Sugar-based peptidomimetics inhibit amyloid β-peptide aggregation

Article abstract of DOI:10.1016/j.ejmech.2011.10.008

Alzheimer's disease is characterized by the oligomerization and amyloid fibril formation of amyloid β-peptide (Aβ). We describe a novel class of small water-soluble Aβ binding peptidomimetics based on two hydrophobic Ala-Val and Val-Leu dipeptides linked

Full text of DOI:10.1016/j.ejmech.2011.10.008

European Journal of Medicinal Chemistry 46 (2011) 5959e5969
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Sugar-based peptidomimetics inhibit amyloid b-peptide aggregation
,
,
Bertrand Dorgeret a 1, Lucie Khemtémourian b 1, Isabelle Correia b, Jean-Louis Soulier a,
**
Olivier Lequin b , Sandrine Ongeri
,
a,
*
a Molécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, IPSIT, LabEx LERMIT, Université Paris-Sud 11, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément,
92296 Châtenay-Malabry Cedex, France
b UPMC Univ Paris 06, Laboratoire des Biomolécules, UMR 7203 CNRS-UPMC-ENS, 4 place Jussieu, 75252 Paris Cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease is characterized by the oligomerization and amyloid fibril formation of amyloid b-
Received 28 July 2011
Received in revised form
30 September 2011
Accepted 4 October 2011
Available online 12 October 2011
peptide (A
hydrophobic Ala-Val and Val-Leu dipeptides linked to a
and carboxyethyl links in C1 and C6 positions. These compounds combine the targeting of hydrophobic
recognition interfaces with an original hydrophilic sugar -breakage strategy. These molecules were
shown, by fluorescence thioflavin-T assays, to dramatically slow down the kinetics of amyloid fibril
formation even at a low peptidomimetics to A ratio of 0.1:1. Electron microscopy images revealed that
the peptidomimetics efficiently reduced the amount of typical amyloid fibrils. NMR saturation transfer
difference experiments indicated that these molecules interact with A aggregated species through their
b
). We describe a novel class of small water-soluble A
b binding peptidomimetics based on two
D-glucopyranosyl scaffold through aminoalkyl
b
b
Keywords:
Amyloid b-peptide
Aggregation
Inhibitor
Peptidomimetics
IAPP
b
hydrophobic amino acid residues. This inhibition effect was found to be sequence-specific since these
molecules did not alter the kinetics of aggregation of another amyloid peptide, IAPP, involved in type 2
diabetes mellitus.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
fibrillation process rather than by discrete oligomeric species [10].
Therefore attractive strategies for treating AD aim at inhibiting the
Alzheimer’s disease (AD) is one of the most common age-related
neurodegenerative disorders, affecting more than 30 million people
in the world, a number that is expected to double every 20 years [1].
AD is associated with the formation of extracellular amyloid fibrils
formation of amyloid fibrils and/or redirecting the aggregation
cascade toward off-pathway species with reduced toxicity [11].
Several approaches have been used to inhibit the aggregation of
soluble A
b
monomers, either by stabilizing native conformations
-strand intermolecular interactions using
-sheet binders [13]. The hydrophobic KLVFF sequence in the
by amyloid
filaments by the protein Tau [2]. The amyloid A
and Ab1e42, are produced by the cleavage of the transmembrane
amyloid protein precursor [3]. In solution, the aggregation process
of A peptides involves a secondary structure transition from
unordered/ -helix [4] to -sheet conformation leading to cross
sheet structure formation with -strands perpendicular to the long
b
-peptides (A
b) and of intraneuronal paired helical
[12] or by preventing b
b
b
peptides, Ab1e40
central region of A
b peptide (residues 16e20) appears to be
b
important for stabilizing intra- and intermolecular interactions
involved in aggregation and amyloid formation [14]. Small peptides
mimicking this hydrophobic sequence were demonstrated to
modulate the kinetics of aggregation [13,15,16]. Furthermore,
b
a
b
b-
b
fibril axis [5]. Distinct morphological species including oligomers,
protofibrils, and fibrils are formed during the aggregation process
[6]. Soluble oligomeric Ab forms are thought to be mainly respon-
sible for neurological toxicity [7e9]. However recent studies
suggest that the toxicity of amyloid peptides is mediated by the
variants of this core domain incorporating
acids such as N-methyl amino acids [17], proline [18] or
isobutyric acid [19] have proved to be valuable molecules for
inhibiting A peptide aggregation by destabilizing the pathological
-sheet conformation.
We describe herein a novel class of small water-soluble pepti-
domimetics based on flexible, sugar-based scaffolds, as inhibitors of
aggregation (Fig. 1). These compounds incorporate two short
hydrophobic dipeptide units, Ala-Val and Val-Leu that were chosen
to interact with the hydrophobic regions of A . These sequences are
attached to a -glucopyranosyl scaffold via an aminoalkyl and
b
-sheet breaker amino
a-amino-
b
b
* Corresponding author. Tel.: þ33 146835737; fax: þ33 146835740.
** Corresponding author. Tel.: þ33 144273113; fax: þ33 144277150.
E-mail addresses: olivier.lequin@upmc.fr (O. Lequin), sandrine.ongeri@u-psud.fr
(S. Ongeri).
A
b
b
1
These authors contributed equally to this work.
D
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.008

5960
B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
HO
conferring peptide resistance to proteolytic cleavage [20], and
promoting -turn conformations [20,21]. Saccharides and sulfated
glycosaminoglycans have been shown to bind to A and interfere
with fibril formation [22,23]. However their role in fibril formation
is controversial. Negatively charged saccharides are generally
described as Ab aggregation promoters but low molecular weight
saccharides and glycosaminoglycans can also be neuroprotective.
The glucosidic moiety in piceid, a resveratrol analog appeared as an
OH
b
HO
O
O
b
H
N
H
N
O
O
O
O
N
H
n
N
H
BocNH
O
O
O
Val 4
Leu 5
Sug
ar A
Ala 1
A 3
Val 2
1
n = 3 C1 = β
n = 2 C1 = β
2
4
n = 3 C1 = α
n = 2 C1 = α
important determinant of its Ab disaggregation activity [11,24].
3
However, to the best of our knowledge, sugar-based peptidomi-
metics have not yet been described as inhibitors of amyloid
formation.
Fig. 1. Structure of peptidomimetics 1e4 containing D-glucopyranosyl sugar amino
acid derivatives.
Four peptidomimetic molecules (Fig.1) differing by the length of
the linker in position 1 (n ¼ 2, 3) and the anomeric carbon config-
a carboxyethyl links in C1 and C6 positions, respectively (Sugar AA
3, Fig. 1). The carboxy and aminoalkyl linkers in our peptidomi-
metics were chosen to confer flexibility that may be necessary for
uration (
decrease the rate of A
binding assay and transmission electron microscopy. The
conformation of these molecules and their interaction properties
with A were investigated by NMR spectroscopy.
a or b) were synthesized. These molecules were shown to
b
fibril formation by a fluorescence thioflavin-
T
optimal interaction of amino acid residues with the
b-sheet Ab
target. The design of these peptidomimetics was inspired by
previous strategies of Soto [18] and others [13e17] who described
b
synthetic five residues
b-sheet breaker pseudopeptides as inhibi-
tors of A aggregation. The originality of our peptidomimetics
b
2. Results and discussion
consists in the introduction of a central sugar moiety to perturb the
regular interstrand hydrogen-bond network and destabilize
2.1. Chemistry
b
-sheet formation locally. We anticipated that these inhibitor
peptidomimetics would operate mainly by binding to A through
their four hydrophobic amino acids at one hydrogen-bonding face
and simultaneously hindering the interaction with other A
peptides thanks to the central sugar moiety acting as a -sheet
breaker element. The introduction of a carbohydrate in peptides
can also have a multifaceted impact on the properties of peptides
such as modulating the hydrophilicity/hydrophobicity balance,
b
The synthesis of compounds 1e4 is described in Scheme 1. The
intermediate 8 was prepared from the commercially available
methyl-a-D-glucopyranoside 5 by successive tritylation of the
primary hydroxyl in C6, benzylation of the three hydroxyls in C2, C3
and C4 and then hydrolysis of the trityl protection of 7 [25,26]. A
Michael addition of 8 on tert-butylacrylate was carried out in the
presence of a catalytic amount of tetrabutyl ammonium bromide
b
b
OTr
O
OH
O
OTr
O
OH
O
(a)
(b)
(c)
BnO
BnO
HO
HO
HO
HO
BnO
BnO
HO
OMe
HO
BnO
BnO
OMe
OMe
OMe
8
5
6
7
O
H
N
O
O
OH
O
O
O
O
N
H
(d)
(g)
(e)
(f)
O
BnO
BnO
O
O
BnO
BnO
O
BnO
BnO
O
O
BnO
9
BnO
OMe
BnO
OH
OH
10
11
O
O
H
H
N
N
O
O
O
O
N
H
N
(i)
(h)
O
H
O
BnO
BnO
O
O
BnO
BnO
O
O
O
BnO
12 n = 3
13 n = 2
BnO
14 n = 3
15 n = 2
O
N3
NH2
n
n
O
H
O
H
N
O
O
N
O
O
N
H
N
H
(j)
(k)
NHBoc
O
O
O
BnO
BnO
O
BnO
BnO
1-4
O
H
O
H
O
BnO
BnO
O
N
O
N
NHBoc
N
H
n
n
O
O
20 n = 3 C1 =
21 n = 3 C1 =
22 n = 2 C1 =
23 n = 2 C1 =
16 n = 3 C1 =
17 n = 3 C1 =
18 n = 2 C1 =
19 n = 2 C1 =
Scheme 1. Synthesis of peptidomimetics 1e4. Reagents and conditions: a) TrCl, Et3N, DMF, rt, 92%. b) BnBr, NaH, DMF, rt, 75%. c) p-TsOH, MeOH/CH2Cl2, rt, 70%. d) tert-butylacrylate,
TBAB (15% m/m), 20% aqueous NaOH, rt, 90%. e) CF3SO3H, H2O, AcOH, 80 ꢀC, 55%. f) HeVal-LeueOMe, HBTU, HOBt, DIPEA, DMF, rt, 94%. g) Cl3CCN, NaH, CH2Cl2, rt then azidoalcohol,
BF3$OEt2, CH2Cl2, ꢁ20 ꢀC, 59%, ratio
a
:
b
¼ 1:1 for 12 and 49%, ratio
a
:
b
¼ 1:1 for 13. h) PPh3, THF/H2O, rt, 78% for 14 and 76% for 15. i) N-BoceL-Val, HBTU, HOBt, DIPEA, DMF, rt, then
epimers separation by gel chromatography, 91% (
a:
b
¼ 40:60) for 16e17 and 85% (
a:b
¼ 45:55) for 18e19. j) TFA, CH2Cl2, rt, 100% then N-Boc-L-Ala, HBTU, HOBt, DIPEA, DMF, rt, 68%
for 20, 88% for 21, 76% for 22, and 75% for 23. k) H2, Pd(OH)2, CH2Cl2/MeOH, 100% for 1, 92% for 2, 96% for 3 and 89% for 4.

B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
5961
Table 1
(TBAB) and in a 20% aqueous sodium hydroxide solution to intro-
duce, in good yield (90%), a carboxy ethyl link on the primary
hydroxyl group in C6 (compound 9, Scheme 1) [27,28].
