Structure-activity relationships of β-hairpin mimics as modulators of amyloid β-peptide aggregation

Accepted Manuscript

Structure-activity relationships of β-hairpin mimics as modulators of amyloid β-peptide

aggregation

Nicolo Tonali, Julia Kaffy, Jean-Louis Soulier, Maria Luisa Gelmi, Emanuela Erba,

Myriam Taverna, Carine van Heijenoort, Tap Ha-Duong, Sandrine Ongeri

PII:

S0223-5234(18)30422-7

10.1016/j.ejmech.2018.05.018

EJMECH 10429

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 30 January 2018

Revised Date: 11 May 2018

Accepted Date: 12 May 2018

Please cite this article as: N. Tonali, J. Kaffy, J.-L. Soulier, M.L. Gelmi, E. Erba, M. Taverna, C. van

Heijenoort, T. Ha-Duong, S. Ongeri, Structure-activity relationships of β-hairpin mimics as modulators

of amyloid β-peptide aggregation, European Journal of Medicinal Chemistry (2018), doi: 10.1016/

j.ejmech.2018.05.018.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to

our customers we are providing this early version of the manuscript. The manuscript will undergo

copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please

note that during the production process errors may be discovered which could affect the content, and all

legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Structure-activity Relationships of β-Hairpin Mimics as Modu-

lators of Amyloid β-Peptide Aggregation

Nicolo Tonali,^aJulia Kaffy,*^aJean-Louis Soulier,^aMaria Luisa Gelmi,^bEmanuela Erba,^bMyriam Ta-

verna,^cCarine van Heijenoort,^dTap Ha-Duong,^aand Sandrine Ongeri *^a

a

BioCIS, Univ. Paris-Sud, CNRS, Université Paris Saclay, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex,

France.

^bDISFARM-Sez. Chimica Generale e Organica “A. Marchesini”, Università degli Studi di Milano, via Venezian 21, 20133

Milano, Italy

^cProtéines et Nanotechnologies en Sciences Séparatives, Institut Galien Paris-Sud, Univ. Paris-Sud, CNRS, Université Paris

Saclay, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France

d

Equipe Biologie et Chimie Structurales, Dept Chimie et Biologie Structurales et Analytiques, CNRS, ICSN, Université

Paris Saclay, 1 avenue de la terrasse, 91190 Gif sur Yvette FRANCE

Corresponding authors : E-mail: sandrine.ongeri@u-psud.fr; Tel : 0033 146835737.

E-mail: julia.kaffy@u-psud.fr; Tel : 0033 146835743.

ABSTRACT:

Aggregation of amyloid proteins is currently involved in more than 20 serious human diseases that are actually

untreated, such as Alzheimer’s disease (AD). Despite many efforts made to target the amyloid cascade in AD,

finding an aggregation inhibiting compound and especially modulating early oligomerization remains a relevant

and challenging strategy. We report herein the first examples of small and non-peptide mimics of acyclic beta-

hairpins, showing an ability to delay the fibrillization of amyloid-β (Aβ_1-42) peptide and deeply modify its early

oligomerization process. Modifications providing better druggability properties such as increased hydrophilicity

and reduced peptidic character were performed. We also demonstrate that an appropriate balance between flexi-

bility and stability of the β-hairpin must be reached to adapt to the different shape of the various aggregated forms

of the amyloid peptide. This strategy can be investigated to target other challenging amyloid proteins.

KEYWORDS amyloid, Alzheimer’s disease, peptidomimetics, β-Hairpin, oligomerization, fibrillization.

design drugs targeting the amyloid cascade in AD but

they have failed in clinical trials.[4] An ongoing

promising strategy to find a therapy for AD involves

the search for compounds that can inhibit Aβ_1-42ag-

gregation, to be used very early before the symptoms

appear and before brain pathology (amyloid-plaques

and tau tangles).[4c]

Introduction

Currently more than 30 human proteins are implicated

in a range of degenerative disorders owing to their

misfolding and misassembly into various aggregate

structures and leading to at least 20 serious human

diseases named amyloidosis.[1] The self-assembled

insoluble aggregates are characterized by highly or-

dered cross-

disease (AD) is associated with the aggregation of the

amyloid- (Aβ_1-42) peptide into senile plaques in the

brain.[2] Approximately 47 million people worldwide

are living with AD and this number is expected to

reach 75 million by 2030, and 135.5 million by

2050.[3] Yet, despite significant means implemented,

no causal treatment exists nor for AD nor for the other

amyloid diseases. Many efforts have been made to

β

structures.[1] In particular, Alzheimer’s

However, designing small molecular inhibitors that

disrupt protein−protein interactions mediating the

aggregation is still a challenge.[1] Indeed, pro-

tein−protein interactions occur over large surface

areas that are difficult to characterize. Furthermore,

many different forms of aggregates are produced

while their structures and toxicity are far to be eluci-

dated.[5] In particular, while the monomeric form of

β

A

β_1-42is currently declared as non toxic, fibrils are

ACCEPTED MANUSCRIPT

able to generate damaging redox activity and promote

pounds 3-7, Figure 1): i) the substitution of the C-

terminus peptide arm with a peptidomimetic, ii) the

introduction of a di- or tripeptide peptide sequence at

the N-terminus. In particular, according to our previ-

ous results with sugar based peptidomimetics (com-

the nucleation of toxic oligomers.[6] Recent studies

demonstrate that soluble transient oligomers preced-

ing fibril formation are highly toxic species constitute

a major target to prevent the death of neurons.[7]

A large number of small molecules (e.g., tramipro-

sate, inositol, curcumin, epigallocatechin-3-gallate

and resveratrol) have been reported as Aβ_1-42inhibi-

tors and have been studied in clinical trials.[5,8]

However, the majority of these agents bind to Aβ_1-42

with low affinity, without selectivity and have no or

unknown effect on the most toxic oligomeric species.

Recently various monoclonal antibodies (such as

Solanezumab) have been produced to inhibit Aβ_1-42

aggregation by targeting soluble species of the pep-

tide. However they failed to show clinical efficacy in

phase II or III clinical trials probably mainly because

of their failure to cross the blood-brain barrier (BBB)

due to their large size.[4a]

pound 2,

Figure 1),[14] the 5-amino-2-

methoxybenzhydrazide (Amb) peptidomimetic strand

linked to a Lys residue was selected for the C-

terminal arm of the compounds and Val-Ala or Ala-

Val-Ala was introduced at the N-terminus. Finally,

the introduction of different substituents on the amino

group at C-4 of the piperidine ring was also planned

for assessing their effect on the activity.

Figure 1

2. Results and discussion

2.1 Synthesis

The synthesis of the newly designed peptidomimetics

3-5 is described in scheme 1, starting from scaffolds 8

and 9. The tosyl scaffold 8 was prepared according to

our published method, based on a key multicompo-

nent reaction using N-benzylpiperidinone, tosyl azide

and proline methyl ester.[12,13] We adapted this pro-

cess to the synthesis of the nosyl scaffold 9, replacing

tosyl azide by nosyl azide. This new synthetic proto-

col is fully described in the supporting information

(Scheme 1S).

Peptide-based drug discovery could be a serious op-

tion for addressing new therapeutic challenging can-

didates. The number of approved peptides is dramati-

cally increasing, in particular because they often offer

greater efficacy, selectivity, specificity and a reduced

risk of unforeseen side‐reactions compared to small

organic molecules.[9] Modified peptides and pep-

tidomimetics can also be designed to decrease the

susceptibility to proteases and to cross the BBB. A

variety of small peptides or modified peptides that

The coupling reaction between the peptidomimetic

strand 10 [14] and compounds 8 and 9 provided the

desired products 11 and 12 in good yield (88 and

65%, respectively), using HATU and HOAt as cou-

pling reagents, in the presence of collidine in DMF.

Other coupling agents were also tested in parallel for

the coupling of 10 with 8, such as DMTMM in the

presence of NMM, or EDC in the presence of HOBt

and DIPEA but were less efficient (compound 11 was

obtained in 18% and 43% of yield, respectively). Af-

ter the Fmoc cleavage using 20% piperidine in DMF,

the construction of the N-terminus arm was then per-

formed using the Boc strategy. The coupling of N-

Boc-L-Val-OH and compounds 11 or 12 was per-

formed using HATU and HOAt, in the presence of

collidine in DMF, to afford compounds 13 and 14 in

60 and 75% yields respectively. After the Boc cleav-

age, the second amino acid (N-Boc-L-Ala-OH) was

coupled affording compounds 15 and 16 in 83 and

75% yield, respectively. The Cbz protecting group of

15 was then cleaved by hydrogenolysis using hydro-

gen in the presence of 10% Pd/C in MeOH, and the

resulting product 17 was finally Boc deprotected in

the presence of 4M HCl in dioxane to afford the final

compound 3. The final compound 5 was obtained

from 16, in 60% yield, by following the same hydro-

inhibit aggregation of Aβ and reduce its toxic effects

have been already described.[10,4a] However, the use

of pre-structured peptides, in particular small acyclic

β-hairpins has been very rarely explored as β-sheet

binders and inhibitors of aggregation.[11] We firmly

believe that the greater flexibility of such compounds

with respect to the cyclic peptides, allows them to

adapt to the different shape of the various aggregated

forms of the amyloid peptide. As a result, they can

better bind to the sequences involved in the β-sheet

structures of the amyloid peptide.

We have recently begun to demonstrate this concept

by reporting a novel class of

β-hairpin peptidomimet-

ics, built on a piperidine-pyrrolidine semi-rigid

β-turn

inducer [12] and bearing two recognition pentapeptide

sequences, designed on oligomeric and fibril struc-

tures of Aβ_1-42(compound 1, Figure 1).[13] This study

was the first example of acyclic small β-hairpin mim-

ics possessing such a highly efficient anti-aggregation

activity.

We propose now to design more druggable com-

pounds, particularly with a better hydrophobicity/

hydrophilicity balance and reduction of the peptidic

character. To reach this target, starting from com-

pound 1, we planned three main modifications (com-

ACCEPTED MANUSCRIPT

genation/hydrolysis procedures. On the other hand,

could not be reached at 313 K. We next examined the

temperature dependence of amide proton chemical

shifts, as it can provide information on the network of

hydrogen bonds and their relative stabilities.[16]

None of the amide protons of the major isomer

showed small negative temperature coefficient, except

for the NH(b) proton (∆δHN/∆T value of –4.47 ppb/K

for 3 and –4 ppb/K for 4, Tables S3 and S8, support-

ing information), presumably because of its hydrogen

bonding with the methoxy group of the aromatic unit,

as already reported by Nowick for the Hao motif.[17]

In both conformers of 3, the temperature coefficients

of the Lys and Val amide proton fell within interme-

diate values (∆δHN/∆T value of –6.33-7.33 ppb/K,

Tables S3 and S4), suggesting their partial engage-

ment in intramolecular hydrogen bonds. Similarly, the

NH of Lys and Ala in compound 4 had intermediate

∆δHN/∆T values (Tables S8 and S9).

The vicinal J_NH-Hαcoupling constant also yields direct

information on the main chain ϕ dihedral angle,

through the Karplus relationship. In the two conform-

ers of both compounds 3 and 4, the coupling constants

exhibited values between 7.4-8.7 Hz. As expected for

extended conformation, positive difference between

J_NH-Hαvalues in the random coil and the values deter-

mined experimentally were found, reflecting ϕ angle

values around -120° (Tables S5 and S6 for 3 and S10

for 4, supporting information).¹⁸Chemical shift devia-

tions (CSD) are defined as the difference between

experimental chemical shifts and corresponding ran-

dom coil values and are considered as good de-

scriptors of backbone conformational space for each

residue.[19] We calculated the CSD for the natural

amino acids present in the molecule (Tables S5 and

S6 for 3 and S10 for 4, supporting information). In

both conformers, the C_αand the carbonyl group were

upfield shifted (negative CSD), indicating that an

extended conformation for the peptide and pep-

tidomimetic arms predominates. The downfield shift-

ed H_αand NH (positive CSD, > 0.1 ppm) of Val and

Lys indicated that they are more implicated in the β-

conformation than the N-terminal Ala in 3. Similarly,

for compound 4, Val-2, Ala and Lys CSD values in-

dicated that they are more implicated in the β-

conformation than the N-terminal Val-1. In the

ROESY experiments performed at 278 K, strong se-

quential and medium intra-residual Hα-HN ROEs,

which are characteristic of an extended backbone

conformation, were detected for the dipeptide and

tripeptide sequence in 3 and 4, respectively. ROEs

between the di- or tripeptide and the peptidomimetic

arms in the major conformers of both 3 and 4 were

observed (Figures 2A and 3A). ROEs between H2 of

the 5-amino-2-methoxybenzhydrazide (Amb) motif

the Boc cleavage of 15 followed by the coupling with

N-Boc-L-Val-OH afforded compound 18 in 57%

yield. Hydrogenolysis of the Cbz group followed by

the Boc cleavage afforded the final compound 4.

Scheme 1

The synthesis of compounds 6-7 is described in

scheme 2, starting from the Boc scaffold 19 whose

synthesis is fully described in the supporting infor-

mation (Scheme 2S, see supporting information). The

direct cleavage of the nosyl group on the scaffold

derivative S1 was only partially satisfactory, whatever

the used conditions (thiophenol/K₂CO₃in acetonitrile

in the presence of DMSO, heating at 50°C or using

ultrasounds or microwaves; thioglycolic acid/LiOH in

DMF at room temperature or at 70°C). Thus, the

nosylamide was converted first to the Boc-nosylimide

S4 in order to activate the nosyl group towards the

attack and cleavage by thiophenol.[15] Indeed, the

cleavage of the nosyl group, giving S5, was then sat-

isfactory and the Boc scaffold 19 was easily obtained

after hydrogenolysis of S5. N-Fmoc-L-Val-OH and N-

Cbz-L-Ala-OH were successively coupled, using

HATU and HOAt as coupling reagents and collidine

as the base to afford compound 21 (Scheme 2). Sa-

ponification of the methyl ester of 21, afforded prod-

uct 22 (92% yield). The coupling reaction between 22

with the peptidomimetic strand 10, using HATU and

HOAt, did not provide the desired product 23. Satis-

factory results were obtained using the triazine-based

reagent DMTMM (1.1 eq.) in the presence of NMM

in DMF (61% yield). The two Cbz protecting groups

of 23 were cleaved by hydrogenolysis to afford prod-

uct 6 that was subjected to the Boc cleavage to afford

the final compound 7 (70% yield).

