K N Dahlgren

A M Manelli

W B Stine

L K Baker

G A Krafft

K Ono

M M Condron

L Ho

J Wang

W Zhao

G M Pasinetti

D B Teplow

J Biol

Chem

T Lim

F Chu

W Yang

S A Beech

G M Frautschy

J Cole

Neurosci

F Yang

G P Lim

A N Begum

O J Ubeda

M R Simmons

S S Ambegaokar

P Chen

R Kayed

C G Glabe

N Reichert

B Ketter

V Nejadnik

M Guenzler

D Miloslavsky

Y Wang

J Lu

I C Lull

E Tudor

M Liu

Grundman

General Introduction to Protein Structure, the concept of Foldamers, Alzheimer's disease and the treatment strategies

In this chapter the different secondary structure elements of proteins and their importance in nature will be briefly described, as well as the use of synthetic mimics of these elements together with their oligomers called Foldamers. Alzheimer's disease, a widespread neurodegenerative disease caused by the misfolding of the amyloid beta peptide will be introduced, together with evidence of its pathology and treatment strategies investigated by different research groups worldwide.

this PhD possible, for teaching me so much, for having greatly contributed to my growth as a chemist and as a person.

I would like to express my gratitude to Prof. Maria Luisa Gelmi and to Dr. Gilles Guichard that have agreed to take part of the jury of my thesis.

In terms of collaborations, I would like to thank:

Prof. Cesare Gennari (University of Milan) for his advices.

Prof. Olivier Lequin and Dr. Isabel Correia in the University Pierre et Marie Curie (UMR CNRS 7203) for their precise NMR conformational analysis.

My sincerely thanks to Dr. Julia Kaffy for the biological evaluation and for her kindness during my stay in Paris, and to Dr. Guillaume Bernadat for computational studies and his patience in teaching me the basics of molecular modeling.

A special thanks to Jean-Louis for his passion and advices in chemistry, Claire (Paris) and Marta (Como) for NMR, Sonia (Como) and Karine (Paris) for Mass spectroscopy and microanalysis.

I have to dedicate a special thanks to Dr. Roberto Fanelli, thank you for your support in every moment of these three years.

I would like to thank all of my friends in Italy and France, thank you for all the fun that we had together inside and outside of the laboratory.

My parents receive my greatest wholeheartedly thank for their support, understanding, helpfulness and encouragement they gave me all through this experience.

Merci beaucoup a vous tous

Grazie mille a tutti Proteins are the most important class of organic molecules present in the cells, making up 50% or more of their dry weight. These macromolecules are responsible for most of the complex functions that make life possible, for instance: enzymatic catalysis, transport and storage, coordinated motion, mechanical support, immune protection, generation and transmission of nerve impulses, growth control, and differentiation.

Despite the difference in function, all proteins are constituted of smaller building blocks, namely amino acids that are covalently joined together by amide bonds to form polypeptide chains. In nature, twenty, so called proteinogenic amino acids, and all belonging to the L series (i.e. all, but cysteine, with the S absolute configuration at the stereocenter), are normally found in the protein chains. The diversity in structure, function and general properties of proteins are all determined by the sequence of amino acids that make up its primary linear sequence. However, the long linear polymer of amino acids (primary structure) is not sufficient to explain the biological activity of proteins. In fact, protein activity can be achieved once the correct primary sequence folds into a unique and precise three-dimensional conformation. The folded conformation obtained is the thermodynamically most stable shape. Therefore, the structure of proteins can be classified into four main levels (Figure 1 (iii) Tertiary structure: results from the three-dimensional, folded and biologically active conformation of proteins. (iv) Quaternary structure: is the arrangement of polypeptide chains (also called subunits) in the space to form a functional multimeric protein. Hydrophobic interactions, hydrogen bonds, salt bridges, hydration and disulfide bonds are forces that hold the subunits together and stabilize the secondary, tertiary and quaternary structure of proteins.

Protein Secondary structures

Secondary structure comes up from the geometry of the bond angle between amino acids as well as hydrogen bonds between nearby amino acids. It can be divided into: helical structure, sheet like structure, and turns or bends.

α-Helix

The α-helix was first described by Linus Pauling and Robert Corey in 1951 as a structure predicted to be stable and favourable on the basis of the exact geometrical parameters he had calculated for the peptide unit from small molecule crystal structure.

The α-helix is a right-handed rod-like structure in which the inner part is formed by the coiled polypeptide main chain and the surface by the side-chains projecting outwards in a helical arrangement. In natural proteins the torsion and dihedral angle values associated with α-helical conformations range from -57 to -70 º for Φ, and from -35 to -48º for Ψ. More importantly this arrangement allows some of the backbone atoms to form hydrogen bonds that occur between the carbonyl oxygen (acceptor) of one residue and the amide hydrogen (donor) of a residue four ahead in the polypeptide chain. These hydrogen bonds are straight, strong, and parallel to the long axis of the helix. The regular α-helix (Figure 1.2)1 has 3.6 residues per turn with each residue offset from the previous residue by 1.5 Å (this parameter is called the translation per residue distance). With these two characteristics the pitch (translation distance between any two corresponding atoms on the helix) of the α-helix is 5.4 Å (i.e. 3.6 x 1.5). In natural proteins the lengths of α helices vary from a few to several tens of residues, with an average of about 10 residues. The most frequently α-helix in proteins is the right handed helix as mentioned above. A left-handed α helix is also sterically possible, but since the side chains are too close to the main chain, this conformation is unstable and rarely present in natural polypeptides. A less common helical secondary structure is the 3 10 helix. Having three residues per turn, the carbonyl on residue i is hydrogen-bonded to the amide NH on residue i+3, forming 10-membered repetitive rings.

A huge number of methods have been investigated to stabilize a helical secondary structure, and these can be helpful in the design of helices in peptide sequences: (a) introduction of Ala residues; (b) design of covalent or non-covalent interactions between side chains residues; (c) addition of non peptide templates to initiate helix formation; (d) introduction of charged residues near the N or C termini to electrostatically stabilize the helix; (e) replacement of one of the main chain intramolecular hydrogen bonds with a covalent linkage. 2

β-Strand and β-Sheets

The β-strand is a linear or zig zag arrangement of amino acids, with amide bonds being almost coplanar, and whose side chains are positioned alternatively above or below the plane of the peptide backbone (labeled B on the Ramachandran plot, Figure 1.3). year as the α helix, and named 'β' because it was the second structure they elucidated. 4 This conformation utilises the whole hydrogen bonding capacity of the polypeptide backbone. Generally, the residues with high intrinsic β-sheet propensities are Val>Ile>Thr>Tyr>Trp>Phe>Leu, whereas Cys>Met>Gln>Ser>Arg are more or less neutral to adopt β-sheet conformations. Characteristic average values for the Φ and Ψ angles of β-strand residues in antiparallel β-sheets are -139 o and +135 o , respectively and about -120°, +115° in parallel sheets.

β-pleated sheets are of two types:

1. The antiparallel orientation, in which adjacent hydrogen bonded polypeptide chains run in opposite directions.

2. The parallel β-pleated sheet, in which neighbouring hydrogen bonded polypeptide chains extend in the same direction (Figure 1.4).

-strands can combine into either a pure parallel sheet, a pure antiparallel sheet, or a mixed sheet with some strand pairs running parallel and some antiparallel. In general parallel β-sheet is more regular but less stable than antiparallel, and it almost never occurs in sheets of less than five total strands, whereas antiparallel β structure often occurs as a twisted ribbon of just two strands.

Antiparallel association of β-strands leads to co-linear N-H•••O=C hydrogen bonds and, consequently, to a network of 10-and 14-membered H-bonded rings, while a parallel association of strands leads to deviation from co-linearity and to a network of 12-membered H-bonded rings. Another amazing characteristic of β-sheets is the ability to produce amphipathic structures. This ability arises from the fact that the residues side chains in β-strands point at right angles alternately above and below the plane of the propagating polypeptide chain. As a consequence, patterns in amino acid character in the primary sequence of a segment that adopts a β-structure can lead to sidedness in the character of a β-sheet. By changing polar and nonpolar residues, an amphipathic sheet will be created.

Turns

A reverse turn can be defined as a region of the polypeptide in which hydrogen bonds can form between one main chain carbonyl oxygen and the main chain N-H group 3 residues along the chain (i.e. O i to N i+3 ). Reverse turns are very abundant in globular proteins and generally occur on the surface of the molecule. The stability of the native state of protein can be influenced by turns due to their intrinsic preference to sample favourable Φ and Ψ space and their side-chain folding process.

Generally as turns tend to be on the solvent-exposed surface of proteins, the hydrophilic residues are more likely to occur than hydrophobic residues, so Gly, Pro, Asn, and Asp are particularly favourable amino acids involving in turns. Reverse turns comprise the widely distributed β-turns, as well as the less prevalent γ-turns and α-turns, and may include well-defined loops, such as Ωloops. [START_REF]After the terminate of the reaction a quick chromatographic purification (petroleum ether/EtOAc, 6/4) was performed to remove the hydrazo-derivative and the resulting crude residue was then purified by flash chromatography on silica gel (CH 2 Cl 2 /CH 3 OH, 99/1) to afford product 2.175 as a white foam[END_REF] Turns are classified on the basis of Φ and Ψ angles of the chain backbone, the position of the stabilizing hydrogen bond, and the length of the chain where the turn occurs. 6 The γ-turn consists of three consecutive residues with an intra-turn hydrogen bond formed between the main chain CO i and NH i+2 and is the second most characterized and commonly found turn, after the β-turn. There are two types of γ-turns (Figure 1.5): inverse and classic which differ in that the main-chain atoms of the two forms are related by mirror symmetry (just as type I and I' or type II and II' β-turns are). Of the two, classic ones are far less common and inverse γ-turns tend not to give rise to polypeptide chain reversal. This kind of turn is characterized by the fact that the middle residue (i+1) does not participate in H-bonding whilst the first and third residues can form the final and initial H-bonds of the antiparallel β-stands, resulting in a 7-membered hydrogenbonded ring. The change in direction of the polypeptide chain caused by a γ-turn is reflected in the values of Φ and Ψ for the central residue. As a result of the size and conformational flexibility, Gly is a favoured residue in this position although other amino acids can be found. The most common type of turn is the β-turn where the hydrogen bond occurs between the i and i+3 residues. In proteins and both linear and cyclic peptides it is more common to find four residue turns, meaning β-turns. According to the last revaluation of the β-turn classification, nine distinct structural types were suggested based on the Φ and Ψ torsion angles in residues i+1 and i+2 (Figure 1.6). 7 β-turns, may (classical β-turn) or may not (open β-turn) be stabilized by an intramolecular Hbond formed between the C=O of the first residue (i) and the N-H of the fourth residue (i+3) (abbreviated as 4 → 1 H-bond), giving rise to a 10-membered H-bonded ring. Venkatachalam's analysis proposed two main classes of four-residue β-turn that has been in use up to date: namely type I and II. The main difference among these two types is the relative orientation of the peptide bond between residues i+1 and i+2 (corner sides), which, consequently modifies the side chain positions. In type I turns, that are the most prevalent in naturally occurring proteins, both the i+1 and i+2 positions accommodate L-residues, however proline preferentially fits in the i+2 position.

In a type II turn, the i+1 position can accommodate an L-residue (usually proline) and the i+2 position favours a glycine, small polar L-residues, or a D-residue because of steric clash with a side chain in the L-configuration. Proline in the i+1 position is a strong sequence determinant for either a type I or II turn because of the restriction on the Φ angle from the cyclic side chain. 8 7 D. J. Hill, M. J. Mio, R. B. Prince, T. S. Hughes, J. S. Moore, Chem. Rev. 2001, 101, 3893-4012. 8 E. G. Hutchinson, J. M. Thornton, Protein Sci., 1994, 3, 2207-2216. Types I′ and II′ are the mirror images of types I and II, with the inverse Φ, Ψ values and are energetically equivalent if residues of the opposite chirality occupy corresponding sequence positions. This means that, for example, a D-Pro residue would be favoured in position i+1 of a type I′ turn or in position i+2 of a type II′ turn. On the other hand, the intervening peptide bond (between residue i+1 and i+2) can adopt a cis conformation and still allow the turn to form an i to i+3 hydrogen bond and link to two β-strands. The resulting turn is called a type VI turn.

 i+1  i+1  i+1  i+1 TURN TYPE  i+1  i+1  i+2  i+2 I -

β-Hairpins

β-hairpins (Figure 1.7) are one of the most simplest super-secondary structures that consist of short loop regions (or turns) between antiparallel β-strands. This type of structure has been found in many naturally bioactive cyclic peptides such as gramicidin S and the peptide hormones oxytocin and vasopressin. 7 Favourable energetic contributions to the stability of these structural features have been demonstrated to include cross-strand aromatic-aromatic, aromatic-polar, H-bonding and type I type II

 i+1  i+1  i+2  i+2 type I type II  i+1  i+1  i+2  i+2
salt-bridge interactions in combination with loop conformational propensity and entropy terms dependent on loop length.9 The difference between β-hairpin motifs based on the length and shape of the loop, can be categorized according to the number of residues in the turn and the number of interstrand hydrogen bonds between the residues flanking the turn. The most frequent β-turns used for the construction of β-hairpins are type I′, followed by type II′, while type I occurs rarely. This characteristic can be explained by the fact that type I′ and II′ β-turns have a right-handed twist suitable for the β-strand pairing, whereas type I and II β-turns are left-handed twisted, among them the uncommon structure in protein β-hairpins are type II β-turns.

Foldamers

Protein-protein interactions, which are fundamental to many biological functions, often occur in protein regions, which are organized in secondary structure elements such as helical segments, -sheet or -hairpins. As a consequence, one approach to modulate protein-protein interactions is to reproduce the interacting segments and use them as antagonists of the interactions. In principle, the best approach would be to use short peptides biased to reproduce the correct conformation and interactions. Unfortunately, peptides show several limits in their clinical applications: 1) being composed of amino acids, they are readily degraded by specific or non-specific proteases under physiological conditions; 2) they tend to be quite conformationally flexible, so that they can bind to more than one receptor or receptor subtype, leading to undesired side effects; 3) due to their high molecular mass and to the lack of a specific delivery system, they are characterized by poor bioavailability. 11 Therefore, peptidomimetics mimicking natural peptides are designed to circumvent some of these problems.

The term "foldamer" refers to any polymer with strong tendency to adopt a specific and compact conformation. 12 Proteins and peptides are important classes of natural foldamers that perform diverse important functions in biology, including molecular recognition, information storage, catalysis, and controlled crystallization of inorganic materials. The desire to mimic such functions with synthetic molecules inspires the field of foldamer design. Foldamers are produced in a diverse range of contexts: from folded oligomers synthesized stepwise and structured synthetic polymers to artificial nucleic acids or protein sequences produced by directed evolution methods.

Two parameters can affect the folding. Internal factors consist of the overall shape and rigidity of the molecule and its stability to establish attractive or repulsive intramolecular non-covalent interactions. These factors depend on monomer size and shape, linkage orientation, rotational restrictions, local intramolecular interactions, and interactions between monomers distant from each other in a sequence. External factors include solvent effects such as hydrophobic effects, aggregation, host-guest complexation, and contacts with interfaces. 13 There are two general molecular classes of foldamers, determined by the presence or absence of aromatic units within the monomer unit. Aliphatic foldamers have saturated carbon chains separating amide or urea groups. Examples of this group include the β-, 14 γ- 15 and δ- 16 peptides, oligoureas, 17 azapeptides, 18 pyrrolinones, 19 α-aminoxypeptides 20 and sugar-based peptides. 21 The second class makes use of aromatic spacers within the backbone. The poly-pyrrole/imidazole DNA-binding oligomers provided early examples of heteroaryl oligomers that bind biologically relevant targets (Figure 1.8) 22 . In either case, selecting the initial monomer is typically affected by a variety of factors, including the ease of their synthesis and structural characterization. Oligomers composed exclusively of -amino acids (so-called -peptides) are probably the most thoroughly investigated peptidomimetics. 23 -Peptides are the closest relatives of natural -peptides, under the category of aliphatic foldamers, where an extra methylene is introduced between two peptide bonds that can bear side chains. In this case, the Hbonding features of the peptide bonds are retained, the solvophobic forces can be tuned via the side-chain chemistry, and the backbone stereochemical configuration pattern offers a very efficient tool through which the structure is controlled. The new class of -peptide sequences has a prominent position in the foldamer field, and by using this type of backbone numerous periodic secondary structure motifs are performed. The different chemical and structural characteristics of the α-and the β-peptides resulted in fundamental bio-orthogonality between these systems. Since most α-peptides are degraded by luminal, pancreatic, cytosolic, or lysosomal proteases resulting in bioavailability and relatively short half-life in vivo, a large amount of β-peptides with proteinogenic side chains have been investigated. Recently, different studies in vitro have shown that βpeptide and mixed α,β-peptide sequences become extremely resistant towards enzymatic hydrolysis by many common proteases and peptidases of all kinds (endo-and exopeptidases, serine-, threonine-, cysteine-, aspartyl-, or metalloproteases, peptidases, or amidases). 24 The β-peptides are considered as a privileged class of peptidomimetic foldamers due to their desirable characters for biomedical applications. The ability of foldamers to adopt specific conformations intrinsically brings the possibility to reproduce the biological activity of natural peptides. In particular, β-peptide secondary elements and mainly helical β-peptides have found to be significant as they are capable to modulate important peptide/protein-protein interactions as well as to promote strong interactions with biomembranes and act as mimetics of antimicrobial and cell -penetrating peptides.

The first demonstration of a β-peptidic mimicry of a α-peptidic helix was the inhibition of the lipid-transport protein SR-B1. The protein SR-B1 appears to play a fundamental role in cholesterol metabolism and homeostasis, in liver and steroidogenic tissues. Apparently, SR-B1 also plays a key role in transportation of free and esterified cholesterol from HDL to cells, as well as the reverse process termed reverse cholesterol transp ort. This protein is inhibited by peptides and proteins, folding to, or containing amphipathic helices, in which one face carry polar α-amino acid side chains, and nonpolar ones are on the other face of surface. The β-nanopeptide 1.1, which in its 3 14 -helical form has the hydrophobic β-amino acid side chains of alanine and phenylalanine on one side and the hydrophilic side chains of lysine on the other side, demonstrated the inhibition of cholesterol transport by SR-B1

through the brush-border membrane (CaCo-2 cells). 25 In 2009 Imamura et al. found that the 12-helical β-peptides 1.2, oligomers of trans-2aminocyclopentanecarboxylic acid (ACPC), are potent γ-secretase inhibitors. γ-Secretase, is a 24 D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodivers., 2004, 1, 1111 25 D. Seebach, J. Gardiner, J. Accounts Chem. Res., 2008, 41, 1366-1375. membrane-embedded aspartic protease comprising presenilin (PS), nicastrin, Aph-1 and Pen-2, that is one of the enzymes responsible for the proteolytic processing of amyloid precursor protein (APP) within the transmembrane domain to generate Aβpeptides. Since γ-secretase cleaves hydrophobic membrane protein, this group decided to choose enantiomers of ACPC as the building blocks due to week hydrophobicity of their oligomers and also the capacity of enantiomerically pure oligomers of ACPC to form stable 12-helix structure (defined by 12-membered ring C=O (i) →H- N (i+3) hydrogen bonds), resulting a good mimic of the α-helix in terms of overall shape. 26 Oligomers containing both α-and β-amino acid residues exhibit specific helical secondary structures. The heterogeneous backbone of α/β-peptides introduces a new parameter of structural variation. For oligomers with 1:1 α:β variation, the nature of the helix can be changed by altering the cyclic constraint embedded in the β residues and other structural parameters. At the same time as Imamura, Gellman and Fairlie reported the structure of a complex between an /-peptide foldamer and a protein partner, Bcl-xL.

Bcl-xL is a pro-survival member of the Bcl-2 family that inhibits apoptosis signaling.

Anti-apoptotic Bcl-2 family members display a hydrophobic cleft that accommodates a - Fairlie, Angew. Chem. Int. Ed., 2009, 48, 4318 -4322. This compound contains a depsipeptide unit composed of the L-proline-glycolic acid residue, adopts a reverse turn conformation and promotes hairpin formation (Figure 1.13). 28 The resulting tetrapeptide, generally contains exclusively -amino acid residues, and represented the first autonomously folded -peptide sheet.

helical
Stable -hairpins generated from hybrid peptide sequences containing both and amino acids have also been reported. Balaram and co-workers were the first to describe hairpin-turn formation involving mixed -peptides. They reported -hybrid hexapeptide 1.5 containing the conventional D-Pro-Gly -peptidic turn motif carrying amino acid residues ( 3 Val or  3 Leu) i on opposite sides of the antiparallel sheet part of the hairpin. In this case, a hydrogen bond between the C=O of residue i and the N-H of residue i+3 of the -turn is formed resulting in a 10-membered H-bonded ring (Figure Seebach and co-workers have also reported hairpin-turn secondary structures of mixed -peptides containing novel types of turn structures with 9 -and 10-membered H-bonded rings. The hexapeptide 1.6 consists of  3 -homo-amino,  2 -homo-amino, and -amino acids with a central  2 - 3 segment. Despite its small size, this peptide adopted stable hairpin structure showing a turn with a 10-membered H-bonded ring induced by the  2 / 3 unit. 30 The enzymatic stability and the variety of secondary structures discovered push the door wide open for the application of -peptides as peptidomimetics in medicinal chemistry. The simplicity of this building block allows the Nowick's group to prepare many different structures forming well-defined turns, with distinct NMR properties (NOEs, magnetic anisotropy, and J-coupling) that indicate their folding. In order to prepare water-soluble molecules that fold into well-defined structures in aqueous solution they designed molecular hairpins like hairpin 1.17 in which ornithine and Hao residues are separated by pairs of hydrophobic amino acids that adopt folded -sheet (Figure 1.24). More recently, on the basis of -hairpin 1.17 they established macrocyclic -sheets composed of two δ-linked ornithine -turn mimics, a pentapeptide in the "upper" strand, the amino acid Hao and two -amino acids in the "lower" strand. Hao is a relatively rigid tripeptide -strand mimic that serves as a template for the folding of the upper strand and blocks the lower strand to minimize edge-to-edge aggregation. The two -amino acids in the lower strand allow tuning of folding and solubility of the cyclic modular -sheets without changing the upper strand. They described this macrocycle 1.18 as one of the best inhibitors synthesized in their laboratories (Figure 1.25) that suppressed aggregation of tau-protein-derived peptide AcPHF6 at 1.0 equiv. 43 The hydrophobic surface created by the valine, isoleucine, and leucine residues at positions R 1 , R 3 , and R 7 on this macrocycle -sheet peptide is necessary for inhibition.