Inhibition of Ab1e40 fibrillation by sugar-based peptidomimetics 1e4 at a 1:1
:peptidomimetic ratio.
Ab
Compound
Lag extension
Plateau reduction
(% inhibition)a
The C1 methoxy group and the tert-butyl ester were cleaved by
(fold increase)a
a trifluorosulfonic acid solution in acetic acid at 80 ꢀC [29] in 55%
1
2
3
4
3.0 ꢂ 1.0
3.4 ꢂ 1.0
3.4 ꢂ 1.0
3.4 ꢂ 1.0
52 ꢂ 15
60 ꢂ 15
40 ꢂ 15
22 ꢂ 10
yield (ratio
a
:
b
¼ 1:1), and the obtained product 10 was coupled
with the dipeptide HeVal-LeueOMe using classical coupling
reagents (HBTU, HOBt) to give the intermediate compound 11 in
excellent yield (94%). Addition of compound 11 on tri-
a
Parameters are expressed as mean ꢂ SE, n ¼ 2e6 (see Experimental section for
chloroacetonitrile using NaH as a base, provided the
a-imidate
the calculation of the lag extension of the plateau reduction).
according to the literature and to the 1H NMR spectrum (H1:
d
¼ 6.35 ppm and J1,2 ¼ 3 Hz) [30]. Nucleophilic substitution reac-
a stereoselective reaction. However, we were interested in both
isomers so we pursued the following synthetic steps. The azido
group of 12 and 13 was reduced via a Staudinger reaction in 78%
and 76% yields respectively. The obtained amines 14 and 15 were
tions by azidopropanol (prepared from bromopropanol [31]) and by
azidoethanol (prepared from bromoethanol [32]) as glycosyl
acceptors were carried out with BF3$OEt2 as Lewis acid at ꢁ20 ꢀC
[30,33] to provide 12 and 13 respectively in 59% and 49% yield for
the two steps from 11. These “Schmidt” conditions [33] are
described to yield essentially the
case, the and epimers of 12 and 13 were obtained in equal
proportion and could not be separated at this stage. The peptidic
chain in C6 could maybe interfere during the mechanism of
nucleophilic substitution of the azidoalcohol and prevent
coupled with N-Boce
L-Val, using HBTU and HOBt as coupling
reagents, to afford after separation of
a
and isomers compounds
b
b-isomer [30]. However, in our
16 and 17 in 56% and 35% yields respectively, and compounds 18
and 19 in 45% and 40% yields respectively. The rest of the synthesis
a
b
was carried out separately for the two
groups were cleaved by TFA in quantitative yield and the free
amines were coupled with N-Boce -Ala using HBTU and HOBt as
a and b isomers. The Boc
L
coupling reagents to obtain compounds 20 and 21 in 68% and 88%
yields respectively from 16 and 17, and to obtain compounds 22 and
23 in 76% and 75% yields respectively from 18 and 19. Finally, the
cleavage of benzyl groups by catalytic hydrogenation using Pd(OH)2
as catalyst afforded the desired final compounds 1, 2, 3 and 4 in
100%, 92%, 96% and 89% yields, respectively.
2.2. Fluorescence spectroscopy
The ability of compounds 1e4 to inhibit amyloid fibril formation
was first tested on Ab1e40 peptide. The fluorescence-detected thi-
oflavin-T (ThT) binding assay is the standard method used to
monitor the time course of fibril formation [34]. The final ThT
fluorescence intensity is dependent on the amount of fibrillar
material formed, the binding constant for ThT, and the quantum
yield of the bound dye. The fluorescence assay of Ab1e40 at
a concentration of 20 mM shows a typical fibrillation process with
a lag phase of approximately 12 h followed by a growth phase and
a final plateau (Fig. 2A).
Fig. 2A shows that peptidomimetic 1 at Ab1e40:1 ratios of 1:0.1
and 1:1 increases the lag phase of Ab1e40 from 12 to 26 and 41 h
respectively. In addition, the final fluorescence intensity is signifi-
cantly reduced relative to Ab1e40 alone by a factor of 2 (Fig. 2A).
Control experiments indicate that, under the experimental condi-
tions used, the ThT fluorescence of the peptidomimetic remains
low (Fig. 2A, dash dot). Similar results were observed with pepti-
domimetics 2e4, namely a reduction of fibril formation accompa-
nied by an increase in the lag time. The results for all
peptidomimetics are summarized in Table 1 with respect to their
effect upon both lag extension and equilibrium plateau reduction.
All compounds exhibit strong inhibitory capabilities, increasing the
Table 2
Effects on IAPP fibrillation induced by sugar-based peptidomimetics 1e4 at a 1:10
IAPP:peptidomimetic ratio.
Compound
Lag extension
Plateau reduction
(% inhibition)a
(fold increase)a
Fig. 2. (A) Representative ThT fluorescence assay showing the fibril formation of
A
b1e40 in the absence (solid) or in the presence of the peptidomimetic 1 at a Ab:pep-
1
2
3
4
1
1
1
5 ꢂ 5
tidomimetic ratio of 1:0.1 (solid with open circle) and 1:1 (solid with open square). ThT
traces are shown for peptidomimetic 1 alone (dash). (B) Representative ThT fluores-
cence assay showing the fibril formation of IAPP in the absence (solid) or in the
presence of the peptidomimetic 1 at a Ab:peptidomimetic ratio of 1:1 (solid with open
square) and 1:10 (solid with open circle). ThT traces are shown for peptidomimetic 1
9 ꢂ 5
1 ꢂ 4
Slight promotion
Slight promotion
a
Parameters are expressed as mean ꢂ SE, n ¼ 6 (see Experimental section for the
(dash).
calculation of the lag extension of the plateau reduction).

5962
B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
Fig. 3. Negatively stained electron microscopy images of fibril formation of Ab1e40alone (top) and in the presence of the peptidomimetic 1 (down) at a 1:1 ratio in 10 mM Tris$HCl,
100 mM NaCl buffer at pH 7.4 after 1 day (A and D), 2 days (B and E) and 2 weeks of incubation (C and F). Panel F shows the rare isolated fibrils observed (scale bars, 500 nm).
lag time by a factor of approximately 3 and reducing the equilib-
rium plateau by 20e60%. These results indicate that the peptido-
mimetics inhibit Ab1e40 fibril formation but that the length of the
aminoalkyl link and the configuration of the anomeric center have
only little influence on these inhibition properties.
Next, we wanted to investigate if the inhibition effect observed
on Ab1e40 was specific. For that purpose, the effects on another
amyloid peptide, IAPP (Islet Amyloid Polypeptide), involved in type
II diabetes mellitus were evaluated [35]. This peptide shares similar
features in length (37 residues), sequence (w20% identity) and
morphology of amyloid fibrils. Fluorescence data show a fast
small and large spherical aggregates (Fig. 3A and B), while after 2
weeks TEM images reveal dense mats of fibrils with the classical
morphology(Fig. 3C). In contrast, images ofAb1e40:1 mixture at a 1:1
ratio, collected after 1 and 2 days show almost no aggregates
(Fig. 3DeE). In addition, after 2 weeks of incubation, very few fibrils
are observed. The population of Ab1e40:1 mixture fibrillar assem-
blies consists of thin fibrils that, in contrast with Ab40 alone, do not
appear tohavea strongpropensity to cluster. (Fig. 3F). Similar results
were observed with Ab1e40:1 at a 1:0.1 ratio (data not shown).
fibrillation process for IAPP (5 mM) with a lag phase of w3 h in
agreement with previous results [36].
However, the peptidomimetics 1e4 do not inhibit the IAPP fibril
formation even at a high IAPP:peptidomimetic ratio of 1:10 (Fig. 2B
and Table 2). The lag time is not reduced after addition of the
peptidomimetics and the final fluorescence intensity remains the
same. In addition, our results show that peptidomimetic 4 slightly
promotes aggregation as evidenced by a decrease in the lag time
and an increase in the equilibrium plateau relative to the control.
Overall, our data demonstrate that the peptidomimetics 1e4 do not
inhibit IAPP fibril formation, whereas they are potent inhibitors of
Ab1e40 fibrillation even at low ratios of inhibitors.
2.3. Transmission electron microscopy
The reduction of the final fluorescence intensity of Ab1e40
induced by the peptidomimetics should not necessarily be inter-
preted in terms of the amount of amyloid formed. Indeed, the
changes in the binding constant or the quantum yield of the dye can
also contribute to any observed differences. Thus, this is critical to
assay any potential inhibitors by an independent method. Trans-
mission electron microscopy (TEM) studies conducted as a function
of time confirm that the peptidomimetics significantly affect Ab1e40
fibril formation but do not affect IAPP fibril formation. Aliquots of
each reaction mixture were collected at different time points, and
TEM images recorded. After 1e2 days, TEM images of Ab1e40 show
Fig. 4. Negatively stained electron microscopy images of fibril formation of IAPP alone
(top) and in the presence of the peptidomimetic 1 (down) at a 1:10 ratio in 10 mM
Tris$HCl, 100 mM NaCl buffer at pH 7.4 after 3 h (A and C) and 24 h of incubation (B and
D) (scale bars, 500 nm).

B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
5963
Fig. 5. 2D NOESY spectra of peptidomimetic 1 in the absence (A) and in the presence (B) of Ab1e40. Positive signals are shown with blue contour levels and negative signals with red
contour levels. NOE correlations indicated by arrows correspond to intramolecular NOEs of peptidomimetic 1. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
Next, TEM images of IAPP alone and IAPP:1 mixture at a 1:10
ratio were collected after 3 and 24 h. In both cases with and without
the peptidomimetic, the images reveal dense mats of fibrils with
the classical morphology (Fig. 4AeD). Our data demonstrate that
the peptidomimetics dramatically slow down the aggregation of
peptidomimetic 1. These results tend to indicate that peptidomi-
metic 1 does not strongly interact with monomeric Ab1e40 peptide.
However the 2D NOESY spectra of peptidomimetic 1 (1 mM)
showed significant differences in NOEs upon addition of Ab1e40
peptide (0.25 mM). Indeed, while the NOE correlations are char-
A
b1e40 and efficiently reduce the amount of typical amyloid fibrils,
acterized by positive or near zero values in the absence of Ab1e40,
whereas they do not affect the kinetics of IAPP fibril formation
neither decrease the amount of IAPP fibrils.
the addition of Ab1e40 leads to the appearance of negative NOEs for
the HN/aliphatic protons correlations of peptidomimetic 1 (Fig. 5).
2.4. NMR spectroscopy
NMR studies carried out in aqueous solution showed that pep-
tidomimetics 1e4 are highly soluble in water and are not prone to
aggregation, as indicated by the observation of small proton reso-
nance line widths and the absence of significant chemical shift
dependency upon concentration variation (0.25e2.8 mM range).