Scheme 2

2.2. Conformational studies

Conformational studies of compounds 3 and 4 were

conducted by NMR in water, which is more challeng-

ing for intra-molecular hydrogen bond formation in

comparison with aprotic organic solvents such as

chloroform, but that is closer to physiological condi-

tions. The observed proton and carbon chemical shifts

are displayed in Tables S1 and S2 for compound 3

and in table S7 for compound 4 (supporting infor-

mation). Compounds 3 and 4 were present in solution

as a mixture of two conformers in 3:1 and 4:1 ratio,

respectively. The dynamic equilibrium between the

two conformers was confirmed by the occurrence of

splitting for some protons (see supporting infor-

mation). The coalescence between the two forms

ACCEPTED MANUSCRIPT

and both H

(H2 Amb/Me_Val and H2 Amb/H

Amb/Me_Val-2 and H2 Amb/H _Val-2 in 4). ROEs

were also detected between the methoxy group of the

benzhydrazide motif and Hα of Val

β

and methyl protons of Val were observed

to be dependent on the amount of fibers formed (Ta-

ble 1).

Each compound was tested at a 10-fold excess (100

µM) and at a 1/1 ratio (10 µM). Compounds 3 and 4

were also tested at the substoichiometric ratio of 0.1/1

(compound/Aβ_1-42).

β

_Val in 3 and H2

β

(Hα_Val/OMe_Amb in 3 and Hα_Val-2/OMe_Amb

in 4). Furthermore, in compound 3 a ROE between

H2 of the benzhydrazide motif and the methyl of Ala

(H2 Amb/Me_Ala) was noticed. In both compounds 3

and 4, we observed ROEs between the aromatic pro-

ton H2 of the tosyl group and both Hα and H4 of Pro,

suggesting that the tosyl protecting group is stacked

onto the proline ring. Three-dimensional structures of

3 and 4 were then generated using simulated anneal-

ings with restrained distances inferred from the six

and five NMR ROEs previously described and report-

ed in Figures 2A and 3A for compound 3 and 4, re-

spectively. As shown in Figures 2B and 3B, both

compounds 3 and 4 exhibited a partially folded β-

hairpin conformation in which the piperidine-

pyrrolidine and lysine form the β-turn while Val in

compound 3 or Val2 in compound 4 side chain make

hydrophobic interactions with the benzhydrazide ar-

omatic ring. However, the N-terminal residues (Ala in

compound 3 or Val2-Ala in compound 4) are not ob-

served in contact with the C-terminal acetyl group,

which would form a more extended β-hairpin. No

particular « inter-arm» hydrogen bond was observed

for stabilizing the β-hairpin. On the other hand, the

carbonyl group of the alanine residue is close to and

oriented toward the amine groups of both pyrrolidine,

lysine and hydrazine, avoiding the formation of a

fully extended β-hairpin. All these data suggest that

compounds 3 and 4 adopt, a similar partial β-hairpin

conformation.

Significant differences in activity on the aggregation

process were observed between all the evaluated

compounds (Table 1, Figure 4 and Figure S6 support-

ing information). The most significant activity on the

aggregation process of Aβ_1-42was obtained with com-

pound 3, which is the analogue of the glycopep-

tidomimetic 2,[14] composed of the same two arms. 3

displayed a comparable activity to 2 at the 1/1 ratio

(extension of the t_1/2by 36% and decreased of the

fluorescence plateau by 38% for 3 compared to 48%

and 29% for 2). However, the activity of 3 was slight-

ly lower than 2 at a 10-fold excess: delaying the rate

of aggregation by almost 80% and decreasing the

fluorescence plateau by almost 70% (vs no aggrega-

tion for 2). Compound 17, the analogue of 3, protect-

ed by a terbutoxycarbonyl group on the N-terminal

amine of Ala residue, was less active (extension of the

t_1/2by 59% and23% respectively for ratios 10/1 and

1/1 of 17/Aβ_1-42and no effect on the fluorescence

plateau). The totally protected precursor 15 was inac-

tive at the ratio 1/1 (15/Aβ_1-42) and self-aggregated at

the 10-fold excess (100 µM). These results indicate

that both free amines, either on the N-terminal posi-

tion and on the side chain of both arms, are beneficial

for the activity.[13,14]

The elongation of the N-terminus arm, by adding a

third hydrophobic amino acid (Val), had more than

halved the inhibitory activity of compound 4 with

respect to 3. Indeed, at the 10 fold excess concentra-

tion relative to Aβ₁₄₂, 4 increased the t_{1/2 only}by 30%

and reduced the fluorescence plateau by 32%, com-

pared to 76% and 66% for 3. At the 1:1 ratio, 4 dis-

played no activity. A supplementary ThT fluores-

cence assay was performed by first, adding compound

3 after 3 hours or after 19h, when presumably oligo-

mers are in formation or are already large species

respectively. Finally, compound 3 was added after 44

hours, when presumably essentially fibrils are present

(Figure S7 in supporting information). A similar ac-

tivity was obtained with compound 3 added at the

beginning of the kinetics or after 3 hours. However no

effect was observed when compound 3 was added

after 44 hours. When 3 was added after 19h, no effect

was observed at the 1:1 ratio and the fluorescence

observed at the 10:1 (3/Aβ_1-42) ratio after 19h, was

maintained throughout the rest of the kinetics. This

result indicates that 3 does not disaggregate large

oligomers and preformed fibrils but rather prevent the

early oligomerization process.

Figures 2 et 3

2.3 Evaluation of the inhibition of fibrillization

The anti-amyloidogenic activity of compounds 3-7

was investigated using the in vitro thioflavin-T (ThT)

assay. ThT is a dye that fluoresces upon binding to β-

sheet rich species and thus, is widely used to monitor

the kinetics of aggregation of amyloid proteins.²⁰For

A

β_1-42alone (at a concentration of 10 µM), the fluo-

rescence curve followed a typical sigmoidal shape

with a lag phase around 4-6 h, an elongation phase

and a final plateau reached after 12-20 h (Figure 4,

purple curve). Two parameters were derived from the

ThT curves: t_1/2, defined as the time at which the fluo-

rescence has reached 50% of its maximum, is a meas-

ure of the aggregation process rate; and F, the fluo-

rescence value of the final plateau which is assumed

ACCEPTED MANUSCRIPT

As these peptidomimetics were soluble in aqueous

media, we verified that their activity on A ₁₋₄₂aggre-

(Figure 5). Differences were observed in quantity of

β

aggregates formed. At 42 h, a very dense network of

fibers displaying a typical morphology was observed

for Aβ_1-42alone (Figure 5a). In the sample containing

3, the network of fibers, displaying the same mor-

phology, was significantly less dense than in the con-

trol experiment after 42 h (Figure 5b). The same

trends were observed with compound 5, but to a lesser

extent (Figure 5c). These results validated the ThT-

fluorescence data, indicating that 3 significantly

slowed down the aggregation of Aβ_1-42and efficiently

reduced the amount of typical amyloid fibrils, and

that 5 is slightly less efficient.

gation was maintained when solubilized in water

(without DMSO). Indeed, the most active compound

3, dissolved in water (0.5% v/v), displayed a compa-

rable or even slightly higher activity in the ThT fluo-

rescence assay (at ratio 10/1 of 3/Aβ_1-42) to that ob-

served when solubilized in the same amount of

DMSO (t_1/2increased by 96% and F decreased by

81% in the case of a solubilization in water vs 76%

and 66% in DMSO). The same result was obtained for

the tripeptide analogue, 4, the t_1/2being increased by

58% and F decreased by 35% when the sample was

solubilized in water compared to 30% and 32% in

DMSO. This observation suggests that DMSO may

perturb the secondary structure of 3, reducing its af-

Table 1

Figure 4

Figure 5

finity for Aβ₁₋₄₂

.

Focusing on the pharmacomodulation induced by the

substituent at C-4 of the piperidine ring, the amino-

phenyl-sulfonylamino derivative 5, the terbutoxycar-

bonylamino compound 6 and the primary amine 7

were tested. Compound 5 retained some activity,

however slightly less than that of the tosyl derivative

3. For the Boc compound, 6, the ability to inhibit the

aggregation process was even lower (t_1/2increased by

29% and F decreased by 43%, ratio 10/1 of 6/Aβ_1-42).

The activity of compound 7 almost disappeared (only

F reduction of 18% at the same ratio). These results

suggest that both the aromatic ring branched to the

primary amine of the pyrrolidine of the scaffold

through a sulfamide bond, and its para substitution by

a methyl group rather than an amine are crucial for

the inhibition of Aβ_1-42aggregation. These data sug-

gest that, the interaction of compounds 3 and 5 to Aβ_1-

2.4 Evaluation of the inhibition of oligomerization

We recently developed a capillary electrophoresis

(CE) method to monitor the early steps of the oli-

gomerization process over time.[21] This method

allows to follow the formation of small soluble oli-

gomers and to evaluate the impact of small molecules

on three kinds of species, (i) the monomer of Aβ_1-42

(peak ES), (ii) different small metastable oligomers

grouped under peak ES’ and (iii) transient later spe-

cies corresponding to oligomers larger than do-

decamers (peak LS).[13,14,21] The control kinetics

(Aβ_1-42, 100 µM) showed that overtime, the peak of

monomer ES decreases in favor of the peaks of LS,

and that soluble species are no longer visible after 6 h

(Figure 6A). Moreover, ES’ species are hardly visible

while spikes, corresponding likely to insoluble and

larger oligomers, are clearly visible as soon as 2 h.

The electrophoretic profiles obtained in the presence

of compound 3 indicate that 3 significantly modified

the aggregation process. 3 was able to maintain the

non-toxic monomer ES overtime. Indeed, ES is still

present after 12 h (Figure 6B): 49% and 31% of the

monomer are still present after 6 h and 12 h respec-

tively in the presence of 3, while it was no longer

detected in the control sample after 6 h (Figure 6C).

Furthermore, LS migrated slower and higher number

of spikes were observed from the beginning of the

kinetics (0-2 h) and on each electrophoretic profile.

These two features suggest another pathway of Aβ_1-42

aggregation in the presence of 3.

peptide is also probably driven by hydrophobic

42

and/or aryl-aryl interactions involving the scaffold

moiety.

Finally, to confirm that the entire molecule of

β-

hairpin was essential to interact and inhibit Aβ_1-42

aggregation, the scaffold 25 was also evaluated by the

Tht-fluorescence assay (Table 1). It displayed a mod-

est activity and only at the high 10/1 ratio (25/Aβ_1-42).

F decreased by 23% and no effect was observed on

the t_1/2. The activity of the peptidomimetic arm 24 has

been already reported by us [14] and it is significantly

lower than for the entire

β-hairpin mimic (t_1/2in-

creased by only 7% and F decreased by 44% at the

24/Aβ_1-42ratio of 10/1 vs +76% and -66% in the case

of 3).

Transmission electron microscopy (TEM) analyses

were performed on the most promising compounds 3

and 5 according to the Tht-fluorescence assay results.

Images were recorded at 42 h of fibrillization kinetics

with samples containing 100 µM of each compound

corresponding to the compound/Aβ_1-42ratio of 10/1

Figure 6

Conclusion

ACCEPTED MANUSCRIPT

We described the design and the synthesis of five new

modifications that provide better druggability proper-

ties. This strategy can now be envisaged to design

inhibitors of other types of amyloid-forming proteins.

acyclic peptidomimetics based on a piperidine-

pyrrolidine β-turn inducer bearing a di- or a tri- pep-

tide N-terminus arm and a peptidomimetic arm con-

taining a 5-amino-2-methoxybenzhydrazide unit at

the C-terminus. Compound 3 is the most active com-

pound of this series. It is able to greatly delay the

fibrillization process of Aβ_1-42, as demonstrated by

Tht fluorescence spectroscopy and TEM images. It is

also able to modify the early oligomerization steps

and to maintain the presence of the non toxic mono-

mer of Aβ_1-42. The results obtained in this study al-

lowed us to establish some informative structure-

activity relations. 1) With respect to compound 1, the

reduction of the peptidic character by both the short-

ening from pentapeptide arms to a di- or tripeptide,

and the substitution of the C-terminus peptide arm by

a peptidomimetic arm, maintained a satisfactory ac-

tivity of Aβ_1-42aggregation inhibition. Moreover,

compounds 3 and 4 were hydrosoluble. 2) The length

of the peptide arm dramatically influences the aggre-

gation inhibition activity, a dipeptide being more ad-

vantageous than a tripeptide sequence. 3) Concerning

the substituent on the amino group at C-4 of the piper-

idine ring of the β-turn mimic, the tosyl residue is

crucial for an inhibitory activity, as the Boc com-

pound 6 and the free amine derivative 7 are almost

inactive. The more polar aminophenyl-sulfonylamino

derivative 5 is much less active than 3. We hypothe-

size that the tosyl moiety of 3 interacts with aromatic

residues of Aβ_1-42through hydrophobic and aryl-aryl

interactions. 4) Compounds 15 and 23, being the di-

and mono-N-protected analogues of 3, exhibited a

strong decrease of activity. This result supports our

previous hypothesis on the importance of establishing

ionic interactions between the amino groups and acid-

ic residues of Aβ_1-42.[13,14] The whole β-hairpin

construct is necessary to strongly delay the Aβ_1-42

aggregation kinetics, as the isolated scaffold 25 and

the peptidomimetic arm 24 displayed only very mod-

est activity. 6) The conformational studies allowed us

to hypothesize that a fully extended β-hairpin struc-

ture is more convenient and can further inhibit the

early oligomerization and fibrillization process. In

fact, we observed that compound 3 exhibits a partially

folded β-hairpin conformation while the more active

reference compound 1 was able to adopt a more stable

β-hairpin conformation.