-Hairpin

43 J. Zheng, A. M. Baghkhanian, J. S. Nowick, J. Am. Chem. Soc., 2013, 135, 6846-6852. On 3 November 1906, Alois Alzheimer presented interesting results obtained from the histological examination of one of his patient's brain at the 37th meeting of the Society of Southwest German Psychiatrists in Tübingen, Germany. The detailed analysis that were performed in psychiatric hospital in Munich, revealed the presence of intraneuronal fibrils, the deposition of a pathological metabolic substance, that was described as a tangle pathology by Alzheimer for the first time. 46 Today, 100 years later, the presence of neurofibrillary tangles and amyloid deposits are still the disease-defining parameters.

ALZHEIMER'S DISEASE

Introduction

In the initial stages of the disease the semantic memory-the meaning of a word-and procedural memory-how to dress or how to ride a bicycle-are relatively spared. Memory is made up of electrical impulses along the neuronal connections in the brain, formed by the opening and closing of synapses and facilitating for a specific time frame a specific circuit that encodes specific information. The formation of episodic memory requires, in particular, neuronal connections of small areas of the entorhinal cortex and of the hippocampus in the medial temporal lobe (hippocampus and the parahippocampal gyrus). The huge amount of data which is acquired by seeing, hearing, and feeling then is processed in the neocortex and transported from almost all neocortical areas to the entorhinal region (Figure 1.27). This associated data is then transmitted to the hippocampus from where it is back again into a deep entorhinal layer, and form there information is distributed again back to the neocortex. Although the entorhinal cortex and the medial temporal lobe are only a tiny fraction of the whole cerebral cortex, they are a key interface and the functioning of their connections is essential for the formation of long-term memory. Alzheimer's is a progressive disease, where dementia symptoms gradually worsen over a number of years. In its early stages, memory loss is mild, but with late-stage Alzheimer's, individuals lose the ability to carry on a conversation and respond to their environment. Those with Alzheimer's live an average of eight years after their symptoms become noticeable to others, but survival can range from four to 20 years, depending on age and other health conditions. The most common early symptom of Alzheimer's is difficulty remembering newly learned information because Alzheimer's changes typically begin in the part of the brain that affects learning. As Alzheimer's advances through the brain it leads to increasingly severe symptoms, including disorientation, mood and behaviour changes; deepening confusion about events, time and place;

unfounded suspicions about family, friends and professional caregivers.

47 R. Jakob-Roetne, H. Jaconsen, Angew. Chem. Int. Ed., 2009, 48, 3030-3059

Pathogenesis of Alzheimer's disease

The etiology of AD is a complex process resulting from a combination of genetic and neurobiological factors and is often compared with an incomplete puzzle: the Alzheimer's puzzle. In a normal mature neuron, tubulin is present in over tenfold excess of tau, and thus practically all tau protein is microtubule bounded in the cell (Figure 1.31). In neurons affected in AD, abnormally phosphorylated cytosolic tau neither binds to tubulin nor promotes microtubule assembly. Instead, this protein inhibits the assembly and disrupts the microtubule organization. Amyloid fibrils are self-assembled, filamentous protein or peptide aggregates with cross-β molecular structures, meaning that they contain ribbon like β-sheets in which the β-strands run approximately perpendicular to the long fibril axis and the inter strand hydrogen bonds run parallel the long fibril axis (Figure 1.32). The cross- architecture provides very great stability to fibrils, as it allows the formation of a continuous array of hydrogen bonds. 56 width, morphology such as curvature and persistence length, surface characteristics such as periodic twists, and higher-order assembly.

There has been increasing evidence that the amyloid state might be thermodynamically more stable than the functional native states of many protein molecules even under physiological conditions. Aβ peptides (39-43 amino acids in length) derive from the proteolytic cleavage of amyloid precursor protein (APP) by enzymes called secretases. As the conversion of proteins from their soluble states to the amyloid form involves the formation of intermolecular contacts, the thermodynamic stability of the amyloid state is increasingly favored at higher concentrations. 58 As brain of AD patients, forming both localized and diffuse deposits (called plaques) in the brain tissue and brain vasculature.

The APP gene resides on chromosome 21, the multiple expression of which not only underlies Down's syndrome but also is associated with increased risk of AD. APP is a type I, single-pass transmembrane protein that contains three potential sites for cleavage by various secretases, which are designed α, β, and γ. 59 Two pathways were described for APP cleavage. Non amyloidogenic pathway consist the cleavage by α-secretase leading to the production of soluble amyloid precursor protein α (sAPPα) and the carboxyl terminal fragment C83. On the other hand the cleavage by βand γ-secretase in this pathway produce sAPPβ and Aβ through successive cleavage by β-secretase (also known as BACE for β-amyloid precursor protein site cleaving enzyme) and γ-secretase complex that is made up of several substituents. The amyloidogenic pathway instead produces Aβ of several different lengths from 37 to 43 amino acids. The N-terminus of amyloid-β is located in the extracellular domain of APP, 28 amino acids from the transmembrane region, and its C terminus is in the transmembrane region. The and γ-secretases activity gives rise to the N and C termini. 60 The predominantly produced species is Aβ40. A trace amount of Aβ42 is also formed in a ratio of approximately 99 to 1 (Figure 1.33). Aβ42 has a high β-strand propensity and so greater tendency to aggregate and is therefore more toxic to neurons than Aβ40. The distribution of these residues, as well as the six Gly residues are significant for the chemical and physical properties of Aβ. 64 The Phe19 in the A antiparallel β-sheet is a key in determining the properties of the fibril. Minor alterations in sequence, such as replacement of Val or Phe with Leu or Ala, can abolish fibril formation and that the amyloidogenic properties of short peptides can be destroyed by introduction of adjacent sequence motifs such as β-turns. These data indicate that both the amino acid sequence as such and its structural context affect the ability to form amyloid fibrils.

Aβ peptides are produced as soluble monomers and undergo oligomerization and amyloid fibril formation via an unclear process. Several studies proposed that monomeric Aβ in solution exists in an equilibrium between an α-helical and a β-sheet conformation and from this mixture only the β- early protofibrils. 66,67 Hecht was then performed random mutagenesis on positions 41 and 42 (Ile and Ala) to investigate the difference in aggregation behavior of Aβ40 and Aβ42. Their results indicate that the hydrophobicity of residues at these positions plays a dominant role in promoting aggregation. They also demonstrated that rather than longer peptide sequence which is thought to be the cause of aggregation, it is the hydrophobicity and β-sheet propensity of residues 41 and 42 that cause the enhanced amyloidogenicity of Aβ42 relative to Aβ40. 68 Historically, the amyloid cascade hypothesis posits that the deposition of the amyloid-β peptide in the brain parenchyma is a crucial step that ultimately leads to Alzheimer's disease. Until recently, much credit was warranted to this hypothesis but the latest research results suggest that the oligomers, and not the fibrillar forms, are the most toxic species both in vitro and in vivo. 69,70 The amyloid cascade hypothesis now suggests that synaptotoxicity and neurotoxicity may be mediated by such soluble forms of multimeric amyloid-β peptide species. It is also shown that extracellular applications of spherical oligomeric forms of Aβ42 and several other disease-related amyloidogenic proteins and peptides cause an immediate and large increase in cytosolic free One way which Alzheimer's harms the brain is decreasing levels of acetylcholine, a chemical messenger that's important for alertness, memory, thought and judgment. Cholinesterase inhibitors are a type of drug that raises the amount of acetylcholine available to nerve cells by preventing its breakdown in the brain. There are three FDA-approved cholinesterase inhibitors in the market.

Although these drugs were approved by FDA but they have diverse side effects also in some patients. In principle all the key steps in pathways leading to the formation of pathological species can be considered as good targets for blocking disease onset and/or progression. The following factors might be accounted for the difficulties in finding effective ways to overcome the protein aggregation problem: 1) limited knowledge of the molecular mechanisms for protein aggregation,

2) few promoting agents (either exogenous or endogenous) or events have been identified, 3)

73 alzheimers.org.uk 74 http://www.alz.org/alzheimers_disease_standard_prescriptions.asp enzymes and chaperones involved in disease onset also have physiological roles, precluding their inhibition, 4) the involvement of more than one specific event in diverse steps, and 5) lack of suitable in vitro and in vivo assays for mimicking the complex human pathological scenario and for monitoring the formation of different prefibrillar species are currently available.

In the case of Alzheimer's disease, observations from post-mortem tissues revealed that tau pathology that was explained in previous section, correlates better with the severity of dementia.

Despite the evidence that tau pathology contributes to disease progression, therapeutic approaches have been concentrated almost entirely on modulating Aβ production or accumulation. These interventions include: reduction of APP processing by blocking the β or γ secretases; prevention of Aβ aggregation; and enhancing the clearance of amyloid plaques or sAβ (Figure 1.36). 75 In this way β-secretase (BACE-1) inhibition, and γ-secretase inhibition have been investigated as potential therapeutic approaches for prevention of Aβ accumulation. Another strategy that was investigated is the activation of the α-secretase, having shown many side effects. 76 Blocking the production of Aβ by inhibiting the first proteases required for its generation, βsecretase, may prove to be the next step in blocking AD progression or even preventing the disease.

Several studies have confirmed β-secretase as a therapeutic target for AD. BACE-1 inhibitors were initially designed as substrate analogs mimicking the APP-cleavage sequence, with a non hydrolyzable peptide bond, such as hydroxyethylene, statine analogue, and hydroxyethylamine isosteres. Inhibitor structures were designed to fit the active site pockets, and were further modified to reduce their size and improve their pharmacological properties. GlaxoSmithKline was the first to develop the use of the hydroxyethylamine derivative GSK188909, a BACE-1 inhibitor, to lower Aβ levels in the brain of APP transgenic mice (Figure 1.37). 77 GSK188909 (1.20) is a small potent, selective, non-peptidic and orally bioavailable compound, although its brain penetration is relatively low. Tsai, J. Biol. Chem., 2004, 279, 1942-1949. 80 S. M. Paul, Sci. Transl. Med., 2011, 3, 84ps20. cleavage site of APP. 81 On the other hand, BACE-1 appears to play an important role in the developing central and peripheral nervous system, and also requires for the maintenance and repair of these systems, particularly in response to injury and inflammation. Furthermore, it was discovered that neuregulin-1, a peptide that is essential for the normal development of the nervous system and the heart, is also a substrate of BACE and a complete inhibition of BACE-1 could affect the myelination of neurons, due to the operating of neuregulin-1. However, BACE-1 inhibitor CTS-21166, produced by pharmaceutical company CoMentis, did not influence the myeline sheath in their mouse model, which is an important prelude to obtain a successful inhibitor to treat AD. 82 Although still unclear, the negative trial results may be attributed to several factors including the stimulation of GS due to a rebound effect, thus resulting in overall stimulation of Aβ production, or the inhibition of GS resulting in the inhibition of processing of not only APP but also other substrates relevant for cognition. Since the discovery of the NSAID series, other small molecules, both NSAID and non-NSAID derived, have been explored with improved in vitro potency. Both in vitro and in vivo data support the conclusion that GSMs do not interfere with Notch processing, but instead, via slight shifts in the cleavage site on APP, lower Aβ42 production to produce the other normal Aβ peptide products. 88 Thus, modulation of γ-secretase may represent the most selective approach to treat Alzheimer's disease via a decrease in Aβ42 production, more selective that an inhibitor of either βor γ-secretase. Modulators do differ both in chemical structure and in their effects on APP processing, producing the desired decrease in Aβ42 and a variety of changes in other Aβ peptides.

Understanding the molecular basis of these varying profiles may shed further light on the biology of γ-secretase.

A considerable research done in this area suggests that the self-assembly of amyloidogenic proteins generally occurs through the following steps: Several phenolic compounds have been reported, such as wine-related polyphenols (myricetin (Myr), morin, and tannic acid, and so on), curcumin (Cur), ferulic acid (FA), nordihydroguaiaretic acid (NDGA), Flavonoids, and rosmarinic acid (RA) which shown especially strong anti-Aβ aggregation effects in vitro. 91 Cole's group showed that curcumin decreases the amounts of soluble and insoluble A as well as lessening plaque burden in many affected brain regions using Tg2576 mice. 92 Later, they reported that curcumin (Figure 1.43) inhibits the formation of A oligomers and fibrils, binds plaques, and reduces plaque burden in vivo. 93 structure. 94 Their results suggest that oleuropein (Figure 1.44) reduces the exposure of the hydrophobic regions in Aβ1-42. Furthermore, oleuropein inhibits Aβ1-42 oligomerization as detected by an oligomer-specific monoclonal antibody. 95 The major advantage of using small molecules is their generally good drug-like properties, which may enable to use these compounds by the oral route and get substantial quantities of them to reach the brain. However, their usefulness as amyloid inhibiting drugs has been compromised by their common lack of specificity, toxicity and their unknown mechanism of action in most of the cases. While small-molecule aggregation inhibitors have been reported, none have been developed into a clinically useful therapeutic.

Therapeutic peptides offer several advantages over small organic molecules that make up traditional medicines. The first advantage is that often representing the smallest functional part of a protein, they offer greater efficacy, selectivity and specificity (limited non-specific binding to molecular structures other than the desired target) than small organic molecules. A second advantage is that the degradation products of peptides are amino acids, thus minimizing the risks of systemic toxicity (minimization of drug-drug interactions). Third, because of their short half-life, Another approach under the category of peptide based inhibitors is the use of β-sheet breakers.

-sheet breakers are synthetic rationally-designed peptides specifically created to break -sheets.

The self-recognition sequence Aβ 16-20 (KKLVFF), in the central region of the Aβ peptide proved to be important in the Aβ-Aβ interaction, because it binds β-sheets and initiates aggregation (nucleation site). In particular, the phenylalanine residues present in this sequence represent a key structural element in the self-assembly process, providing high affinity and selectivity for the molecular recognition. Substitution of hydrophilic for hydrophobic residues in the central hydrophobic region of A (17-21) impairs fibril formation, and peptides designed to mimic the structure of this region could inhibit aggregation.

The first group to make use of a core section of Aβ as a structural starting point was Tjernberg and co-workers, who showed that Aβ (16-20) was able to bind full-length peptide and thus prevent its assembly into fibrils. 106 Soto and co-workers were then designed a series of inhibitors containing A amino acids 17-20 (LVFF), and then chemically modified to obtain more active and stable pentapeptides with the basic sequence LPFFD (named also iAβ5 Although, the peptidic molecules impose serious problems for administration and delivery, especially for compounds needed to act on the brain, thus need to pass BBB. In addition, peptides are rapidly degraded, resulting in the need for more frequent administrations and the use of large doses. Nevertheless, the great advance on peptide chemistry permits to use a variety of strategies to minimize these weaknesses. 119 The peptidomimetic hairpins have also been developed by Nowick and co-workers demonstrating considerable biological activity. Their synthesis and structures are reported in the first part of this chapter, their ability to inhibit Aβ oligomerization also measured (see section 1.2.2).

An alternative tactic is to focus on Aβ clearance using antibodies that currently is under study.

Indeed, the results from preclinical work using this approach with various mouse AD models have been so promising that at least 13 different Aβ immunotherapy trials are currently in progress. Finally, it is notable that the targets of antibodies do not have to be the amyloidogenic protein itself. Bodin et al. recently demonstrated that antibodies against serum amyloid protein (SAP), which associates with amyloid deposits of several different proteins, efficiently clear amyloid via phagocytosis in a mouse model. 126 A key aspect of this work is the parallel use of a small molecule that binds and promotes the clearance of circulating SAP. This clearance enables the antibody to reach SAP in the amyloid deposits.

Recently, two Phase III trials Bapineuzumab and Solanezumab in patients with mild-tomoderate AD have provided encouraging evidence with respect to the efficacy of this intervention. 127 For instance, in one of these key trials, the administration of bapineuzumab, a monoclonal antibody targeting Aβ in the brain, by intravenous infusion every 13 weeks for 78 weeks led to treatment differences in biomarkers. 128 128 S. Salloway, R. Sperling, N. C.Fox, K. Blennow, W. Klunk, M. Raskind, M. Sabbagh, L. S. Honig, A. P.

DIKETOPIPERAZINE (DKP)

Structure and biological activity

2,5-Diketopiperazines (DKPs), the smallest cyclic peptides (Figure 2.1), are an important class of biologically active compounds 129 and their research has been fundamental to many aspects of peptide chemistry. They were once believed to be only protein artifacts or degradation products, but thanks to the wide spectrum of their biological properties 130 that point to several therapeutic applications, they have acquired again a huge interest among chemists and pharmacologists. Due to the limits in utilising peptides, the structural similarity of 2,5-diketopiperazines to peptides open a new way for medicinal chemists in designing molecules with similar characteristics. Altering the physical properties, reducing the susceptibility to metabolic amide bond cleavage and inducing conformational rigidity are the results of constraining the nitrogen atom of an α-amino amide into a DKP ring. These changes in structural and physical properties, as well as the presence of groups acting as donors and acceptors of hydrogen bonds enhance favourable interactions with biological targets. Moreover, since they present up to four positions (N1, N4, C3, C6) for introducing different functional groups and two positions for controlling the stereochemistry of molecules it can be possible to prepare a large number of different DKPs. These simple heterocyclic scaffolds can be obtained from readily available α-amino acids using conventional synthetic procedures, 133,134 solid phase 135,136 and microwave-assisted organic synthesis. 137 Considering these structural properties, the importance of these constrained bifunctional cyclopeptides as privileged structures for the discovery of new lead compounds can be understood;

in fact DKPs represent a useful toolset in the area of combinatorial biomedical chemistry, 138 de novo design of proteins 139 and functional molecular devices. Furthermore, DKP scaffolds became interesting as organocatalysts and have also been used in the asymmetric hydrocyanation of aldehydes 140 and imines 141a (although these results were latert somehow questioned) 13b and more recently in the conjugate addition of aldehydes to nitroolefins. 142 Some of the most important biological activities of diketopiperazines are related to the inhibition of plasminogen activator inhibitor-1 (PAI-1) and alteration of cardiovascular and bloodclotting functions. 143 PAI-1 is the main physiological inhibitor of the serine proteases, urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). When PAI-1 is quantitatively and qualitatively disrupted, a variety of modulated functions are implicated in several pathological situations such as coronary heart disease, atherosclerosis, thrombosis, and cancer progression associated with increased levels of circulating PAI-1. PAI-1 is unique amongst serpins in that the protein can spontaneously convert from its active conformation into a latent, inactive form that is unable to irreversibly bind to tPA. The reactive center loop (RCL), which is responsible for the inhibitory activity of PAI-1 is exposed in the active form, but is inserted into the major -sheet A in the latent confirmation. Diketopiperazines are the most potent PAI-1 inhibitors known, acting through a mechanism involving conformational changes, which inactivate PAI-1 loopcomplementarity, preventing its binding to the target protein. There were reported four molecules that were designed structurally based on DKPs as inhibitors of PAI-1. 144 Other strategies, such as introduction of structural rigidity, were also applied to metabolite 2.1, leading to compounds like 2.4 that also shows an interesting PAI-1 inhibitory activity (IC 50 0.3 mM).

Tumour cell pro-coagulant activity has been considered to favour metastatic processes by encasing malignant cells in a fibrin coat, which protects them from the host immunological system.

Thus, components of the blood-clotting system are potential targets for a new class of therapeutic agents designed to selectively inhibit tumour growth and prevent metastasis. Haematological studies showed that cyclo(L-His-L-Tyr) DKP 2. Several advances in the synthesis of DKP scaffolds on solid phase were also achieved, as a convenient way to build combinatorial libraries on a small scale in medicinal chemistry. But due to the problems relating the scale up of solid-phase reactions, this procedure is less useful for largescale synthesis of 2,5-diketopiperazines. 2,5-Diketopiperazine formation via dipeptide ester cyclization is the most commonly used procedure to build this ring system. Formation of dipeptide unit is an easy process from commercially available α-amino acids using a large number of coupling reagents. This route generally utilizes an α-amino acid protected as amino functionality

(2.28) and α-amino acid ester (2.29). The resulting coupled product (dipeptide 2.30) undergoes the deprotection step to give the free amino dipeptide system (2.31), which can then cyclize to afford compound 2.32 as a desired molecule.

Scheme 2.1.

The cyclization rate depends on the conformation of amide bond. Generally to achieve molecule 2.32 the amide bond must adopt a cis orientation, in the case it is prevented sterically or electronically, the yield of this step can decrease. Numerous methods have been developed to improve the yield of cyclization step when the reaction occurs with difficulty, although careful selection of reaction conditions is required to limit racemization. One way that is often used to force the formation of the ring is heating the system in an acidic medium. 159 The use of basic conditions is also possible, but it increases the risk of racemisation at the stereocenters. 160 Cyclization of amino dipeptide esters can be also carried out under thermal conditions, by refluxing using high boiling solvents such as toluene or xylene for 24h. 161 Refluxing the formate salt of dipeptide in toluene-sec-BuOH is also shown to be an efficient method leading to the formation of ring system. 162 However, for many DKPs, simple reflux of dipeptidyl methyl esters in low-boiling solvents such as methanol is normally effective (Scheme 2.2). 163

Scheme 2.2. Solution-phase synthesis: a) MeOH, reflux, 14h

The use of microwaves to improve the yield of ring formation was investigated by Luthman et al. They described this method as a new general and efficient solution-phase synthetic way which, instead of other methods, is independent on the amino acid sequence and leads to no epimerization. 164 Moreover, a comparison with classic thermal heating was performed showing that microwave-assisted heating for 10 min using water as solvent was the most efficient method of cyclization, giving moderate to excellent yields (63-97%) of 2,5-DKPs. In other approaches, microwave irradiation of N-Boc dipeptide esters directly caused N-Boc deprotection and cyclization to 2,5-diketopiperazines in a fast and efficient manner.