The analysis of chemical shifts, vicinal coupling constants and ROEs
shows that the linker segments and the dipeptide units are highly
flexible in solution. The amide protons exhibit large temperature
dependency of their chemical shifts (ꢁ10.1 to ꢁ6.4 ppb/ꢀC)
suggesting that they are not sequestered from solvent. The large
3 a
J
a coupling constants (ꢃ7.5 Hz) of Val and Leu residues suggest
HN,H
that extended conformations are significantly populated for these
hydrophobic residues. This is also confirmed by the analysis of
intraresidual and sequential HaeHN ROEs. The temperature coef-
ficients and the absence of long-range ROEs indicate that pepti-
domimetics alone do not form stable
intramolecularly or intermolecularly.
b-sheets either
Since these peptidomimetics showed very similar results in
terms of conformation and inhibition properties, a single peptido-
mimetic was selected for further NMR studies in aqueous solution
in the presence of Ab1e40. 1D and 2D NMR experiments realized on
different mixtures of peptidomimetic 1 and Ab1e40 did not show
significant chemical shift changes of peptidomimetic resonances. It
has been shown that the NMR spectrum of Ab1e40 is dominated by
the monomeric species, the oligomeric and fibril species being
NMR-invisible [37]. In our case, we did not observe any significant
chemical shift changes of Ab1e40 resonances upon addition of
Fig. 6. (A and B) Aliphatic region of 1D 1H NMR spectra of peptidomimetic 1 (0.6 mM)
alone (A) and in the presence of 30
mixture of peptidomimetic 1 (0.6 mM) and Ab1e40 (30
1 h (C) and 2 weeks (D). The dispersion peaks observed in (C) are difference artefacts.
m
M Ab1e40 (B). (C and D) 1D STD spectra of the
mM) recorded after a period of

5964
B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
The detection of negative NOE correlations clearly indicates that
peptidomimetic 1 is able to interact with Ab1e40 species. In the
absence of chemical shift modifications of Ab1e40 resonances, as
aforementioned, we postulate that the negative transferred NOEs
arise from the interaction with aggregated, NMR-invisible Ab1e40
species.
To confirm this hypothesis, the interaction of peptidomimetic 1
with Ab1e40 was further examined by saturation transfer difference
(STD) experiments [38] at different concentrations. No STD signal
was observed on peptidomimetic 1 alone (1 mM concentration). In
the presence of Ab1e40 (0.25 mM), STD signals were observed for
the methyl resonances of Ala, Leu, and Val residues of peptidomi-
metic 1, proving that 1 interacts with Ab1e40 species through its
hydrophobic amino acids. Similar experiments were carried out at
were performed on a PerkineElmer CHN, Analyser 2400. Mass
spectra were obtained using a Bruker Esquire electrospray ioniza-
tion apparatus. IR spectra were recorded on a Bruker Vector 22 FT-
IR spectrometer. NMR standard spectra were recorded on a Bruker
AMX 200, (1H, 200 MHz, 13C, 50 MHz) or an Ultrafield AVANCE 300
(1H, 300 MHz, 13C, 75 MHz) or a Bruker AVANCE 400 (1H, 400 MHz,
13C, 100 MHz). Chemical shift
d are in parts per million and the
following abbreviations are used: singlet (s), doublet (d), triplet (t),
multiplet (m), broad singlet (br s).
4.1.1. Synthesis of peptidomimetics 1e4
4.1.1.1. BoceAla-Val-[(C1)b-glucose-(n3)]-Val-LeueOMe 1. A solu-
tion of 20 (75 mg, 0.07 mmol) and Pd(OH)2 (18 mg, mass 25%) in
MeOH/DCM 3:1 (4 ml) was stirred under hydrogen atmosphere for
3 h then the mixture solution was filtered on Celite and evaporated
under reduced pressure to afford 1 as a yellowish white solid
lower concentrations of Ab1e40 (30
mM) corresponding to an
A
b1e40:1 ratio of 1:20. STD signals were not observed initially
(Fig. 6C), indicating that the aggregated species of Ab1e40 inter-
acting with peptidomimetic 1 were not present at the very begin-
ning in these diluted Ab1e40 samples. However the STD signals
were observed after a period of about 2 weeks (Fig. 6D). This time
dependence of STD signal can be explained by the slow conversion
of monomeric Ab1e40 to aggregated species and confirms that
peptidomimetic 1 interacts preferentially with aggregated Ab1e40
species.
(57 mg, 100%). IR (neat):
n
¼ 3273 (OeH), 2963 (NeH), 1645 (C]O),
1633 (C]O); MS (ESI, ion polarity positive): m/z: 828 [M þ Na]þ;
HRMS (TOF, ion polarity positive): calcd for C37H67N5O14Na:
828.4582; found: 828.4583. The NMR assignment is shown in
Table S1 (Supplementary data).
4.1.1.2. BoceAla-Val-[(C1)a-glucose-(n3)]-Val-LeueOMe 2. A solu-
tion of 21 (143 mg, 0.13 mmol) and Pd(OH)2 (36 mg, mass 25%) in
MeOH/DCM 3:1 (6.6 ml) was stirred under hydrogen atmosphere
for 3 h then the mixture solution was filtered on Celite and evap-
orated under reduced pressure to afford 2 as a white solid (99 mg,
92%). C37H67N5O14 þ 2.25H2O: calcd C 52.50, H 8.51, N 8.27; found C
3. Conclusions
In summary, we have described a novel class of water-soluble
aggregation inhibitors based on a flexible sugar amino acid inser-
ted in the center of a short hydrophobic peptidic sequence to
52.48, H 8.22, N 8.26; IR (neat):
n
¼ 3273 (OeH), 3034 (NeH), 2963
(NeH), 1645 (C]O), 1633 (C]O); MS (ESI, ion polarity positive): m/
z: 828 [M þ Na]þ; HRMS (TOF, ion polarity positive): calcd for
C37H67N5O14Na: 828.4582; found: 828.4583. The NMR assignment
is shown in Table S2 (Supplementary data).
destabilize locally the
These compounds combine the targeting of hydrophobic recogni-
tion interfaces with an original hydrophilic sugar -breakage
b-sheet structure of Ab aggregated species.
b
strategy. These peptidomimetics dramatically slow down the
aggregation of Ab1e40 even at low concentrations (peptidomimetic
to Ab1e40 ratios of 0.1:1) and efficiently reduce the amount of
typical amyloid fibrils. Noteworthy, this inhibition effect is
sequence-specific since these molecules do not alter the kinetics of
aggregation of IAPP amyloid peptide. These peptidomimetics do
4.1.1.3. BoceAla-Val-[(C1)b-glucose-(n2)]-Val-LeueOMe 3. A solu-
tion of 22 (126 mg, 0.12 mmol) and Pd(OH)2 (32 mg, mass 25%) in
MeOH/DCM 3:1 (6 ml) was stirred at rt, under hydrogen atmo-
sphere for 4 h. The mixture solution was filtered on Celite and the
filtrate was evaporated under reduced pressure to afford 3 as
not strongly interact with unordered/
a
-helix Ab1e40 monomer, as
a
white
solid
(99 mg,
96%);
m.p.
decomposition;
suggested by the absence of NMR chemical shift perturbation. This
points toward a distinct mechanism of action from other inhibitors
C36H65N5O14 þ 2.0H2O: calcd C 52.22, H 8.40, N 8.46; found C 52.20,
H 7.78, N 8.20; IR (neat):
n
¼ 3279 (OeH), 2957 (NeH), 1635 (C]O);
such as
D-Trp-Aib, which was shown to interact with A
b
monomer
MS (ESI, ion polarity positive): m/z: 814 [M þ Na]þ; HRMS (TOF ESI,
ion polarity positive): calcd for C36H65N5O14Na: 814.4426; found:
814.4395. The NMR assignment is shown in Table S3
(Supplementary data).
using a similar NMR approach [19]. However, trNOEs and STD
experiments revealed that these peptidomimetics are in dynamic
equilibrium with complexes involving A
inhibition effect probably results from binding to A
b
species. Therefore the
aggregated
b
species. Further experiments are necessary to decipher the binding
4.1.1.4. BoceAla-Val-[(C1)a-glucose-(n2)]-Val-LeueOMe 4. A mixture
mode of these peptidomimetic inhibitors to Ab1e40 species and to
of 23 (105 mg, 0.1 mmol) and Pd(OH)2 (27 mg, mass 25%) in MeOH/
DCM 3:1 (5 ml) was stirred at rt, under hydrogen atmosphere for 4 h.
The mixture solution was then filtered on Celite and the filtrate was
evaporated under reduced pressure to afford 4 as a white solid
(70 mg, 89%); m.p. decomposition; C36H65N5O14 þ 2.0H2O: calcd C
analyze if the A
b aggregation cascade is redirected toward off-
pathway species [11].
4. Experimental
4.1. Chemistry
52.22, H 8.40, N 8.46; found C 51.92, H 7.68, N 8.04; IR (neat):
n
¼ 3295
(OeH), 2971 (NeH), 1641 (C]O); MS (ESI, ion polarity positive): m/z:
814 [M þ Na]þ; HRMS (TOF ESI, ion polarity positive): calcd for
C36H65N5O14Na: 814.4426; found: 814.4435. The NMR assignment is
shown in Table S4 (Supplementary data).
Usual solvents were purchased from commercial sources and
dried and distilled by standard procedures. Azidopropanol was
prepared from bromopropanol [31] and azidoethanol was prepared
from bromoethanol [32] according to methods reported in the
literature. HeVal-LeueOMe was prepared according to classical
literature reported coupling method using isobutyl-chloroformiate.
Pure products were obtained after flash chromatography using
4.1.2. Synthesis of intermediates 6e15
4.1.2.1. Methyl 6-O-(triphenylmethyl)-a-D-glucopyranoside 6. A solu-
tion of methyl- -glucopyranoside 5 (1.0 g, 5.2 mmol), trityl chlo-
a-D
ride (1.5 g, 5.7 mmol) and triethylamine (1.3 ml, 9.3 mmol) in dry
DMF (15 ml) was stirred overnight at room temperature under
nitrogen. After 24 h, the solvent was evaporated under reduced
Merck silica gel 60 (40e63
mm). TLC analyses were performed with
0.25 mm 60 F254 silica plates (Merck). Element analyses (C, H, N)

B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
5965
pressure and the residue was dissolved in EtOAc (20 ml) and water
(20 ml). After the separation of the layers, aqueous layer was
extracted with EtOAc (3 ꢄ 10 ml). The combined organic layers were
dried with MgSO4, filtered and evaporated under reduced pressure.
The crude residue was purified by chromatography on a silica gel
column (cyclohexane/EtOAc, 4:6 then 2:8) to give compound 6 as
filtered and evaporated under reduced pressure. The crude residue
was purified by chromatography on a silica gel column (cyclo-
hexane/EtOAc, 8:2) to afford compound 9 as a colorless oil (5.49 g,
1
90%). H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.23e7.17 (m, 15H,
Harom), 4.88e5.51 (m, 7H, 3 ꢄ PhCH2 and H1b), 3.88 (t, 3J(H, H) ¼
10.0 Hz, 1H, H3), 3.60e3.43 (m, 7H, H2, H4, H5, H6, OCH2), 3.35 (s,
3H, OCH3), 2.38 (m, 2H, CH2eCO), 1.31 (s, 9H, CH3eBoc); 13C NMR
1
a white solid (2.1 g, 92%). H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.47e7.30 (m, 15H, Harom), 4.71 (d, 3J(H, H) ¼ 3.6 Hz, 1H, H1b),
(100 MHz, CDCl3, 25 ꢀC):
d
¼ 138.6, 138.2, 137.9, 128.1, 128.0, 127.8,
127.7, 127.6, 127.4, 127.3, 97.9, 81.8, 80.2, 79.6, 77.3, 75.4, 74.7, 73.1,
69.8, 69.2, 66.9, 54.8, 35.9, 27.8; IR (neat):
3.68 (m, 1H, H5), 3.65 (dd, 3J(H, H) ¼ 3.6, 9.1 Hz, 1H, H2), 3.45 (t, 3J(H,
H) ¼ 9.1 Hz, 1H, H3), 3.39 (m, 3H, OCH3), 3.37 (m, 1H, H4), 3.31 (m,
n
¼ 1731 (C]O);
13
2H, H6); C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 143.9, 128.7, 127.8,
C35H34O8: calcd C 70.92, H 7.48; found C 70.96, H 7.45; MS (ESI, ion
127.0, 99.2, 86.7, 74.4, 72.0, 71.3, 70.4, 63.9, 55.0; m.p. 153e155 ꢀC
polarity positive, MeOH): m/z: 616 [M þ Na]þ.