Experimental

Chemistry

General Experimental Methods. Solvents were

purchased from commercial sources and dried and

distilled by standard procedures. Protected amino

acids,

N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-

b]pyridin-1-ylmethylene]-N-methylmethanaminium

hexafluorophosphate N-oxide (HATU), 4-(4,6-

dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

chloride

(DMTMM(Cl^-)),

and

1-Hydroxy-7-

azabenzotriazole (HOAt) were purchased from com-

mercial sources. Compounds 1,[13] 2 [14] 8 [13], 24

[14] and 25 [13] were prepared according to pub-

lished methods. The syntheses of compounds 9 and

18 are described in the supporting information. Pure

products were obtained after liquid chromatography

using Merck silica gel 60 (40-63 µm). TLC analyses

were performed on silica gel 60 F₂₅₀(0.26 mm thick-

ness) plates. The plates were visualized with UV light

(λ = 254 nm) or revealed with a 4% solution of phos-

phomolybdic acid in EtOH or with a solution of nin-

hydrin in EtOH. Melting points were determined on a

Kofler melting point apparatus. NMR spectra were

recorded on an ultrafield Bruker AVANCE 300 (¹H,

300 MHz, ¹³C, 75 MHz), a Bruker AVANCE 400 (¹H,

400 MHz, ¹³C, 100 MHz), a Bruker AVANCE I

600MHz (¹H, 600 MHz, ¹³C, 150 MHz) or a Bruker

AVANCE III 800MHz (¹H, 800 MHz, ¹³C, 200 MHz)

spectrometers, the last two equipped with a z-gradient

TCI cryprobe. Chemical shifts

δ are in ppm and the

following abbreviations are used: singlet (s), doublet

(d), doublet of doublet (dd), doublet of triplet (dt),

triplet (t), triplet of doublet (td), quintuplet (qt), mul-

tiplet (m), broad multiplet (bm), and broad singlet

(bs). Mass spectra were obtained using a Bruker Es-

quire electrospray ionization apparatus. HRMS were

obtained using a TOF LCT Premier apparatus (Wa-

ters), with an electrospray ionization source. The puri-

ty of compounds 3-7 was determined by HPLC using

the 1260 Infinity system (Agilent Technologies) and a

column SUNFIRE (C18, 3.5 µm, 150 mm X 2.1 mm);

mobile phase: H₂O + 0.1% formic acid/CAN gradient

from 5 to 100% in 20 min. or from 1 to 60% in 20

min.; detection at 230, 254, 310 nm; flow rate 0.25

mL/min.

Thus, this work demonstrates that a compromise be-

tween flexibility of the β-hairpin, hydrosolubility and

reduction of the peptidic character can be reached.

These peptidomimetics are the first examples of small

and non-peptide analogues of acyclic beta-hairpins,

which have preserved anti-aggregation activity after

(

2S)-N-[(1S)-1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-amino-pentyl]-1-

[(3R,4R)-1-[(2S)-2-[[(2S)-2-

aminopropanoyl]amino]-3-methyl-butanoyl]-4-(p-

tolylsulfonylamino)-3-piperidyl]pyrrolidine-2-

ACCEPTED MANUSCRIPT

1

carboxamide trihydrochloride 3. Compound 15

(64.0 mg, 0.058 mmol, 1.0 eq.) was dissolved in

MeOH (4.0 mL) and 10% Pd/C (10 mg) was added.

The reaction was kept 2 hours under stirring and un-

der hydrogen atmosphere at room temperature. The

catalyst was then removed over a celite pad and the

solvent was evaporated under vacuum. The resulting

product was dissolved in dioxane (2.0 mL) and a 4 N

solution of HCl in dioxane (0.293 mL, 30.0 eq.) was

added. The reaction was kept 2 hours under stirring at

room temperature. Diethyl ether was then added to

allow precipitation of product 3 that was isolated as

the hydrochloride salt after filtration. The crude was

purified by recrystallization in MeOH and diethyl

ether to afford the pure compound 3 as a white pow-

der (19.0 mg, 0.019 mmol, 50%). mp = 221–223 °C;

19%). mp = 229–231 °C; H NMR (H₂O/ D₂O, 800

MHz): = 10.27 (1H, bs), 10.09 (1H, bs), 9.59 (1H,

δ

s), 8.89 (1H, d, J = 7.4 Hz), 8.46 (1H, d, J = 6.2 Hz),

8.18 (1H, d, J = 8.1 Hz), 7.82 (3H, bs), 7.79 (1H,

bs),7.59-7.54 (3H, m), 7.33 (3H, bs,), 7.27-7.18 (3H,

m), 6.92 (1H, d, J = 10.2 Hz), 4.34 (1H, m), 4.25 (1H,

m), 4.24 (1H, m), 4.12 (1H, m), 3.92 (1H, m), 3.83

(1H, m), 3.69 (3H, s), 3.64 (1H, m), 3.49-3.47(2H,

m), 2.84-2.76 (5H, m), 2.29 (1H, m), 2.26 (1H, m),

2.15 (3H, s), 1.91 (1H, m), 1.88 (3H, s), 1.81 (1H, m),

1.71 (1H, m), 1.70 (2H, m), 1.67 (1H, m), 1.65 (1H,

m), 1.49 (2H, m), 1.30 (1H, m), 1.28 (2H, m), 1.19

(1H, m), 1.05 (3H, d, J = 7.5 Hz), 0.73-0.71 (6H, m),

0.57-0.54 (6H, m); ¹³C NMR (H₂O/ D₂O, 200 MHz):

δ

= 174.1, 172.9, 171.8, 171.6, 170.8, 169.0, 166.3,

155.1, 145.3, 135.3, 130.1, 129.8, 128.4, 126.6, 124.7,

118.4, 112.7, 58.3, 56.0, 54.6, 52.4, 51.7, 49.3, 48.9,

46.8, 43.6, 40.9, 39.2, 39.1, 30.1, 30.1, 29.9, 29.9,

29.7, 26.3, 23.8, 22.4, 21.9, 20.6, 18.4, 16.9, 17.5,

16.6, 16.3; IR (neat): ν_max= 3194, 2966, 1652,

1539, 1494, 1456, 1250, 1161 cm^-1; HRMS (TOF

ESI, ion polarity positive): m/z [M + H]⁺calcd

for C₄₆H₇₂N₁₁O₁₀S 970.5184, found: 970.5181; HPLC

1H NMR (H₂O/ D₂O, 600 MHz):

δ = 10.42 (1H, bp),

10.31 (1H, bs), 9.79 (1H, s,), 9.09 (1H, d, J = 7.5 Hz),

8.52 (1H, d, J = 7.5 Hz), 8.06 (3H, bs), 7.95 (1H, bs),

7.88-7.84 (3H, m), 7.57-7.56 (4H, m), 7.47 (2H, m),

7.22 (1H, d, J = 9.4 Hz), 4.60 (1H, m), 4.52 (1H,

m),4.23 (1H, m), 4.22 (1H, m), 4.11 (1H, m), 3.96

(3H, s), 3.93 (1H, m), 3.72 (1H, m), 3.48 (1H, m),

3.15 (1H, m), 3.04 (2H, m), 3.01 (1H, m), 2.85 (1H,

m), 2.60 (1H, m), 2.50 (1H, m), 2.44 (3H, s), 2.18

(3H, s), 2.11 (1H, m), 2.06 (1H, m),1.98 (2H, m), 1.94

(1H, m), 1.90 (1H, m), 1.76 (2H, m), 1.63 (1H, m),

1.53 (2H, m),1.48 (1H, m), 1.46 (3H, d, J = 7.5 Hz),

purity (XBridge C18, 3.5

µm, H₂O + 0:1% form.

ac./ACN, gradient 5–100% in 20 min): TR = 10:47

min, 92%

(2S)-N-[(1S)-1-[[(5-acetamido-2-methoxy-

0.82-0.83 (6H, m);¹³C NMR (H₂O/ D₂O, 133 MHz):

δ

benzoyl)amino]carbamoyl]-5-amino-pentyl]-1-

[(3R,4R)-4-[(4-aminophenyl)sulfonylamino]-1-

[(2S)-2-[[(2S)-2-aminopropanoyl]amino]-3-methyl-

butanoyl]-3-piperidyl]pyrrolidine-2-carboxamide

trihydrochloride 5 .The same procedure was used as

that described for the synthesis of compound 3: from

16 (25.0 mg, 0.022 mmol, 1.0 eq.) in MeOH (4.0 mL)

= 173.1, 171.9, 171.3, 170.7, 166.5, 164.9, 155.1,

145.6, 135.6, 130.2, 130.2, 128.4, 126.7, 124.7, 118.7,

112.9, 56.3, 55.1, 52.2, 52.1, 48.8, 44.2, 44.1, 41.4,

39.3, 37.0, 31.4, 30.3, 30.2, 30.1, 29.7, 26.4 , 24.1,

22.5, 21.8, 20.6, 18.5, 16.7, 16.5; IR (neat): ν_max

=

3194, 2926, 1652, 1546, 1493, 1457, 1252, 1157;

HRMS (TOF ESI, ion polarity positive): m/z [M

+ H]⁺calcd for C₄₁H₆₃N₁₀O₉S 871.4500, found:

to afford, after recrystallization in MeOH and diethyl

ether, the pure compound

(13.0 mg, 0.013 mmol, 60%). mp = 233-235 °C; H

NMR (DMSO-d₆, 400 MHz): = 10.50 (1H, bs),

5 as a pale yellow powder

Na]⁺

calcd

for

1

871.4497,m/z

C₄₁H₆₂N₁₀O₉NaS, 893.4320, found: 893.4316; HPLC

purity (XBridge C18, 3.5 m, H₂O + 0:2% form.

[M

+

δ

µ

10.20 (1H, bs), 9.96 (1H, s), 9.22 (1H, bs), 8.69 (1H,

bs), 8.30 (3H, bs), 8.22 (3H, bs), 8.02 (1H, bs), 8.00

(1H, s), 7.75 (1H, m), 7.61 (2H, m), 7.10 (1H, d, J =

8.8 Hz), 6.86 (2H, m), 4.59 (1H, m), 4.48 (1H, m),

4.46 (1H, m), 4.44 (1H, m), 4.19 (2H, m), 3.97-3.86

(2H, m), 3.84 (3H, s), 3.66 (1H, m), 3.15-3.09 (3H,

m), 2.79 (2H, m), 2.49 (1H, m), 2.10 (1H, m), 2.06-

2.05 (2H, m), 2.02 (3H, s), 1.89-1.63 (6H, m), 1.47-

1.20 (8H, m), 0.87-0.82 (6H, m);¹³C NMR (DMSO-

ac./ACN, gradient 5–100% in 20 min): TR = 10:08

min, 100%.

(2S)-N-[(1S)-1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-amino-pentyl]-1-

[(3R,4R)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-

methyl-butanoyl]amino]propanoyl]amino]-3-

methyl-butanoyl]-4-(p-tolylsulfonylamino)-3-

piperidyl]pyrrolidine-2-carboxamide

trihydro-

d₆, 100 MHz):

δ = 169.9, 169.5, 169.6, 169.4, 168.3,

chloride 4. The same procedure was used as that de-

scribed for the synthesis of compound 3: from 17

(50.0 mg, 0.042 mmol, 1.0 eq.) to afford, after recrys-

tallization in MeOH and diethyl ether, the pure com-

pound 4 as a white powder (9.0 mg, 0.008 mmol,

163.6, 152.8, 149.9, 132.7, 128.6, 128.2, 123.6, 121.4,

120.9, 115.1, 112.4, 64.8, 61.5, 56.3, 53.8, 51.9, 50.5,

47.5, 42.6, 38.3, 38.1, 31.0, 29.7, 29.6, 29.4, 29.3,

26.1, 23.8, 23.0, 22.0, 19.5, 18.1, 17.3; IR (neat):

ν_max=

3220, 2970, 1636, 1546, 1494, 1304, 1254 cm¹;

ACCEPTED MANUSCRIPT

and diethyl ether to afford the pure compound 7 (12.0

mg, 0.014 mmol, 70%) as a white powder. mp = 226-

HRMS (TOF ESI, ion polarity positive): m/z [M

+ H]⁺calcd for C₄₀H₆₂N₁₁O₉S 872.4453, found

872.4440; HPLC purity (Sunfire C18, 3.5 µm, H₂O +

0.1% form. ac./ACN – gradient 5-100% in 20 min):

TR = 7.31 min, 97%.