Another method to obtain diketopiperazines is the Ugi reaction, that was first described in 1959.

This synthetic pathway comprises a small number of multicomponent steps which allows the substitutions on the secondary amino group. 165 Using an isonitrile, acid (or amino acid), aldehyde, and amine, a dipeptide is produced in high yield and optical purity. 166 This method has been widely used to prepare 2,5-DKP libraries, because the reaction is performed in one pot without the use of expensive coupling reagents. The main disadvantage using this type of reaction for the formation of 2,5-diketopiperazines is its difficulty in the cyclization of dipeptide. In this route rather than a simple dipeptide ester cyclization, a C-terminal amide was formed that is more difficult to cyclize.

In Scheme 2.4 was reported an alternative way in the preparation of trisubstituted DKPs such as 2.43 which is a stereospecific synthesis. However, it was not possible to control the stereochemistry of the chiral centre located in an α carbon of the tertiary amine. 168

Scheme 2.4. Ugi reaction: a)

1 eq Et 3 N, MeOH; b) t-Bu-isonitrile, N-Boc-R-indanylglycine; c) 4 N HCl/dioxane, 4h; d) Et 3 N
The problem of stereocontrol might be solved by using a symmetric ketone as the starting carbonyl compound. For instance, in the discovery and synthesis of pharmacologically active compounds such as aplaviroc, the desired product was obtained using Ugi-type reaction (Scheme 2.5). 169 For the one-pot synthesis of symmetric 1,4-disubstituted-DKPs, phase transfer catalysis seems to be a valid method starting from a suitable amine and chloroacetyl chloride in the presence of an aqueous base. 171 Recently, the O,N-acyl transfer attracted great attention as a valuable method for the efficient synthesis of difficult peptide sequences and racemization-free segment condensation.

Using this strategy, there were also prepared hexa-and pentasubstituted diketopiperazines under mild conditions. This approach also allows to achieve oligomers that carry different functional group on each monomer. In reactions involving amino acids, the DKP formation is a quite common side reaction. For example, attempts to perform nucleophilic substitutions in substituted pyrimidinic rings, by ethyl glycine chloride in the presence of a strong base such as DABCO resulted in the glycine DKP produced by predominant auto-condensation of ethyl glycine. 174 However the solution phase synthesis has the advantage in which few number of steps are needed, solid-phase synthesis has been used also to prepare DKPs with the chance to have variations in the resin type, protecting groups and cleavage system used. In multistep procedures, the first peptide bond resulting dipeptide is generated in situ then is attached to the solid support, and it remains bound through the carboxylic extremity up to the cycling step, that is then followed by separation from the matrix or release of the N-protective group of the amino acid. 175 A generic solid-phase protocol is shown in Scheme 2.8 for obtaining DKPs under mild conditions, using the phenacyl ester (O-Pac) bond to attach the carboxylic amino acid terminal to the resin. Deprotection of the Boc-amino group and coupling with the next amino acid afforded dipeptide 2.62, which was subsequently deprotected and cyclized by intramolecular aminolysis. 175 Solid-phase synthesis of unsaturated 3-substituted DKP, employing a carbamate bond between the dipeptide N-terminal and the resin, was also described. Our group has also developed a bifunctional DKP scaffold able to act as -hairpin inducer; this will be presented and discussed in section 2.1.6.

DKPs in cyclic peptidomimetics

In order to stabilize the peptidic residue into a -turn conformation, a rigid template is used to constrain the backbone of a cyclic peptide forming an external -turn inducers. Considerable examples in this field using diketopiperazine scaffold were reported by Robinson and coworkers. 190 This group investigated the role of diketopiperazine templates based on proline on stabilizing the turn and hairpin conformations in cyclic peptides containing the Asn-Pro-Asn-Ala (NPNA)

sequence. This tetrapeptide motif was found as a repeated unit in the circumsporozoite (CS) surface protein of the malaria parasite Plasmodium falciparum which has a tendency to adopt type I -turn in aqueous solution that appears to be important for immune recognition of the folded CS protein.

Several bifunctional bi-and tricyclic diketopiperazine scaffolds were prepared as illustrated in A molecular rod 2.83 (Figure 2.23) was synthesized starting from the bis-amino acid 4-amino-4carboxy proline 2.82. 195 Starting from commercially available trans-4-hydroxy-L-proline, this substituted proline was obtained in multigram quantity after nine synthetic steps. The preparation of molecular rod 2.83 occurred in two stages: a first "assembly" stage where the linear oligomer was grown, coupling the proline COOH to the primary amine in 4-position, by solid-phase peptide synthesis. A "rigidification" stage followed after cleavage from the resin. In this step, through an intramolecular aminolysis reaction the diketopiperazines were undergo the cyclization, in which the 193 a) M. E. Pfeifer, J. A. Robinson, Chem. Commun., 1998, 1977-1978; b) M. E. Pfeifer, K. Moehle, A. Linden, J. A.

Robinson, Helv. Chim. Acta., 2000, 83, 444-464. 194 C. E. Schafmeister, Z. Z. Brown, S. Gupta, Acc. Chem. Res., 2008, 41, 1387-1398. 195 C. G. Levins, C. E. Schafmeister, J. Am. Chem. Soc., 2003, 125, 4702-4703. secondary amine of each monomer attacked the methyl ester of the previous monomer.

Interestingly, when the bis-amino acid 2.82 was functionalized by reductive alkylation with different aldehydes at the primary amine group, the bis-peptide 2.84 resulting from the coupling of the corresponding homochiral monomers, adopted a left-handed helical arrangement, and the nitrogen substituents superimposed well on the side chains of a model α-helical peptide (Figure 2.24).196 in which the imidazole group of the histidine residue is catalytically active as a base, afforded Rmandelonitrile in 90% ee in only 30 min. 206

Scheme 2.10. Asymmetric addition of hydrogen cyanide to benzaldehyde catalysed by dipeptides A rationale for the stereoinduction was provided, whereby the role of histidine is to direct the attack of cyanide to the si-face of benzaldehyde while the re-face is blocked by the aromatic ring of phenylalanine residue. Further work reported how time, temperature, solvent and crystallization of the catalyst, all play a significant role in an enantioselective reaction. A similar diketopiperazine was used in the asymmetric hydrocyanation of imines (Strecker reaction), 207,208 although in this case the results have been questioned. 208 The organocatalyzed enantioselective conjugate addition of aldehydes and ketones to electron poor olefins and particularly nitroolefins has gained momentum in the last few years, and many short peptides featuring a terminal proline residue have been applied as selective catalysts. The efficacy of these compounds has been attributed to their ability to adopt a well-defined threedimensional secondary structure, suitable for the transfer of stereochemical information.

Four peptidomimetics (2.111-2.114) containing the cis-DKP or trans-DKP scaffolds formally derived from the head-to-tail cyclization of L-aspartic acid and either (R)-or (S)-2,3diaminopropionic acid and either L-Pro or D-Pro were synthesized. To rationalize these results, a conformational study of these peptidomimetics was undertaken. A Monte Carlo/Energy Minimization (MC/EM) conformational search for the enamine intermediate derivatives revealed conformations where one face of the enamine moiety is shielded by the diketopiperazine ring and the carboxylic acid is likely to act as a hydrogen-bond donor, activating the nitroolefin acceptor (-NO 2 ) and directing its approach from the less hindered enamine face.

Beta-strand mimetics

General introduction

Peptide -strand is the simplest peptide structural element, which is normally not found in isolated peptides, but at least paired in hydrogen bonded structures, forming -sheets in proteins.

The association of a -strand from the ligand with a strand or a -sheet in the binding partner plays an important role in the protein-protein interactions (PPIs) which are essential at almost every level of organization and communication in living cells. 210 Interactions between the hydrogen-bonding edges of -strands in different protein chains constitute an important mode of PPIs, with hydrogen bonds being formed between -strands belonging to different protein chains. These interactions are frequent in protein dimers and other quaternary structures, both in interaction among different proteins and in protein aggregation. Although these PPIs occur in non-pathological processes, they are also involved in a wide range of diseases, such as cancer, 211 parasitic, fungal and viral infections, 212,213 inflammatory, immunological and respiratory conditions, 214 and cardiovascular 215 and degenerative disorders, including Alzheimer's disease. 216 Therefore, a lot of effort is being dedicated to the dissection of protein interaction sites, the identification of motifs common to protein interaction interfaces, to mapping protein-protein contact surfaces, and hence to the search of external modulators such as -strand mimics.

The simplest approach in the design of -strand mimetics would be to use short peptides corresponding to strand/sheet regions of proteins. However, it is known the disadvantage of using short peptides, due to their conformational flexibility and poor pharmacological profiles. Methods to increase the conformational stability involve the restriction of the peptide freedom by incorporation of conformational constraints into the peptide backbone, or through different types of cyclizations. 217 To design and develop molecules that selectively mimic the -strand, it is necessary to consider the differences between size, shape, and composition of a -strand versus sheets, turns, and helices Recently small molecules able to mimic -strands have been investigated as enzyme inhibitors and antagonists with important potential application in medicine. Due to the high tendency of these strands to aggregate, only a few examples of monomeric -strand mimics were reported.

The replacement of amino acid components with a wide variety of constraints such as disulfides, double bonds, N-methyl amino acids, D-amino acids and aromatic or heterocyclic rings, leads to the preparation of conformationally and metabolically stable peptidomimetics or nonpeptidic mimics which are able to mimic -strands. While significant advances have been made in the synthesis of conformationally constrained -sheet mimetics, there is a pressing need for small molecules that mimic -strands.

Approaches to categorizing the general classes of -strand mimetics are potentially variable. In this section some examples of macrocyclic peptidomimetics, alicyclic compounds terminating with aliphatic and aromatic carbocycles are reported.

Macrocyclic peptidomimetics

Macrocycles which are highly constrained structural mimics of tri-and tetrapeptide components of a linear peptide substrate or inhibitor can be prepared through condensing peptide side chains to the main chain. These peptidomimetics fix the receptor bound conformation of bioactive peptides in an extended conformation, which results in functional mimicry.

Macrocyclic -strand mimetics have been increasingly used in the development of protease Constraining peptide sequences into macrocycles has been a method of choice to make amide bonds less recognizable by other proteases. Thus, such macrocycles are not only resistant to peptidases of the gut, bloodstream, or cells but can also penetrate cell membranes and exhibit potent antiviral activity in cell culture. 223 For instance, macrocycle 2.121 provides ~17-fold less HIV-1 protease inhibition than its acyclic counterpart but displays more potent antiviral activity.

This observation suggests that this macrocyclic inhibitor may have improved cell permeability and/or resistance to cellular enzymes. In this field macrocyclic receptors incorporating carboxylic acid binding site specifically for peptides with a free carboxylic acid terminus, have been also developed. As an example, the macrocycle structure 2.122 can be mentioned, which contains a diaminopyridine unit at the base of an open, bowl-shaped cavity (Figure 2.37). To interact with peptide guest molecule the hydrogenbonding source was needed which was provided by an additional amide functionality around the rim of the bowl-shaped structure. NMR spectroscopic studies concluded that this macrocylce was a flexible receptor, able to bind Cbz-β-alanyl-D-alanine substrate within the macrocyclic cavity via a series of well-defined hydrogen bonds making the peptidic substrate an extended conformation. 225 Based on the results obtained by Nowick and co-workers, our group developed β-strand peptidomimetics, which were shown to be extremely effective in the formation of intermolecular βsheets with the terminal parts of HIV-1 proteases and in the inhibition of their dimerization, as well as to increase the proteolytic stability with respect to proteases. 237 In this study, our group created a few proteolysis-resistant inhibitors of PR-dimerization by replacing the peptidic strands with peptidomimetics. In particular, compound 2.139 with peptidic arms (VLV sequence) acts as an antidimer with good inhibitory activity (K id = 80 nM), where by changing one peptidic arm with peptidomimetic strand (compound 2.140) they obtained more significant results. This compound is still fully active against wild-type PR (K id = 0.2 μM for 2.140, compared to K id = 0.56 μM for 2.139). This suggests that peptidomimetic molecular tongs are candidates to successfully overcome the resistances presently encountered with classical protease inhibitors. In the next section the synthesis of several -strands will be discussed as well as their incorporation into rigid scaffold to form -hairpin structures as potential inhibitors of A peptide aggregation associated with Alzheimer's disease.

Alicyclic compounds

Design and synthesis of hairpin mimics

Design of the hairpin mimics

The formation of peptide and protein aggregates through the interaction of β-sheets has increasingly drawn attention since it occurs in many widespread human diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, and Huntington's disease (HD). Mimicking -strands to antagonize -sheet formation or recognition represent a new therapeutic strategy toward the prevention or treatment of diseases associated with -sheet structures and, in particular, AD, in which the protein aggregation process involves a secondary structure transition from unordered/α-helix to β-sheet rich conformation, leading to cross β-sheet structure formation (see chapter 1). In this case, drug design consists in the investigation of protein-protein interactions associated with this disease followed by the design of small molecules that can mimic or bind to one of the interacting proteins involved in this disease.

There are two possible strategies for treating AD: inhibiting the formation of oligomers or redirecting the aggregation cascade toward off-pathway species with reduced toxicity (fibrils). 238,239 Recently In the project of this thesis, we decided to follow another strategy using β-hairpin mimics.

Based on the few recent published data on β-hairpin mimics in particular on the macrocyclic structures of Nowick, 241 as inhibitors of protein aggregation (see chapter 1), we hypothesized that pre-structuring the peptidomimetic molecules might increase their affinity for Aβ peptides and thus increase their aggregation inhibitory activity. strand was aimed at increasing the metabolic stability of the molecule vis a vis of its potential use in biological assays as amyloid aggregation inhibitor. As peptidomimetic strand, we decided to incorporate into our sequence 5-amino-2-methoxybenzhydrazide, which is a part of the -strand mimic (" ao" unit) reported by Nowick and co-workers. 242 The introduction of 5-amino-2methoxybenzhydrazide into -strand mimics was shown to be extremely effective in the formation of intermolecular β-sheets with the terminal parts of HIV-1 proteases and in the inhibition of their dimerization, as well as to increase the metabolic stability with respect to proteases. 243 Finally, as for the peptide sequence, four residues Gly-Val-Val-Ile were introduced. The sequence GVVI was chosen because it is the sequence 38-41 of A Indeed, with the view to increase the affinity of this series of hairpin mimics towards Aβ 1-42 , we provided to these molecules the possibility to engage also electrostatic interactions with Aβ 1-42 . For this purpose, the N-terminal residue was deprotected in all the final molecules in order to promote ionic interactions with acidic residues of Aβ 1-42 . Indeed, several computational and experimental studies on Aβ 1-42 have shown that, in addition to the hydrophobic interactions involving in particular the 16-21 sequence (KLVFFA), the formation of a salt-bridge between amino acids Asp23 and Lys28 might stabilize a turn motif involving residues 24-28. 246 An interaction with Glu22 might be also promoted and beneficial for the activity of the molecules, as suggested by T.

Schrader's work. 246b Our group previously observed that ionic interactions improved the inhibitory activity of glycopeptides. 247 The studied criteria that can influence the activity on early oligomerization and fibrillation have been:

1-The -hairpin structure : cis versus trans-DKP scaffold; the presence of two arms linked to the scaffold versus only one arm; the effect of the isolated tetrapeptide and peptidomimtic arms) A CF 3 group was also introduced into our peptidomimetic strands, following the same synthetic pathway, to test its pharmacomodulation properties. The preparation of our fluorinated strands was obtained using trifluoroacetic anhydride instead of acetic anhydride, used for the preparation of strands 2.148 a/b as was illustrated in Scheme 2.15.

Scheme 2.15. Synthesis of the fluorinated Val-and Lys-peptidomimetics; i) (CF 3 CO) 2 O, TEA, THF, 89%; ii) TFA, CH 2 Cl 2 , quantitative yield; iii) Boc-Val-OH, HBTU, HOBt, DIPEA, DMF, overnight, 80%; iv) Nɛ-Cbz-Nα-Boc-L-Lys-OH, HBTU, HOBt, DIPEA, DMF, overnight, quantitative yield; v) = vi) TFA, CH 2 Cl 2 , r.t., quantitative yield.

II) Synthesis of hydrazide based peptidomimetic

Our group in Paris had designed peptidomimetics containing carbohydrazide (CONHNHCO) fragments in replacement of amino acid residues. As a result, the peptidomimetics became more resistance to cleavage by peptidases. Furthermore, these new strands, by decreasing the peptidic character of molecules, increased the hydrosolubility yielding compounds with efficient ability to inhibit PR and two multimitated mutated HIV-1 protease. 249 The synthetic procedure designed to obtain the new peptidomimetic arms involved the synthesis of a dihydrazide motif 2.154.

III) Synthesis of fluorinated analogues of of 5-amino-2-methoxybenzoic hydrazide derivatives

Fluorine in medicinal chemistry has exploded during the past 25 years and especially in the last decade. The origins of organic fluorine chemistry date back to the 1890s when the Belgian chemist Fédéric Swarts discovered the Cl/F exchange chemistry of the inorganic antimony trifluoride (SbF 3 ), a hydrofluoric acid (HF) widely used in dyeing and pottery. Then he prepared many polyhalogen compounds containing fluorine and established a foundation for others to follow. The most notable example of this influence was the application of dichlorodifluoromethane and congeners as the next generation of refrigerants to replace toxic NH 3 and SO 2 . 250 In contrast to fluoroaliphatics, the introduction of fluorine on the aromatic nucleus had an earlier and less eventful history. p-Fluorobenzoic acid was prepared from its precursor diazonium salt and concentrated HF, and this was followed by other successes. 251 However, the first practical method 2.162 totally protected (Scheme 2.17). For the latter coupling the common coupling reagents HOBt, HBTU, in the presence of DIPEA in DMF were applied, but the desired product was obtained with a low yield of 35%. In order to improve the yield of the reaction a different coupling reagent, DMTMM(BF 4 ), in the presence of N-methylmorpholine was employed, and the product 2.162 was obtained in a higher yield.

Scheme 2.17. The synthetic procedure for the preparation of new fluorinated peptidomimetic; i) NH During my period in Paris, I developed the synthesis of the fluorinated peptidomimetic 2.157a.

The synthesis was challenging due to the presence of a fluorine atom and the very reactive hydroxyl group on the aromatic ring which interferes with most steps of the synthesis. For the synthesis of peptidomimetic 2.157b, I could obtain the precursor 2.166 which can then be transformed into the target compound following the path depicted in Scheme 2.20.

Synthesis of 2,5-diketopiperazine scaffold

The synthesis of DKP1 and DKP2 (bearing a benzyl group at nitrogen N-4, Scheme 2.21) was performed according to procedures already developed in our laboratories. 252 In addition, a different 2,5-diketopiperazine scaffold namely DKP9, was also synthesized. In this scaffold the two side chains are in a cis configuration (and S,S absolute configuration) and bears a benzyl group at Nitrogen N-1 (Figure 2.51).

Synthesis of DKP1 and DKP2

The diketopiperazine ring was obtained by cyclization of the depeptide methylester derived from protected and functionalized L-aspartic and L-or D-serine (Scheme 2.21).

Scheme 2.21. Retrosynthesis approach to DKP1 and DKP2 Thus, aspartic acid was protected orthogonally to Boc and methylester on the β carboxylic acid functionality as an allylester. L-aspartic acid was esterified by treatment with acetyl chloride in allyl alcohol to give aspartic acid β-allyl ester hydrochloride 2.167. The reaction of acetyl chloride with an alcohol is used to generate HCl in situ leading to the formation of allyl acetate, which can be removed if necessary by evaporation under reduced pressure. This procedure was reported to be selective for the β-carboxylic group esterification, but some bis-allylation can occure. In order to minimize the α-carboxylic acid esterification the reaction should be performed at low temperature (0-5 o C). The amino group was then protected as N-tert-butoxycarbonyl derivative, under standard conditions, giving N-(tert-butoxycarbonyl)-aspartic acid β-allyl ester 2.168. For the other building block it was thought that the OH of the serine side chain should not interfere with the subsequent coupling. Therefore, only the α-carboxylic acid group was protected as its methyl ester. Serine methyl ester hydrochloride 2.169 was obtained by esterification of the corresponding free amino acid in refluxing methanol, in the presence of acetyl chloride. The serine α-amino acid was then alkylated through a reductive amination resulting N-benzylserine methyl ester 2.170. The presence of a tertiary amide decrease the energy difference between cis and trans isomers facilitating the cyclization step led to the formation of DKP. The hydrochloride salt was treated with benzaldehyde in methanol, in the presence of iPr 2 EtN to obtain the corresponding imine that was subsequently reduced the sodium borohydride (Scheme 2.22). In order to minimize racemization during the recent step, special attention should be given to both the temperature (should be remain -5-0 o C) and the reaction time. This fact was studied in detail by NMR spectroscopy for compounds 2.172a and 2.172b: (a) in Scheme 2.24. Proposed mechanism for diketopiperazine formation: i) TFA, CH 2 Cl 2 ; ii) 4 equiv iPr 2 EtN, 2-propanol.

To improve the coupling reaction between two building blocks providing the isopeptide, in addition to Caprino's reagent ( ATU, OAt), which is very useful to avoid epimerization on the α proton during amino acid coupling, another coupling reagent for the scale up of the reaction was used. Since an ester bond is formed instead of an amide bond, more appropriate and better valued conditions were provided, using the coupling reagent EDC (1-ethyl-3(3-dimethylaminopropyl) carbodiimide) in the presence of a catalytic amount of DMAP (4-dimethylaminopyridine).

Synthesis of DKP9

As was mentioned before the prepration of the cis DKP1 displays some critic and difficult steps, such as using the H 3 N that can be dangerous, and difficult purification after the Mitsunobu reaction, also due to competitive elimination. For these reasons, our group decided to investigate another synthetic pathway to provide a cis diketopiperaznie scaffold starting from two amino acids, L-asparagine and L-aspartic acid following an easier procedure providing the new DKP9 (2.178) as illustrated in Scheme 2.31.