(lit. [39]: m.p. 154.5e155.5 ꢀC); IR (neat):
n
¼ 3429 (OeH); C26H29O6:
calcd C 71.54, H 6.47; found: C 71.35, H 6.42; MS (ESI, ion polarity
4.1.2.5. 6-O-[2-(carboxyethyl)]-2,3,4-tri-O-benzyl-a-D-glucopyranoside
positive): m/z: 459 [M þ Na]þ.
10. To a solution of 9 (1.2 g, 2.02 mmol) in acetic acid (23 ml) was
added a solution of 2 N trifluorosulfonic acid (4.0 ml, 8.08 mmol). The
reaction mixture was stirred for 5 h at 80 ꢀC then water (20 ml) and
DCM (30 ml) were added. The different layers were separated and the
aqueous layer was extracted with EtOAc (4 ꢄ 20 ml). The combined
organic layers were dried over MgSO4, filtered and evaporated under
reduced pressure. The crude residue was purified by chromatography
on a silica gel column (cyclohexane/EtOAc/AcOH, 8:2:0 then 20:75:5)
4.1.2.2. Methyl 2,3,4-tri-O-benzyl-6-O-(triphenylmethyl)-a-D-gluco-
pyranoside 7. To a solution of 6 (1.41 g, 3.22 mmol) in DMF (15 ml)
were added NaH (970 mg, 24.2 mmol) and benzyl bromide (2.4 ml,
19.3 mmol). The reaction mixture was stirred at rt overnight. Excess
of NaH was destroyed by addition of MeOH (20 ml) and water
(10 ml). MeOH was evaporated under reduced pressure and DCM
(10 ml) was added. After the separation of the layers, the aqueous
layer was extracted with DCM (2 ꢄ 5 ml). The combined organic
layers were dried over MgSO4, filtered and evaporated under
reduced pressure. The crude residue was purified by chromatog-
raphy on a silica gel column (cyclohexane/EtOAc, 4:6 then 2:8) to
afford compound 7 as a colorless oil (1.71 g, 75%). 1H NMR
to afford compound 10 as a yellowish oil (2.0 g, 55%, ratio
a
:
b
¼ 1:1).
1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.40e7.25 (m, 15H, Harom), 5.16
(d, 3J(H, H) ¼ 3.5 Hz, 0.5H, H1a), 4.99e4.57 (m, 6.5H, H1 and
b
3 ꢄ PhCH2), 3.96 (m, 1H, H3), 3.65e3.31 (m, 6H, H2, H4, H5, H6, OCH2),
2.51 (t, 3J(H, H) ¼ 6.2 Hz, 2H, CH2eCO); 13C NMR (100 MHz, CDCl3,
25 ꢀC):
d
¼ 176.3, 176.1, 138.7, 138.5, 138.4, 138.3, 138.1, 137.9, 128.5,
(400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.57e6.95 (m, 30H, Harom), 5.04e4.77
128.5, 128.2, 128.1, 128.0, 128.0, 127.8, 127.7, 97.5, 91.2, 84.5, 83.2, 81.7,
80.0, 75.8, 75.0, 74.8, 74.3, 73.2, 70.0, 69.9, 66.6, 66.6, 34.7; IR (neat):
(m, 7H, 3 ꢄ PhCH2 and H1b), 4.07 (m, 1H, H3), 3.91 (m, 1H, H5), 3.73
(m, 2H, H4, H2), 3.61 (m,1H, H6), 3.54 (s, 3H, OCH3), 3.29 (m,1H, H6);
n
¼ 3369 (OeH), 1719 (C]O); C30H34O8 þ 0.34H2O: calcd C 68.15, H
13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 143.9, 138.8, 138.3, 138.0,
6.61; found C 68.16, H 6.74; MS (ESI, ion polarity positive, MeOH): m/
z: 545 [M þ Na]þ.
128.8, 128.4, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 127.4, 127.1, 126.9,
126.2, 97.9, 86.3, 82.3, 80.3, 78.2, 75.9, 74.9, 73.3, 70.3, 62.7, 54.9; IR
(neat):
n
¼ 3080 (CaromeH); 3030 (CaromeH); 1069 (CeO); 1028
4.1.2.6. Synthesis of compound 11. To a solution of 10 (1.8 g,
3.44 mmol) in DMF (25 ml) were added HBTU (1.83 g, 4.82 mmol),
HOBt (0.65 g, 4.82 mmol), DIPEA (3 ml, 17.2 mmol) and HeVal-Leu-
eOMe (1.71 g, 4.82 mmol). The reaction mixture was stirred over-
night at rt then the solvent was removed under reduced pressure and
the residue was dissolved in EtOAc. This organic layer was washed
with water (25 ml), an aqueous 10% citric acid solution (25 ml), an
aqueous saturated NaHCO3 solution (25 ml) and brine (25 ml). The
organic layer was dried over MgSO4, filtered and evaporated under
reduced pressure. The crude residue was purified by chromatography
on a silicagel column (cyclohexane/EtOAc, 2:8 then 0:10) to afford the
desired product 11 as a yellowish solid (2.43 g, 94%). 1H NMR
(CeO); MS (ESI, ion polarity positive, MeOH): m/z: 729 [M þ Na]þ.
4.1.2.3. Methyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside 8. A solution
of 7 (1.75 g, 2.48 mmol) and p-TsOH (50 mg, 0.26 mmol) in DCM/
MeOH 1:2 (25 ml) was stirred overnight. Then the solvent was
evaporated under reduced pressure and the crude residue was dis-
solved with DCM (10 ml). This solution was washed with water
(2 ꢄ 10 ml), aqueous 10% Na2CO3 solution (10 ml) and aqueous
saturated NaCl solution (10 ml). The organic layer was dried over
MgSO4, filtered and evaporated under reduced pressure. The crude
residue was purified by chromatography on a silica gel column
(cyclohexane/AcOEt, 7:3 then 6:4) to afford 8 as white solid (806 mg,
(300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.99 (m, 1H, NH), 7.65 (m, 1H, NH),
1
70%). H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.30e7.42 (m, 15H,
7.39e7.26 (m, 15H, Harom), 6.69 (m, 1H, NH), 5.23e4.52 (m, 8H, H1,
3 ꢄ PhCH2 and CHaeLeu), 4.25e4.12 (m, 2H, H3 and CHaeVal),
3.86e3.19 (m, 10H, H2, H4, H5, H6, OCH2 and OCH3), 2.53 (m, 2H,
CH2eCO), 2.03 (m, 1H, CHbeVal), 1.62e1.58 (m, 3H, CH2beLeu and
CHgeLeu), 0.95e0.85 (m, 12H, CH3eVal and CH3eLeu); 13C NMR
Harom), 5.02 (d, 3J(H, H) ¼ 11.0 Hz, 1H, H15), 4.92e4.82 (m, 6H,
3 ꢄ PhCH2), 4.60 (d, 3J(H, H) ¼ 3.0 Hz, 1H, H1b), 4.04 (t, 3J(H, H) ¼
9.0 Hz,1H, H3), 3.67e3.78 (m, 3H, H5, H6), 3.52e3.58 (m, 2H, H2, H4),
3.39 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3, 25 ꢀC):
d
¼ 138.7,138.1,
138.1, 128.4, 128.3, 128.0, 127.9, 127.9, 127.8, 127.5, 98.1, 81.9, 79.9,
(75 MHz, CDCl3, 25 ꢀC):
d
¼ 174.9, 172.7, 172.0, 138.6, 138.0, 128.6,
77.4, 75.7, 74.9, 73.3, 70.7, 61.7, 55.1; m.p. 187e189 ꢀC; IR (neat):
128.4, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 98.1, 97.0, 92.1, 91.1, 84.6,
84.1, 83.3, 82.8, 81.2, 75.4, 75.1, 74.9, 74.8, 74.4, 70.8, 70.9, 68.8, 68.1,
66.2, 66.1, 65.7, 59.0, 58.9, 58.7, 52.3, 50.9, 41.0, 36.4, 29.9, 26.9, 21.9,
n
¼ 3467 (OeH); C28H32O6: calcd C 72.39, H 6.94; found C 72.39, H
7.17; MS (ESI, ion polarity positive, MeOH): m/z: 487 [M þ Na]þ.
19.2, 18.7, 18.6; m.p. 136e138 ꢀC; IR (neat):
n
¼ 3273 (OeH), 3034
4.1.2.4. Methyl 6-O-[2-(carboxy-tert-butyl)ethyl]-2,3,4-tri-O-benzyl-
a-D-glucopyranoside 9. To a suspension of 8 (5.0 g, 10.8 mmol) in
(NeH), 2963 (NeH), 1745 (C]O), 1633 (C]O); C42H56N2O10: calcd C
67.36, H7.54, N3.74;found C 67.16,H 7.66, N 3.43;MS(ESI, ionpolarity
positive): m/z: 771 [M þ Na]þ; HRMS (TOF ESI, ion polarity positive):
calcd for C42H56N2O10Na: 771.3833; found: 771.3841.