1

228 °C; H NMR (DMSO-d₆, 400 MHz):

δ = 10.60

(1H, s), 10.20 (1H, s), 10.00 (1H, s), 8.97 (3H, bs),

8.51 (1H, bs), 8.30 (1H, bs), 8.25 (3H, bs), 8.05 (3H,

bs), 8.04 (1H, s), 7.71 (1H, m),7.11 (1H, d, J = 8.8

Hz), 4.52-4.44 (3H, m), 4.08 (1H, m), 3.89 (1H, m),

3.86 (3H, s), 3.39 (1H, m), 3.37 (1H, m), 3.12 (1H,

m), 3.06 (1H, m), 2.73 (2H, m), 2.70 (1H, m), 2.69

(1H, m), 2.48 (1H, m), 2.09 (1H, m), 2.03 (3H, m),

1.90-1.59 (8H, m), 1.36-1.22 (7H, m), 0.84-0.78 (6H,

acetic acid; tert-butyl N-[(3R,4R)-3-[(2S)-2-[[(1S)-

1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-amino-

pentyl]carbamoyl]pyrrolidin-1-yl]-1-[(2S)-2-[[(2S)-

2-aminopropanoyl]amino]-3-methyl-butanoyl]-4-

piperidyl]carbamate 6. Compound 22 (63.0 mg,

0.06 mmol, 1.0 eq.) was dissolved in MeOH (4.0 mL)

and 10% Pd/C (10 mg) was added. The reaction was

kept 2 hours under stirring and under a hydrogen at-

mosphere at room temperature. The catalyst was then

removed over a celite pad and the solvent was evapo-

rated under vacuum. The crude was diluted in a min-

imum amount of MeOH and 2.0 eq. of acetic acid

were added. The amine was isolated as an acetate salt

by precipitation in diethyl ether. The crude product

was purified by recrystallization in MeOH and diethyl

ether to afford the pure compound 6 (25.0 mg, 0.03

m); ¹³C NMR (DMSO-d₆, 100 MHz):

δ = 171.3,

171.1, 169.7, 168.5, 168.0, 163.8, 153.2, 133.1, 124.2,

121.9, 120.9, 112.9, 65.2, 58.4, 56.7, 54.1, 50.5, 50.4,

48.2, 44.3, 42.5, 38.3, 37.9, 30.8, 30.5, 29.4, 28.4,

26.0, 24.2, 23.4, 22.0, 19.1, 17.6, 16.8; IR (neat):

ν_max

=

3235, 2967, 1634, 1547, 1494, 1252 cm^-1;

HRMS (TOF ESI, ion polarity positive): m/z [M

+ H]⁺calcd for C₃₄H₅₇N₁₀O₇717.4412, found

717.4409; HPLC purity (X Bridge C18, 3.5 µm, H₂O

+ 0.1% formic acid /ACN – gradient 1-60% in 20

min): TR = 7.75 min, 85%.

1

mmol, 50%) as a white powder. H NMR (DMSO-d₆,

Procedure A for coupling reactions using HATU

and HOAt: To a solution of the carboxylic acid (1.2

eq.) in dry DMF (10 mL) at 0°C under nitrogen at-

mosphere, were successively added collidine (6.0

eq.), HATU (2 eq.) and HOAt (2 eq.). The solution

was let stirring at 0 °C for 1h. and then the solution of

the amine (free or as a salt, 1 eq.) in DMF (5 mL)

was added to the previous solution. The reaction mix-

ture was stirred at room temperature overnight. The

solvent was evaporated under vacuum and the residue

was taken up with EtOAc or CH₂Cl₂. The organic

phase was successively washed with water, saturated

aqueous NaHCO₃and brine, dried over Na₂SO₄, fil-

tered and concentrated under vacuum to afford the

crude product which was purified by column chroma-

tography on silica gel.

400 MHz):

δ = 10.03 (1H, s), 8.25 (1H, bs), 8.03 (1H,

s), 7.85-7.61 (2H, bs), 7.10 (1H, m), 6.94 (3H, bs);

4.47-4.32 (7H, m), 3.93-3.84 (5H, m), 3.33-2.64 (4H,

m), 2.73 (2H, m), 2.24 (1H, m), 2.06 (1H, m), 2.02

(3H, s), 1.83-1-56 (8H, m), 1.43-1.23 (6H, m), 1.34

(9H, s), 1.09 (3H, s), 0.75, 0.67 (6H, m); ¹³C NMR

(DMSO-d₆, 100 MHz):

δ = 174.2, 173.1, 172.5,

169.4, 168.0, 155.1, 152.8, 142.6, 138.9, 132.7, 123.4

121.6, 112.4, 78.1, 63.0, 58.7, 56.2, 53.4, 52.8, 50.5,

49.8, 43.4, 39.0, 38.6, 32.2, 31.6, 30.5, 28.6, 28.2,

27.5, 24.2, 23.8, 22.9, 22.4, 19.4, 17.9, 17.7; IR.

HRMS (TOF ESI, ion polarity positive): m/z [M

+ H]⁺calcd for C₃₉H₆₅N₁₀O₉817.4936, found

817.4939; HPLC purity (X Bridge C18, 3.5 µm, H₂O

+ 0.1% formic acid /ACN – gradient 5-100% in 20

min): TR = 9.13 min, 86%.

Procedure

B

for coupling reactions using

DMTMM: To a solution of the carboxylic acid (1

eq.) in dry DMF under nitrogen atmosphere and at

0ºC, were added DMTMM(Cl^-) (1,1 eq.) and NMM (4

eq.). After 30 min, a solution of the free amine or its

salt (1 eq.), in DMF was added and the reaction mix-

ture was stirred at 0°C for 1 h and at room tempera-

ture overnight. The solvent was evaporated under

vacuum and the residue was taken up with EtOAc.

The organic phase was successively washed with

water, saturated NaHCO₃and brine, dried over

Na₂SO₄, filtered and concentrated under vacuum to

afford the crude product which was purified by col-

umn chromatography column on silica gel using

EtOAc 100% to EtOAc/MeOH 95:5 as eluent.

(2S)-N-[(1S)-1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-amino-pentyl]-1-

[(3R,4R)-4-amino-1-[(2S)-2-[[(2S)-2-

aminopropanoyl]amino]-3-methyl-butanoyl]-3-

piperidyl]pyrrolidine-2-carboxamide tetrahydro-

chloride 7. Compound 6 (20.0 mg, 0.02 mmol, 1.0

eq.) was dissolved in dioxane (2.0 mL) and a 4 N

solution of HCl in dioxane (0.15 mL, 30.0 eq.) was

added. The reaction was kept 2 hours under stirring at

room temperature. Diethyl ether was then added to

allow precipitation of product 7 that was isolated as

the hydrochloride salt after filtration.. The crude

product was purified by recrystallization in MeOH

ACCEPTED MANUSCRIPT

bs), 7.13 (1H, m), 6.28 (1H, bs),5.00 (2H, s), 4.54

(1H, m), 4.35 (2H, m), 4.21 (1H, m), 3.85 (3H, s),

3.38 (1H, m), 3.20 (1H, m), 3.01(2H, m), 2.94(1H,

m), 2.70 (1H, m), 2.46 (1H, m),2.37 (1H, m), 2.33

(1H, m), 2.02 (3H, s), 2.00 (1H, m),1.85-1.65 (6H,

m), 1.59 (1H, m),1.44-1.23 (2H, m),1.42 (1H, m),1.32

(2H, m), 1.22 (1H, m); ¹³C NMR (DMSO-d₆, 100

(3R,4R)-(9H-fluoren-9-yl)methyl 3-((S)-2-((((S)-1-

(2-(5-acetamido-2-methoxybenzoyl)hydrazinyl)-6-

(((benzyloxy)carbonyl)amino)-1-oxohexan-2-

yl)oxy)carbonyl)pyrrolidin-1-yl)-4-(4-

methylphenylsulfonamido)piperidine-1-

carboxylate 11. Compound 11 was synthesized ac-

cording to the general procedure A from compound 8

(452 mg, 0.93 mmol, 1.2 eq.) and compound 10 (454

mg, 0.77 mmol, 1.0 eq.) to give, after purification by

column chromatography on silica gel using EtOAc

100% to EtOAc/MeOH 95:5 as eluent, compound 11

(713 mg, 0.67 mmol, 88%) as a white solid. mp =

MHz):

δ = 174.6, 171.1, 168.1, 165.7, 163.4, 156.2,

152.8, 149.4, 147.6, 139.7,137.8, 137.3, 132.7, 129.2,

127.9, 127.9, 127.7, 127.3, 124.4, 123.5, 121.5,

121.3,120.4, 120.1, 112.6, 69.4, 66.8, 63.2, 59.6, 59.6,

54.5, 50.7, 47.0, 44.7, 44.3, 40.1, 34.6, 32.4, 32.0,

30.5, 29.0, 24.5, 23.8, 22.8; IR (neat): ν_max

=3298,

1

2934, 1693, 1528, 1477, 1449, 1347 cm^-1; HRMS

(TOF ESI, ion polarity positive): m/z [M + H]⁺calcd

for C₅₅H₆₂N₉O₁₃S 1088.4188, found 1088.4180; m/z

[M + Na]⁺calcd for C₅₅H₆₁N₉O₁₃NaS 1110.4007,

found 1110.4021.

113-115 °C; R_f= 0.60 (EtOAc/MeOH 90:10); H

NMR (DMSO-d₆, 400 MHz):

δ = 10.56 (1H, bs),

10.08 (1H, bs), 9.95 (1H, s), 8.19 (1H, bs), 8.01 (1H,

s), 7.84 (2H, m),7.77 (1H, m), 7.72 (2H, d, J = 7.9

Hz), 7.56 (3H, m), 7.37 (2H, m), 7.35 (2H, m), 7.34

(5H, m), 7.30 (2H, m),7.20 (1H, bs), 7.11 (1H, m),

5.00 (2H, s), 4.54 (1H, m), 4.36 (2H, m), 4.24 (1H,

m), 3.86 (1H, m), 3.84 (3H, s), 3.83 (1H, m), 3.57

(1H, m), 3.21 (1H, m), 3.00 (2H, m), 2.79 (1H, m),

2.48 (1H, m), 2.36 (3H, s ), 2.23 (1H, m), 2.03 (3H,

s), 2.01 (1H, m), 1.83 (1H, m),1.73 (2H, m), 1.72 (1H,

m), 1.69 (1H, m H₁₁), 1.64 (1H, m), 1.57 (1H, m),

1.45 (2H, m H₂₄), 1.43 (1H, m), 1.35 (2H, m), 1.06

tert-butyl

N-[(1S)-1-[(3R,4R)-3-[(2S)-2-[[(1S)-1-

[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-

(benzyloxycarbonyla-

mino)pentyl]carbamoyl]pyrrolidin-1-yl]-4-(p-

tolylsulfonylamino)piperidine-1-carbonyl]-2-

methyl-propyl]carbamate 13. The N-Fmoc protect-

ed compound 11 (682 mg, 0.64 mmol, 1.0 eq.) was

dissolved in a 20% solution of piperidine in DMF (10

mL) and let under stirring at room temperature for 2

h. After evaporation of DMF and piperidine in excess,

the free amine was coupled with Boc-NH-Val-OH

(278 mg, 1.28 mmol, 2.0 eq.) in DMF (10.0 mL) ac-

cording to the procedure A to give, after purification

by column chromatography on silica gel using EtOAc

100%, then EtOAc/MeOH 95:5 as eluent, compound

13 (400 mg, 0.39 mmol, 60%) as a white solid. mp =

(1H, m); ¹³C NMR (DMSO-d₆, 100 MHz):

δ = 170.8,

168.4, 163.7, 156.5, 154.5, 153.2, 152.8, 143.8, 142.6,

140.8, 138.7, 137.3, 132.7, 129.6, 128.3, 128.2, 127.1,

126.5, 124.8, 123.7, 121.4, 120.7, 120.1, 112.5, 66.9,

65.1, 58.9, 56.5, 56.1, 53.4, 50.3, 47.1, 44.9, 41.9,

40.1, 32.8, 32.3, 32.0, 30.9, 29.2, 24.7, 23.8, 23.2,

20.9; IR (neat): ν_max= 3294, 2943, 1656, 1610, 1521,

1450 cm^-1; HRMS (TOF ESI, ion polarity positive):

m/z [M + Na]⁺calcd for C₅₆H₆₄N₈O₁₁NaS 1079.4313,

found 1079. 4354.