Scheme 2.31.The retrosynthesis pathway for preparation of DKP9

The free amino functionality of natural amino acid L-asparagine was protected as N-tertbutoxycarbonyl derivative, under the standard conditions giving the protected compound 2.179.

The In this reaction the temperature has an important role, as to the reaction at room temperature proceed in a lower yield. With the two building blocks in our hands, we performed the coupling reaction using the Carpino's reagent ( ATU, OAt) in DMF. Unfortunately this reaction did not take place, thus we used the other coupling reagent that was shown to be more efficient in our coupling reactions. The DMTMM(BF 4 ) was used in the presence of NMM in DMF, without any traces of desired product. Finally, the symmetric anhydride of compound 2.181 was generated using DCC in CH 2 Cl 2 affording the anhydride intermediate 2.187 that was isolated and immediately used in the coupling reaction with the compound 2.183, providing the dipeptide 2.184 in a moderate yield after 3 days (Scheme 2.37).

Synthesis of Hairpins

As described in the beginning of this section, our goal is to build -hairpin structures utilizing the rigid cis DKP scaffold, that acts as a good promoter of reverse turn inducer, a peptidomimetic strand, which is able to stabilize the structure of our molecules and also is able to interact with the Aβ peptide blocking the formation of β-sheet structures and thus inhibiting further aggregation of this protein associated with Alzheimer's disease, and finally a peptidic strand composed of four amino acids.

The synthesis of first hairpin was performed using the peptidomimetic containing Val amino acid 2.148a. The coupling reaction between the -strand and the DKP 1 scaffold (2.85) provided the desired product using the common coupling reagents for sterically hindered systems, such as HATU and HOAt. Unfortunately, the synthesis of compound 2.188 was realized in a poor yield of <20%.

Scheme 2.39. The first coupling reaction in the preparation of Hairpin; i) HATU, HOAt, DIPEA, DMF, overnight, <20%.

An investigation of alternative coupling agents was then undertaken: HOBt/EDC. The construction of the lower peptidic arm was then performed via solution peptide synthesis using Boc protected amino acids. In each coupling step, the Boc protected amino acid was introduced using HATU and HOAt in DMF, leading to the formation of the expected compound in high yields. The first amino acid is the L-Isoleucine that was introduced after the cleavage of Boc in product 2.188, providing compound 2.189 in a good yield. By using this procedure (Boccleavage/amino acid coupling) we continued our synthesis to obtain the first hairpin (Scheme 2.41).

Scheme 2.41. The synthesis of peptidic arm to prepare the final product, i) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Ile-OH, DMF, 0 °C to r.t., overnight, 78%; ii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 83%; iii) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 74%; iv) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Gly-OH, DMF, 0 °C to r.t., overnight, 77%.

Following this procedure (peptide coupling Boc strategy) the first -Hairpin 2.192 totally protected was obtained. The cleavage of Boc using TFA in dichloromethane gave the deprotected compound 2.193 (Scheme 2.42) ready for the biological test and also the conformational NMR study that will be discus in the next section. The synthesis of the second hairpin containing the Lys-peptidomimetic was then performed. The same coupling strategy, i.e. DMTMM(BF 4 ) in the presence of NMM in DMF, was utilized also in this case and the desired product was obtained in a very good yield of 64% (compared to 17% using HATU and HOAt).

Scheme 2.43. First coupling for the preparation of second -Hairpin; i) DMTMM(BF 4 ), NMM, DMF, overnight, 64%.

The peptidic arm was coupled similarly to the previous above reported hairpin, using peptide synthesis with Boc strategy. Also in this case the final molecule was obtained in a good yield (Scheme 2.43). The final hairpin was then deprotected: the Cbz protecting group was cleaved first by hydrogenation using Pd/C, H 2 in MeOH, and then after filtration the resulting product was subjected to Boc depretection in the presence of TFA in dichloromethane (Scheme 2.44).

Scheme 2.44. The synthesis route providing second β-hairpin; ii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Ile-OH, DMF, 0 °C to r.t., overnight, 62%; iii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r. At this point, we decided to change the peptidic arm from GVVI to KLVF, maintaining the cis DKP scaffold, to control the effect of peptide sequence in the inhibitory activity of our -hairpins.

In order to realize this, the fluorinated peptidomimetic 2.152a was used to investigate the effect of fluorine on the bioactivity of this molecule. The synthesis started from the coupling between the peptidomimetic arm 2.152a and the DKP1 scaffold 2.85 using the same conditions used previously, DMTMM(BF 4 ), NMM in DMF (Scheme 2.45).

Scheme 2.45. The coupling between fluorinated -strand and the DKP scaffold; i) DMTMM(BF 4 ), NMM, DMF, overnight, 40%.

The peptidic strand was prepared by peptide synthesis Boc strategy using Boc protected amino acids (Phe, Val, Leu, Lys), HATU, HOAt as coupling reagents. We then turned our attention to peptidomimetics containing the trans-diketopiperazine DKP2.

The synthesis of the trans diketopiperazine was achieved as described above. Peptides containing this scaffold were studied by NMR, revealing that the peptide sequences containing DKP2 tend to adopt an extended structure as opposed to cis-DKP1, which promotes a hairpin structure. As we obtained good results from our hairpins as modulators of Aβ aggregation process, we were curious to study the effect DKP2 on the peptidomimeitc conformation and if extended structures could still ensure good biological activity. To achieve this idea we performed the synthesis of compounds containing DKP2 (2.86) as our scaffold, the peptidomimetic strands that were already used with CH 3 and also CF 3 groups and as peptidic arm we used only the GVVI sequence.

Initially, compounds 2.214 and 2.215, containing the Valine and the peptidomimetic arm derivatized as acetamide and trifluoroacetamide, respectively, were prepared. The yield of each step was higher than the same reaction with cis DKP, affording our final molecules in good yields.

The synthetic route for the preparation of these molecules is illustrated in Scheme 2.48 (for CH 3 & CF 3 ).

Scheme 2.48. The synthesis of two new molecules containing DKP2; i) DMTMM(BF 4 ), NMM, DMF, overnight, 0 °C to r.t., 54% (R′= C 3 ), 52% (R′= CF 3 ); ii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Ile-OH, DMF, 0 °C to r.t., overnight, 98% (R′= C 3 ), 98% (R′= CF 3 ); iii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 80% (R′= C 3 ), 85% (R′= CF 3 ); iv) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 60% (R′= C 3 ), 90% (R′= CF 3 ); v) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Gly-OH, DMF, 0 °C to r.t., overnight, 67% (R′= C 3 ), 65% (R′= CF 3 ).

The coupling reaction with peptidomimetic strand using trans diketopiperazine, was realized with higher yield than the same reaction with cis DKP1. By this way there were obtained two new molecules 2.216, 2.217 that were deprotected to provide the final molecules in an acidic condition using TFA in dichloromethane.

Scheme 2.49. The deprotection step providing 2.216 & 2.217 as new molecules containing DKP2; v) TFA, CH 2 Cl 2 , 5h, quantitative yield.

In the same way, the synthesis of peptidomimetics containing a lysine in the upper strand were prepared. The compounds were then purified by preparative HPLC to remove the impurities present in our samples. The synthetic pathway was reported in scheme 2.50.

Scheme 2.50. The synthesis of compounds containing DKP2 and Lys-peptidomimetic; i) DMTMM(BF4), NMM, DMF, overnight, 0 °C to r.t., 44% (R′= C 3 ), 53% (R′= CF 3 ); ii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Ile-OH, DMF, 0 °C to r.t., overnight, 96% (R′= C 3 ), 75% (R′= CF 3 ); iii) TFA, CH 2 Cl 2 , 0 °C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 64% (R′= C 3 ), 67% (R′= CF 3 ); iv) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 52% (R′= C 3 ), 83% (R′= CF 3 ); v) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Gly-OH, DMF, 0 °C to r.t., overnight, 62% (R′= C 3 ), 87% (R′= CF 3 ).

Compounds 2.228 and 2.229 were obtained after the cleavage of Cbz and Boc protecting groups. In this case, and for the first time, we faced the problem of low solubility of these compounds in methanol to perform hydrogenation. To overcome this problem, the compounds were solubilised in a small amount of DMF and then MeOH was added, before hydrogenation under H 2 at atmospheric pressure. The reaction was left overnight for completion; finally Boc cleavage afforded the desired compounds (Scheme 2.51).

Scheme 2.51. vi) Pd/C, H 2 , DMF/MeOH 1:4, overnight; then TFA, CH 2 Cl 2 .

Unexpectedly, using methanol as solvent, the presence of a by-product could be identified in the reaction mixture. This compound isolated by HPLC, and identified by MS, revealed a molecular ion corresponding to the expected m/z value plus 28 a.m.u. This was interpreted as the nitrogen of lysine being doubly methylated during the hydrogenation. For this reason, in all the following hydrogenation reactions a mixture of H 2 O/THF was used to avoid the alkylation of free amine.

The last molecule that we decided to synthesize is the analog of compound 2.205 differing for the presence of the trans DKP2 2.86 and the peptidic arm where the FVLK sequence was introduced. The synthesis started from compound 2.207 which is a common intermediate in the synthesis of other ligands. The peptidic arm was then built by the peptide synthesis in solution.

Compound 2.233 was deprotected to afford 2.234 as a final product totally deprotected. The cleavage of Cbz by hydrogenation following by Boc cleavage (Scheme 2.52) provide the desired product that was then purified by preparative HPLC.

Scheme 2.52. The synthesis; i) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Phe-OH, DMF, 0 °C to r.t., overnight, 75%; ii) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 81%; iii) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Leu-OH, DMF, 0 °C to r.t., overnight, 64%; iv) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, N ɛ -Cbz-N α -Boc-L-Lys-OH, DMF, 0 °C to r.

Solid phase peptide synthesis

In order to prepare the peptidic arm we performed the synthesis of two tetrapeptide that were used in our molecules by solid phase peptide synthesis with a Biotage Initiator™ Microwave Synthesizer using the Rink Amide MHBA Resin and the Fmoc strategy (Scheme 2.53).

Each coupling step was carried out at 70 °C, temperature reached irradiating the vial with microwaves. The use of microwaves allowed to speed up the coupling reaction on the solid phase:

in less than 1 h 30 min it was possible to link a residue to the resin bead and to perform the capping, Fmoc-deprotection and washing steps. The Biotage Initiator™ Microwave Synthesizer is a semi-automatic instrument and, although every addition has to be effected by an operator, it is possible to set every kind of synthetic program, modifying all the parameters for each single step (solution or solvent to use and in which volume, reaction temperature and time, rpm for the vortex, use of inert atmosphere or not) and the operations order. Starting from the Fmoc-protected bead, the first step bead deprotection with 25% piperidine in DMF. Subsequently, the procedure for each amino acid coupling consisted of i) coupling with the activated amino acid, ii) capping with a solution 25% Ac 2 O in DMF, and iii) Fmoc deprotection with a solution 25% piperidine in DMF.

Every coupling cycle ends with various resin washes in MeOH and DMF. The activation of the Fmoc-protected amino acid was performed with DIC, HOAt, DIPEA in DMF. The last step was the full cleavage of the peptide from the resin and removal of all protecting groups present on residue side chains affording compounds 2.235 and 2.236 as our tetrapeptides fully deprotected. A relatively low δ is a characteristic of a proton involved in an intramolecular H-bonding. The formation of a single H-bonding has a rather deshielding effect. When more H-bonds are formed with the solvent, higher δ values are generally observed. Moreover, high Δδ/ΔT coefficient absolute values are indicative of NH protons involved in an equilibrium between intramolecular Hbonds and H-bonds with the solvent. More than one stable conformation can thus be expected.

COSY, TOCSY (revealing the proton cross-peaks in the same spin system) and NOESY (cross peaks due to dipolar coupling resulting from through space interactions) experiments allow the complete characterization of the synthesized compounds and assignment of all the proton chemical shifts, thus giving information on the primary structure. Medium-and long-range NOE contacts, detected from both NOESY and ROESY experiments, are significant in the determination of the preferential conformations adopted in solution.

Usually, the NMR data can be used as an indirect source of structural information, from which the overall 3D structure can be calculated. For diverse compounds, hybrid MC/MD simulations are thus performed, starting from conformers that possess random torsion angle values and fitting them to the conformational restrains derived from the NMR measurement.

In this chapter, first will be presented the NMR conformational analysis and computational studies of compounds 2.193 and 2.217 that were performed by Professor Olivier Lequin and Dr.

Isabel Correia in the University Pierre et Marie Curie (UMR CNRS 7203) and Dr. Guillaume

Bernadat in the University of Paris Sud (UMR CNRS 8076), respectively. A description of the techniques used for biological tests and the operating conditions of Thioflavin-T fluorescence assay, transmission electron microscopy assay and capillary electrophoresis, will be presented.

NMR conformational analysis

As was described in the second chapter, DKP1 scaffold can be seen as a conformationally constrained dipeptide formed by a β 2 and a β 3 -amino acid. Extensive investigation on β-peptides indicated that these are able to adopt stable secondary structures such as helices and sheets. 258 In particular, the stabilization of β-peptide-hairpins sequences was shown to occur in oligo-β-peptides containing the dipeptide sequence formed by a β 2 -amino acid (C2-substituted) followed by a β 3amino acid (C3-substituted). 259 The 10-membered H-bonded ring which formed between the C=O of the β 3 -amino acid and the NH of the β 2 -amino acid, involves the intramolecular hydrogen bonding pattern. When DKP1 was introduced in short peptide sequences (e.g. Val-Ala-DKP-1-Val-Ala) 260 or in foldamers composed of repeating units of DKP1, the same structural feature was observed. 261 In the case of compound 2.193, the use of a longer peptide sequence on one arm and of a β-strand peptidomimetic, which was reported to act mainly in constrained cyclic structure, did not grant the formation of a stable hairpin.

The conformation of 2.193 was detected in a protic solvent, which is more challenging for intramolecular hydrogen bond formation in comparison with aprotic organic solvents such as chloroform. Unfortunately, 2.193 turned out to be poorly soluble in aqueous solution but could be solubilised in methanol at millimolar concentrations.

Complete 1 H and 13 C resonance assignments were obtained from the analysis of 2D 1 H-1 H TOCSY and ROESY, and 2D 1 H- 13 C HSQC and HMBC experiments (Table 3.1). As the 1 H spectra exhibited good chemical shift dispersion at 298 K, most experiments were recorded at this temperature. However the hydrazide proton NH b showed a broad signal and NH c was broadened beyond detection. Cooling the sample down to 270 K gave rise to peak sharpening of hydrazide protons and in particular the NH c proton could be observed. Remarkably, due to the presence of a second set of chemical shifts, weak additional resonances could be detected on the spectra. This minor species, representing less than 5%, turns out to be in equilibrium with the major form, as exchange peaks were observed on 2D ROESY spectra between the NH protons of two forms. The origin of this exchange phenomenon was not characterized but could be due to a rotation of 180° The vicinal 3 J HN-Hα coupling constant also yields direct information on the main chain  dihedral angle, through the Karplus relationship. The coupling constants of the four aliphatic residues (Table 3. Finally, the analysis of H-HN ROE correlations (Figure 3 Furthermore, the ROE between hydrazide NH b proton and methoxy protons supports the formation of a hydrogen bond between the two groups (Figure 3.1). Finally, the long-range ROE between the aromatic H 6 proton and the Gly 1 H protons is not expected in a regular -sheet and can be ascribed to fraying at the N-terminus of the -strand. The temperature dependence of amide proton chemical shifts was also analysed, as it can provide information on the network of hydrogen bonds and their relative stabilities (Table 3.3).

Amide protons that are involved in intramolecular hydrogen bonds typically exhibit small temperature dependence ( HN /T > -4.5 ppb/K) in aqueous 263 indicating that 2.217 undergoes an oligomerization equilibrium. 1 H and 13 C resonance assignments were obtained from the analysis of 2D Weak additional resonances could be detected on the spectra, corresponding to a second set of chemical shifts. This minor species, representing around 3%, turns out to be in equilibrium with the major form, as exchange peaks were observed on the 2D ROESY spectrum between the aromatic protons of two forms. The origin of this exchange phenomenon could be due to 180° rotation around the Ar-NH or Ar-CO bonds, occurring in slow exchange on the The conformational preferences of 2.217 residues were inferred from the analysis of 1 H and 13 C chemical shift deviations (CSD), defined as the differences between experimental chemical shifts and corresponding random coil values, together with vicinal 3 JHN-H coupling constants and ROEs (Table 3 The Val and Ile residues tend to adopt extended backbone conformations, as indicated by upfield shifted C carbons (negative CSD values between -3.5 and -1.7 ppm), and downfield shifted H protons (positive CSD values, Val 3 excepted). These extended conformations are further supported by the large 3 JHN-H coupling constants (> 8 Hz) and the analysis of 2D

ROESY spectrum revealing strong sequential and medium intraresidual H-HN correlations.

Importantly, no long-range ROEs could be detected between the two peptidic arms, proving that 2.217 does not adopt a -hairpin conformation. The amide protons also show a large variation of their chemical shift with temperature (HN/T between -9 and -6 ppb/K) confirming that they are not involved in intramolecular hydrogen bonds.

Computational study

Molecular Modelling is concerned with the description of the atomic and molecular interactions that govern microscopic and macroscopic behaviours of physical systems. The two most widely used methods for atomic-level modelling are Monte Carlo statistical mechanics (MC) and molecular dynamics (MD).

Both procedures typically have the same system setup including representation of molecules as collections of atom-centred interaction sites, utilization of classical force fields for the potential energy terms, and implementation of periodic boundary conditions. The principal differences are in the modes of sampling the configuration space available to the system. For MC, a new configuration is generated by selecting a random molecule, translating it, rotating it, and performing any internal structural variations. Application over enough configurations yields properly Boltzmann-weighted averages for structure and thermodynamic properties. For MD, new configurations are generated by application of Newton's equations of motion to all atoms simultaneously over a small time step to determine the new atomic positions and velocities. In both cases, the force field controls the total energy (MC) and forces (MD), which determine the evolution of the systems.

Molecular modelling was employed in order to evaluate the propensity of compound characteristics found by NMR in methanol to aqueous conditions was a reasonably safe assumption.

An identical protocol was applied to the trans derivative, 2.217, but no structure containing a significant number of key hydrogen bonds could be observed in the results.

Among the most structured geometries found, conformations exhibiting one arm fully extended could be observed, but the other one adopted a random, globular structure.

Biological evaluation

The synthesized final products and key intermediates were all tested to investigate their influence on Aβ 1-42 aggregation kinetics. The ability of compounds to inhibit amyloid fibrils formation was first examined by a thioflavin T (ThT) fluorescence assay. The effect of the more promising compounds (from ThT assay) were then evaluated by transmission electron miscroscopy (TEM) analyses in order o validate the results of ThT and by electrophoresis capillary in order to study the effect on the early oligomerization process.

Thioflavin T (ThT) fluorescence assay

Thioflavin T (ThT) is a benzothiazole-containing dye first noted to bind amyloid by Vassar and Culling in 1959, 268 and later noted to interact with patient-derived amyloids by Naiki and coworkers (Figure 3.4). 269,270 The same phenomena was observed with synthetic A fibrils in vitro, The red curves in this figure show the standard behavior of a negative test: corresponding to the compounds alone (100 µM). 2.148a and 2.152a do not exhibit any inherent fluorescence that would compromise ThT measurements. In presence of Aβ 1-42 , at both ratios compound: Aβ 1-42 of 10:1 and 1:1, both compounds display no effect on the t 1/2 but a significative effect on the fluorescence plateau with an increase of 49 % for 2.148a at ratio of 1:1 (and surprisingly no effect at higher ratio of 10:1) and of 24 % for 2.152a at ratio of 10:1. compound:Aβ 1-42 ratio of 10:1, the compound 2.192 appears to have no effect on the aggregation kinetics. Indeed, the green fluorescence curve seems to be the exact addition of the red and the dark blue curves corresponding to an addition of fluorescence of the compound alone with the amyloid peptide. There is no effect observable at compound:Aβ 1-42 ratios of 1:1 and 0.1:1.

Compound 2.193 the ratios 1:1 and 1:0.1 (Aβ 1-42 :compound) was observed, as it is shown in Figure 3.9 with fuchsia pink and light blue curves. In the case of final product without CF 3 , the deprotected compound, 2.216, does not represent a significant activity. Nevertheless, at the ratio of 1:10 Aβ 1-42 :compound a fiddling decrease on the fluorescence plateau was noted (about -30 %) associated with an increase of the t 1/2 by a factor of 1.6. Surprisingly, the deprotected final compound with CF 3 displays an opposite behaviour since it accelerates the aggregation at the ratio of 1:10 Aβ 1-42 :compound (t This behavior suggests that the presence of CF 3 in this series of compounds is necessary and has an enormous influence on the oligomerization process.

Compound 2.233 of compound 2.163 that has a free hydroxyl group, the situation is different. In both ratios of 10:1 and 1:1 in the presence of peptide a decrease in the fluorescence plateau could be observed, which probably means that there is an interaction between the compound and the Aβ 1-42 peptide (green and fuchsia pink curves).

Compound 2.157a In the Aβ sample incubated with 2.217 a network of comparable density, composed of significantly thicker fibers was observed. peptide in order to evaluate the activity of different compounds on the oligomerization process.

Capillary Electrophoresis

This method rather focused on more advanced stages of the oligomerization than those we are interested in. 280 Our group in Paris recently proposed an improved method to monitor overtime easily, reproducibly and efficiently the very early steps of the oligomerization process. 281 The method have been also slightly modified (the electrosmotic flow modificator diaminobutane was not added into the buffer) in order to evaluate the impact of small molecules on the three kinds of decreased in favor of the peaks of oligomers ES′ and LS, and that insoluble species, forming spikes in the profile, appeared after 6 h (Figure 3.24). for the control sample. Furthermore, this compound promoted the formation of new and large oligomer species and aggregated species (grouped under the peak LS′ and new species + spike) even from the beginning of the kinetics and even at a low Aβ 1-42 : 2.193 ratio of 1:1. It is noteworthy that compound 2.193 did not show activity on Aβ 1-42 fibrillation in the ThT-fluorescence assay at a Aβ 1-42 :compound ratio 1:1, while it shows a dramatic effect on the early oligomerization at this ratio.