tert-butylacrylate (3.13 ml, 21.6 mmol) were added TBAB (0.52 g,
1.62 mmol) and a 20% aqueous solution of sodium hydroxide
(47 ml). The reaction mixture was stirred for 4 h then water
(100 ml) and EtOAc (100 ml) were added. The different layers were
separated and the aqueous layer was extracted with EtOAc
(2 ꢄ 100 ml). The combined organic layers were dried over MgSO4,
4.1.2.7. Synthesis of compound 12. To a solution of 11 (805 mg,
1.1 mmol) in DCM (5 ml) were added successively NaH (56 mg,
1.4 mmol) and trichloroacetonitrile (931 mg, 6.5 mmol). The

5966
B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
1
reaction mixture was stirred 3 h at rt then the solvent was removed
under reduced pressure and the residue was dissolved in DCM
(7 ml). Azidopropanol (131 mg, 1.3 mmol) and BF3$OEt2 (153 mg,
1.1 mmol) were added and the reaction mixture was stirred 3 h
at ꢁ20 ꢀC then quenched by addition of ice. The quenched reaction
was extracted by DCM and the organic layer was washed by water
(1 ml), an aqueous 5% NaHCO3 solution (1 ml) and brine (1 ml). The
organic layer was dried over MgSO4, filtered and evaporated under
reduced pressure. The crude residue was purified by chromatog-
raphy on a silica gel column (DCM/OEt2, 8:2) to afford the desired
as a yellow oil (355 mg, 78%). H NMR (300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.36e7.23 (m,15H, Harom), 6.96 (d, 3J(H, H) ¼ 8.4 Hz,1H, NHeVal),
6.36 (d, 3J(H, H) ¼ 7.8 Hz,1H, NHeLeu), 4.99e4.49 (massif, 7.5H, H1
,
b
3 ꢄ PhCH2, CHaeLeu), 4.38 (d, 3J(H, H) ¼ 7.8 Hz, 0.5H, H1a), 4.15 (t,
3J(H, H) ¼ 8.0 Hz,1H, CHaeVal), 3.96 (m, 1H, H3), 3.84e3.39 (m,14H,
H2, H4, H5, H6, OeCH2eCH2eCH2eN3, OeCH2eCH2CO, OCH3), 2.47
(m, 2H, OCH2eCH2eCO), 2.07 (m, 1H, CHbeVal), 1.81 (m, 2H,
OCH2eCH2eCH2NH2), 1.67e1.53 (m, 3H, CH2beLeu and CHgeLeu),
1.01e0.89 (m, 12H, CH3eVal and CH3eLeu); 13C NMR (75 MHz,
CDCl3, 25 ꢀC):
d
¼ 173.2, 172.3, 171.7, 171.4, 171.3, 138.8, 138.5, 138.3,
product 12 as a colorless oil (531 mg, 59%, ratio
a
:
b
¼ 1:1). 1H NMR
138.2, 177.9, 128.5, 128.4, 128.1, 128.0, 127.9, 127.9, 127.8, 103.9, 97.1,
84.5, 82.1, 80.1, 77.7, 74.4, 70.1, 75.0, 75.2, 75.1, 74.9, 73.3, 70.2, 69.8,
67.7, 67.4, 67.2, 66.4, 58.9, 52.3, 51.1, 50.8, 41.3, 41.0, 39.3, 36.7, 30.5,
(400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.34e7.26 (m, 15H, Harom), 6.69 (d,
3J(H, H) ¼ 8.9 Hz, 1H, NHeVal), 6.30 (d, 3J(H, H) ¼ 8.2 Hz, 1H,
NHeLeu), 5.00e4.52 (massif, 7.5H, H1b, 3 ꢄ PhCH2, CHaeLeu), 4.37
(d, 3J(H, H) ¼ 7.8 Hz, 0.5H, H1a), 4.20 (m, 1H, CHaeVal), 3.96 (m, 1H,
H3), 3.76e3.37 (m, 14H, H2, H4, H5, H6, OeCH2eCH2eCH2eN3,
OeCH2eCH2CO, OCH3), 2.48 (m, 2H, OCH2eCH2eCO), 2.10 (m, 1H,
CHbeVal), 1.85 (m, 2H, OCH2eCH2eCH2N3), 1.62e1.52 (m, 3H,
30.5, 24.8, 22.8, 22.0, 21.9, 19.3, 18.8,18.4; IR (neat):
n
¼ 3306 (NeH),
1744 (C]O), 1641 (C]O); MS (ESI, ion polarity positive): m/z: 806
[M þ H]þ; HRMS (TOF ESI, ion polarity positive): calcd for
C45H64N3O10: 806.4592; found: 806.4590.
CH2beLeu and CHgeLeu), 0.97e0.86 (m, 12H, CH3eVal and
4.1.2.10. Synthesis of compound 15. Same procedure as described
for 14 from compound 13 (711 mg, 0.87 mmol) to afford the desired
product 15 as a pale yellow oil (688 mg, 76%). 1H NMR (300 MHz,
13
CH3eLeu); C NMR (75 MHz, CDCl3, 25 ꢀC):
d
¼ 173.1, 171.3, 170.9,
138.8, 138.5, 138.3, 138.2, 138.1, 128.4, 128.3, 128.0, 127.9, 127.9,
127.8, 127.7, 127.6, 127.5, 103.6, 97.0, 84.6, 82.2, 81.9, 80.1, 75.6, 75.6,
75.0, 74.9, 74.9, 74.6, 73.1, 67.0, 69.8, 69.4, 67.5, 67.2, 66.6, 64.8, 59.9,
58.5, 53.4, 52.2, 48.5, 48.3, 41.3, 37.0, 36.7, 30.6, 30.5, 29.2, 28.8,
CDCl3, 25 ꢀC):
d
¼ 7.72 (m, 1H, NH), 7.33e7.25 (m, 15H, Harom), 6.94
(d, 3J(H, H) ¼ 8.4 Hz, 1H, NH), 6.80 (m, 1H, NH), 6.55 (d, 3J(H, H) ¼
7.8 Hz, 1H, NH), 5.00e4.51 (m, 7.5H, H1b, 3 ꢄ PhCH2 and CHaeLeu),
4.34 (d, 3J(H, H) ¼ 7.8 Hz, 0.5H, H1a), 4.18 (m, 1H, CHa-Val), 3.97 (m,
1H, H3), 3.80e2.89 (massif, 16H, NH2, H2, H4, H5, H6, OCH2CH2NH2,
OCH2 and OCH3), 2.48 (m, 2H, CH2eCO), 2.09 (m, 1H, CHbeVal),
1.65e1.52 (m, 3H, CH2beLeu and CHgeLeu), 1.03e0.87 (m, 12H,
24.9, 22.7, 21.9, 19.2, 18.4; IR (neat):
n
¼ 3290 (NeH), 2097 (N3), 1745
(C]O), 1640 (C]O); MS (ESI, ion polarity positive): m/z: 832
[M þ H]þ, 654 [M þ Na]þ; HRMS (TOF ESI, ion polarity positive):
calcd for C45H61N5O10Na: 854.4316; found: 854.4319.
CH3eVal and CH3eLeu); 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d
¼ 173.1,
4.1.2.8. Synthesis of compound 13. To a solution of 11 (1.5 g,
2.0 mmol) in DCM (8 ml) were added successively NaH (90 mg,
2.2 mmol) and trichloroacetonitrile (1.74 g, 12 mmol). The reaction
mixture was stirred 3 h at rt then the solvent was removed under
reduced pressure and the residue was dissolved in DCM (14 ml).
Azidoethanol (209 mg, 2.4 mmol) and BF3$OEt2 (284 mg, 2 mmol)
were added and the reaction mixture was stirred 3 h at ꢁ20 ꢀC then
quenched by addition of ice. The quenched reaction was extracted
by DCM and the organic layer was washed by water (3 ml), an
aqueous 5% NaHCO3 solution (3 ml) and brine (3 ml). The organic
layer was dried over MgSO4, filtered and evaporated under reduced
pressure. The crude residue was purified by chromatography on
a silica gel column (DCM/Et2O, 8:2) to afford the desired product 13
171.7, 171.3, 171.1, 138.7, 138.3, 138.2, 138.1, 128.4, 128.4, 128.3, 128.0,
127.9, 127.9, 127.6, 104.5, 97.2, 84.5, 82.0, 81.9, 80.0, 74.3, 75.6, 75.0,
75.0, 74.3, 73.2, 70.6, 70.0, 69.7, 67.8, 67.3, 59.7, 58.7, 52.2, 51.0, 50.7,
41.5, 41.2, 36.8, 36.3, 30.3, 30.3, 24.8, 22.8, 21.9, 19.2, 18.9, 18.3; IR
(neat):
n
¼ 3262 (NeH), 3206 (NeH), 3178 (NeH), 1745 (C]O), 1641
(C]O); C42H56N2O10 þ 3.6H2O: calcd C 61.68, H 8.02, N 4.90; found
C 61.72, H 7.76, N 4.77; MS (ESI, ion polarity positive): m/z: 792
[M þ H]þ; HRMS (TOF ESI, ion polarity positive): calcd for
C44H62N3O10: 792.4435; found: 792.4456.
4.1.3. Synthesis of compounds 16 and 17
To a solution of N-BoceL-Val (172 mg, 0.79 mmol) in DMF
(15 ml) were added HBTU (300 mg, 0.79 mmol), HOBt (107 mg,
0.79 mmol), DIPEA (0.21 ml, 1.2 mmol) and 14 (319 mg, 0.4 mmol).
The reaction mixture was stirred for 40 h at rt then the solvent was
removed under reduced pressure and the residue was dissolved in
EtOAc (25 ml). This solution was washed with water (25 ml), an
aqueous 10% citric acid solution (25 ml), an aqueous saturated
NaHCO3 solution (25 ml) and brine (25 ml). The organic layer was
dried over MgSO4, filtered and evaporated under reduced pressure.
The crude residue was purified by chromatography on a silica gel
column (cyclohexane/AcOEt, 35:65 then 20:80) to afford 16 as
a white solid (222 mg, 56%) and 17 as a pale yellow solid (138 mg,
35%).
as a colorless oil (810 mg, 49%, ratio
a
:
b
¼ 1:1). 1H NMR (400 MHz,
CDCl3, 25 ꢀC):
d
¼ 7.37e7.26 (m, 15H, Harom), 6.77 (m, 1H, NHeVal),
6.46 (m, 1H, NHeLeu), 5.00e4.44 (m, 7.5H, H1b, 3 ꢄ PhCH2 and
CHaeLeu), 4.43 (d, 3J(H, H) ¼ 7.6 Hz. 0.5H, H1a), 4.22 (m, 1H,
CHaeVal), 3.96 (m, 1H, H3), 3.81e3.41 (m, 14H, H2, H4, H5, H6,
OCH2CH2N3, OCH2 and OCH3), 2.48 (m, 2H, CH2eCO), 2.29 (m, 1H,
CHbeVal), 1.65e1.51 (m, 3H, CH2beLeu and CHgeLeu), 0.98e0.87
(m, 12H, CH3eVal and CH3eLeu); 13C NMR (75 MHz, CDCl3, 25 ꢀC):
d
¼ 173.0, 171.3, 171.3, 170.9, 170.8, 138.8, 138.5, 138.4, 138.3, 138.1,
128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5,
127.5, 103.6, 97.2, 84.5, 82.1, 81.6, 81.6, 79.9, 75.6, 75.6, 74.9, 74.8,
74.6, 73.1, 69.8, 69.4, 68.1, 67.4, 67.1, 70.1, 58.4, 52.1, 51.0, 50.7, 50.6,
41.2, 36.9, 36.7, 30.7, 30.6, 24.8, 22.7, 21.9, 19.2, 18.4, 18.2, IR (neat):
4.1.3.1. Compound 16. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
n
¼ 3270 (OeH), 2102 (N3), 1745 (C]O), 1638 (C]O); MS (ESI, ion
d
¼ 7.26e7.22 (m,15H, Harom), 7.09 (br s,1H, OCH2CH2CH2eNH), 6.77
polarity positive): m/z: 818 [M þ H]þ; HRMS (TOF ESI, ion polarity
(d, 3J(H, H) ¼ 6.5 Hz, 1H, NHeLeu), 6.64 (d, 3J(H, H) ¼ 7.7 Hz, 1H,
NHeVal), 5.24 (br s, 1H, NHeBoc), 4.90e4.50 (m, 7H, 3 ꢄ PhCH2,
CHaeLeu), 4.33 (d, 3J(H, H) ¼ 7.9 Hz 1H, H1a), 4.25 (m, 1H, CHaeVal),
4.02 (m, 1H, CHaeBocVal), 3.88 (m, 1H, OeCHHeCH2eCH2eNH),
3.72e3.58 (m, 9H, H3, H6, OeCHHeCH2eCHHeNH, OeCHHeCH2CO,
OCH3), 3.48e3.33 (m, 4H, H2, H4, H5, OeCH2eCH2eCHHeNH), 3.18
(m, 1H, OeCHHeCH2CO), 2.37 (m, 2H, OCH2eCH2eCO), 2.08e1.78
(m, 4H, OCH2eCH2eCH2NH, 2 ꢄ CHbeVal), 1.61e1.49 (m, 3H,
CH2beLeu and CHgeLeu), 1.39 (s, 9H, CH3eBoc), 0.95e0.85 (m, 18H,
positive): calcd for C44H59N5O10Na: 840.4160; found: 840.4160.