1

151-153 °C; R_f= 0.55 (EtOAc/MeOH 90:10); H

NMR (DMSO-d₆, 400 MHz):

δ = 10.55 (1H, bs),

(3R,4R)-(9H-fluoren-9-yl)methyl 3-((S)-2-(((S)-1-

(2-(5-acetamido-2-methoxybenzoyl)hydrazinyl)-6-

(((benzyloxy)carbonyl)amino)-1-oxohexan-2-

yl)carbamoyl)pyrrolidin-1-yl)-4-(4-

nitrophenylsulfonamido)piperidine-1-carboxylate

12. Compound 12 was synthesized following the pro-

cedure described according to method A from

10.04 (1H, bs), 9.95 (1H, s), 8.21 (1H, bs), 8.00 (1H,

s), 7.77 (1H, m), 7.73 (2H, d, J = 7.8 Hz), 7.57 (1H,

bs), 7.38 (2H, m), 7.36-7.34 (5H, m), 7.21 (1H, bs),

7.12 (1H, m), 6.70 (1H, s), 5.01 (2H, s), 4.52 (1H, m),

4.32 (1H, m), 4.11 (1H, m), 3.88 (1H, m), 3.86 (3H,

s), 3.37 (1H, m), 3.29 (1H, m), 3.00 (2H, m), 2.95

(1H, m), 2.64 (1H, m), 2.36 (3H, s), 2.21 (1H, m),

2.05 (1H, m), 2.02 (3H, s), 1.85 (3H, m), 1.82-1.58

(5H, m), 1.45 (2H, m), 1.40 (1H, m),1.35 (2H,

m),1.33 (9H, s), 1.25 (1H, m), 0.79-0.72 (6H, m); ¹³C

compound 9 (246 mg, 0.41 mmol, 1.2 eq) in DMF (5

mL) and compound 10 (235 mg, 0.34 mmol, 1.0 eq)

in (10 mL) to give after purification by column chro-

matography, compound 12 (239 mg, 0.22 mmol,

65%) as a pale yellow solid. mp = 142-144 °C: R_f=

NMR (DMSO-d₆, 100 MHz):

δ = 175.7, 169.9, 168.0,

1

163.1, 162.1, 156.5, 156.1, 152.8, 142.6, 138.4, 137.3,

132.7, 129.6, 128.3, 127.7, 126.6, 126.0, 123.3, 121.5,

120.9, 112.5, 77.1, 65.1, 62.6, 58.0, 55.8, 54.7, 52.8,

50.0, 42.4, 39.9, 39.2, 32.8, 31.8, 31.6, 29.8, 29.5,

28.5, 28.1, 23.6, 23.8, 22.3, 20.9,19.1, 17.8; IR (neat):

0.50 (EtOAc/MeOH 95:5); H NMR (DMSO-d₆, 400

MHz):

δ = (1H, bs), 10.08 (1H, bs), 9.95 (1H, s),8.36

(2H, d, J = 8.8 Hz), 8.26 (1H, bs), 8.09 (2H, d, J = 8.7

Hz), 8.01 (1H, s), 7.88 (2H, m), 7.83 (2H, m), 7.73

(1H, m), 7.42 (2H, m), 7.36-7.34 (7H, m), 7.24 (1H,

ACCEPTED MANUSCRIPT

ν_max

=

3280, 2938, 2870, 1652, 1520, 1454 cm^-1;

30.0 eq.) and the reaction mixture was let under stir-

ring at room temperature for 2 h. The solvent was

evaporated, toluene (2 x 10 mL) was added followed

by evaporation, and then ether was added and evapo-

rated to afford the corresponding TFA salt. This TFA

salt in solution in DMF (5.0 mL) in the presence of

collidine (0.13 mL, 1.0 mmol, 4.0 eq.) was coupled

with Boc-NH-Ala-OH (95 mg, 0.5 mmol, 2.0 eq.)

following the procedure A to give after purification

by column on silica gel using EtOAc 100% then

EtOAc/MeOH 95:5 as eluent, compound 15 (228 mg,

0.21 mmol, 83%) as a white solid. mp = 149-151 °C:

R_f= 0.25 (EtOAc/MeOH 95:5); ¹H NMR (DMSO-d₆,

HRMS (TOF ESI, ion polarity positive): m/z [M +

H]⁺calcd for C₅₁H₇₂N₉O₁₂S 1034.5021, found

1034.5016.

[4-[[(3R,4R)-3-[(2S)-2-[[(1S)-1-[[(5-acetamido-2-

methoxy-benzoyl)amino]carbamoyl]-

5(benzyloxycarbonylamino)pentyl]carbamoyl]pyrr

olidin-1-yl]-1-[(2S)-2-(tert-butoxycarbonylamino)-

3-methyl-butanoyl]-4-

piperidyl]sulfamoyl]phenyl]azinic acid 14. The

Fmoc protected compound 12 (209 mg, 0.19 mmol,

1.0 eq.) was dissolved in a 20% solution of piperidine

in DMF (10 mL) and the reaction mixture was let

stirring at room temperature for 2 h. After concentra-

tion, the free amine in DMF (5 mL) was coupled

with Boc-NH-Val-OH (83 mg, 0.38 mmol, 2.0 eq.)

was dissolved in DMF (10.0 mL) following the pro-

cedure described according to method A to give after

purification by column chromatography, compound

14 (152 mg, 0.14 mmol, 75%) as a pale yellow solid.

mp = 146-148 °C; R_f= 0.60 (EtOAc/MeOH 90:10);

400 MHz):

δ = 10.58 (1H, bs); 10.06 (1H, bs), 9.95

(1H, s), 8.22 (1H, bs,), 8.01 (1H, s), 7.76 (1H, m),

7.74 (2H, m), 7.63 (1H, bs), 7.58 (1H, bs),7.37 (2H,

m), 7.34-7.29 (5H, m), 7.21 (1H, bs), 7.12 (1H, m),

6.95 (1H, bs), 5.00 (2H, s), 4.52 (1H, m), 4.48 (1H,

m), 4.33 (1H, m), 3.95 (1H, m), 3.86 (1H, m), 3.85

(3H, s), 3.37 (1H, m) 3.29, (1H, m), 3.00 (2H, m),

2.98 (1H, m), 2.65 (1H, m), 2.36 (3H, s), 2.24 (1H,

m), 2.04 (1H, m), 2.03 (3H, s),1.85 (3H, m), 1.81-

1.71 (2H, m), 1.67 (1H, m), 1.59 (1H, m), 1.44 (2H,

m), 1.43 (1H, m),1.34 (2H, m), 1.35 (9H, s), 1.28 (1H,

m),1.26 (1H, m),1.12 (3H, s), 0.74-0.72 (6H, m); ¹³C

¹H NMR (DMSO-d₆, 400 MHz):

δ = 10.54 (1H, bs),

10.06 (1H, bs),9.95 (1H, s), 8.38 (2H, d, J = 8.7 Hz),

8.25 (1H, bp), 8.12 (2H, d, J = 8.8 Hz), 8.03 (1H, s),

7.72 (1H, m), 7.36-7.34 (5H, m),7.21 (1H, bs), 7.12

(1H, m), 6.72 (1H, s), 6.28 (1H, bs),4.99 (2H, s), 4.51

(1H, m), 4.32 (1H, m),4.09 (1H, m), 3.85 (4H, m),

3.39 (2H, m), 2.99 (3H, m), 2.66 (1H, m), 2.27 (1H,

m), 2.05 (1H, m), 2.02 (3H, s), 1.90 (1H, m),1.82-

1.60 (7H, m), 1.44-1.40 (3H, m), 1.32 (11H, m), 1.28

(1H, m), 0.79-0.72 (6H, m); ¹³C NMR (DMSO-d₆,

NMR (DMSO-d₆, 100 MHz):

δ = 174.5, 172.5, 170.5,

169.3, 168.0, 163.1, 156.1, 155.0, 152.8, 142.7, 138.5,

137.3, 132.7, 129.6, 128.3, 127.7, 126.6, 126.9, 123.7,

121.5, 120.6, 112.5, 78.1, 65.1, 62.9, 58.4, 56.2, 53.2,

52.8, 50.4, 49.8, 43.8, 40.3, 39.9, 33.7, 33.6, 31.8,

30.7, 30.2, 29.9, 28.2, 24.5, 23.8, 22.7, 20.9, 19.4,

17.5, 17.9; IR (neat): ν_max= 3298, 2932, 1652, 1626,

1518, 1455 cm^-1; HRMS (TOF ESI, ion polarity posi-

tive): m/z [M + H]⁺calcd for C₅₄H₇₇N₁₀O₁₃S

1105.5392, found 1105.5410; m/z [M + Na]⁺calcd for

C₅₄H₇₆N₁₀O₁₃NaS 1127.5212, found 1127.5217;

HPLC purity (Sunfire C18, 3.5 µm, H₂O + 0.2%

form. ac./ACN – gradient 5-100% in 20 min): TR =

17.83 min, 99%.

100 MHz):

δ = 173.6, 172.1, 170.1, 168.0, 164.0,

156.8, 156.1, 153.4, 150.0, 147.6, 137.4, 133.3, 127.7,

128.3, 127.7,126.6, 124.5, 123.9, 123.3, 121.1, 112.0,

78.0, 65.1, 62.7, 58.6, 56.2, 55.0, 53.5, 50.1, 43.3,

40.1, 39.8, 33.0, 31.9, 31.7, 30.4, 28.7, 28.1, 24.2,

23.8, 22.7, 21.1, 19.4, 18.0; IR (neat): ν_max=3296,

2939, 1684, 1527, 1477, 1450, 1348 cm^-1; HRMS

(TOF ESI, ion polarity positive): m/z [M + H]⁺calcd

for C₅₀H₆₉N₁₀O₁₄S 1065.4715, found 1065.4714; m/z

[M + Na]⁺calcd for C₅₀H₆₈N₁₀O₁₄NaS 1087.4535,

found 1087.4546.

[4-[[(3R,4R)-3-[(2S)-2-[[(1S)-1-[[(5-acetamido-2-

methoxy-benzoyl)amino]carbamoyl]-5-

(benzyloxycarbonyla-

mino)pentyl]carbamoyl]pyrrolidin-1-yl]-1-[(2S)-2-

[[(2S)-2-(tert-

tert-butyl

N-[(1S)-2-[[(1S)-1-[(3R,4R)-3-[(2S)-2-

butoxycarbonylamino)propanoyl]amino]-3-

methyl-butanoyl]-4-

[[(1S)-1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-

(benzyloxycarbonyla-

mino)pentyl]carbamoyl]pyrrolidin-1-yl]-4-(p-

tolylsulfonylamino)piperidine-1-carbonyl]-2-

methyl-propyl]amino]-1-methyl-2-oxo-

ethyl]carbamate 15. To a solution of the N-Boc pro-

tected compound 13 (256 mg, 0.25 mmol, 1.0 eq.) in

DCM (5.0 mL) was added TFA (0.56 mL, 7.5 mmol,

piperidyl]sulfamoyl]phenyl]azinic acid 16. To a

solution of the N-Boc protected compound 14 (125

mg, 0.12 mmol, 1.0 eq.) in DCM (8.0 mL) was added

TFA (0.45 mL, 6.0 mmol, 50.0 eq.) and the reaction

mixture was let stirring at room temperature for 2 h.

The solvent was evaporated, then toluene (2 x 10

mL)) was added followed by evaporation, and then

ACCEPTED MANUSCRIPT

ether was added and evaporated to afford the corre-

17 as a white powder (52.0 mg, 0.050 mmol, Yield

1

sponding TFA salt. The solution of the previous TFA

salt and collidine (0.10 mL, 0.72 mmol, 6.0 eq.) in

DMF (5.0 mL) was coupled with Boc-NH-Ala-OH

(46 mg, 0.24 mmol, 2.0 eq.) following the procedure

described according to method A to give after purifi-

cation by column chromatography, compound 16

(100 mg, 0.09 mmol, 75%) as a pale yellow solid. mp

87%). H NMR (DMSO-d6, 400 MHz): δ = 10.00

(1H, bs), 8.27-8.18 (2H, bs), 8.05 (1H, s), 7.74 (3H,

m), 7.65 (1H, bs), 7.35 (2H, m), 7.11 (1H, m), 6.95

(1H, bs), , 4.48-4.33 (3H, m), 3.94-3.63 (5H, m),

3.34-3.30 (2H, m), 2.95 (1H, m), 2.74 (2H, m), 2.64

(1H, m), 2.36-2.23 (4H, m), 2.04-2.02 (4H, m), 1.83-

1.56 (9H, m); 1.431.27 (13H, m), 1.10-1.07 (4H, m),

0.80-0.70 (6H, m); ¹³C NMR (DMSO-d6, 400 MHz):

1

= 148-150 °C; R_f= 0.55 (EtOAc/MeOH 95:5; H

NMR (DMSO-d₆, 400 MHz):

δ

= 10.54 (1H, bs),

: δ = 174.2, 173.0, 172.5, 169.4, 168.0, 162.3, 155.1,

10.05 (1H, bs), 9.94 (1H, s), 8.40 (2H, d, J = 8.5 Hz),

8.24 (1H, bs), 8.11 (2H, d, J = 8.5 Hz), 8.09 (1H, bp),

8.04 (1H, s), 7.71 (1H, m),7.63 (1H, bp), 7.34 (5H,

m), 7.22 (1H, bs), 7.12 (1H, m), 6.95 (1H, bs),5.00

(2H, s), 4.51 (1H, m), 4.46 (1H, m), 4.32 (1H, m),

3.95 (1H, m), 3.85 (4H, m), 3.69 (1H, m), 3.39 (1H,

m), 3.38 (1H, m), 3.01 (2H, m), 2.96 (1H, m), 2.30

(1H, m), 2.04 (1H, m), 2.02 (3H, s), 1.90-1.62 (8H,

m), 1.43-1.42 (3H, m), 1.36 (9H, s), 1.33 (2H, m),

1.29 (1H, m), 1.10 (3H, s), 0.77-0.72 (6H, m); ¹³C

152.8, 142.6, 138.9, 132.7, 129.6, 126.5, 123.4, 121.6,

, 112.4, 111.6, 78.1, 62.9, 58.5, 56.2; 53.2, 52.8, 50.5,

49.8, 43.4, 39.3, 38.9, 33.2, 32.2, 31.6, 30.4, 30.3,

28.2, 27.5, 24.2, 23.8, 22.4, 20.9, 19.4, 17.9, 17.7;

HRMS (TOF ESI, ion polarity positive): m/z [M +

H]⁺Calcd. for C46H70N10O11S 971.5025, found:

971.5021; calcd for [M + Na]⁺993.4844, found:

993.4814; HPLC purity (SUNFIRE C18, 3:5

µm,

H2O + 0:2% form. ac./ACN, gradient 5–100% in 20

min): TR = 11:12 min, 82%.