The electrophoretic results obtained in the presence of derivative 2.205 indicated that it dramatically delayed the aggregation process and stabilized the monomer ES which was still present after 6h (Figure 3.26). Small and large oligomeric species were only slightly visible after 4 and 6h. These results suggest that 2.205 prevent the presence of small and toxic oligomeric species. In this thesis we reported the synthesis and biological evaluation of a new class of compounds designed to inhibit or modulate the Aβ 1-42 aggregation process. These molecules contain a rigid diketopiperazine scaffold (DKP), cis or trans, that bears one peptidic and one peptidomimetic arms.

The peptide sequence and the peptidomimetic arm are held together in a suitable position to form two interacting β-strands by the use of DKP scaffold, a reverse-turn mimic, which forces a peptide chain to fold back upon itself. One of the two strands is composed of amino acid residues important for stabilizing intra-and intermolecular interactions involved in aggregation and amyloid formation.

A library of 8 bifunctional 2,5-diketopiperazine scaffolds (DKP1-8) was prepared, derived from L-or D-aspartic acid and L-or D-Ser. In the work presented here we used the DKP1 which was shown previously to act as a reverse turn inducer element, the DKP2 capable of inducing an extended structure and finally we designed a new diketopiperazine scaffold in a cis configuration (DKP9) derived from L-asparagine and L-aspartic acid. This new synthetic route let us not only to synthesize a cis DKP without problems that we faced during the preparation of the DKP1, but also permits us to scale up the reaction.

Different Nowick based peptidomimetic strands were synthesized which were incorporated to DKP scaffold. To perform a pharmacomodulation, a CF 3 group was introduced in these strands revealing significant effects on the aggregation process evaluated by ThT fluorescence assay.

Moreover, the fluorinated analogues of the Nowick based peptidomimetics were designed and one of the target molecules was synthesized but unfortunately does not show any activity toward the Aβ 1-42 aggregation.

For the peptide sequence, two series of four residues were introduced, GVVI and KLVF. The first sequence was chosen from the classical hydrophobic residues (Val, Ile, Gly) encountered in natural β-sheet structures, and in particular in the hydrophobic sequences playing a crucial role in promoting and stabilizing the protein aggregation. The second sequence, KLVF, was selected due to its importance in amyloid structuration and aggregation.

We designed two series of molecules, utilizing cis 2,5-diketopiperazine scaffold let us to prepare β-hairpin mimics at which were linked two peptidic and peptidomimetic arms. One of the interesting compounds, β-hairpin 2.193 which contain Val-peptidomimetic arm and GVVI peptide sequence was further studied by NMR conformational analysis confirming the hairpin structure.

The biological evaluation of this compound was followed using ThT fluorescence assay demonstrating that this hairpin delays the kinetics of aggregation by increasing the lag phase by a factor of 2.5. In order to confirm the ability of this compound in modulating the aggregation kinetics, the transmission electron microscopy (TEM) analysis also was performed and results Chapter 5

Experimental Section

Chemistry and Biological part and brine (1×), dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure to afford the crude product.

General procedure C for coupling reactions:

DMTMM (BF 4 ) (1equiv) and NMM (3 equiv) were added to a solution of the N-protected amino acid in DMF (0.1 M), under a nitrogen atmosphere and at 0 ºC. After 30 min, a solution of the TFA salt of the peptide in DMF was added and the reaction mixture was stirred at 0 ºC for 1 h and at r. 9, 163.6, 140.3, 129.4, 126.7, 122.8, 112.4, 55.9; IR νmax : 3258, 1725, 1611, 1514, 1342, 1177. 0, 165.8, 158.2, 156.0, 153.6, 133.2, 125.9, 122.8, 117.8, 112.0, 79.6, 57.1, 56.6, 33.4, 28.5, 24.7, 17.9 1.1 eq) and HOBt (1.37 g, 10.19 mmol, 1.1 eq) were successively added to the reaction mixture that was left under stirring and argon atmosphere at room temperature for 24 h. DMF was evaporated under reduced pressure and the residue was dissolved in EtOAc (200 mL). The organic phase was washed with 10 % aqueous citric acid (50 mL), 10 % aqueous K 2 CO 3 (50 mL), and water (100 mL). The organic phase was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product obtained was purified by flash chromatography on silica (EtOAc, 100% 158.6, 156.3, 155.6, 153.6, 136.6, 133.1, 128.4, 128.0, 126.1, 122.5, 117.8, 112.4, 79.8, 66.5, 56.5, 52.0, 40.7, 34.0, 29.5, 28.6, 24.6, 21.9 N 11.96; found C 59.33, H 6.75, N 11.74; IR ν max : 3312, 1663, 1627, 1530, 1494, 1246, 1168, 1015. 56, 154.46 (q), 129.16, 125.23, 123.42, 117.71, 79.11, 56.07, 28.07 N 11.07; found C 47.65, H 4.69, N 10.76; IR ν max : 3373, 3205, 1713, 1662, 1494, 1227, 1148, 836. , 159.3, 155.6, 130.2, 126.8, 124.6, 118.4, 112.1, 57.5, 56.7,

N'-(2-Methoxy-5-nitrobenzoyl)

tert-Butyl-2-(Hydrazinecarbonyl)-Hydrazinecarboxylate (2.154)

A solution of compound 2.153 (5 g, 19.82 mmol) and hydrazine monohydrate (9.6 mL, 198.2 mmol, 10 eq) in methanol (40 mL) was heated under reflux for 1.5 h and then cooled at room temperature. Upon removal of the solvent under reduced pressure, the resulting residue was triturated in Cyclohexane. To a solution of compound 2.154 (7.69 g, 40.44 mmol) in dry THF (100 mL), pyridine (8.15 mL, 101.10 mmol, 2.5 eq) was added dropwise under the nitrogen atmosphere. The mixture was stirred for 10 minute and at 0 o C was added phenyl chloroformate (5.6 mL, 44.48 mmol, 1.1 eq) followed by stirring at room temperature for 1h. The solvent was evaporated and the crude was diluted with EtOAc and washed with water (100 mL), 10% aqueous citric acid (50 mL), 10% aqueous K 2 CO 3 (50 mL). The organic layer was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue obtained was purified by flash chromatography on silica gel (EtOAc/cyclohexane 80:20) to afford compound 2.155 (5.38 8, 155.8, 154.9, 150.8, 129.3, 125.1, 121.5, 78.9, 28 : 3260, 1717, 1662, 1633, 1556, 1489, 1281, 1244, 1152. ), 8. 14-8.11 (m, 3H), 7.86-7.83 (m, 2H), 7.77-7.71 (m, 2H), 7.61-7.53 (m, 3H), 7.50-7.44 (m, 2H), 1.44 (s, 9H); 13 C NMR (75 MHz, DMSO) δ 166.3, 164.5, 162.9, 156.1, 155.8, 152.8, 134.8, 134.3, 133.3, 132.4, 130.4, 129.4, 128.8, 128.2, was heated to reflux. After 2.5 h reflux was then stopped and the flask was cooled to room temperature. 200 mL of Et 2 O were added provoking the precipitation of the resulting salt which was filtrated on a glass funnel and dried under high vacuum affording 2.169 as a white solid (8.79 g, 99%). mp: 162-164 °C (lit. 160-165 °C); [] 20 D = +3.98 (S-, c: 1.0, CH 3 OH); 1 H NMR (400 MHz, D 2 O) δ 4.25 (t, J=3.8 Hz, 1H), 4.08 (dd, J 1 =12.6 Hz, J 2 =4.3 Hz, 1H), 3.98 (dd, J 1 =12.6 Hz, J 2 =3.4 Hz, 1H), 3.83 (s, 3H); 13 C NMR (100 MHz, D 2 O) δ 169.3, 59.6, 55.1, 54.1; S: IR ν max : 3358, 2928, 1750, 1595, 1510, 1260, 1095, 1040; R: IR ν max : 3366, 2924, 1748, 1595, 1508, 1256, 1094, 1036.

tert-butyl-2-(3-amino

(R)-or (S)-N-benzylserine methyl ester (2.170) 6 g (38.6 mmol) of S-or R-serine methylester hydrochloride 2.169 were dissolved in 90 mL MeOH, and the mixture was cooled to -10 o C. 9.4 mL of iPr 2 EtN (54 mmol, 1.4 eq) and 0.79 mL (7.71 mmol, 1eq) of freshly distilled benzaldehyde were successively added dropwise. The reaction eas stirred for 4 hours at r.t. The temperature was then lowered again to -10 o C and 2.9 g (76.6 mmol, 2 eq) of NaBH 4 were added portionwise over 30 min. The reaction mixture was again let react for half an hour, then, reaction was quenched by adding at low temperature HCl 4 M until no more gas formation was observed. The mixture was washed 3 times with Et2O. The organic phases were combined and extracted with HCl 4 M twice. The aqueous phases were then combined and neutralized by careful addition of saturated NaHCO 3 solution until a pH=8 was reached. The aqueous phase was then extracted 4 times with Et 2 O. The organic phase was separated and dried over Na 2 SO 4 , filtered, concentrated under reduced pressure and dried under vacuum affording 2.170 as a transparent oil (7.35 g, 91%). R f : 0.2 (EtOAc/hexane 1:1); [] 20 D = +39.4 (R-, c :1.0, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 7. 38-7.26 (m, 5H), 3.91 (d, J=13.0 Hz, 1H), 3.82 (dd, J 1 =10.9 Hz, J 2 =4. 4 Hz, 1H), 3.79-3.75 (m, 4H), 3.66 (dd, J 1 =10.8 Hz, J 2 =6.2 Hz, 1H), 3.46 (dd, J 1 =6.1 Hz, J 2 =4.5 Hz, 1H), 2.78 (s, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 173. 6, 139.3, 129.0, 128.8, 127.8, 62.8, 62.2, 52.6, 52.4; S: IR ν max : 3320, 2951, 1736, 1454, 1202, 1142, 1057; R: IR ν max : 3320, 2951, 1738, 1202, 1142, 1057.

4-allyl 1-[2-(benzylamino)-3-methoxy-3-oxopropyl] N-(tert-butoxycarbonyl)-aspartate (2.172a, b)

To a solution of either (R)-or (S)-N-benzylserine methyl ester 2.170 (734 mg, 3.51 mmol, 1eq) and EDC.HCl (3.4 g, 17.6 mmol, 5 eq) in dry CH 2 Cl 2 (20 mL) at 0 o C under N 2 atmosphere, -allyl (2R)-N-(tert-butoxycarbonyl) aspartate ester 2.168 (1.92 g, 7.01 mmol, 2eq) was added as a solution in CH 2 Cl 2 . After 30 min, DMAP (214 mg, 1.76 mmol, 0.5 eq) was added in one portion.

The mixture was stirred at 0 o C for 2 h and for additional 4 h at r.t. The resulting mixture was diluted with EtOAc (70 ml) and washed with KHSO 4 1 M aqueous solution (2x40 ml), aqeous NaHCO 3 (2x40 ml) and brine (2x40 ml), dried over Na 2 SO 4 , and volatiles were removed under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane/EtOAc 7:3) to afford the desired product as a transparent oil (734 mg, 45%). R f : 0.30 (hexane/EtOAc 6:4); a) [] 20 D = -2.65 (c 1.0, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 7.34-7.24 (m, 5H), 5.93-5.83 (m, 1H), 5.48 (d, J=8.2 Hz, 1H), 5.30 (d, J=17.2 Hz, 1H), 5.23 (d, J=10.4 Hz, 1H), 4.61-4.51 (m, 3H), 4.48-4.32 (m, 2H), 3.88 (d, J=13.1 Hz, 1H), 3.75 (s, 3H), 3.73 (d, J=13.1 Hz, 1H), 3.55 (t, J=4.7 Hz, 1H), 2.99 (dd, J 1 =17.0 Hz, J 2 =4.3 Hz, 1H), 2.85 (dd, J 1 =17.0 Hz, J 2 =4.7 Hz, 1H), 2.21 (br s, 1H), 1.45 (s,9H); 13 C NMR (100 MHz, CDCl 3 ) δ 172.8, 171.0, 155.7, 139.6, 132.1, 128.9, 128.7, 127.6, 119.1, 80.6, 66.2, 66.2, 59.5, 52.7, 52.2, 50.3, 37.1, 28.7; IR ν max : 3358, 2928, 1738, 1500, 1454, 1385, 1163, 1053. b) [] 20 D = +11 (c 1.0, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 7. 34-7.26 (m, 5H), 5.93-5.83 (m, 1H), 5.47 (d, J=8.2 Hz, 1H), 5.32 (d, J=17.2 Hz, 1H), 5.25 (d, J=10.4 Hz, 1H), 4.66-4.52 (m, 3H), 4.46 (dd, J= 4.6, 11.0 Hz, 1H), 4.35 (dd, J= 4.8, 11.0 Hz, 1H), 3.89 (d, J=13.1 Hz, 1H), 3.80-3.68 (m, 4H, 3H (OMe) + 1H), 3.55 (t, J=4.7 Hz, 1H), 3.01 (dd, J 1 =17.0 Hz, J 2 =4.3 Hz, 1H), 2.86 (dd, J 1 =17.0 Hz, J 2 =4.7 Hz, 1H), 1.96 (s, 1H, OH), 1.45 (s,9H); IR ν max : 3362, 2978, 1740, 1500, 1455, 1368, 1167, 1053 : C 59.47, H 6.94, N 6.03; found C 59.07, H 7.01, N 5.91. completion (overnight). The reaction mixture was cooled down to 0 o C and filtered under vacuum.

The filter cake was washed with ethyl acetate and dried in vacuum at 65 o C to obtain the 2.180 (430 mg, 65%). mp: 210 o C; 1 H NMR (400 MHz, CD 3 OD) δ 4.05 (t, J= 5.4 Hz, 1H), 3.17-3.14 (m, 2H) : 3507, 3349, 2968, 1688, 1534, 1404, 1364, 1016.

Nα-Boc, Nβ-Cbz aminopropionic acid (2.181)

To an ice-cold stirred solution of 2.180 (800 mg, 3.91 mmol) in water (40 mL) was added NaHCO 3 (821.2 mg, 9.77 mmol, 2.5 eq) slowly in portions. To this mixture, CbzCl (2.012 g, 5.87 mmol, 1.68 mL, 1.5 eq) was added in one stretch and stirred vigorously overnight at room temperature. Toluene was removed under reduced pressure and the aqueous layer was acidified to pH 2 with KHSO 4 1 M solution. The compound was extracted into EtOAc, organic layer were collected, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford 2.181 as a white foam (892 mg, 67%). mp: 180-182 o C; 1 H NMR (400 MHz, CDCl 3 ) δ 7.34-7.27 (m, 5H), 5.41 (br s, NH, 1H), 5.11 (s, 2H), 4.14-4.11 (m, 1H), 3.66-3.60 (m, 2H), 1.45 (s, 9H); 13 C NMR ( 50MHz, CDCl 3 ) δ 173.2, 157.2, 155.8, 136.0, 128.2, 127.8, 80.0, 66.7, 54.0, 42.5; LC-MS calcd : 338.1478, found: 339.2566 [M + +H].283

N-benzyl-dimethyl aspartate (2.183)

At 0 o C to 45 mL of anhydrous methanol, 10.9 mL (150.3 mmol) of thionyl chloride was added dropwise. The solution was stirred at 0 o C for 30 min and then 5 g (37.5 mmol) of Asp was added.

The reaction mixture was left under stirring overnight, then the solvent was evaporated under reduced pressure and the residue was triturated with petroleum ether repeatedly to provide 2.182 (7 g, 95%) as a white solid that was used for the next step.

To a solution of 2.182 (1 g, 5.06 mmol) in 15 mL of dry methanol were added triethyl amine (900 μL, 7.08 mmol, 1.4 eq) and freshly distilled benzaldehyde (510 μL, 5.06 mmol). The reaction mixture was left for 4 h at r.t. under stirring. The temperature was then lowered to -10 o C and 382 mg (10.12 mmol, 2 eq) of NaBH 4 were added portionwise over 30 min. The reaction mixture was again let react for half an hour, then, reaction was quenched by adding at low temperature HCl 4 M until no more gas formation was observed. The mixture was washed 3 times with Et2O. The organic phases were combined and extracted with HCl 4 M twice. The aqueous phases were then combined and neutralized by careful addition of saturated NaHCO 3 solution until a pH=8 was reached. The aqueous phase was then extracted 4 times with Et 2 O. The organic phase was separated and dried over Na 2 SO 4 , filtered, concentrated under reduced pressure and dried under vacuum affording 2.183 as a colorless oil (1.08 g, 85%). The characterization was reported in literature. 284 formed and stirring continued for 2 h at r.t. The mixture was filtered on a cotton wool to remove DCU. The white DCU residue was washed with cold CH 2 Cl 2 twice and the filtrate was concentrated under reduced pressure and dried under high vacuum to afford symmetric anhydride 2.163 as a white solid, which was used without further purification. N-benzyl-aspartic dimethyl ester 2.159 (57.2 mg, 0.227 mmol, 0.7 eq) was dissolved in dry CH 2 Cl 2 (3 mL) and the mixture was cooled to 0 o C. A solution of symmetric anhydride in 3 mL of dry CH 2 Cl 2 was then added dropwise. The reaction mixture was let to react for 3 days at r.t. The solvent was evaporated under reduced pressure, and then the crude was purified by flash column chromatography (hexane/acetone 8:2) affording 2.184 as a white solid (76 mg, 60%). R f : 0.26 (hexane/acetone 7:3); mp: 83-85 o C ; 1 H NMR (400 MHz, CD 3 OD) δ 7.47-7.23 (m, 10H), 5.08 (s, 2H), 5.01 (d, J= 16.6 Hz, 1H), 4.94-4.91 (m, 1H), 4.57 (d, J=16.6 Hz, 1H), 4.31 (t, J=4 Hz, 1H), 3.62 (s, 3H, OMe), 3.57 (s, 3H, OMe), 3.48-3.43 (m, 2H), 1.87-1.84 (m, 1H), 1.74-1.70 (m, 1H), 1.40 (s, 9H); 13 C NMR (100 MHz, CD 3 OD) δ 168.7, 162.2, 155.3, 151.0, 146.9, 139.9, 134.0, 73.2, 71.6, 59.7, 42.1, 35.7, 31.6, 21.0; IR ν max : 3278, 2948, 1715, 1523, 1345, 1120.

N-Cbz-DKP 9-COOMe (2.178)

The dipeptide 2.184 (50 mg, 0.087 mmol) was deprotected according to general procedure A.

The corresponding trifluoroacetate salt was then dissolved in iPrOH 1 mL and was added DIPEA

Compound 2.228

Compound 2.226 (35 mg, 0.028 mmol) was dissolved in dry DMF 1 mL and then were added methanol 4 mL and Pd/C (7 mg, 20% weight). The reaction flask was flushed three times with hydrogen, and the reaction was stirred, under hydrogen atmosphere, for 5 h at r.t. The mixture was filtered through an HPLC filter, then the filtrated solution was evaporated under reduced pressure to afford product without Cbz protecting group that undergoes the deprotection reaction according to general procedure A. The reaction is left overnight under stirring to obtain product 2.228 as a yellow solid (30mg, 87% over two step). mp: 180-182 o C; 1 H NMR (400 MHz, DMSO) δ 10.31 (s, NH, 1H), 10.00 (s, NH, 1H), 9.96 (s, NH, 1H), 8.42 (d, J= 12 Hz, NH, 1H), 8.29-8.25 (m, NH, 2H), 8.17 (s, NH, 1H), 8.09-8.06 (m, NH, 3H), 7.99 (d, J= 4 Hz, 1H), 7.82-7.79 (m, 3H), 7.73 (dd, J= 4, 8 Hz, 1H), 7.35-7.28 (m, 5H), 7.11 (d, J= 8 Hz, 1H), 5.26 (d, J= 16 Hz, 1H), 4.47-4.37 (m, 3H), 4.23-4.15 (m, 2H), 4.02 (d, J= 16 Hz, 1H), 3.86 (s, 3H), 3.73-3.64 (m, 3H), 3.55-3.51 (m, 1H), 2.86-2.76 (m, 2H), 2.02 (s, 3H), 1.98-1.91 (m, 2H), 1.78-1.69 (m, 2H), 1.61-1.56 (m, 2H), 1.47-1.36 (m, 3H), 1.13-1.06 (m, 1H), 0.87-0.76 (m, 18H); 13 C NMR (101 MHz, DMSO) δ 171. 9, 170.8, 170.5, 170.0, 169.3, 168.1, 166.6, 166.0, 165.8, 163.5, 158.7, 158.3, 152.8, 136.5, 132.7, 128.6, 127.8, 127.4, 123.6, 121.4, 121.1, 112.5, 58.5, 58.2, 57.6, 56.8, 56.3, 50.9, 50.4, 46.6, 40.2, 38.9, 38.8, 36.6, 36.5, 31.7, 31.1, 30.1, 26.7, 24.1, 23.8, 22.2, 19.3, 19.1, 18.5, 17.9, 15.3, 10.8 N 13.37, Found C 49.54, H 6.87, N 12.45; IR ν max : 3364, 2363, 1652, 1423, 1032, 987, 752.

LIST OF ABBREVIATIONS

AA

  .1): (i) Primary structure, i.e. is the linear sequence of amino acids which are linked together by peptide bonds to form a polypeptide chain. (ii) Secondary structure: referring to the local conformation of the polypeptide chain. This structure can be determined by hydrogen bond interactions between the carbonyl group of one amino acid and the amide hydrogen of a different one placed at the right distance. The most common structures are α-helix and β-pleated sheet.

Figure 1 . 1 .

 11 Figure 1.1. Four levels of protein structure a) primary, b) secondary, c) tertiary, and d) quaternary

Figure 1 . 2 .

 12 Figure 1.2. Regular -helix

Figure 1 . 3 .