4.1.2.9. Synthesis of compound 14. A solution of 12 (470 mg,
0.56 mmol) and triphenylphosphine (297 mg, 1.1 mmol) in THF/
water 9:1 (2.8 ml) was stirred for 24 h. The solvent was removed
under reduced pressure and the crude residue was purified by
chromatography on a silica gel column (DCM/Et2O, 2:8 then DCM/
MeOH/aqueous sol NH4OH, 87:9:4) to afford the desired product 14

B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
5967
13
2 ꢄ CH3eVal and CH3eLeu); C NMR (75 MHz, CDCl3, 25 ꢀC):
36.7, 36.6, 31.3, 30.5, 28.3, 24.8, 22.7, 21.9, 19.3, 19.1, 18.4, 17.8; m.p.
d
¼ 173.0, 171.4, 138.6, 138.4, 138.1, 128.4, 128.4, 128.0, 127.8, 127.7,
160e162 ꢀC; C54H78N4O13: calcd C 65.43, H 7.93, N 5.65; found C
127.6,103.8, 84.6, 82.2, 81.0, 77.6, 75.6, 75.0, 74.8, 69.7, 67.7, 67.7, 59.2,
58.4, 52.1, 50.9, 40.8, 36.8, 36.4, 31.7, 30.9, 29.7, 28.4, 24.8, 22.8, 21.8,
65.41, H 7.94, N 5.55; IR (neat):
n
¼ 3281 (NeH), 2955 (NeH), 1640
(C]O); MS (ESI, ion polarity positive, MeOH): m/z: 992 [M þ H]þ;
HRMS (TOF, ion polarity positive): calcd for C54H78N4O13Na:
1013.5463; found: 1013.5447.
19.1, 18.7; m.p. 140e142 ꢀC; IR (neat):
n
¼ 3310 (NeH), 2961 (NeH),
1637 (C]O); MS (ESI, ion polarity positive): m/z: 1006 [M þ H]þ;
HRMS (TOF ESI, ion polarity positive): calcd for C55H80N4O13Na:
1027.5620; found: 1027.5619.
4.1.5. Synthesis of protected peptidomimetics 20e23
4.1.5.1. Compound 20. A solution of 16 (124 mg, 0.123 mmol) in
DCM/TFA 1:1 (0.6 ml) was stirred for 2 h at rt then the solvent was
removed under reduced pressure and the residue was dissolved in
4.1.3.2. Compound 17. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.37e7.26 (m,15H, Harom), 6.82e6.79 (br s, 2H, OCH2CH2CH2eNH
and NHeLeu), 6.45 (br s, 1H, NHeVal), 5.20 (br s, 1H, NHeBoc),
5.03e4.50 (m, 8H, H1
DMF (1.5 ml). To the resulting solution were added N-BoceL-alanine
,
3 ꢄ PhCH2, CHaeLeu), 4.22 (m, 1H,
(47 mg, 0.25 mmol), HBTU (94 mg, 0.25 mmol), HOBt (33 mg,
0.25 mmol) and DIPEA (0.09 ml, 0.50 mmol). The reaction mixture
wasstirredfor48 hatrtthenthesolventwasremovedunderreduced
pressure and the residue was dissolved in EtOAc (8 ml). This solution
was washed with water (8 ml), an aqueous 10% citric acid solution
(8 ml), an aqueous saturated NaHCO3 solution (8 ml) and brine
(8 ml). The organic layer was dried over MgSO4, filtered and evapo-
rated under reduced pressure. The crude residue was purified by
chromatography on a silica gel column (DCM/MeOH, 98:2) to afford
20 as a white solid (91 mg, 68%). 1H NMR (300 MHz, CDCl3, 25 ꢀC):
b
CHaeBocAla), 4.07e3.96 (m, 2H, H3, CHaeVal), 3.75e3.62 (m, 8H, H5,
H6, OeCHHeCH2eCH2eNH, OeCH2eCH2CO, OCH3), 3.54e3.39 (m,
5H, H2, H4, H6, OeCHHeCH2eCHHeNH), 3.25 (m, 1H,
OeCH2eCH2eCHHeNH), 2.47 (m, 2H, OCH2eCH2eCO), 2.07e1.82
(m, 4H, OCH2eCH2eCH2NH, 2 ꢄ CHbeVal), 1.64e1.51 (m, 3H,
CH2beLeu and CHgeLeu), 1.42 (s, 9H, CH3eBoc), 0.95e0.85 (m, 18H,
13
2 ꢄ CH3eVal and CH3eLeu); C NMR (75 MHz, CDCl3, 25 ꢀC):
d
¼ 171.3, 171.2, 139.0, 138.1, 128.5, 128.4, 128.4, 128.1, 128.0, 127.8,
127.5, 97.4, 84.6, 82.1, 81.0, 77.9, 75.7, 75.0, 70.0, 67.2, 59.2, 58.4, 52.2,
50.9, 41.1, 37.5, 36.7, 31.7, 30.9, 29.7, 28.4, 24.8, 22.8, 21.9, 19.4, 19.2,
d
¼ 7.64e7.41 (br s,1H, NH), 7.37e7.26 (m,15H, Harom), 6.87e6.76 (br
18.4; m.p.144e146 ꢀC; IR (neat):
n
¼ 3304 (NeH), 2952 (NeH),1645
s, 2H, NH), 5.26 (br s, 1H, NH-Boc), 4.93e4.16 (m, 11H, H1a, H3,
3 ꢄ PhCH2, CHaeLeu4, CHaeVal3, CHaeAla1), 3.87e3.13 (m, 15H, H2,
H4, H5, H6, CHaeVal2, OeCH2eCH2eCH2eNH, OCH2eCH2eCO,
OCH3), 2.37 (m, 2H, OCH2eCH2eCO), 2.10e1.82 (m, 4H,
OCH2eCH2eCH2NH, 2 ꢄ CHbeVal), 1.66e1.51 (m, 3H, CH2beLeu and
CHgeLeu), 1.43 (s, 9H, CH3eBoc), 1.33 (d, 3J(H, H) ¼ 6.9 Hz, 3H,
CH3eAla), 0.97e0.89 (m, 18H, 2 ꢄ CH3eVal and CH3eLeu); 13C NMR
(C]O); MS (ESI, ion polarity positive): m/z: 1006 [M þ H]þ; HRMS
(TOF, ion polarity positive): calcd for C55H80N4O13Na: 1027.5620;
found: 1027.5627.
4.1.4. Synthesis of compounds 18 and 19
Same procedure as described for 16 and 17 from compound 15
(457 mg, 0.58 mmol) to afford 18 as a white solid (256 mg, 45%) and
19 as a yellowish solid (226 mg, 40%).
(75 MHz, CDCl3, 25 ꢀC):
138.4, 138.0, 128.5, 128.4, 128.1, 127.9, 127.8, 127.6, 104.2, 84.5, 80.3,
77.3, 75.6, 75.1, 74.9, 74.8, 58.5, 69.4, 67.9, 67.7, 59.1, 58.5, 52.3, 50.9,
40.8, 35.9, 30.9, 30.1, 28.3, 24.8, 22.8, 21.8, 19.3, 19.1, 18.0, 18.8; m.p.
d
¼ 173.9,173.1,171.7,171.7,171.3,155.8,138.5,
4.1.4.1. Compound 18. 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.30e7.25 (m,15H, Harom), 7.17 (br s,1H, OCH2CH2eNH), 7.06 (br s,
decomposition; IR (neat):
n
¼ 3281 (NeH), 2963 (NeH),1637 (C]O);
1H, NHeVal), 6.80 (br s, 1H, NHeLeu), 5.29 (br s, 1H, NHeBoc),
4.94e4.53 (m, 7H, and3 ꢄ PhCH2, CHaeLeu), 4.38 (t, 3J(H, H) ¼ 7.8 Hz,
1H, H1a), 4.25 (m,1H, CHaeVal), 3.97 (m,1H, CHaeBocVal), 3.82e3.36
(m, 15H, H2, H3, H4, H5, H6, OCH2CH2NH, OeCH2eCH2CO, OCH3), 2.55
(m, 2H, OCH2eCH2eCO), 2.13e1.99 (m, 2H, 2 ꢄ CHbeVal), 1.66e1.56
(m, 3H,CH2beLeu andCHgeLeu),1.43(s, 9H,CH3eBoc),0.99e0.87 (m,
MS (ESI, ion polarity positive): m/z: 1076 [M þ H]þ; HRMS (TOF ESI,
ion polarity positive): calcd for C58H85N5O14Na: 1098.5991; found:
1098.5964.
4.1.5.2. Compound 21. Same procedure as described for 20 from
compound 17 (197 mg, 0.196 mmol) to afford 21 as a pale yellow
13
18H, 2 ꢄ CH3eVal and CH3eLeu); C NMR (75 MHz, CDCl3, 25 ꢀC):
solid (172 mg, 88%). 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.34e7.25
d
¼ 172.9, 171.8, 171.2, 171.3, 155.9, 138.5, 138.3, 138.0, 128.4, 128.4,
(massif, 15H, Harom), 7.02 (br s, 1H, OCH2CH2CH2eNH), 6.88e6.80
(m, 3H, NHeLeu, NHeAla, NHeVal), 5.14 (br s, 1H, NHeBoc),
128.0, 127.8, 127.7, 127.6, 103.8, 84.5, 82.2, 77.5, 75.6, 75.0, 74.0, 74.6,
69.8, 67.7, 67.4, 59.9, 58.9, 52.2, 51.0, 40.8, 40.0, 36.5, 36.4, 31.6, 30.5,
28.3, 24.8, 22.7, 21.8, 19.2, 19.1, 18.7, 17.8; m.p. 144e146 ꢀC;
C54H78N4O13 þ 6.0H2O: calcd C 59.0, H 8.25, N 5.10; found C 58.78, H
5.02e4,53 (m, 8H, H1
,
H3, 3 ꢄ PhCH2), 4.32e4.26 (m, 2H,
b
CHaeLeu4, CHaeVal3), 4.16 (m, 1H, CHaeAla1), 3.96 (t, 3J(H, H) ¼
9.3 Hz,
1H,
CHaeVal2),
3.78e3.64
(m,
8H,
H2,
7.29, N 4.95; IR (neat):
n
¼ 3283 (NeH), 2957 (NeH),1636 (C]O); MS
OeCH2eCH2eCH2eNH, OeCH2eCH2CO, OCH3), 3.54e3.23 (m, 6H,
H4, H5, H6, OeCH2eCH2eCH2eNH), 2.37 (t, 3J(H, H) ¼ 5.4 Hz, 2H,
OCH2eCH2eCO), 2.08 (m, 2H, OCH2eCH2eCH2NH), 1.86e1.82 (m,
2H, 2 ꢄ CHbeVal), 1.66e1.53 (m, 3H, CH2beLeu and CHgeLeu), 1.43
(s, 9H, CH3eBoc), 1.33 (d, 3J(H, H) ¼ 6.9 Hz, 3H, CH3eAla), 0.94e0.86
(m, 18H, 2 ꢄ CH3eVal and CH3eLeu); 13C NMR (75 MHz, CDCl3,
(ESI, ion polarity positive): m/z: 992 [M þ H]þ; HRMS (TOF ESI, ion
polarity positive): calcd for C54H78N4O13Na: 1013.5463; found:
1013.5443.