NMR (DMSO-d₆, 100 MHz):

δ = 174.6, 172.7, 172.3,

170.7, 169.6, 168.3, 163.8, 156.1, 153.1, 149.5, 147.4,

137.3, 132.7, 127.9, 127.8, 124.5, 123.4, 121.4, 120.6,

112.5, 78.4, 65.1, 63.1, 58.5, 56.2, 53.4, 52.6, 50.2,

49.6, 43.3, 40.1, 39.5, 33.0, 31.9, 31.6, 30.3, 30.2,

28.9, 28.1, 24.2, 23.8, 22.7, 19.2, 17.5, 17.8; IR

tert-butyl

N-[(1S)-1-[[(1S)-2-[[(1S)-1-[(3R,4R)-3-

[(2S)-2-[[(1S)-1-[[(5-acetamido-2-methoxy-

benzoyl)amino]carbamoyl]-5-

(benzyloxycarbonyla-

mino)pentyl]carbamoyl]pyrrolidin-1-yl]-4-(p-

tolylsulfonylamino)piperidine-1-carbonyl]-2-

methyl-propyl]amino]-1-methyl-2-oxo-

(neat): ν_max

= 3297, 2933, 1653, 1527, 1494, 1454,

1348 cm^-1; HRMS (TOF ESI, ion polarity positive):

m/z [M + H]⁺calcd for C₅₃H₇₄N₁₁O₁₅S 1136.5087,

found 1136.5095; Anal. calcd for C₅₃H₇₃N₁₁O₁₅S.1.5

H₂O: C 54.72, H 6.60, N 13.25; found C 54.70, H

6.25, N 12.41; HPLC purity (Sunfire C18, 3.5 µm,

H₂O + 0.2% form. ac./ACN – gradient 5-100% in 20

min): TR = 17.68 min, 100%.

ethyl]carbamoyl]-2-methyl-propyl]carbamate 18.

To a solution of the N-Boc protected compound 15

(228 mg, 0.21 mmol, 1.0 eq.) in DCM (5.0 mL) was

added TFA (0.46 mL, 6.19 mmol, 30.0 eq.) and the

reaction mixture was let stirring at room temperature

for 2 h. The solvent was evaporated, toluene (2 x 10

mL) was added followed by evaporation, and then

ether was added and evaporated to afford the corre-

sponding TFA salt. The solution of the previous TFA

salt and collidine (0.11 mL, 0.84 mmol, 4.0 eq.) in

DMF (5.0 mL) was coupled with Boc-NH-Val-OH

(91 mg, 0.42 mmol, 2.0 eq.) following the procedure

described according to method A to give after purifi-

cation by column chromatography, compound 18

(144 mg, 0.12 mmol, 57%) as a white solid. mp =

153-155 °C; R_f= 0.40 (EtOAc/MeOH 95:5); ¹H NMR

(S)-6-(2-(5-acetamido-2-

methoxybenzoyl)hydrazinyl)-5-((S)-1-((3R,4R)-1-

((S)--2-((S)-2-((tert-

butoxycarbonyl)amino)propanamido)-3-

methylbutanoyl)-4-(4-

methylphenylsulfonamido)piperidin-3-

yl)pyrrolidine-2-carboxamido)-6-oxohexan-1-

aminium acetate 17.

Compound 15 (64.0 mg, 0.058 mmol, 1.0 eq.) was

dissolved in MeOH (4.0 mL) and 10% Pd/C (13 mg)

was added. The reaction was kept 2 hours under stir-

ring in hydrogen atmosphere at room temperature.

The catalyst was then removed over a celite pad and

the solvent evaporated under vacuum. The crude was

diluted in a minimum amount of MeOH and 1.0 eq. of

acetic acid were added. Product 17 was isolated as the

acetate salt by precipitation in diethyl ether. The

crude product was purified by crystallization in

MeOH and diethyl ether to afford the pure compound

(DMSO-d₆, 400 MHz): δ = 10.56 (1H, bs), 10.05 (1H,

bs), 9.95 (1H, s), 8.23 (1H, bs), 8.00 (1H, s), 7.87

(1H, bs), 7.86 (1H, bs), 7.76 (1H, m), 7.74 (2H, m),

7.68 (1H, bs), 7.37 (2H, m), 7.36-7.16 (6H, m), 7.12

(1H, m), 6.67 (1H, bs), 5.01 (2H, s), 4.52 (1H, m),

4.44 (1H, m), 4.30 (1H, m), 4.34 (1H, m), 3.87 (1H,

m),3.85 (3H, s), 3.79 (1H, m), 3.38 (1H, m),3.29 (1H,

m), 3.00 (2H, m), 2.96 (1H, m), 2.65 (1H, m), 2.36

(3H, s), 2.24 (1H, m), 2.05 (1H, m),2.03 (3H, s), 1.91

(2H, m), 1.84 (2H, m), 1.69 (2H, m),1.65 (1H,

m),1.59 (1H, m), 1.43-1.23 (16H, m), 1.13 (3H, s),

ACCEPTED MANUSCRIPT

0.83-0.73 (12H, m); ¹³C NMR (DMSO-d₆, 100 MHz):

= 174.2, 171.8, 170.9, 170.5, 169.4, 168.0, 163.3,

dure described according to method A to give 21 (170

δ

mg, 0.27 mmol, 69%) as a white solid. In this case

purification by column chromatography on silica gel

used c-Hex/EtOAc 6:4 as eluent. mp = 97-99 °C; R_f=

156.1, 155.4, 152.8, 142.6, 138.6, 137.3, 132.7, 129.6,

128.3, 127.7, 126.5, 126.6, 123.7, 121.5, 120.6, 112.5,

78.0, 65.1, 63.3, 59.4, 58.9, 56.2, 53.6, 53.2, 50.3,

47.9, 43.8, 40.5, 40.0, 33.7, 32.1 30.8, 30.4, 30.0,

29.0, 28.1, 24.7, 23.8, 23.2, 20.9, 19.4, 17.9, 19.2,

17.7, 18.3; IR HRMS (TOF ESI, ion polarity posi-

tive): m/z [M + H]⁺calcd for C₅₉H₈₆N₁₁O₁₄S

1204.6076, found 1204.6078; m/z [M + Na]⁺calcd for

C₅₉H₈₅N₁₁O₁₄NaS 1226.5896, found 1226.5890;

HPLC purity (XBridge C18, 3.5 µm, H₂O + 0.2%

form. ac./ACN – gradient 5-100% in 20 min): TR =

17.60 min, 90%.

1

0.50 (c-Hex/EtOAc 6:4); H NMR (DMSO-d₆, 400

MHz): δ = 7.82 (1H, bs), 7.42 (1H, bs),7.34-7.30 (5H,

m), 6.49 (1H, bs), 5.01 (2H, s), 4.54 (1H, m), 4.28

(1H, m), 4.09 (1H, m), 3.83 (2H, m), 3.65 (1H, m),

3.60 (3H, s), 3.11 (1H, m), 2.95 (1H, m), 2.78 (1H,

m), 2.57 (1H, m), 2.45 (1H, m), 2.01 (2H, m), 1.93

(1H, m), 1.80 (1H, m), 1.67 (2H, m), 1.38 (9H, s),

1.29 (1H, m), 1.18 (3H, d, J = 7.1 Hz), 0.82, 0.81 (6H,

m); ¹³C NMR (DMSO-d₆, 100 MHz):

δ = 175.5,

172.2, 169.3, 156.7, 155.5, 137.0, 128.3, 128.0, 127.8,

77.7, 65.4, 60.2, 59.9, 59.0, 53.0, 51.5, 50.1, 46.8,

42.7, 39.9, 30.8, 30.3, 29.1, 28.2, 23.5, 18.1, 17.7,

(S)-methyl

1-((3R,4R)-1-((S)-2-((((9H-fluoren-9-

yl)methoxy)carbonyl)amino)-3-methylbutanoyl)-4-

((tert-butoxycarbonyl)amino)piperidin-3-

18.0; IR (neat): ν_max=3306, 2970, 1708, 1624, 1504,

1453, 1221, 1167 cm^-1; HRMS (TOF ESI, ion polarity

positive): m/z [M + H]⁺calcd for C₃₂H₅₀N₅O₈

632.3659, found 632.3663.

yl)pyrrolidine-2-carboxylate 20. Compound 20 was

synthesized following the procedure described ac-

cording to method A from 19 (264 mg, 0.8 mmol, 1.0

eq.) in DMF (5 mL) and Fmoc-NH-Val-OH (543 mg,

1.6 mmol, 2.0 eq.) in DMF (5.0 mL) to give com-

pound 20 (296 mg, 0.46 mmol, 58%) as a white solid.

In this case purification by column chromatography

on silica gel used c-Hex/EtOAc 6:4 as eluent. mp =

(S)-1-((3R,4R)-1-((S)-2-((S)-2-

(((benzyloxy)carbonyl)amino)propanamido)-3-

methylbutanoyl)-4-((tert-

butoxycarbonyl)amino)piperidin-3-yl)pyrrolidine-

2-carboxylic acid 22. Compound 21 (150 mg, 0.24

mmol, 1.0 eq.) was dissolved in MeOH (8.0 mL) and

NaOH 2 M (0.6 mL, 1.2 mmol, 5.0 eq.) was added

dropwise to the solution. The reaction was stirred at

60° C for 2 h. The solvent was evaporated and the

solid obtained was solubilized in water. The mixture

was acidified with 10% solution of KHSO₄until pH =

2-3. A part of the product was extracted from the wa-

ter phase with EtOAc and another part of the desired

compound was recuperated by solving the solid resi-

due of the water phase with MeOH. The two com-

bined organic phases were dried over Na₂SO₄and

concentrated under vacuum to yield compound 22

1

104-106 °C; R_f= 0.40 (c-Hex/EtOAc 6:4); H NMR

(DMSO-d₆, 400 MHz):

δ = 7.88 (2H, m); 7.74 (2H,

m); 7.51 (1H, bs,); 7.41 (2H, m); 7.32 (2H, m); 6.45

(1H, bs); 4.31-4.21 (5H, m), 4.07 (1H, m), 3.81 (1H,

m), 3.64 (4H, m), 3.07 (1H, m),2.96 (1H, m), 2.78

(1H, m), 2.57 (1H, m), 2.47 (1H, m), 2.04 (2H, m),

1.98 (1H, m),1.78 (2H, m), 1.65 (2H, m), 1.38 (9H, s),

0.88, 0.83 (6H, m); ¹³C NMR (DMSO-d₆, 100 MHz):

δ

= 175.1, 169.9, 156.1, 155.2, 143.8, 140.7, 127.6,

127.0, 125.3, 120.0, 77.5, 65.7, 60.1, 59.7, 59.3, 55.4,

51.5, 46.7, 47.1, 44.2, 39.9, 31.1 29.8, 29.1, 28.2,

23.7, 19.5, 18.1; mp = 104-106 °C; R_f= 0.40 (c-

Hex/EtOAc 6:4); IR (neat): ν_max

= 3324, 2967, 1707,

(137 mg, 0.22 mmol, 92%) as a white solid. mp =

1

1506, 1219, 1166 cm^-1; ; HRMS (TOF ESI, ion po-

larity positive): m/z [M + H]⁺calcd for C₃₆H₄₉N₄O₇

649.3601, found 649.3597.

129-131 °C; H NMR (DMSO-d₆, 30 MHz):

δ

= 8.31

(1H, bs), 7.87 (1H, bs), 7.44 (1H, bs), 7.35-7.22 (5H,

m), 6.80 (1H, bs), 5.02 (2H, s), 4.55 (1H, m), 4.28

(1H, m), 4.09 (1H, m), 3.84-3.81 (2H, m), 3.57 (1H,

m), 3.13 (1H, m), 3.04 (1H, m), 2.82 (1H, m), 2.56

(1H, m), 2.49 (1H, m), 2.06 (1H, m), 1.91 (1H, m),

1.83 (1H, m), 1.71 (1H, m), 1.62 (1H, m), 1.38 (9H,

s), 1.24 (2H, m), 1.18 (3H, d, J = 7.1 Hz), 0.82- 0.76

(S)-methyl

1-((3R,4R)-1-((S)-2-((S)-2-

(((benzyloxy)carbonyl)amino)propanamido)-3-

methylbutanoyl)-4-((tert-

butoxycarbonyl)amino)piperidin-3-yl)pyrrolidine-

2-carboxylate 21.The N-protected compound 20 (253

mg, 0.39 mmol, 1.0 eq.) was dissolved in a 20% solu-

tion of piperidine in DMF (10 mL) and the reaction

mixture was let stirring at room temperature for 2 h.

After concentration, the free amine in DMF (5 mL)

was coupled with CbZ-NH-Ala-OH (174 mg, 0.78

mmol, 2.0 eq.) in DMF (5.0 mL) following the proce-

(6H, m); ¹³C NMR (DMSO-d₆, 100 MHz):

δ = 178.7,

(C₆); 172.1, 169.4, 156.5, 155.5, 136.9, 127.8, 127.4,

126.4, 77.7, 65.0, 61.4, 61.0, 59.6, 52.7, 49.6, 46.9,

42.8, 39.8, 29.8, 29.6, 27.9, 28.6, 23.8, 19.2, 17.4,

17.9; IR (neat): ν_max=1702, 1627, 1547, 1453, 1225,

1160, cm^-1; HRMS (TOF ESI, ion polarity positive):

m/z [M + H]⁺calcd for C₃₁H₄₈N₅O₈618.3503, found

ACCEPTED MANUSCRIPT

618.3514; m/z [M + Na]⁺calcd for [M + Na]⁺

C₃₁H₄₇N₅O₈Na 640.3322, found 640.3322.

ppm). The chemical shifts deviations were calculated

as the differences between observed chemical shifts

and random coil values reported in water reported by

Wishart et al. (¹H, ¹³C and ¹⁵N random coil NMR

chemical shifts of the common amino acids).[19e]

Vicinal coupling constants were extracted from 1D ¹H

spectrum and from 2D ¹H-¹H COSY at 278 K and 298

K.

tert-butyl

N-[(3R,4R)-3-[(2S)-2-[[(1S)-1-[[(5-

acetamido-2-methoxy-benzoyl)amino]carbamoyl]-

5-

(benzyloxycarbonyla-

mino)pentyl]carbamoyl]pyrrolidin-1-yl]-1-[(2S)-2-

[[(2S)-2-

(benzyloxycarbonylamino)propanoyl]amino]-3-

methyl-butanoyl]-4-piperidyl]carbamate 23.