 13 Figure 1.3. Ramachandran plot 3 A β strand is not a stable structure and it tends to interact with other β strands forming more stable secondary structure called β sheet. It was discovered by Pauling and Corey in 1951, the same

Figure 1 . 4 .

 14 Figure 1.4. Types of -pleated sheets

Figure 1 . 5 .

 15 Figure 1.5. Torsion angles of classic and inverse -turns

Figure 1 . 6 .

 16 Figure 1.6. Torsion angles of classic -turns.

Figure 1 . 7 .

 17 Figure 1.7. Ribbon representation of a -hairpin 10

Figure 1 . 8 .

 18 Figure 1.8. Examples of Foldamer framework 1.2.1. Helix and -sheet like -peptides

Figure 1 . 9 .

 19 Figure 1.9. -Nanopeptide (3 14 -helical form)

Figure 1 .

 1 Figure 1.10. 12-Helical -peptide as inhibitor of γ-secretase

  segment from the Bcl-2 homology 3 (BH3) domain of a pro-apoptotic binding partner. Peptide-and non-peptide-based scaffolds have been explored as BH3 domain mimics to antagonize Bcl-2 family interactions, including efforts to identify helical band -peptide foldamer ligands for Bcl-xL. The 15-mer -peptide 1.3 (Figure 1.11), which contains an N-terminal segment with alternating  residues and a C-terminal  segment, binds tightly to Bcl-xL.26 Y. Imamura, N. Watanabe, N. Umezawa, T. Iwatsubo, N. Kato, T. Tomita, T. Higuchi, J. AM.CHEM. SOC., 2009, 131, 7353-7359.

Figure 1 . 11 .

 111 Figure 1.11. Structures of -peptides 1.3 The first four  residues in 1.3 have a five-membered ring constraint that promotes helical folding. The structure shows that 1.3 adopts a helical conformation and is oriented within the BH3 recognition channel in a similar way to natural BH3 domains. The helix formed by Bcl-xL-bound 1.3 features a regular pattern of backbone C=O (i)•••H-N(i+4) H-bonds over nearly the entire length of the oligomer. 27

Figure 1 .

 1 Figure 1.12. a) X-ray structures of human Bcl-xL (magenta) complexed with foldamer 1.3 (green), b) Stereoview of foldamer 1.3. This H-bond pattern underlies the -helix formed by pure -residue peptides, and is also characteristic of the 14/15-helix, a secondary structure favored by 1:1 alternating  backbones in which -residues have a five-membered ring constraint (Figure 1.12).Besides helical arrangements, -peptides, like the -peptidic prototypes, can also adopt non-aggregating extended-chain structures and can assemble to parallel and antiparallel pleated sheets (as a part of hairpin turn). During the last decade significant progresses have been made toward the goal of preparing -peptides with sheet-like conformations.

Figure 1 . 13 .

 113 Figure 1.13. Folded β-peptide sheet Initial studies performed in Gellman's laboratory suggest that α,β-disubstituted βamino acid residues with appropriate relative stereochemistry at C α and C β as in compound 1.4 are particularly well suited for the formation of polar antiparallel β-peptide sheets.

  and -Sheet mimetics -Hairpin motifs in naturally occurring peptides and proteins feature a remarkable degree of structural diversity. The diversity can occur due to variations in the loop size, hairpin register, occurrence of the -bulges within the -strands, and of course due to sequence variation. In 1988, Kemp et al. reported the first example of antiparallel -sheet formation with urea functionalized peptide conjugates of 2,8-diaminoepindolidione 31 (Figure 1.15). The presence of two D-Ala-Pro loops led to the three-stranded antiparallel sheet 1.7 in which two -hairpins share a common peptidomimetic strand. 32

Figure 1 . 15 .

 115 Figure 1.15. Three stranded antiparallel sheet As years go by, research on -sheet structure molecules became more interesting to researchers and numerous developments were performed in this field. König and co-workers developed a model system containing Gellman's -turn fragment (D-Pro-Gly), a peptidic and a peptidomimetic -strand formed by methoxypyrrole amino acids-MOPAS (Figure 1.16). The system adopts a

Figure 1 . 17 .Figure 1 . 18 .

 117118 Figure 1.17. Different hydrogen-bonding patterns in N-acetylated hexapeptide mimics -Hairpin loops are prominent in the antigen-binding sites of antibodies and the ligand binding sites of many cytokine receptors and polypeptide growth factors, as well as in many integrins and viral proteins, and in a wide variety of smaller host-defence peptides such as defensins.

Figure 1 . 19 .Figure 1 . 20 .Figure 1 . 21 . 14 (Figure 1 . 22 .Figure 1 .

 119120121141221 Figure 1.19. Hairpin mimetic based on antibody CDR loops

Figure 1 .

 1 Figure 1.24. -Hairpin using δ-linked ornithine and Hao unit

Figure 1 . 25 .

 125 Figure 1.25. The generic structure of Macrocyclic peptideTheir study demonstrates the hydrophobic surfaces between the -sheet layers are important in inhibiting amyloid aggregation. They suggest that this principle is applicable to larger amyloids such as those formed by full-length tau protein or -amyloid peptides. They also prepared a 54membered ring comprising a heptapeptide -strand (upper strand) one Hao unit flanked by two dipeptides (the lower strand) and two δ-linked ornithine ( δ Orn) turns. They described that the flexibility of the dipeptides flanking Hao in the lower strand would better accommodate the flatness of the Hao template and thus minimize the kinks in the -strands that they previously observed in 42-membered ring macrocycle 1.18. They designed the macrocycle 1.19 to display exposed heptapeptide -strand so that these -strands can recognize and bind their parent amyloid proteins (Figure1.26). Actually, they showed the good inhibitory activity of these well-defined -sheets in the aggregation of amyloid protein in Alzheimer's disease.44

Figure 1 .

 1 Figure 1.26. 54-membered macrocycle -sheet mimic

  Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes mellitus, and several other neurodegenerative and systemic disorders are protein-misfolding diseases characterized by the accumulation of insoluble protein deposits. Many protein aggregation diseases affect the nervous system; among these one of the more challenging is AD. Alzheimer's disease is the most common form of dementia that causes memory loss in the elderly. In 2013, there were 35 million persons afflicted with AD worldwide, a number expected to double by 2050.45

Figure 1 .

 1 Figure 1.27. Brain areas and circuits involved in memory process 47 Loss of these connections directly causes clinical symptoms of episodic memory impairment which is a hallmark of AD at the beginning stage of the disease. In the brain of AD patients severe pathological changes present by the first symptoms of deficit in episodic memory. These changes develop gradually over years in the key areas of memory formation and can be confirmed by diverse techniques. Early longitudinal imaging studies based on computed tomogram techniques followed the reduction of the thickness of the medial temporal lobe in normal aging and in AD patients that shows a tiny reduction. Modern magnetic resonance imaging (MRI) techniques not only confirmed the volume reduction in the gray matter (neurons) but demonstrated also a correlation between the entorhinal cortex atrophy and episodic memory impairment in the patients. In addition to the structural MRI techniques, positron-emission tomography (PET) studies measuring Aβ deposition with C-PIB or other PET ligands and functional techniques such as glucose utilization and functional MRI (fMRI) are used today to follow the progress of the disease and to potentially allow early diagnosis. 47

  The neuropathological hallmarks are neuronal loss in regions related to memory, neurotransmitter depletion, and synaptic alteration which are found in the AD patient's brain. During the last two decades the role of protein aggregation in neurodegenerative diseases has been extensively investigated, suggesting that cumulative protein misfolding and aggregation is a leading cause of neuronal dysfunction and death in these types of diseases.49 Microscopically, the most common findings are abnormal protein deposits, including senile neuritic plaques (SNPs) and neurofibrillary tangles (NFTs). The extracellular accumulation of insoluble aggregates of β-amyloid protein (Aβ) leads to the formation of senile plaques, whereas NFTs occur intracellulary and are composed of paired helical filaments of hyperphosphorylated tau protein.Tau is one of the microtubules associated proteins that are thought to have a role in the stabilization of neuronal microtubules. Beyond Aβ, Tau also appears to be the earliest markers of AD and forms the neurofibrillary changes (neuropil threads, neurofibrillary tangles, neuritic plaques). 50 It consists of three major domains: an amino terminal projection domain, a carboxy terminal domain of microtubule-binding (MTB) repeats and a short tail sequence (Figure1.28).

Figure 1 .

 1 Figure 1.28. Representation of the functional domains of the largest tau isoform (441 amino acids) Tau is predominantly found in axons as a result of an incompletely understood mechanism. Under physiological conditions, it has also been localized to dendrites in a much lower concentrations. Tau has 84 putative phosphorylation sites, of which 45 are serines, 35 threonines, and 4 tyrosines. Tau is a hydrophilic protein that has been characterized in solution by circular dichroism representing a random coiled protein structure.

Figure 1 .

 1 Figure 1.29. Normal function of tau protein that stabilizes microtubules 51Hyperphosphorylated tau accumulates in the somatodendritic component of neurons, aggregates and eventually forms NFTs (Figure1.30). Evidence has been provided that soluble hyperphosphorylated tau cause neuronal dysfunctions, such as mitochondrial respiration and axonal transport before its aggregation.52 Alterations in the amount or the structure of tau protein can affect stabilization of microtubules and other processes related to this protein. The phosphorylation of tau regulates its activity to bind to microtubules and stimulate their assembly. For the optimal function of tau a normal level of phosphorylation is required, whereas the hyperphosphorylated state makes tau to lose its biological activity.At cellular level, abnormal phosphorylation of tau introduces diversity changes in several processes which are directly regulated by the suitable organization of the microtubule network.

Figure 1 .

 1 Figure 1.30. Hypothesis of aggregation process of tau protein 53

Figure 1 .

 1 Figure 1.31. Difference of healthy and Alzheimer's microtubule 54

Figure 1 .

 1 Figure 1.32. 3D structure of amyloid β fibrils 56 Research on amyloid fibril structures began in laboratory of Robert Tycko in 1998. At that time, relatively little was known about the molecular structures of amyloid fibrils, firstly because amyloid fibrils are inherently noncrystalline and insoluble, thus direct structure determination by X-ray crystallography and liquid state nuclear magnetic resonance (NMR) is impossible. 57 In over 14 years, the knowledge on amyloid fibrils has advanced due to the huge amount of research effort being devoted to the structure of fibrils and each -strands in details using different peptidic models. Electron microscopy and atomic force microscopy (AFM) are the two most widely used microscopy techniques used in the study of amyloids. Both provide a nanometer-resolution perspective of the ultrastructural characteristics of amyloids. This includes amyloid fiber length and

Figure 1 . 33 .

 133 Figure 1.33. Amyloidogenic and non-Amyloidogenic pathway for App cleavage 61

  contains six negatively charged residues (D1, E3, D7, E11, E22, D23) and three positively charged residues (R5, K16, K28), yielding a net charge of -3. The three His residues (H6, H13, H14) have PK a s close to physiological pH. There is a special significance to the hydrophobic region flanked by positively and negatively charged residues, namely KLVFFAE (Aβ16-23), and to the position of K28 in an otherwise hydrophobic expanse.

Figure 1 .

 1 Figure 1.34. Amyloid β peptide (1-42) 63

Figure 1

 1 inhibitors, b) Memantine (antagonist of the NMDA receptor).

Figure 1 . 36 .

 136 Figure 1.36. Therapeutic strategies targeting A

Figure 1 .

 1 Figure 1.39. γ-secretase inhibitors in clinical trial A considerable number of γ-secretase inhibitors (GSIs) were prepared with the similar structure and functional properties to DAPT showing poorly understood mechanism of action. There were also several GSIs undergoing clinical trials, of which LY-450139 (Figure 1.40) 85 was in phase III clinical trials. Compound 1.23 was shown to be associated with an increased risk in skin cancer.

Figure 1 . 42 .

 142 Figure 1.42. Grape seed polyphenolic extract (Mega-Natural-Az)

Figure 1 . 43 .

 143 Figure 1.43. Curcumin as a well inhibitor of A oligomerization

Figure 1 . 44 .

 144 Figure 1.44. Polyphenol based compound find in the extra virgin olive oilFlavonoids are the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants being present in several fruits and vegetables, beverages like tea and wine, and seeds like cocoa beans and grape seeds. Flavonoids may be divided into several subclasses which among them the flavanol monomers, known as catechins, are found in high concentration in the tea plant leaves. The major tea catechins include (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-EC gallate (ECG) and (-)-EGC gallate (EGCG).96 In addition to the antioxidant properties of EGCG, this compound shows promising effects on tumour growth in cell and mouse models. Thus, a possible application in cancer therapy has been extensively investigated.97 EGCG (1.31) reduces the toxicity of Aβ in pheochromocytoma and neuroblastoma cell models98 and decreases the formation of amyloid deposits.99,100 An ongoing clinical trial is investigating if EGCG positively affects the course of AD as assessed by Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) in early state AD patients co-medicated with acetylcholine esterase inhibitors.101

Figure 1 .

 1 Figure 1.45. EGCG structure, polyphenol inhibitor Non-polyphenol inhibitors include small molecules like alkaloids, glycosides and phenazines. Most non-polyphenol inhibitors contain one or several aromatic rings, which are key factors to the inhibitory effect. For example Scherzer-Attali and co-workers synthesized two molecules that contain a tryptophan and a naphthoquinone, termed NQTrp and Cl-NQTrp 102 (Figure 1.46), both of which are capable of completely inhibiting the fibrillization of amyloidogenic proteins, including Aβ, α-syn (Parkinson's disease), IAPP (type II diabetes), lysozyme, calcitonin and insulin. 103

Figure 1 . 46 .

 146 Figure 1.46. Molecules built with Tryptophan & Naphthoquinone

  102 R. Scherzer-Attali, D. Farfara, I. Cooper, A.Levin, T. Ben-Romano,D. Trudler, M. Vientroy, R. Shaltiel-Karyo, D. E. Shalev, N. Segey-Amzaleq, E. Gazit, D. Segal, D. Frenkel, Neuro. Biol. Dis., 2012, 46, 663-672. 103 R. Scherzer-Attali, R. Shaltiel-Karyo, Y.H. Adalist, D. Segal, E. Gazit, Proteins, 2012, 80, 1962-1973. few peptides accumulate in tissues (reduction of risks of complications caused by their metabolites). 104 Cyclic peptides (CPs) have been developed as a new class of amyloid inhibitor over the past five years. They have been shown to be powerful and specific inhibitors of amyloid formation. CPs are metabolized more slowly than their non-cyclic equivalents because of their resistance to enzymatic degradation. However, CPs can self-assemble as a result of hydrophobic interactions, which decreases their inhibition efficiency. D,L--Cyclic peptides can self-assemble into nanotubes. These cyclic peptides stack on top of each other with an approximately 4.8 Å spacing to form a hollow -sheet tubular structure. These tubular structures share conformational and functional features with amyloid fibrils. The structural similarity of amyloid fibrils and D,L- CP tubes may indicate the cross-reaction of their monomers. By screening a focused library of six residue D,L--CPs, Richman et al. found that such CPs interact with monomeric A and inhibit the aggregation of A. D,L--CP hexamers were synthesized with varying side chains at positions R1-5 (Figure 1.47).

Figure 1 .

 1 Figure 1.47. A cyclic D,L--CP hexapeptide (upper panel) and the substituted groups (lower panel) in R 1-5

  smallest and most potent compound able to alter the fibril morphology and reduce A cellular toxicity.A different approach that has been used to inhibit A aggregation is the incorporation of Nmethyl amino acids into peptides as disrupting elements. For the first time methylated β-sheet breaker was described by Hughes et al. after it has been suggested that amino acid sequence 'GSNKGAIIGLM' (Aβ 25-35) resembles a biologically active region of Aβ that forms large βsheet fibrils and is highly cytotoxic. This group synthesized six N-methylated derivatives of this sequence that as there were homologues to Aβ, were expected to bind the wild type form and prevent further aggregation of monomers. As expected these N-methylated peptide variants had significant influence on Aβ aggregation. Notably, N-methyl-Gly-33 was shown to be powerful in inhibiting of aggregation and cytotoxicity.110 Recently, it has been demonstrated by Doig and coworkers that N-methylated peptides (meptides) of the A16-20 have an inhibitory activity in A aggregation. As they act as a β-sheet breaker, one side of these compounds present a hydrogenbonding "complementary" face to the protein, with the other having N-methyl groups in place of backbone NH groups, thus presenting a "blocking" face which is able to destabilize the β-sheet formation.111 Meredith and co-workers also investigated N-methylated peptides of a region corresponding to 16-22 and later 16-20 of the amyloid "core domain" region.112 Using a twodimensional NMR and circular dichroism spectroscopy they found a pentapeptide with two Nmethyl amino acids, Ac-K(Me)LV(Me)FF-NH 2 , which has the intended structure of an extended strand and is extremely stable. This compound effectively inhibits A polymerization and also disassembles preformed A fibrils. Bearing methyl, the resistance of this compound toward protease was increased in comparison to the un-methylated Aβ 16-22 peptides. In 2006, Kokkoni et al. optimized meptides based on the KLVFF sequence in a large approach based on five peptide libraries. Peptide length, methylation sites, end-blocking, side chain identity and chirality were varied in their investigation. The most interesting compound, judged by Aβ fibrillization and cell cytotoxicity inhibition activity, was D-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH 2 and rules could be stated to predict peptide performance. Ideal inhibitors should be D-peptides, possess a free N-but an amidated C-terminus and residues one to four should be large, branched hydrophobic side chains. Only one methylated amino acid was essential for the design of this type of inhibitors. 113 In 1999, Findeis et al. started the development of new β-sheet breaking peptides derived from 15-residues Aβ peptides. The inhibitory activity of these peptides was enhanced by modification of their amino terminus, where different organic reagents were attached. In library designed in this group the size of inhibitors as well as the amino acid sequence were optimized to result compound cholyl-LVFFA-OH as a good initiative compound. The acyl-D-amino acid analogue and the amide analogue of this compound not only inhibited the Aβ polymerization potently but were stable also in monkey CSF for at least 24h. 114,115 In 2009, Fryman-Marom et al. introduced the D-enantiomeric β-sheet breaker NH 2 -D-Trp-Aib-OH, which combined an indole and α-aminobutyric acid (Aib). Aib has been shown to induce helical conformations and to disrupt β-sheet structures. 116 This compound specifically interacted with untoxic low molecular weight Aβ oligomers and inhibited their growth to larger, cell toxic Aβ forms in vitro. The compound was stable, safe, orally bioavailable, and crossed the blood brain barrier (BBB). It reduced also the amount of plaques in the brains of AD transgenic mice and improved their cognitive performance. 117 Under the same class of inhibitors containing peptides it can be specified the important use of peptide hairpins. In 2009, Teplow et al. were demonstrated a 13-residue peptide hairpin, PP-Leu, as an inhibitor (Figure 1.48) that blocks the formation of the extended β-sheets necessary for Aβ fibril growth, disrupts the structure of preformed fibrils, and potently inhibits Aβ oligomerization.Importantly, PP-Leu shows substantial protease resistance.118

Figure 1 .

 1 Figure 1.48. (a) Primary structure and (b) ball-and-stick model of PPLeu In 2012, Andersen et al. described hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites that can modulate the aggregation of two unrelated amyloidogenic systems: human pancreatic amylin (hAM) and α-synuclein (α-syn), associated with type II diabetes and Parkinson's disease, respectively.

  Solomon and colleagues began this line of work in 1996 that anti-Aβ monoclonal Abs can dissolve Aβ aggregates in vitro and prevent Aβ monomers from aggregating. The AN1792 Aβ vaccine was designed for this purpose; however, it failed in Phase II clinical trials due to the development of meningoencephalitis.120 Using active immunization of Aβ42 in a transgenic mouse model overexpressing human amyloid precursor protein,Schenk et al. demonstrated in 1999 that the generated anti-Aβ antibodies were able to prevent Aβ plaque formation in young animals as well as to reduce Aβrelated neuropathological changes in old animals. The presented data suggested that the antibodies were able to cross the BBB and act within the central nervous system.121 The only antibodies against the N-terminal region of Aβ recognized and promoted clearance of Aβ plaques. This finding indeed correlates very well with previous in vitro observations and the fact that the Nterminus is not involved in the core of Aβ amyloid fibrils and therefore should be accessible to antibody binding. With emerging insights about the critical roles of prefibrillar aggregates in Alzheimer's disease, the attention has been directed towards antibodies that recognize conformational epitopes on oligomeric species. Significant advances in this area were made by Glabe and co-workers. They were first to demonstrate that it is possible to achieve selective binding to soluble Aβ oligomers. interestingly, the polyclonal A11 antibodythat they obtained also recognize soluble oligomers of other amyloid forming proteins and it reduces their cytotoxicity, suggesting that a common conformational epitope might be involved in cytotoxic mechanisms.122 Further work resulted in the isolation of antibodies with selective binding to fibrils and fibrillar oligomers and to annular protofibrils, and they recognize also similar species formed by proteins with different primary structure.However, not all amyloid/oligomer binding antibodies display broad recognition patterns. A monoclonal antibody raised against protofibrils of Aβ with the Arctic mutation was shown to be selective for protofibrillar Aβ and not bind other Aβ species or amyloid of other proteins, demonstrating the existence of unique epitopes of Aβ aggregates that are distinct from more general amyloid epitopes.123 This antibody was also shown to inhibit Aβ aggregation and to effectively clear soluble Aβ aggregates in vivo.124 An interesting finding regarding the oligomer selectivity of anti-Aβ antibodies was recently reported by Lindhagen-Persson et al. They showed that a monoclonal IgM antibody (with 10 antigen binding regions) raised against monomeric Aβ40 preferentially binds oligomeric Aβ. The antibody was also shown to inhibit fibril growth and reduce Aβ-induced cytotoxicity. Furthermore, the authors demonstrated that oligomer selectivity seems to be a common property among IgM autoantibodies against Aβ isolated from humans.125

Figure 2 . 1 .