4.1.4.2. Compound 19. 1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.33e7.25 (m, 15H, Harom), 6.92 (br s, 1H, OCH2CH2eNH), 6.77
25 ꢀC):
d
¼ 173.1, 172.7, 171.3, 170.0, 166.0, 155.0, 138.9, 138.2, 128.5,
(br s, 1H, NHeVal), 6.47 (br s, 1H, NHeLeu), 5.10 (sl, 1H, NHeBoc),
4.99e4.51 (m, 8H, H1b, 3 ꢄ PhCH2 and CHaeLeu), 4.21 (m, 1H,
CHaeVal), 3.98e3.93 (m, 2H, H3, CHaeBocVal), 3.78 (m, 1H, H5),
3.70e3.65 (m, 7H, H2, H6, OeCH2eCH2NH, OCH3), 3.57e3.48 (m,
5H, H6, OCH2eCH2eNH, OeCH2eCH2CO), 3.37 (t, J ¼ 9.6 Hz, 1H, H4),
2.47 (m, 2H, OCH2eCH2eCO), 2.13e1.99 (m, 2H, 2 ꢄ CHbeVal),
1.65e1.51 (m, 3H, CH2beLeu and CHgeLeu), 1.42 (s, 9H, CH3eBoc),
0.98e0.85 (m, 18H, 2 ꢄ CH3eVal and CH3eLeu); 13C NMR (100 MHz,
128.5, 128.4, 128.1, 128.0, 127.9, 127.6, 97.4, 82.1, 80.0, 78.1, 75.8, 75.1,
73.4, 71.2, 70.0, 67.3, 67.3, 58.5, 58.4, 52.2, 57.4, 57.1, 41.2, 37.2, 36.9,
31.0, 30.9, 29.5, 28.3, 24.2, 22.8, 22.0, 19.4, 19.2, 17.8, 18.4; m.p.
decomposition; IR (neat):
n
¼ 3294 (NeH), 2968 (NeH), 1636 (C]
O); MS (ESI, ion polarity positive): m/z: 1076 [M þ H]þ; HRMS (TOF
ESI, ion polarity positive): calcd for C58H85N5O14Na: 1098.5991;
found: 1098.5939.
CDCl3, 25 ꢀC):
d
¼ 173.0, 171.8, 172.2, 171.1, 155.9, 138.7, 138.1, 138.0,
4.1.5.3. Compound 22. Same procedure as described for 20 from
compound 18 (135 mg, 0.136 mmol) to afford 22 as a white solid
128.5, 128.4, 128.0, 127.9, 127.8, 127.7, 127.6, 97.1, 82.0, 79.9, 78.0,
75.6, 75.0, 73.3, 70.3, 70.1, 70.1, 67.1, 59.7, 58.7, 52.2, 50.8, 41.1, 39.0,
(138 mg, 76%). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.36e7.25 (m,

5968
B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
16H, Harom, NH), 7.14 (d, 3J(H, H) ¼ 9.0 Hz, 1H, NH), 6.90 (d, 3J(H,
H) ¼ 7.2 Hz, 1H, NH), 6.78 (d, 3J(H, H) ¼ 9.3 Hz, 1H, NH), 5.36 (br s,
1H, NHeBoc), 4.95e4.55 (m, 7H, H1a, 3 ꢄ PhCH2), 4.38e4.26 (m, 3H,
CHaeLeu, CHaeVal4, H3), 4.17 (m, 1H, CHaeVal2), 3.96 (m, 1H,
CHaeAla1), 3.84e3.25 (m, 14H, H2, H4, H5, H6, OCH2CH2NH,
OeCH2eCH2CO, OCH3), 2.59 (m, 2H, OCH2eCH2eCO), 2.14e1.96 (m,
2H, 2 ꢄ CHbeVal), 1.68e1.51 (m, 3H, CH2beLeu and CHgeLeu), 1.43
(s, 9H, CH3eBoc), 1.33 (d, 3J(H, H) ¼ 6.9 Hz, 3H, CH3eAla1),
0.98e0.90 (m, 18H, 2 ꢄ CH3eVal and CH3eLeu); 13C NMR (100 MHz,
HCl, 100 mM NaCl at pH 7.4. The concentration of IAPP was held
constant at 5 M and the peptidomimetics were added in equi-
molar amounts to yield 1:10 and 1:1 molar ratio mixtures. For
b1e40, the ThT was measured on aliquots of Ab1e40 incubations (20
M) made in 10 mM Tris/HCl, 100 mM NaCl at pH 7.4. For the
m
A
m
studies on the effects of the peptidomimetics on Ab1e40 fibrillo-
genesis at different time points, stock solutions of peptidomimetics
were mixed with
a stock solution of Ab1e40 at different
Ab1e40:peptidomimetics ratios (1:1 and 1:0.1) in a mixture of 10 mM
CDCl3, 25 ꢀC):
d
¼ 172.7, 171.2, 170.9, 138.5, 138.3, 137.9, 128.5, 128.4,
ThT and 10 mM Tris/HCl, 100 mM NaCl at pH 7.4. The ThT assays
were performed between 2 and 6 times, on different days, using
different IAPP and Ab1e40 stock solutions.
128.1, 128.1, 127.9, 127.8, 127.6, 104.1, 84.5, 82.2, 77.6, 75.6, 75.1, 74.9,
74.7, 70.0, 67.8, 58.5, 58.2, 52.1, 50.8, 41.1, 40.3, 36.8, 31.8, 30.9, 28.3,
24.8, 22.7, 22.0, 19.3, 19.2, 18.5, 18.1; m.p. decomposition; IR (neat):
The assessment of inhibition of amyloid peptide aggregation by
peptidomimetic was determined as followed. Amyloid peptide
aggregation assays were performed in the absence and presence of
peptidomimetic and fibril formation was monitored via ThT fluo-
rescence to evaluate each compound’s ability to alter both the lag
time to aggregate formation and the equilibrium plateau indicative
of the quantity of aggregate formed. The extension of the lag time
was evidenced as an increase in the experimental lag time in the
presence of the peptidomimetic relative to the lag time observed
n
¼ 3273 (NeH), 2964 (NeH), 1638 (C]O); MS (ESI, ion polarity
positive): m/z: 1063 [M þ H]þ; HRMS (TOF ESI, ion polarity posi-
tive): calcd for C57H83N5O14Na: 1084.5834; found: 1084.5862.
4.1.5.4. Compound 23. Same procedure as described for 20 from
compound 19 (177 mg, 0.18 mmol) to afford 23 as a pale yellow
solid (142 mg, 75%). 1H NMR (400 MHz, CDCl3, 25 ꢀC):
d
¼ 7.33e7.24
(m, 15H, Harom), 7.17 (m, 1H, NH), 6.95 (d, 3J(H, H) ¼ 8.7 Hz, 1H, NH),
6.87 (d, 3J(H, H) ¼ 8.1 Hz,1H, NH), 6.81 (d, 3J(H, H) ¼ 8.7 Hz,1H, NH),
4.87e4.53 (m, 8H, H1b, H3, 3 ꢄ PhCH2), 4.33 (t, 3J(H, H) ¼ 8.1 Hz, 1H,
CHaeLeu), 4.24 (t, 3J(H, H) ¼ 7.2 Hz, 1H, CHaeVal4), 4.16 (m, 1H,
without the peptidomimetic, and is evaluated as
a ratio,
tA þ peptidomimetic/tAb. The reduction of the equilibrium plateau was
b
evidenced as a decrease in the experimental fluorescence plateau
with the peptidomimetic, relative to the fluorescence plateau
observed without the peptidomimetic, and is evaluated as the
percentage decrease from the experimental fluorescence plateau
CHaeVal2), 3.97e3.25 (m, 15H, H2, H4, H5, H6, OCH2CH2NH, CHa
-
eAla1, OeCH2eCH2CO, OCH3), 2.52 (m, 2H, OCH2eCH2eCO),
2.09e2.01 (m, 2H, 2 ꢄ CHbeVal), 1.66e1.54 (m, 3H, CH2beLeu and
CHgeLeu), 1.43 (s, 9H, CH3eBoc), 1,31 (d, 3J(H, H) ¼ 7.2 Hz, 3H,
CH3eAla1), 0.94e0.89 (m, 18H, 2 ꢄ CH3eVal and CH3eLeu); 13C
with the peptidomimetic, (FAb ꢁ FA þ peptidomimetic)/FAb ꢄ 100%.
b
NMR (100 MHz, CDCl3, 25 ꢀC):
128.5, 128.4, 128.0, 127.9, 127.8, 127.6, 97.3, 82.0, 79.9, 78.1, 75.7,
74.9, 73.4, 70.5, 70.4, 67.6, 67.2, 58.5, 58.4, 52.1, 50.8, 41.2, 39.3, 36.8,
31.3, 31.0, 28.3, 24.8, 22.7, 22.0, 19.3, 19.2, 18.4; m.p. decomposition;
d
¼ 173.1, 172.8, 171.2, 171.0, 138.1,
4.4. Transmission electron microscopy
TEM was performed at the Institut de Biologie Intégrative
(IFR83) at the University Pierre et Marie Curie. Peptides were
incubated under the same conditions as in the ThT assay. Aliquots
IR (neat):
n
¼ 3275 (NeH), 2957 (NeH), 1639 (C]O); MS (ESI, ion
polarity positive): m/z: 1063 [M þ H]þ; HRMS (TOF ESI, ion polarity
(20 mL) of this mixture were adsorbed onto carbon-coated 300-
positive): calcd for C57H83N5O14Na: 1084.5834; found: 1084.5801.
mesh copper grids for 2 min. The grids were then blotted and
dried. Next, the grids were negatively stained for 45 s on 2.5%
uranyl acetate in 1:1 ethanol/water, and again blotted and dried.
The grids were examined using a ZEISS 912 Omega electron
microscope operating at 80 kV.
4.2. Amyloid peptide preparation
The syntheses of Ab1e40 and IAPP were performed at the Institut
de Biologie Intégrative (IFR83) at the University Pierre et Marie
Curie.
A
b1e40 and IAPP with an amidated C-terminus were
4.5. NMR studies
synthesized using Fmoc chemistry. The peptides were then purified
by HPLC and characterized by MALDI-TOF mass spectrometry. IAPP
and Ab1e40 were dissolved in hexafluoro-isopropanol (HFIP) at
a concentration of 1 mM and 0.5 mM respectively, and incubated
for at least 1 h to dissolve any preformed aggregates. Next, HFIP was
evaporated with dry nitrogen gas followed by vacuum desiccation
for at least 1 h. The resulting peptide film was then dissolved in
DMSO for the microscopy and ThT experiments to obtain stock
solutions of IAPP (0.2 mM) and Ab1e40 (0.4 mM). For those exper-
iments, we used the same concentration of DMSO (2.5% in the final
volume) and hence we were able to compare for those experiments
the shape of the curve and the lag time.
