 7.00e7.13 (m, 7H), 8.03 (s, 2H).
Financial support of this work by University of Ferrara, by
`
Ministero dell’Universita e della Ricerca Scientifica e Tecno-
logica (MURST), Associazione Italiana per la Ricerca sul
1
HMB-Tic-Ser-Leu-VE (26). H NMR (CDCl3): d 1.01 (s,
`
Cancro (AIRC), and Istituto Superiore di Sanita (progetto
6H), 1.30 (t, 3H), 1.49 (m, 2H), 1.79 (m, 1H), 2.03 (s, 1H),
2.34 (s, 3H), 2.96e3.17 (m, 2H), 3.87 (m, 2H), 4.11 (m,
1H), 4.19 (q, 2H), 4.27e4.51 (m, 4H), 4.84 (m, 1H), 5.12 (s,
1H), 5.92 (d, J ¼ 16.3, 1H), 6.85 (d, J ¼ 16.3, 1H), 6.89e
7.10 (m, 7H), 8.01 (s, 2H).
AIDS). English revision of the text was carried out by Anna
Forster.
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