 21 Figure 2.1. General structure of 2,5-diketopiperazin. 2,5-Diketopiperazines have two s-cis secondary amide groups by which an array of hydrogen bonds can be formed horizontally within the ring plane. Due to the rigid cyclic structure bearing amide groups, these compounds are poorly soluble in organic solvents. In the solid state, most of the N-unsubstituted DKPs adopt a linear tape orientation (Figure 2.2a), 131 while the presence of one N-substituent makes up one dimensional hydrogen bonded tapes through an organized non-reciprocal hydrogen-bonded network (Figure 2.2b) or formation of hydrogen-bonded dimers (Figure 2.2c). 132

Figure 2 . 3 .

 23 Figure 2.3. Diketopiperazines as PAI-1 inhibitors Metabolite 2.1 isolated from Streptomyces sp., as the first low-molecular-weight PAI-1inhibitor, was the model for designing structural changes in order to obtain more active products (Figure2.3). Using bioisosteric substitutions and combinatorial chemistry approaches, DKP 2.2 and 2.3 containing different side chains connected by exocyclic double bonds were synthesized and showed both in vivo and in vitro PAI-1 inhibitory activity (IC 50 3.5 mM and 0.2 mM, respectively).

Figure 2 . 7 .

 27 Figure 2.7. Diketopiperazines with antimicrobial activityGypsetin 2.18 was found to be a competitive ACAT (acyl coenzyme A: Cholestrol acyltransferase) inhibitor with respect to oleoyl-CoA.150 ACAT enzyme has been identified as the rate-limiting enzyme in the absorption of cholesterol. Thus the inhibition of ACAT (Figure2.8) has received much attention due to its potential in moderating the effect of elevated cholesterol level.

Figure 2 . 8 .

 28 Figure 2.8. ACAT inhibitor gypsetin Diketopiperazines also have activities as antiviral, 151 antifungal, 152 antibacterial, 153 and antihyperglycaemic agents 154 and affinities for calcium channels and opioid, 155 GABAergic, 156 Serotoninergic 5-HT 1A 157 and oxytocin receptors. 158

Figure 2 . 9 .

 29 Figure 2.9. Different biological activities of 2,5-diketopiperazine

Figure 2 .

 2 Figure 2.10. Possible disconnections

Figure 2 . 11 .

 211 Figure 2.11. General structure of di-(2S,4R)-4-aminoproline diketopiperazine two-armed receptorsFive different receptors were prepared by this route. To have a proper distance between the two side arms was used 4-aminoproline as a suitable scaffold to avoid intramolecular recognition between the two tripeptide arms and collapse of the receptor. The resulting two-armed receptors were screened against an encoded 178 resin-bound tripeptide library with the general structure Ac-AA3-AA2-AA1-NH(CH 2 ) 6 -CONH-PS. The screening revealed highly selective binding properties in organic solvents; in particular, each of the five structurally similar receptor prototypes selects for different tripeptides within the library. Particularly the di-(2S,4R)-4-aminoproline diketopiperazine adopts a highly preorganized turn like conformation which imparts a defined orientation to the tripeptide side arms. These two-armed receptors were also evaluated as selective and sensitive coatings for gravimetric gas sensors. They can be used to detect and differentiate simple alcohols such as methanol, ethanol, n-propanol, and propan-2-ol through subtle changes in their structure.179 Water soluble diketopiperazines were also synthesized in the laboratories of this group, showing well affinity to bind to arginine-rich peptides in aqueous solution. The authors equipped the arms of the DKP receptors with aspartic acids (Figure2.12) to provide water solubility and ability to bind arginine properties (receptor 2.69a,b). The termini of the arms were either acetylated (receptor 2.69a) or functionalized with succinic acid which provided for an additional carboxylic acid functional group (receptor 2.69b).180

Figure 2 . 13 .

 213 Figure 2.13. Diketopiperazine-based anion receptor

Figure 2 .

 2 Figure 2.14. -turn There is a significant discussion in literature on the construction of -turn mimic and the classification of different types of mimics including: a) internal -turn mimics; b) -hairpin mimics (in which a rigid scaffold induces reversal of the peptide chain); and c) external -turn inducers (Figure 2.15).

Figure 2 . 15 .

 215 Figure 2.15. Classification of -turn mimics Several examples of the first class of β-turn mimics including scaffolds bearing side chains with the ability to mimick a peptide reverse turn were reported in literature. Internal -turn mimics are the first class of -turn mimics containing scaffolds displaying side chains with trajectories mimicking a peptide reverse turn. Golebiowski and co-workers have developed a high-throughput organic synthesis protocol for the efficient preparation of bicyclic DKP-containing systems (2.71, Figure 2.16) starting from racemic piperazine-2-carboxylic acid. 182

Figure 2 . 17 .

 217 Figure 2.17. DKP-based internal -turn mimics examplesRecently Burgess and co-workers described the use of diketopiperazine scaffolds in antagonists of tropomysin receptor kinase C.186 The use of amino acid side chains corresponding to those present in the -turn regions of the parent neurotrophin ligands assisted the selectivity of the compounds toward this particular receptor. The diketopiperazines used in this work are functionalized at N1 and C3, and the substituents at these positions were calculated to overlap well with the side chains of the i+1 and i+2 residues of a type I -turn.

Figure 2 . 18 .

 218 Figure 2.18. DKP mimics of a type I -turn

Figure 2 . 19 .

 219 Figure 2.19. example of DKP-based -turn mimic An alternative way is possible to build two different functionalities in the lateral chains of the two amino acids forming the DKP core either by using an amine deriving from Lys, Orn or diaminobutyric acid or utilizing a carboxylic acid deriving from Asp or Glu. Davies and coworkers reported the synthesis of diketopiperazine scaffold 2.75 (Figure 2.20), capable of mimicking a -turn, although no synthesis or conformational studies of peptides containing scaffold 2.75 have been reported. 189

Figure 2 . 20 .

 220 Figure 2.20. Diketopiperazines as -turn mimetics

FigureFigure 2 . 21 .

 221 Figure2.21. 191,192

Figure

  Figure 2.23.

Figure 2 . 1 . 6 .

 216 Figure 2.24. Bis-peptide with a left-handed helical arrangement

Figure 2 . 25 .

 225 Figure 2.25. Structure of bifunctional DKP1-8 scaffoldsImportantly, the bifunctional scaffold 2.85 (DKP1), formed from L-aspartic acid and (S)-2,3diaminopropionic acid, bears the amino and carboxylic acid functionalities that are in a cis configuration and so can be seen as a -turn mimic and promoter of antiparallel -sheet. According to these great properties, was performed the synthesis of several peptidomimetics by solution phase peptide synthesis (Boc strategy). Conformational analysis of these derivatives was carried out by a combination of 1 H-NMR spectroscopy (chemical shift and NOE studies), IR spectroscopy, CD spectroscopy and molecular modelling, revealing the formation of -hairpin mimics (2.93 and 2.94) comprise 10-and 18-membered H-bonded rings and a reverse turn of the growing peptide chain(Figure 2.26). 197

Figure 2 . 27 .

 227 Figure 2.27. Structure of trimer 2.95 and tetramer 2.96This is best observed comparing the CD spectra of the of the -hairpin mimick 2.94 and the tetramer 2.96 in which the special feature of the -turn structure present in both compounds, i.e. the strong minimum around 200 nm and a weaker one at 225 nm with a negative maximum at 215 nm, is repeated and in the tetrameric structure with a 10 fold magnified intensity (Figure2.28).

Figure 2 .

 2 Figure 2.28. CD spectrum of the tetramer 2.96 and the hairpin peptidomimetic 2.94 2.1.7. [DKP]-integrin ligands

Figure 2 .

 2 Figure 2.29. library of cyclo-[RGD-DKP] integrin ligandsThe cis-derivative cyclo-[DKP-1-RGD] 2.97 inhibited biotinylated vitronectin binding to the purified α V β 3 receptor at a micromolar concentration (3.9 ± 0.4 μM), while trans-derivatives 2.98-2.103 ranged from submicro-to subnanomolar concentrations (220 -0.2 nM). To investigate the

Figure 2 .

 2 Figure 2.30. Cyclo-[DKP-RGD]-Paclitaxel conjugates

Figure 2 . 31 .

 231 Figure 2.31. Cyclic isoDGR peptidomimetics containing the DKP scaffoldThe isoAsp-Gly-Arg (isoDGR) sequence is a tripeptidic sequence that origins from a spontaneous post-translational modification of the Asn-Gly-Arg (NGR) motif of the extracellular matrix protein fibronectin, 201 affording a gain of protein function and the creation of a new adhesion binding site for integrins.200 With regards to the biochemical, spectroscopic and computational investigations, isoDGR sequence can fit into the RGD-binding pocket of α v β 3 integrin, establishing the same electrostatic clamp as well as additional polar interactions.201 Two cyclic isoDGR peptidomimetics (Figure2.31) containing the bifunctional diketopiperazine scaffolds mentioned above were synthesized, their conformational properties investigated and their affinity to α v β 3 and α v β 5 integrin receptors measured.201 The cyclo-[DKP-isoDGR] ligands 2.108 and 2.109 compare favourably with the cyclic peptidomimetic cyclo-[(3S,6R)DKP-RGD],202,203 as well as with other isoDGR cyclopeptides.204

(Figure 2 . 33 .

 233 Figure 2.33. Relative sizes of a) extended -strand b) antiparallel -sheet c) -helix d) -turn 218

Figure 2 .

 2 Figure 2.35. Macrocyclic BACE-1 inhibitor examples

224

 224

Figure 2 . 36 .

 236 Figure 2.36. Macrocyclic peptidomimetic example inhibitor of HIV-1 protease

Figure

  Figure 2.37.

Figure 2. 42 .

 42 Figure 2.42.An interesting variation to the design of an aromatic scaffold was observed in the crystal structure of isocoumarin 2.133 bound to porcine pancreatic elastase. The coumarin ring opens in situ to provide a substituted phenyl ring covalently bound to Ser195 and acting as a scaffold to position the side chains into the S1 and S2′ enzyme pockets (Scheme 2.12).232

Figure 2 . 43 .

 243 Figure 2.43. Schematic representation of hydrogen bonding in 2.134As was mentioned in chapter 1, Nowick and co-workers have reported a huge amount of work in this field. The mimetics that were synthesized, are able to duplicate the hydrogen-bonding functionality of a dipeptide and a tripeptide respectively. -sheet-like structures have been also developed in this group by the use of one of these two building blocks mentioned above as a strand which have been localized along one edge allowing the strand to interact with the -strand of other molecules.234

Figure 2 .

 2 Figure 2.44. Nowick -strand peptidomimetic examples The evidence of sheet formation confirms the extended nature of the scaffold and suggests new approaches to -strand mimetics with high potency in medicinal chemistry. For instance, in 2001 -strand mimetic 2.138 was prepared by Kiso et al. and shown to inhibit dimerization of HIV-1 protease. 236

Figure

  Figure 2.45.

Figure

  Figure 2.46.

  our group in Paris where I have spent a part of my thesis, has developed potential ligands of β-amyloid peptide which could modulate the aggregation of A peptides involved in AD. A novel class of glycopeptide derivatives, based on two hydrophobic dipeptides (Ala-Val and Val-Leu) linked to a hydrophilic D-glucopyranosyl scaffold through aminoalkyl and carboxyethyl linkers in C1 and C6 positions, respectively were designed to bind the hydrophobic sequence of βamyloid peptide KLVFF (Aβ 16-20 ) (Figure2.47). These compounds are supposed to establish hydrophobic interactions with the Aβ peptide, while the glycoside scaffold is supposed to perturb the regular interstrand network of hydrogen bonds, therefore disrupting the interaction with other Aβ peptides acting as -sheet breaker element, and consequently hindering the oligomerization. Among these molecules, compound 2.141 results good inhibitory activity by increasing the lag phase of Aβ 1-40 from 12 to 26 at compound:Aβ 1-40 ratios of 1:1 and 0.1:1 respectively.237 Pharmacomodulations allowed the discovery of sugar-based molecules able either to stabilize the monomeric peptide or to accelerate the aggregation of Aβ 1-42 , preventing the presence of small toxic oligomers.240 This glycopeptides series did not show any favored conformation in solution without the presence of Aβ peptides.

Figure 2 . 47 .

 247 Figure 2.47. Sugar based peptidomimetic design

Figure 2 . 48 .

 248 Figure 2.48. Schematic view of the interaction of inhibitors designed with the crucial sequence in Aβ peptide Our design towards a stable β-hairpin mimic, which could interact and eventually act as a βsheet binder and aggregation inhibitor, involved assembling the bifunctional DKP-1 mentioned previously, a peptidomimetic strand to stabilize the formation of β-sheets and finally a suitable peptide sequence for binding to the aggregating protein (Figure 2.48). Although DKP-1 is able to stabilize hairpin structures when introduced between two peptide sequences as a previous work in our group in Como, the choice to substitute one of the peptide sequences by a peptidomimetic

2-

  The peptidic sequence : GVVI (C-terminal sequence residues 38-41, complementary sequence in fibrils) versus KLVF (residues 16-19, central hydrophobic sequence) 3-The groupment CH 3 versus CF 3 at the C-terminal part of the molecules 4-The possibility of forming electrostatic interactions : cleavage of the N-terminal part of the molecules; difference between Lys residue versus a Val residue in the peptidomimetic arm.

Figure

  Figure 2.49. The general structure of -hairpins

  was developed by Balz and Schiemann in 1927. Isolated aryldiazonium tetrafluoroborate salts, dried and heated at 100 o C or above, provided good yields of the fluoroaromatic compounds. The major milestone has been the method of choice for the past 82 years, being of special importance for many new pharmaceuticals. The idea of incorporating the fluorine into compounds of medicinal interest was the early examples of molecular modification in drug design.The importance of fluorine in bioorganic and medicinal chemistry has been demonstrated by a large number of fluorinated compounds approved by the FDA for medical use. 138 fluorinecontaining drugs have received FDA approval for human diseases (23of which, however, have been discontinued from the market), while 33 are currently in use for veterinary applications. These statistics make fluorine the "second-favorite heteroatom" after nitrogen in drug design.The pharmacological superiority of fluorinated compounds over their non-fluorinated analogues may be rationalized as follows: the covalently bound fluorine has a significantly larger steric requirement (C-F bond length 138 pm) than hydrogen (C-H bond length 109 pm), fluorinated compounds usually have a sufficient similarity in size and shape to their non-fluorinated analogues to fit a given enzyme receptor, so that they tend to have similar inherent biological activity. On the other hand, carbon-fluorine bond is very strong (485 KJ mol -1 compared with C-H, 416 KJ mol -1 ), so the fluorinated compounds tend to be more resistant to metabolic degradation. The introduction of fluorine also generally confers increased lipophilicity. On the basis of these considerations, the synthesis of -strands containing fluorine and a free adjacent OH was designed. The fluorine atom close to the free hydroxyl makes this proton more acidic and resulted in the increased polarity of hydroxyl. These compounds were designed with the hydroxyl also due to the importance of OH as -sheet breaker element, as discussed in a previous section. Two molecules 2.157a, 2.157b were designed as illustrated in Figure2.50.

Figure 2 . 50 .

 250 Figure 2.50. The designed fluorinated peptidomimeticsThese compounds were designed with the purpose to interact with the central and critical region of -amyloid peptide (A17-21 ), playing an important role in the aggregation process. These molecules are able to interact with the native peptide by forming hydrogen bonds and blocking the formation of -sheet structures resulting in the inhibition of further aggregation. The introduction of the two amino acids Asp and Lys in the design of these compounds was envisaged due to the possible ionic interaction of Asp with the Lys in the peptide sequence (KLVF). The carbohydrazide fragment was also used, as mentioned previously because of its ability to form hydrogen bond and its high resistance toward the cleavage by peptidases.The synthesis of target 2.157a started from the commercially available 3-fluoro-4-hydroxy benzoic acid. The nitration reaction under standard conditions using H 2 SO 4 and ammonium nitrate afforded compound 2.158 which underwent coupling with tert-butyl carbazate forming 2.159 in high yield. For the next step different methods for the reduction of nitro group to amine were applied, such as THF, HCl 2N in the presence of indium powder, or Raney-Ni, ammonium formate in MeOH. Unfortunately, no reaction was observed with any of these methods. Eventually, using H 2 , Pd/C in a catalytic amount in MeOH, i.e. the usual way that was used in the preparation of previous -strand, the desired product 2.160 was obtained in a quantitative yield. A subsequent reaction with benzoic anhydride led to the formation of the fully protected compound 2.161. This protection was required, due to the high reactivity of hydroxyl adjacent to the fluorine. Cleavage of the Boc group and further coupling to N ɛ -Cbz-N α -Boc-L-Lys-OH gave our new peptidomimetic

Figure 2 . 51 .

 251 Figure 2.51. Three diketopiperazine scaffolds

252

  See ref 197.

the 1 H

 1 Figure 2.52. HMBC spectra of isopeptide for 2.172a in CDCl 3 and 2.172b in CD 2 Cl 2 , the long range coupling between the O-CH 2 protons of Ser and the α-carbonyl carbon of Asp

  Scheme 2.42. Final -Hairpin; v) TFA, CH 2 Cl 2 , overnight, quantitative yield.

Scheme 2. 53

 53 Scheme 2.53 Tetrapeptide preparation by solid phase peptide synthesis

  .1) reveals strong sequential and medium intra-residual H-HN ROEs, which is characteristic of extended backbone conformations. The observation of numerous long-range ROEs between the two -strands, strengthened the evidence of a -hairpin conformation. Indeed, 11 interstrand ROEs could be detected, involving both backbone and sidechain protons (Figure 3.1). The set of ROEs indicates that Ile 4 and Val 7 residues face together and that Val 2 makes contacts with the aromatic ring. Between the hydrazide proton NH c and Val 3 amide proton was observed a weak ROE supporting the formation of an antiparallel -sheet and having the anomalously low intensity due to the short transverse relaxation time of NH c proton. A close inspection of ROE-derived distances revealed the existence of preferred conformers of hydrazide/amide bonds on either side of the aromatic unit. The distances between NH a and H 4 , H 6 protons are consistent with the preferred orientation of the acetamido group depicted in Figure 3.1.

Figure 3 . 1 .

 31 Figure 3.1. Chemical structure of 2.193 showing the assigned ROEs

  and alcoholic solvents 264 while those that are not intramolecularly hydrogen-bonded usually exhibit largely negative values of their temperature coefficients. The amide protons of Ile 4 , Asp 6 and Val 7 exhibit the strongest variations, as expected for solvent-exposed groups. In contrast, the NH b and NH c protons near the aromatic unit, together with the amide proton of Dapa 5 , have the smallest variations. However these temperature coefficients tend to be slightly higher than expected and that of Val 3 amide proton falls within intermediate values. Altogether this analysis suggests a partial engagement in intramolecular hydrogen bonds and is indicative of an equilibrium between the major -hairpin conformation and minor open conformations.Finally, the stabilization of the -sheet conformation was investigated that result from intermolecular association at high concentration. The comparison of 1D 1 H spectra recorded at 1.4 mM and 80 M concentrations does not show significant changes in chemical shifts and linewidths, ruling out the possibility of aggregation in the mM range.The conformation of compound 2.217 was also examined in methanol solution as this compound exhibited good solubility at sub millimolar concentration. Notably, significant chemical shift variations were observed on 1D 1 H spectra upon concentration variation (10 M -0.4 mM) 263 T. Cierpicki, J. Otlewski, J.Biomol. NMR., 2001, 21, 249. 264 K. Guitot, M. Larregola, T. K. Pradhan, J. L. Vasse, S. Lavielle, P. Bertus, J. Szymoniak, O. Lequin, P.Karoyan, ChemBioChem., 2011, 12, 1039-1042.

2. 193

 193 to adopt a hairpin structure in water. The choice of water as a solvent was due to the prominent place of this solvent amongst solvents for obvious reasons; two-thirds of our planet is covered in water, chemical reactions tend to be done in aqueous solution, and so on. The relatively strong hydrogen bonding in liquid water causes many of its physical properties to be anomalous. A Monte-Carlo conformational search using OPLS_2005 molecular mechanics force field with an implicit GBSA solvation model265 yielded an initial hairpin-like geometry of this molecule. Stability of this structure was further studied by molecular dynamics and DFT calculations.Although the open end of the molecule seemed to fluctuate (in agreement with the conclusions from the NMR studies), the overall hairpin structure of the compound (evaluated by the lifetime of key hydrogen bonds, Figure3.2) exhibited rather good stability.

Figure 3 . 2 .

 32 Figure 3.2. Evolution of the number of key hydrogen bonds for the hairpin structure over the molecular dynamics trajectory (1 µs) Delightfully, completely extended conformations were observed several times over the course of the dynamics, with coexistence of the 4 key hydrogen bonds and formation of the expected 10, 14, 10 and 14-membered pseudo-rings (Figure 3.3). The hydrogen bond network and the dihedral angles for the β 2 and β 3 -aminoacids (θ = -62 and 61° respectively), indicate that the hairpin geometry observed in 2.193 is in agreement with previous examples of hairpins 266 and foldamers 267 of DKP1.

Figure 3 . 3 .

 33 Figure 3.3. Hairpin structure of 2.193 observed during OPLS_2005/GBSA molecular dynamics in water and refined at the B3LYP/6-31G* level. Average angle and distance values over the molecular dynamics trajectory were found in good agreement with coupling constants and ROE intensities measured described previously. A discrepancy existed between the average distance from H 6 proton belonging to the 5-acetamido-2-methoxy benzohydrazide moiety to H α in Val 2 residue and the strength of the corresponding signal on the ROESY spectrum. However, this result can be accounted for by the locally dynamic character of the molecule, since 10% of the time, this distance actually drops below 2.8 Å. This study indicated that extrapolation of major structural

  271 and further adapted by LeVine as a probe for both quantifying relative amounts of aggregated A in solution and monitoring fibril formation.272 This protocol has been largely unchanged and is perhaps the most widely employed for monitoring Aaggregation. These initial studies revealed the requirement of cross--sheet secondary structure for ThT binding; indeed, denaturation of sheet abolishes ThT binding. Based on its high-yielding fluorescence change in the presence of aggregated A, ThT has played an important role in elucidating the kinetics of A self-assembly.Further, the relationship between A 1-40 and A 1-42 kinetics in vitro was, in part, elucidated using ThT fluorescence.273 Finally, ThT is broadly used in screening for modulators of A aggregation.