Compound 23 was synthesized following the proce-

dure described according to method B from com-

pound 22 (132 mg, 0.22 mmol, 1.2 eq.) solved in

DMF (5.0 mL) and compound 10 (108 mg, 0.18

mmol, 1.0 eq.) in DMF (5.0 mL) to give after purifi-

cation by column chromatography, compound 23

(114 mg, 0.11 mmol, 61%) as a white solid. mp =

Restrained molecular simulations

Simulated annealing were performed using the

GROMACS package (version 5.0.2) [22], with the

Generalized Amber Force Field [23]. The aqueous

solvent was implicitly modelled by a continuum me-

dium with a dielectric constant of 80. Compound to-

pologies and parameters were generated for

GROMACS using the ACPYPE tool [24]. Simulated

annealings consisted in running molecular dynamics

(MD) simulations at a temperature linearly decreasing

from 900 to 300 K over a period of 10 ns. Then the

compounds were simulated at constant temperature

(300 K) using a standard MD protocol over an addi-

tional period of 10 ns for analysis.

1

130-132 °C; R_f= 0.50 (EtOAc/MeOH 9:1); H NMR

(DMSO-d₆, 400 MHz): δ = 10.64 (1H, bs); 10.03 (1H,

bs), 9.96 (1H, s), 8.01 (1H, s), 7.95-7.69 (3H, m), 7.41

(1H, d, J = 7.7 Hz), 7.30-7.34 (10H, m), 7.21 (1H,

bs), 7.11 (1H, t, J = 8.9 Hz), 6.84 (1H, bs), 5.00 (4H,

s), 4.55-4.38 (3H, m), 4.10 (1H, m), 3.97 (1H, m);

3.84 (3H, s), 3.78 (1H, m), 3.63 (1H, m), 3.31 (1H,

m), 3.09 (2H, m); 2.99 (2H, m), 2.79-2.63 (2H, m),

2.07 (1H, s); 2.02 (3H, s); 1.95-1.66 (6H, m); 1.53-

1.44 (3H, m), 1.37-1.33 (11H, s), 1.22 (1H, m); 1.14

(3H, d, J = 7.2 Hz), 0.75- 0.66 (6H, m); ¹³C NMR

Fluorescence-detected Thioflavin-T binding assay

Thioflavin T was obtained from Sigma. Aβ_1-42was

purchased from American Peptide. The peptide was

dissolved in an aqueous 1% ammonia solution to a

concentration of 1 mM and then, just prior to use, was

diluted to 0.2 mM with 10 mM Tris-HCl, 100 mM

(DMSO-d₆, 100 MHz):

δ

=

172.4, 172.2, 171.9,

169.4, 162.5, 156.2, 155.7, 155.3, 152.9, 137.3, 137.1,

132.8, 128.4, 127.7, 127.3, 123.9, 121.6, 120.4, 112.6,

78.4, 65.4, 62.4, 61.2, 55.8, 53.2, 52.8, 50.1, 49.3,

49.6, 45.1, 43.7, 40.3, 39.1, 32.6, 32.0, 30.0, 29.7,

28.7, 23.4, 27.7, 24.3, 22.7, 19.1, 17.1, 17.8; IR

NaCl buffer (pH 7.4). Stock solutions of

β-hairpin

compounds and fragments were dissolved in DMSO

with the final concentration kept constant at 0.5%

(v/v). Thioflavin T fluorescence was measured to

evaluate the development of Aβ_1-42fibrils over time

using a fluorescence plate reader (Fluostar Optima,

BMG labtech) with standard 96-well black microtiter

plates. Experiments were started by adding the pep-

tide (final Aβ_1-42concentration equal to 10 µM) into a

mixture containing 40 µM Thioflavin T in 10 mM

Tris-HCl, 100 mM NaCl buffer (pH 7.4) with and

without the tested compounds at different concentra-

tions (100, 10 and 1 µM for the compounds active at

10 µM) at room temperature. The ThT fluorescence

intensity of each sample (performed in triplicate) was

recorded with 440/480 nm excitation/emission filters

set for 42 hours performing a double orbital shaking

of 10 s. before the first cycle. The fluorescence assays

were performed between 2 and 4 times on different

days, with the same batch of peptide. The ability of

compounds to inhibit Aβ_1-42aggregation was assessed

considering both the time of the half-life of aggrega-

tion (t_1/2) and the intensity of the experimental fluo-

rescence plateau (F). The t_1/2extension is defined as

(neat): ν_max

= 3297, 2935, 2870, 1658, 1625, 1520,

1455, 1245 cm^-1; HRMS (TOF ESI, ion polarity posi-

tive): m/z [M + H]⁺calcd for C₅₅H₇₇N₁₀O₁₃

1085.5672, found 1085.5688; HPLC purity (X Bridge

C18, 3.5 µm, H₂O + 0.1% formic acid /ACN – gradi-

ent 5-100% in 20 min): TR = 16.38 min, 94%.

NMR Conformational studies

Proton NMR spectra were recorded on Bruker spec-

trometers operating at 600 MHz for 3 and at 800

MHz for 4, equipped with a TCI cryoprobe (ICSN,

Institut de Chimie des Substances Naturelles, Gif-sur-

Yvette). ¹H and ¹³C resonances were completely as-

signed using 1D ¹H WATERGATE, 2D ¹H-¹H

TOCSY (mixing time set to 30, 60 or 100 ms), 2D ¹H-

¹H ROESY (200 ms mixing time), 2D ¹H-¹³C HSQC

and 2D ¹H-¹³C HMBC spectra, recorded at 278 K and

298 K. ¹H and ¹³C chemical shifts were calibrated

using the solvent residual peak (H₂O/D₂O, δ ¹H 4.70

ACCEPTED MANUSCRIPT

the experimental t_1/2in the presence of the tested

compound relative to the one obtained without the

compound and is evaluated as the following percent-

Sample preparation: the commercial Aβ_1-42was dis-

solved upon reception in 0.16% NH₄OH (at 2 mg/mL)

for 10 minutes at 20°C, followed by an immediate

lyophilization. The dried sample was then stored at -

20°C until use.

age: t_1/2(Aβ_1-42+compound) – t_1/2(Aβ_1-42) / t_1/2(Aβ_1-42

)

Χ

100. The F reduction is defined as the intensity of

experimental fluorescence plateau observed with the

tested compound relative to the value obtained with-

out the compound and is evaluated as the following

percentage : (FAβ_1-42+compound – FAβ_1-42) / FAβ_1-42

CE experiments were carried out with a PA800 Prote-

omeLab instrument (Beckman Coulter Inc., Brea, CA,

USA) equipped with a photodiode array detector. UV

Detection was performed at 190 nm. The prepared

sample (as previously described) was reconstituted by

dissolution in 20 mM phosphate buffer pH 7.4 con-

taining DMSO (control or stock solutions of the eval-

uated compound dissolved in DMSO). A constant

DMSO/phosphate buffer ratio of 2.5% (v/v) was used

for each sample. The final peptide concentration was

set at 100 µM regardless the peptide/compound ratio.

Χ

100.

Transmission electron microscopy

Samples were prepared under the same conditions as

in the ThT-fluorescence assay. Aliquots of Aβ_1-42(10

µM in 10 mM Tris-HCl, 100 mM NaCl, pH 7.4 in the

presence and absence of the tested compound) were

adsorbed onto 300-mesh carbon grids for 2 min,

washed and dried. The samples were negatively

stained for 45 s. on 2% uranyl acetate in water. After

draining off the excess of staining solution and dry-

ing, the grids were observed using a JEOL 2100HC

TEM operating at 200 kV with a LaB6 filament. Im-

ages were recorded in zero-loss mode with a Gif

Tridiem energy-filtered-CCD camera equipped with a

2k x 2k pixel-sized chip (Gatan inc., Warrendale,

PA). Acquisition was accomplished with the Digital

Micrograph software (versions 1.83.842, Gatan inc.,

Warrendale, PA).

For the CE separation of Aβ oligomers, fused silica

capillary 80 cm (10.2 cm to the detector) ´ 50 mm I.D.

were used. The background electrolyte was a 80 mM

phosphate buffer, pH 7.4.

The separation was carried out under -30 kV at 20°C.

The sample was injected from the outlet by hydrody-

namic injection at 3.44 KPa for 10 s. After each run,

the capillary was rinsed for 5 min with water, 1 min

with SDS 50 mM, 5 min with NaOH 1 M and equili-

brated with running buffer for 5 min.

Capillary electrophoresis

ACCEPTED MANUSCRIPT

Figures and Schemes

Figure 1

+

NH₃

H

O

S

O

H

N

HN

N

+

⁺H₃N

O

GVMLG

NH₃

N

O

H

⁺HN

O

HN

O

CONH₂

KLVFF

O

HO

2

1

OH

O

S

O

S

H

O

N

H

+

N

NH₃

O

+

NH

NH₃

NH⁺

O

NH⁺

O

NH

O

H

N

H

O

H

O

N

H

⁺H₃N

3

4

O

S

H

O

H

RHN

NH⁺

O

H₂N

N

+

N

NH₃

+

NH

N

NH₃

O

NH

NH⁺

O

NH

O

NH

H

O

H

N

H

O

H

O

N

H

O

N

H

⁺H₃N

6 R= tBuOCO

7 R=H

5

15

ACCEPTED MANUSCRIPT

Scheme 1

NHCbz

O

H

R₁

S N

O

HATU

HOAt

NFmoc

H

N

+

H₃N

N

collidine

DMF

H

COOH

O

CF₃COO

O

8 R₁= CH₃

9 R₁= NO₂

10

O

H

R₁

S N

O

R₁

S N

O

NHBoc

N

1) Piperidine 20% in DMF

NFmoc

O

N

O

N

H

O

H

2) N-Boc-L-Val-OH

HATU, HOAt

H

N

H

O

collidine, DMF

13 R₁= CH₃60%

14 R₁= NO₂75%

11 R₁= CH₃88%

12 R₁= NO₂65%

NHCbz

H

NHCbz

NHBoc

O

H

HN

O

R₁

S N

O

H₃C

S N

O

NH

From 15

NH

N

1) TFA, DCM

O

N

O

N

O

N

H

O

H

2) N-Boc-L-Ala-OH

N

2) N-Boc-L-Val-OH

N

HATU, HOAt

H

O

H

O

collidine, DMF

18 57%

1) H₂, Pd/C 10%, MeOH

2) HCl 4 M, dioxane

NHCbz

15 R₁= CH₃83%

NHCbz

16 R₁= NO₂75%

1) H₂, Pd/C 10%, MeOH

2) HCl 4 M, dioxane

4 19%

H₂, Pd/C 10%, MeOH

5 60%

NHBoc

O

H

S N

O

NH

H⁺

N

O

N

O

H

N

H

N

H

O

17 87%

HCl 4 M, dioxane

+

NH₃

3 50%

16

ACCEPTED MANUSCRIPT

Scheme 2

N-Fmoc-L-Val-OH

BocNH

N

BocNH

HATU, HOAt

NH

N

NHFmoc

N

collidine, DMF

O

COOCH₃

20 58%

COOCH₃

19

BocNH

N

1) Piperidine 20% in DMF

O

NaOH 2M, MeOH

N

NHCbz

N

2) N-Cbz-L-Ala-OH

H

O

HATU, HOAt

COOCH₃

21 69%

collidine, DMF

O

DMTMM

NMM

BocNH

H

N

O

H₂N

+

N

NHbz

H

O

H

N

DMF

N

H

O

N

10

COOH

22 92%

NHCbz

O

BocNH

NHCbz

HCl 4 M,

dioxane

NH

N

1) H₂, Pd/C 10%

O

7

6

50%

NH

O

70%

MeOH

H

O

N

2) CH₃COOH, MeOH

H

O

N

H

CbzHN

23 61%

Figure 2

A)

B)

O

H

N

S

N

NH

+

NH₂

H

O

H

O

H

NH⁺

H

N

O

H

O

H

N

HN

N

H

O

3

⁺H₃N

17

ACCEPTED MANUSCRIPT

Figure 3

A)

B)

O

S

H

O

H

+

NH₃

N

NH

H

N

H

O

H

O

NH⁺

H

N

O

H

O

H

N

HN

N

H

O

4

⁺H₃N

18

ACCEPTED MANUSCRIPT

Table 1.

Compounds

(compound/Aβ

ratio)^a

t_1/2

extension ^b

NA^d

F

reduction ^c

−97±1 %

−90±2 %

−87±1 %

−29±9 %

−66±11 %

−81±10 %

−38±16 %

−38±10 %

−32±9 %

−35±10 %

ne

1¹³10/1

1¹³1/1

NA

2¹⁴10/1

NA

2

¹⁴1/1

+48±12 %

+76±16 %

+96±10 %

+36±12 %

ne^e

3 10/1

3 10/1 (water)

3 1/1

3 0.1/1

4 10/1

+30±3 %

+58±10 %

ne

4 10/1 (water)

4 1/1

4 0.1/1

5 10/1

ne

+56±4 %

+27±7 %

+29±9 %

+13±5 %

ne

−54±4 %

ne

5 1/1

6 10/1

−43±7 %

ne

6 1/1

7 10/1

−18±13 %

ne

7 1/1

ne

15 10/1

15 1/1

SAT^f

SAT^e

ne

17 10/1

17 1/1

+59±11 %

+23±2 %

+7±1 %

ne

O

S

ne

O

H

H₂N

N

CF₃CO₂H

N

24 10/1¹⁴

24 1/1¹⁴

25 10/1

25 1/1

−44±8 %

−16±9 %

−23±14 %

ne

NH

O

H

NH⁺

O

24

25

OCH₃

NH₂HCl

ne

^xParameters are expressed as mean ± SE, n=3-6. ^aUnless otherwise indicated, compounds were dissolved in DMSO with the final concentration kept con-

stant at 0.5%. ^bSee the experimental section for the calculation of the t_1/2extension. ^cSee the experimental section for the calculation of the F reduction. ^d

NA = no aggregation. ^ene = no effect. ^fSAT means that a saturation of the fluorescence signal is observed because 15 self-aggregates at 100 µM.