NMR experiments on peptidomimetics 1e4 were recorded on
a Bruker AVANCE III 500 MHz spectrometer equipped with a TCI
triple resonance cryogenic probe with Z-axis gradient. Experiments
were processed with Bruker TOPSPIN 2.0 software. 1H and 13C
resonances were assigned in CDCl3, using 2D COSY, 2D TOCSY
(70 ms mixing time), 2D ROESY (300 ms mixing time), 2D 1He13C
HSQC and 2D 1He13C HMBC spectra. 1D spectra were acquired over
32 K data points using a spectral width of 5000 Hz. Typically, 512
increments were acquired over a spectral width of 5000 Hz. Prior to
Fourier Transformation in t2 and t1 dimensions, time domain data
were zero-filled and multiplied by a
p/3-shifted square sinebell
function. The 1H and 13C resonance assignments are listed in
Tables S1eS4 (Supplementary data). The temperature gradients of
the amide proton chemical shifts were derived from 1D and 2D
TOCSY spectra recorded at 279.5, 284.5, 294.5, 299.5 K. 3J coupling
constants were measured on 1D spectra at 284.5 K.
4.3. Thioflavin-T assay
Thioflavin-T binding assays were used to measure the devel-
opment of fibrils over time. A plate reader (Fluostar Optima,
BmgLabtech) and standard 96-wells flat-bottom black microtiter
plates in combination with a 440 nm excitation filter and a 485 nm
emission filter were used. For the IAPP/peptidomimetics experi-
4.5.1. Conformation studies of peptidomimetics 1e4
The conformation of peptidomimetics 1e4 was studied in
aqueous solution, either in H2O/D2O (90:10 v/v) or in 50 mM
sodium phosphate, 150 mM NaCl, pH 7.0 containing 10% D2O. The
1H resonance assignments are listed in Tables S5eS8. TOCSY
ments, the ThT assay was started by adding 5
stock solution and between 0.5 and 50 L of a 0.2 mM peptidomi-
metics stock solution to a mixture of 10 M ThT and 10 mM Tris/
mL of a 0.2 mM IAPP
m
m

B. Dorgeret et al. / European Journal of Medicinal Chemistry 46 (2011) 5959e5969
5969
[10] (a) A. Jan, O. Adolfsson, I. Allaman, A.-L. Buccarello, P.J. Magistretti, A. Pfeifer,
A. Muhs, H.A. Lashuel, J. Biol. Chem. 286 (2011) 8585e8596;
(b) M.F.M. Engel, L. Khemtémourian, C.C. Kleijer, H.J.D. Meeldijk, J. Jacobs,
A.J. Verkeij, B. de Kruijff, J.A. Killian, J.W.M. Höppener, Proc. Natl. Acad. Sci.
U.S.A. 104 (2008) 6033e6038.
experiments were recorded with a mixing time of 70 ms. ROESY
spectra were acquired with a mixing time of 300 ms. The solvent
signal was suppressed using a WATERGATE sequence.
[11] A. Reza, A. Ladiwala, J.S. Dordick, P.M. Tessier, J. Biol. Chem. 286 (2011)
3209e3218.
4.5.2. Interaction studies of peptidomimetic 1 with Ab1e40
The interaction of peptidomimetic 1 with Ab1e40 was studied in
an aqueous solution of 50 mM sodium phosphate,150 mM NaCl, pH
7.0 containing 10% D2O. 2D NOESYexperiments were recorded with
a mixing time of 0.2 s. 1D STD experiments were recorded using
a cascade of Gaussian shaped selective pulses (50 ms duration, B1
field of 0.16 kHz) for a total duration of 2 s, applied at ꢁ1
and þ30 ppm alternatively.
[12] C. Nerelius, A. Sandegren, H. Sargsyan, R. Raunak, H. Leijonmarck,
U. Chatterjee, A. Fisahn, S. Imarisio, D.A. Lomas, D.C. Crowther, R. Strömberg,
J. Johansson, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 9191e9196.
[13] For a review, see C.I. Stains, K. Mondal, I. Ghosh, ChemMedChem 2 (2007)
1674e1692.
[14] W.P. Esler, E.R. Stimson, J.R. Ghilardi, Y.A. Lu, A.M. Felix, H.V. Vinters,
P.W. Mantyh, J.P. Lee, J.E. Maggio, Biochemistry 35 (1996) 13914e13921.
[15] L.O. Tjenberg, J. Näslund, F. Lindqvist, A.R. Karlström, J. Thyberg, L. Terenius,
C. Nordstedt, J. Biol. Chem. 271 (1996) 8545e8548.
[16] T. Takahashi, H. Mihara, Acc. Chem. Res. 41 (2008) 1309e1318.
[17] D.J. Gordon, K.L. Sciarretta, S.C. Meredith, Biochemistry 40 (2001) 8237e8245.
[18] C. Soto, E.M. Sigurdsson, L. Morelli, R.A. Kumar, E.M. Castano, B. Frangione,
Nat. Med. 4 (1998) 822e826.
Acknowledgements
We thank Claire Troufflard and Amaury Griffon du Bellay for
NMR experiments and help with sugar amino acid derivatives
synthesis, respectively. We thank the Ministère de l’Enseignement
Supérieur et de la Recherche (MESR) for financial support of BD.
Christophe Piesse and Géraldine Toutirais (IFR83-Université Pierre
et Marie Curie, France) are acknowledged for the amyloid peptides
syntheses and the electron microscopy, respectively.
[19] A. Frydman-Marom, M. Rechter, I. Shefler, Y. Bram, D.E. Shalev, E. Gazit,
Angew. Chem., Int. Ed. 48 (2009) 1981e1986.
[20] (a) S.A.W. Gruner, V. Truffault, G. Voll, E. Locardi, M. Stöckle, H. Kessler,
Chem.dEur. J. 8 (2002) 4366e4376;
(b) F. Schweizer, Angew. Chem., Int. Ed. 41 (2002) 230e253;
(c) M.D.P. Risseueuw, M. Overhand, G.W.J. Fleet, M.I. Simone, Tetrahedron:
Asymmetry 18 (2007) 2001e2010.
[21] R.F. Hirschmann, K.C. Nicolaou, A.R. Angeles, J.S. Chen, A.B. Smith 3rd, Acc.
Chem. Res. 42 (2009) 1511e1520.
[22] Y. Miura, K. Yasuda, K. Yamamoto, M. Koike, Y. Nishida, K. Kobayashi, Bio-
macromolecules 8 (2007) 2129e2134.
Appendix. Supplementary data
[23] (a) T. Ariga, T. Miyatake, R.K. Yu, J. Neurosci. Res. 88 (2010) 2303e2315;
(b) J.J. Valle-Delgado, M. Alfonso-Prieto, N.S. de Groot, S. Ventura, J. Samitier,
C. Rovira, X. Fernandez-Busquets, FASEB J. 24 (2010) 4250e4261.
[24] C. Rivière, J.C. Delaunay, F. Immel, C. Cullin, J.P. Monti, Neurochem. Res. 34
(2009) 1120e1128.
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.10.008.
[25] C. Jaramillo, J.-L. Chiara, M. Martin-Lomas, J. Org. Chem. 59 (1994) 3135e3145.
[26] P. Sahai, M. Chawla, R.A. Vishwakarma, J. Chem. Soc., Perkin Trans. 1 (2000)
1283e1290.
References
[27] A. Dupraz, P. Guy, C. Dupuy, Tetrahedron Lett. 37 (1996) 1237e1240.
[28] H. Bazin, A. Bouchu, G. Descotes, J. Carbohydr. Chem. 14 (1995) 1187e1207.
[29] K. Jansson, G. Noori, G. Magnusson, J. Org. Chem. 55 (1990) 3181e3185.
[30] H. Rathore, T. Hashimoto, K. Igarashi, H. Nukaya, S.D. Fullerton, Tetrahedron
41 (1985) 5427e5438.
[1] (a) K. Blennow, M.J. de Leon, H. Zetterberg, Lancet 368 (2006) 387e403;
(b) A. Abbott, Nature 475 (2011) S2eS4.
[2] M. Goedert, M.G. Spillantini, Science 314 (2006) 777e781.
[3] H. Mori, K. Takio, M. Ogawara, D.J. Selkoe, J. Biol. Chem. 267 (1992)
17082e17086.
[31] P.C. Conrad, P.L. Kwiatkowski, P.L. Fuchs, J. Org. Chem. 52 (1987) 586e591.
[32] M. Dowlut, D.G. Hall, O. Hindsgaul, J. Org. Chem. 70 (2005) 9809e9813.
[33] R.R. Schmidt, J. Michel, Angew. Chem. 92 (1980) 763e764;
Angew. Chem. Int., Ed. 19 (1980) 731e732.
[34] H. LeVine 3rd, Methods Enzymol. 309 (1999) 274e284.
[35] P. Westermark, C. Wernstedt, E. Wilander, D.W. Hayden, T.D. O’Brien,
K.H. Johnson, Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 3881e3885.
[36] L. Khemtémourian, G. Lahoz Casarramona, D.P. Suylen, T.M. Hackeng,
J.D. Meeldijk, B. de Kruijff, J.W. Höppener, J.A. Killian, Biochemistry 48 (2009)
10918e10925.
[4] S. Vivekanandan, J.R. Brender, S.Y. Lee, A. Ramamoorthy, Biochem. Biophys.
Res. Commun. 411 (2011) 312e316.
[5] (a) A.T. Petkova, W.M. Yau, R. Tycko, Biochemistry 45 (2006) 498e512;
(b) L. Yu, R. Edalji, J.E. Harlan, T.F. Holzman, A. Pereda Lopez, B. Labkovsky,
H. Hillen, S. Barghorn, U. Ebert, P.L. Richardson, L. Miesbauer, L. Solomon,
D. Bartley, K. Walter, R.W. Johnson, P.J. Hajduk, E.T. Olejniczak, Biochemistry
48 (2009) 1870e1877.
[6] R. Roychaudhuri, M. Yang, M.M. Hoshi, D.B. Teplow, J. Biol. Chem. 284 (2009)
4749e4753.
[7] D.J. Selkoe, Behav. Brain. Res. 192 (2008) 106e113.
[8] K.N. Dahlgren, A.M. Manelli, W.B. Stine, L.K. Baker, G.A. Krafft, M.J. LaDu, J. Biol.
Chem. 277 (2002) 32046e32053.
[37] N.L. Fawzi, J. Ying, D.A. Torchia, G.M. Clore, J. Am. Chem. Soc. 132 (2010)
9948e9951.
[38] B. Meyer, T. Peters, Angew. Chem., Int. Ed. 42 (2003) 864e890.
[39] S.K. Chaudhary, O. Hernandez, Tetrahedron Lett. 20 (1979) 95e98.
[9] K. Ono, M.M. Condron, D.B. Teplow, Proc. Natl. Acad. Sci. U.S.A. 106 (2009)
14745e14750.