19

ACCEPTED MANUSCRIPT

Figure 4.

Figure 5

a)

b)

c)

Figure 6

20

ACCEPTED MANUSCRIPT

Captions

Figure 1. Structures of β-hairpin mimic 1 and glycopeptidomimetic 2 previously reported [13,14] and of the new-

ly designed β-hairpin mimics 3-7.

Scheme 1. Synthesis of compounds 3-5

Scheme 2. Synthesis of compounds 6-7

Table 1. Effects of compounds 3-7, 15, 23, 24 and 25 on Aβ_1-42fibrillization assessed by ThT-fluorescence spec-

troscopy. The concentration of Aβ_1-42is 10 µM. Compounds were tested at 10/1 and 1/1 compound/Aβ_1-42ratios

and compared to the values obtained for Aβ_1-42alone (t_1/2and F). Compounds 3 and 4 were also tested at the sub-

stoichiometric ratio of 0.1/1 (compound/Aβ_1-42). Data for compounds 1, 2 and 24 are extracted from references 13

and 14.

Figure 2. A) Structures of the major conformer of compound 3 showing the assigned ROEs by blue flashes; B)

Structure of 3 generated by simulated annealing using restrained distances inferred from NMR ROEs. Sulfur,

nitrogen, oxygen, atoms and polar hydrogens are coloured in yellow, blue, red, and white, respectively. The fig-

ure was prepared with UCSF Chimera.[25]

Figure 3. A) Structure of the major conformer of compound 4 showing the assigned ROEs by blue flashes. B)

Structure of 4 generated by simulated annealing using restrained distances inferred from NMR ROEs. Sulfur,

21

ACCEPTED MANUSCRIPT

nitrogen, oxygen, atoms and polar hydrogens are coloured in yellow, blue, red, and white, respectively. The fig-

ure was prepared with UCSF Chimera.[25]

Figure 4. Representative curves of ThT fluorescence assays over time showing Aβ_1-42(10 µM) aggregation in the

absence (purple curve) and in the presence of compounds 3 (a) and 4 (b) at compound/Aβ_1-42ratios of 10/1 (green

curves), 1/1 (blue curves) and 0.1/1 (yellow curves). The control curves are represented in red lines and buffer in

grey.

Figure 5. Fibril formation of Aβ_1-42visualized by TEM: negatively stained images recorded after 42 h of incuba-

tion of Aβ_1-42(10 µM in 10 mM Tris.HCl, 100 mM NaCl at pH = 7.4) alone (a) or in the presence of 100 µM of 3

(b) or 5 (d). Scale bars represent 500 nm.

Figure 6. Electrophoretic profile obtained immediately (0 h), 2 h, 6 h and 12 h after sample reconstitution (t₀) of

Aβ_1-42peptide (100 µM) alone (A), in the presence of compound 3 at 3/Aβ_1-42ratio of 10/1 (B). Peak area of the

monomer (ES) related to its peak area in the sample of Aβ_1-42alone at 0 h, 6 h and 12 h (C).

Acknowledgment

The Ministère de l’Enseignement Supérieur et de la Recherche (MESR) is thanked for financial support for N.

Tonali. Marianna Munafo (BioCIS, UMR 8076, Erasmus student from the Università degli Studi di Milano) is

thanked for her help with the synthesis. Ghislaine Frébourg (Institut de Biologie Paris Seine (IBPS)/FR 3631,

Service de Microscopie Electronique, Université Pierre et Marie Curie (UPMC), France) is thanked for her con-

tribution to cryo-electron microscopy. Camille Dejean (BioCIS, UMR 8076) and Jean-François Gallard (ICSN,

UPR2301) are thanked for their help with the NMR experiments. The Laboratory BioCIS is a member of the La-

boratory of Excellence LERMIT supported by a Grant from ANR (ANR-10-LABX-33).

References

1

a) M. Stefani, C. M. Dobson, J. Mol. Med., 2003, 81, 678–699; b) F. Chiti, C. M. Dobson, Annu. Rev.

Biochem., 2006, 75, 333–366; c) A. Aguzzi, T. O’Connor, Rev. Drug Discov., 2010, 9, 237-248; d) Y. S. Eisele,

C. Monteiro, C. Fearns, S. E. Encalada, R. L. Wiseman, E. T. Powers, J. W. Kelly, Nat. Rev., 2015, 14, 759-780;

e) P. C. Ke, M.-A. Sani, F. Ding, A. Kakinen, I. Javed, F. Separovic, T. P. Davis,R. Mezzenga, Chem. Soc. Rev.,

2017, 46, 6492-6531.

2

a) M. Goedert, M. G. A. Spillantini, Science, 2006, 314, 777-780; b) C. Haas, D. J. Selkoe, Nat. Rev.

Mol. Cell. Biol., 2007, 8, 101-112.

3

4

https://www.alz.co.uk/research/WorldAlzheimerReport2016.pdf

a) D. Goyal, S. Shuaib, S. Mann, B. Goyal, ACS Comb. Sci. 2017, 19, 55−80; b) H. Coman, B. Nemes,

Int. J. Gerontol., 2017, 11 , 2-6; c) E. McDade, R. J. Bateman, Nature, 2017, 457, 153-155.

a) F. Belluti, A. Rampa, S. Gobbi, Exp. Opin. Ther. Pat., 2013, 23, 581-596; b) T. Härd, C. Lendel, J.

Mol. Biol., 2012, 421, 441–465; c) A. J. Doig, P. Derreumaux, Curr. Opin. Struct. Biol., 2015, 30, 50–56.

a) J. Mayes, C. Tinker-Mill, O. Kolosov, H. Zhang, B. J. Tabner, D. Allsop, J. Biol. Chem., 2014, 289,

5

6

12052-12062; b) S. I. A. Cohen, S. Linse, L. M. Luheshi, E. Hellstrand, D. A. White, L. Rajah, D. E. Otzen, M.

Vendruscolo, C. M. Dobson, T. P. J. Knowles, Proc. Natl. Acad. Sci. U S A. 2013, 110, 9758-9763; c) M. J. Guer-

rero-Muñoza, D. L. Castillo-Carranza, R. Kayed, Biochem. Pharm., 2014, 88, 468–478.

7

a) C. Haas, D.J. Selkoe, Nat. Rev. Mol. Cell Biol., 2007, 101-112; (b) K. Ono, M. M. Condron, D.B.

Teplow, Proc. Natl. Acad. Sci. U S A., 2009, 106, 14745-14750; (c) P. Prangkio, E. C. Yusko, D. Sept, J. Yang,

M. Mayer, PLoS One, 2012, 7, e47261; (d) P. Cizas, R. Budvytytec, R. Morkuniene, R. Moldovan, M. Broccio,

M. Lösche, G Niaura, G. Valincius, V. Borutait Arch. Biochem. Biophys., 2010, 496, 84-92; e) I. Benilova, E.

Karran, B. De Strooper Nat. Neurosci., 2012, 15, 349-357.

8

Q. Nie,, X.Du, M. Geng, Acta Pharmacol. Sin., 2011, 32, 545−551.

9

a) P. Vlieghe, V. Lisowski, J. Martinez, M. Khrestchatisky, Drug Discov. Today., 2010, 15, 40-56; (b)

F. Albericio, H.G. Kruger, Future Med. Chem., 2012, 4, 1527-1531; c) T. Uhlig, T. Kyprianou, F. G. Martinelli,

C. A. Oppici, D. Heiligers, D. Hills, X. R. Calvo, P. Verhaert, EuPA Open Proteom., 2014, 4, 58-69; d) Keld

Fosgerau, and Torsten Hoffmann, Drug Discov. Today., 2015, 20, 122-128.

22

ACCEPTED MANUSCRIPT

10

a) S. Aileen Funke, D. Willbold, Curr Pharm Des. 2012, 18, 755–767; b) M. Chemerovski-Glikman, M.

Richman, S. Rahimipour in Top. Heterocycl. Chem., 2016, Publisher: Springer Berlin Heidelberg, 1-32; c) C. I.

Stains, K. Mondal, I. Ghosh, ChemMedChem, 2007, 2, 1674-1692; c) T. Takahashi, H. Mihara, Acc. Chem. Res.,

2008, 41, 1309-1318; d) J. Kumar, R. Namsechi, V. L. Sim, PLoS One, 2015, 10, e0129087; e) V. Parthsarathy,

P.L. McClean, C. Hölscher, M. Taylor, C. Tinker, G. Jones, O. Kolosov, E. Salvati, M. Gregori, M. Masserini, D.

Allsop, PLoS One, 2013; 8, e54769; f) P.-N. Cheng, C. Liu, M. Zhao, D. Eisenberg, J. S. Nowick, Nat. Chem.,

2012, 4, 927-933; g) S. Brogi, S. Butini, S. Maramai, R. Colombo, L. Verga, C. Lanni, E. De Lorenzi, S. Lampo-

ni, M. Andreassi, M. Bartolini, V. Andrisano, E. Novellino, G. Campiani, M. Brindisi, S. Gemma CNS Neurosci

Ther. 2014, 20, 624-32.

11

a) G. Hopping, J. Kellock, B. Caughey, V. Daggett, ACS Med. Chem. Lett., 2013, 4, 824−828; b) K. N. L.

Huggins, M. Bisaglia, L. Bubacco, M. Tatarek-Nossol, A. Kapurniotu, N. H. Andersen, Biochemistry, 2011, 50,

8202−8212; c) Y. Hamada, N. Miyamoto, Y Kiso, Bioorg. Med. Chem. Lett., 2015, 25, 1572–1576; d) L.

Vahdati, D. Brinet, G. Bernadat, I. Correia, S. Panzeri, R. Fanelli, O. Lequin, M. Taverna, S. Ongeri, U. Piarulli,

Eur. J. Org. Chem. 2017, 2971–2980.

12-

S. Pellegrino, A. Contini, M. L. Gelmi, L. Lo Presti, R. Soave, E. Erba, J. Org. Chem., 2014, 79, 3094-

3102.

13

S. Pellegrino, N. Tonali, E. Erba, J. Kaffy, M. Taverna, A. Contini, M. Taylor, D. Allsop, M. L. Gelmi, S.

Ongeri Chem. Sci., 2017, 8, 1295 - 1302.

14 J. Kaffy, D. Brinet, J-L Soulier, I. Correia, N. Tonali, K. F. Fera, Y. Iacone, A. R. F. Hoffmann, L.

Khemtemourian, B. Crousse, M. Taylor, D. Allsop, M. Taverna, O. Lequin, S. Ongeri, J. Med. Chem. 2016, 59,

2025−2040.

15-

16

P. E. Maligres, M. M. See, D. Askin, P. J. Reider, Tet. Lett., 1997, 38, 1586-1587.

T. Cierpicki and J. Otlewski, J. Biomol. NMR., 2001, 21, 249-261.

17

J. S. Nowick, K. S. Lam, T. V. Khasanova, W. E. Kemnitzer, S. Maitra, H. T. Mee, R. Liu, J. Am. Chem.

Soc., 2002, 124, 4972–4973.

18

a) G.W. Vuister and A. Bax, J. Am. Chem. Soc. 1993, 115, 7772-7777; b) F. Avbelj, S. G. Grdadolnik, J.

Grdadolnik and R. L. Baldwin, Proc. Natl. Acad. Sci. USA., 2006, 103, 1272-1277.

19

a) L. Szilagyi and O. Jardetzky, J. Magn. Reson., 1989, 83, 441–449; b) S. Spera and A. Bax, J. Am.

Chem. Soc., 1991, 113, 5490–5492; c) D. S. Wishart and B. D. Sykes, Methods Enzymol., 1994, 239, 363-392; d)

D. S. Wishart and B. D. Sykes, J. Biomol. NMR., 1994, 4, 171–180; e) D. S. Wishart, G. C. Bigam, A. Holm, R.

S. Hodges, B. D. Sykes., J. Biomol. NMR., 1995, 5, 67-81; f) L. J. Smith, K. M. Fiebig, H. Schwalbe, C.M.

Dobson, Fold. Des., 1996, 1, R95–R106.

20

H. LeVine, Methods Enzymol., 1999, 309, 274-284.

21

D. Brinet, J. Kaffy, F. Oukacine, S. Glumm, S. Ongeri, M. Taverna, Electrophoresis, 2014, 35, 3302–

3309.

22

23

24

25

M. J. Abraham, T. Murtola, R. Schulz, S. Páll, J.C. Smith, B. Hess, E. Lindahl, SoftwareX, 2015, 1-2, 19-

J. Wang, R.M. Wolf, J. W. Caldwell, P. A. Kollman, D. A Case, J. Comput. Chem., 2004, 25, 1157-1174.

A. W. Sousa da Silva, W. F. Vranken, BMC Research Notes, 2012, 5, 367.

E. F. Pettersen, T.D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, J

Comput. Chem. 2004, 25, 1605-1612.

23

ACCEPTED MANUSCRIPT

Highlights

No causal treatment exists for Alzheimer’s disease

A promising strategy is the use of Aβ_1-42aggregation inhibitors very early before the symptoms and

the brain pathology appear

Peptides often offer greater efficacy, selectivity, and reduced risk of unforeseen side‐reactions

Small peptide beta-hairpins derivatives show anti-aggregation activity at the very early stages

Article Doi

Structure-activity relationships of β-hairpin mimics as modulators of amyloid β-peptide aggregation

DOI: 10.1016/j.ejmech.2018.05.018

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2018.05.018

Full text of DOI:10.1016/j.ejmech.2018.05.018

Products guided by the article

R&D Labs maybe for 6325-93-5

Relevant to this article

Hot Product