Figure 3 . 4 .Figure 3 . 5 .

 3435 Figure 3.4. (a) Thioflavin T structure, and representation of the twisting C-C bond. (b) "Channel" model of ThT binding to fibril-like β-sheets. ThT is proposed to bind along surface side-chain grooves running parallel to the long axis of the βsheet. 274Investigation of the amyloid fibril formation process requires the ability to distinguish the characteristic amyloid β-sheet structure from amorphous aggregates of the monomer or non-

Figure 3 . 6 .Figure 3 . 7 .

 3637 Figure 3.6. Thiolavin-T fluorescence assay of peptidic armsIn order to confirm the importance of the complete molecules in modulating the aggregation kinetics of Aβ 1-42 the two peptidic arms that were used in our molecules were also studied by Thioflavin-T assay. Results obtained indicate that both peptidic arms do not significantly affect the aggregation process.

Figure 3 . 8 .Figure 3 . 9 .

 3839 Figure 3.8. ThT fluorescence assay of deprotected Val-hairpinOnce the Boc moiety was cleaved, the fluorescence result became different from the protected compound, as we expected, revealing a more significant inhibitory activity of the designed hairpin.At the ratio of 1:10 (Aβ 1-42 :compound) the Aβ fibrillation is delayed by increasing the t 1/2 by a factor of 2 with no effect on the fluorescence plateau. No effect was observed at a lower ratio.

Figure 3 . 10 .Figure 3 . 11 .Figure 3 . 12 .

 310311312 Figure 3.10. ThT assay of fluorinated hairpins In the next series of cis-DKP hairpin the sequence KLVF was introduced. Furthermore a CF 3 replaced the CH 3 in the acetamide group because the CF 3 moiety seemed to increase the affinity of the molecule toward Aβ peptide in the trans-DKP series (see below). As it can be noticed from the ThT fluorescence curves, the single arm fluorinated compound 2.200 does not display any activity

Figure 3 . 13 .

 313 Figure 3.13. ThT assay of the tripeptide precursors To verify the importance of whole four amino acids sequence on the Aβ 1-42 fibrillation we continued the ThT fluorescence study on the two tripeptide precursors 2.212 and 2.213 (non

Figure 3 . 15 .

 315 Figure 3.15. ThT fluorescence of complete moleculesThe behavior of the protected final products with/without CF 3 was investigated by ThT fluorescence assay. Results revealed that as the other protected compounds these molecules tend to self-aggregate. It can be seen a slight acceleration on the kinetics of aggregation at the ratio of 10:1 compound:Aβ 1-42 but due to the saturation of the fluorescence signal (green curves), the results are not interpretable.

Figure 3 . 16 .

 316 Figure 3.16. ThT fluorescence results of totally deprotected final products

Figure 3. 18 .Figure 3 . 19 .

 18319 Figure 3.18.In the case of compound 2.233 the trans analogue of the compound 2.204, the totally protected molecule with KLVF sequence at the ratio of 1:1 Aβ 1-42 :compound, shows a very slight acceleration on the kinetics of aggregation (t 1/2 is reduced by a factor of 0.77). At compound:Aβ 1-42 ratio of 10:1 the result is not interpretable due to saturation of the fluorescence signal (green curves) and the similar result was obtained from the compound alone in the presence of ThT revealing the possible self-aggregation of this compound. The evaluation of compound 2.234, the totally N-deprotected final product is under investigation.

Figure 3 . 20 .

 320 Figure 3.20. ThT assay of totally deprotected fluorinated strandFinally, the effect of totally deprotected compound 2.157a on the kinetics of aggregation was studied by ThT assay. The result suggests that the free amino group jointly with free hydroxyl group does not represent a considerable effect on the fluorescence curve in comparison to the control curves (dark blue curves, peptide Aβ alone). It can be notice just a delicate decline on fluorescence plateau but surprinsingly less significant than for the Boc and Cbz protected compound 2.162.

Figure 3 . 22 .

 322 Figure 3.22. Effects of the hairpin 2.217 on Aβ 1-42 fibril formation visualized by TEM. Negatively stained images were recorded at 5 h and 29 h of incubation of Aβ 1-42 (10 µM in 10 mM Tris.HCl, 100 mM NaCl at pH = 7.4) alone (left row) and in the presence of 100 µM of 2.217 (right row). Scale bars, 500 nm. These results indicate the capability of the cis-acetylated hairpin, 2.193, in delaying the aggregation kinetics of the peptide, while the trans-CF 3 one, 2.217, accelerates the process and modify the fibers morphology, which is in agreement with the fluorescence data (Figure 3.22).

Figure 3 . 23 .

 323 Figure 3.23. Effects of the hairpin 2.205 on Aβ 1-42 fibril for-mation visualized by TEM. Negatively stained images were recorded at 46 h of incubation of Aβ1-42 (10 μM in 10 mM Tris.HCl, 100 mM NaCl at pH = 7.4) alone (left row), in the presence of 10 μM of 2.205 (right row). Scale bars, 500 nm. Due to extremely interesting results of compound 2.205 demonstrated by ThT assay, transmission electron microscopy (TEM) analyses, was performed to assert the fluorescence assays, confirming that this product delays Aβ 1-42 aggregation. Images were recorded at 46 h of fibrillation kinetics and at Aβ 1-42 : 2.205 ratio of 1:1. Slight differences were observed in the quantity of aggregates formed in presence of 2.205. After 46 h of aggregation, once the maximum

Figure 3 . 24 .

 324 Figure 3.24. Electrophoretic profile of Aβ 1-42 peptide (100 µM) obtained immediately (0 h), 4 h and 6 h after sample reconstitution (t 0 ).

Figure 3 . 25 .

 325 Figure 3.25. Electrophoretic profile of Aβ 1-42 peptide (100 µM) obtained immediately (0 h), 4 h and 6 h after sample reconstitution (t 0 ) in the presence of compound 2.193 at Aβ 1-42 : compound ratio of 1:1The electrophoretic profile clearly indicated that the kinetics of oligomerization was significantly modified in the presence of the cis-DKP compound 2.193(Figure 3.25). Indeed, at the beginning of the kinetics and after 4 h, the peaks ES and ES' were still present in the electrophoretic profile of the sample containing compound 2.193 while they significantly decreased

Figure 3 . 26 .

 326 Figure 3.26. Electrophoretic profile of Aβ 1-42 peptide (100 µM) obtained immediately (0 h), 4 h and 6 h after sample reconstitution (t 0 ) in the presence of compound 2.205 at Aβ 1-42 : compound ratio of 1:1The electrophoretic profiles obtained in the presence of the trans-DKP derivative 2.217indicates the dramatically acceleration of the aggregation process (Figure3.27). Indeed, at the beginning of the kinetics, the CE profile of the sample containing 2.217 exhibited already the presence of spikes, not visible for the control sample. These results are in agreement with the ThTfluorescence and TEM data revealing that compound 2.217 is an accelerator of aggregation promoting the formation of larger and less soluble species.

  obtained were in agreement with the ThT data. On the other hand, the hairpin 2.205 composed of the fluorinated Val-peptidomimetic arm and KLVF sequence as peptidic arm shows a significant effect on Aβ 1-42 aggregation kinetics. It can effectively delay the kinetics of oligomerization confirmed by ThT fluorescence assay, TEM and CE analysis. So concluding, the hairpins prepared even with the GVVI sequence or KLVF sequence are able to delay the kinetics of aggregation and we can consider them as modulators of Aβ 1-42 aggregation. The presence of a free amine at the Nterminus of the hairpin increased significantly the activity of all the molecules towards the aggregation of A 1-42 , compared to the activity of the protected compounds; in particular, the protected hairpin 2.198 precursor of 2.199, and the protected hairpin 2.204 precursor of 2.205, suggesting that ionic interactions are likely to be established between the terminal amino group and acidic residues of the amyloid peptide (likely the asp 23 which is crucial for the hairpin structure of Aβ oligomeric structure).The DKP2 was generally used in our group as a scaffold with ability to construct an extended structure. The synthesis of the second series of designed molecules containing trans DKP as hairpin analogues, was performed revealing significant biological results. Interestingly, the combination of DKP2, the fluorinated Val-peptidomimetic strand, and the GVVI sequence, compound 2.217, accelerates the aggregation kinetics of Aβ 1-42 , resulting in the formation of fibers that are morphologically different from the fibers formed by Aβ confirmed by ThT assay as well as TEM and CE analysis. Surprisingly, altering the amino acid of peptidomimetic arm from valine to lysine we obtained a more potent compound which delay the aggregation kinetics at ratio 1:10 of Aβ 1-42 :compound 2.202. In summary, promising results were obtained using both DKP scaffolds (cis/trans), different Nowick based peptidomimetic arms along with two different tetrapeptide sequence. The ThT fluorescence assay results confirmed the importance of the whole molecule to modulate the Aβ

  acid tert-butyl ester (2.147b)Compound 2.145 (3 g, 9.27 mmol) was deprotected according to general procedure A. The corresponding trifluoroacetate salt and Nα-Boc-Nε-Z-L-Lys-OH (3.87 g, 10.19 mmol, 1.1 eq) were dissolved in DMF (70 mL). DIPEA(16.15 mL, 92.7 mmol, 10 eq), HBTU (3.86 g, 10.19 mmol,

  To a solution of 2.144(1 g, 3.55 mmol) in dry THF(15 mL) were added Triethylamine (989 μL, 7.1 mmol, 2 eq) and trifluoroacetic anhydride (987 μL, 7.1 mmol, 2 eq) dropwise at 0 °C. The reaction mixture was stirred for 1h at 0 °C. THF was evaporated under reduced pressure and the crude product was purified by flash chromatography on silica gel (EtOAc/cyclohexane 40:60) to afford compound 2.149 as a white solide(1.23 g, 92%). R f : 0.77 (EtOAc/cyclohexane 70:30); mp: 232-234 o C; 1 H NMR (300 MHz, DMSO) δ 9.91 (s, 1H, NH), 9.60 (s, 1H, NH), 8.92 (s, 1H, NH), 7.89 (s, 1H), 7.75 (d, 1H), 7.07 (d, 1H), 3.83 (s, 3H), 1.43 (s, 9H); 13 C NMR (75 MHz, DMSO) δ 164.

  (500mg, 1.32 mmol) was deprotected according to general procedure A. The corresponding trifluoroacetate salt was coupled to Boc-L-Val-OH (573.5 mg, 2.64 mmol, 2 eq) according to general procedure B. The crude product was purified by flash chromatography on silica gel (EtOAc/cyclohexane 1:1

- 5 -

 5 fluoro-4-hydroxybenzoyl) hydrazine carboxylate (2.160) To a solution of 2.159 (271 mg, 0.86 mmol) in CH 3 OH (40 mL) was added Pd/C (27 mg, 10% weight). The reaction flask was flushed three times with hydrogen, and the reaction was stirred, under hydrogen atmosphere, for 1h at r.t. The mixture was filtered through a pad of celite, and the cake was thoroughly washed with methanol (200 mL). The filtrate was evaporated under reduced pressure to afford product 2.160 as a light-grey solid (241 mg, 98%). mp: 91-93 o C; 1 H NMR (300 MHz, DMSO) δ 9.84 (s, 1H), 8.74 (s, 1H), 6.99 (s, 1H), 6.90 (d, J=12.5Hz, 1H), 1.42 (s, 9H); 13 CNMR (75MHz, DMSO) δ 165.4, 155.3, 151.3 (J = 233.2Hz), 134.3 (J = 15.7Hz), 139.8 (J = 6Hz), 109.5, 102.3 (J = 21Hz), 78.9, 28.0; 19 F NMR (200 MHz, DMSO) δ -139.61 (s); HRMS Calc for C 12 H 16 FN 3 O 4 [M+Na] + (308.1023): found 308.1027; IR ν max : 3283, 2360, 2342, 1635, 788.tert-butyl-2-(3-benzamido-5-fluoro-4-hydroxybenzoyl) hydrazine carboxylate (2.161)At room temperature was added benzoic anhydride (317.2 mg, 4 eq) to a solution of compound 2.160 in dry THF (3.5 mL) under the N 2 atmosphere. The reaction mixture was heated to 60 o C for 3 hours and then cooled to r.t. The solvent was evaporated, the residue obtained was dissolved in EtOAc and was added silice to make sample for purification. The sample prepared was purified by silica flash column chromatography (cyclohexane/EtOAc 7:3 to 6:4) to afford 2.161 as a white solid (yield: 86%). R f : 0.73 (cyclohexane/EtOAc 1:1); mp: 66-71 o C; 1 H NMR (300 MHz, DMSO) δ 10.43 (s,NH, 1H), 10.38 (s, NH, 1H), 9.02 (s, NH, 1H

  mg, 0.32 mmol, 1eq) was added to a solution of Nα-Boc, Nβ-Cbz aminopropionic acid 2.157 (220 mg, 0.65 mmol, 2eq) in 5 mL of CH 2 Cl 2 in one portion. A white precipitate (DCU)

  284 C. A. Faler, M. M. Joullié, Tetrahedron Lett., 2008, 49, 6512-6513.

TABLE OF CONTENTS CHAPTER 1

 OFCHAPTER General Introduction to Protein Structure, the concept of Foldamers, Alzheimer's

	disease and the treatment strategies
	1.1 PROTEIN SECONDARY STRUCTURES

  3

  4

  Doctor Barry Reisberg, clinical director of the New York University school of medicine's Silberstein aging and Dementia Research Centre developed a system to classify the progress of this disease in seven stages:48 Stage 1. No impairment (normal function), the person does not experience any memory problems.An interview with a medical professional does not show any evidence of symptoms of dementia. Very mild cognitive decline (may be normal age-related changes or earliest signs of Alzheimer's disease). The person may feel as if he or she is having memory lapses, forgetting familiar words or the location of everyday objects. But no symptoms of dementia can be detected during a medical examination or by friends, family or co-workers.Stage 3. Mild cognitive decline (early-stage Alzheimer's can be diagnosed in some, but not all, patients with these symptoms), friends, family or co-workers begin to notice difficulties. During a detailed medical interview, doctors may be able to detect problems in memory or concentration.

	Stage 2.

Stage 4. Moderate cognitive decline (Mild or early-stage Alzheimer's disease), at this point, a careful medical interview should be able to detect clear-cut symptoms in several areas: memory lack of recent events, weakend ability to perform challenging mental arithmetic and complex tasks.

Stage 5. Moderately severe cognitive decline (Moderate or mid-stage Alzheimer's disease), gaps in memory and thinking are noticeable, and patients begin to need help with day-to-day activities. At this stage, those with Alzheimer's may: be unable to recall their own address or telephone number or the high school or college from which they graduated, become confused about where they are or what day it is, need help choosing proper clothing for the season or the occasion, still remember significant details about themselves and their family, still require no assistance with eating or using the toilet. Stage 6. Severe cognitive decline (Moderately severe or mid-stage Alzheimer's disease), memory continues to worsen, personality changes may take place and individuals need extensive help with daily activities.

Stage 7. Very severe cognitive decline (Severe or late-stage Alzheimer's disease), in the final stage of this disease, patients lose the ability to respond to their environment, to carry on a conversation and, eventually, to control movement. They may still say words or phrases. At this stage, individuals need help with much of their daily personal care, including eating or using the toilet.

They may also lose the ability to smile, to sit without support and to hold their heads up. Reflexes become abnormal, muscles grow rigid, swallowing impaired.

48

There is no single test that can show whether a person has Alzheimer's. While physicians can almost always determine if a person has dementia, it may be difficult to determine the exact cause.

48

Alzheimer's Association Website 2014 Diagnosing Alzheimer's requires careful medical evaluation, including: medical history, mental status testing, physical and neurological exam, Tests (such as blood tests and brain imaging) to intercept other causes of dementia-like symptoms.

  49,62 Not only is Aβ42 more prone to aggregate than Aβ40, but the pathway toward aggregation is also different. The sequence of Aβ(1-42) as is shown in Figure1.34

	59 A. L. Lublin, S. Gandy, Mt Sinai J. Med., 2010, 77, 43-49.

60

See ref.

46. 61 L.D. Estrada and C. Soto, Curr. Top. Med. Chem., 2007, 7, 115-126. 62

L.O. Tjernberg, C. Lilliehook, D. J.

Callaway et al., J. Biol. Chem., 1997, 272, 12601-12605.

1.3.3. Therapeutic strategies

	exist as stable entities, i.e. they evolve into higher order aggregates and, if they are on pathway
	intermediates, convert into fibrils.
	Alzheimer's drugs offer one strategy to help manage memory loss, thinking and reasoning
	problems, and day-to-day function. Unfortunately, Alzheimer's drugs don't work for everyone, and
	they can't cure the disease or stop its progression. As Alzheimer's progresses, brain cells die and
	connections among cells are lost, causing cognitive symptoms to worsen. While current
	medications cannot stop the damage Alzheimer's causes to brain cells, they may help lessen or
	stabilize symptoms for a limited time by affecting certain chemicals involved in carrying messages
	among the brain's nerve cells.
	In the brain, neurons connect and communicate at synapses, where tiny bursts of chemicals
	called neurotransmitters carry information from one cell to another. Alzheimer's disrupts this
	process, and eventually destroys synapses and kills neurons, damaging the brain's communication
	[Ca 2+ ], whereas equivalent amounts of monomeric and fibrillar forms are without any detectable network. The Food and Drug Administration (FDA) has approved two types of drugs specifically
	effect. Several mechanisms have been proposed to account for the Ca 2+ mobilizing actions of to treat symptoms of Alzheimer's disease as it was reported in a table in
	amyloids in their oligomeric and fibrillar aggregation states. These include a direct interaction with
	membrane components to destabilize the membrane structure; insertion into the membrane to form
	a cation-conducting pore; activation of cell surface receptors coupled to Ca 2+ influx; and oxidative
	stress leading to dysregulation of mitochondrial Ca 2+ homeostasis. Among these possibilities, the
	hypothesis of a generalized increase in membrane permeability induced specifically by spherical
	amyloid oligomers. 71
	Although the toxic oligomer hypothesis became one of the major current working hypothesis in
	AD research, the development of effective diagnosis tools and therapies based on this hypothesis
	has not been realized yet due to the lack of a single toxic Aβ species identification. 72 Furthermore,
	the exact mechanisms by which these species contribute to Aβ toxicity in vivo and the nature of the
	toxic species are not yet fully understood. Unless covalently cross-linked, Aβ oligomers do not

  Addition of the carboxylate ensues an irreversible acyl transfer of 2.37 and drives the reaction forward giving the classical Ugi skeleton 2.38.

The mechanism of this reaction is illustrated in Scheme 2.3; it consists in the condensation of an amine with a ketone or aldehyde to give the corresponding Schiff base 2.35 which upon proton activation combines with an isocyanide, giving the 'so-called' intermediate nitrilium ion 2.36. 167 Scheme 2.3. The Ugi reaction mechanism

  In 2008, Bräse reported a microwave-assisted stereoselective one-pot synthesis of symmetrical and unsymmetrical 2,5-diketopiperazines from unprotected amino acids, through a phosphatepromoted one step coupling reaction (Scheme 2.7). Good yields, scalability, and tolerance to several base-stable protecting groups are the characteristics of this method. Simple filtration through a pad of silica provides the pure compounds.173

	Scheme 2.7. Phosphite promoted coupling of amino acids: a) MeOPCl 2 , Et 3 N, toluene, MW: 84-97%

172 Scheme 2.6. Synthesis of hexasubstituted DKPs via O,N-acyltransfer 170 J. F. Demarest, S. S. Sparks, K. Schell, S. Shibayama, C. B. McDanal, L. Fang, K. K. Adkison, A. Shachoy-Clark, S.

C. Piscitelli, J.

Clin. Pharmacol., 2008, 48, 1179-1188. 171 E. O'Reilly, L. Pes, F. Paradisi, Tetrahedron Lett., 2010, 51, 1696-1697. 172 Z. Z. Brown, C. E. Schafmeister, Org. Lett., 2010, 12, 1436-1439.

1.3. DKPs as two-armed receptors

  In this field Wennemers and co-workers have prepared several symmetrical DKP-based twoarmed receptors derived from 4-aminoproline in which the two amino groups (with cis relative configuration) were functionalized with two tripeptide side chains(Figure 2.11).176,177

	Scheme 2.8. Solid-phase synthesis: a) BrCH 2 COBr, AlCl 3 , nitrobenzene/DCM (1:1); b) Boc-AA′-OH, Et 3 N, DMF; c)
	3.5N HCl/AcOH; d) Boc-AA′′-OH, HOBt, DCC, NMM, DMF; e) 10% DIPEA/EtOAc; f) 5% Et 3 N/THF-H 2 O (8:1)
	Notwithstanding the successful use of activated ligands such O-Pac, the bond susceptible to
	nucleophilic attack requires amino acid side-chain group protection. This relative lability is
	create a cleft with an adequate size to accommodate the guest, and ii) create two side-arms with
	correct geometry to tie up the guest with a sufficient number of binding points. For this purpose,
	diketopiperazines with their flat core and lateral chains of diverse size and nature have been
	synthesized and used.

135

173 M. Jainta, M. Nieger, S. Bräse, Eur. J.

Org. Chem., 2008, 32, 5418-5424. 174 D. S.Ermolat'ev, E. V. Babaev, Molecules, 2003, 8, 467-471. 175

D.-X. Wang, M.-T. Liang, G.-J. Tian, H. Lin, H.-Q.

Liu, Tetrahedron Lett., 2002, 43, 865-867.

generally considered responsible for small losses of the elongating peptides. To resolve this problem it was suggested peptide attachment to the resin by the safety-catch technique, because the stable bond is conveniently activated only after ending the peptide synthesis, allowing cleavage and cyclization. The strategy outlined in Scheme 2.9 permits the removal of protecting groups before cleavage, facilitating further workup. The use of safety-catch ligands allowed the solid-phase generation of bicyclic and tricyclic DKP, which show even greater structural diversity. 138 Scheme 2.9. Solid-phase synthesis by safety-catch approach: a) Boc-AA′-OH, DIC, DIPEA; b) Boc-based solid-phase peptide synthesis; c) TFMSA, TFA; d) DIPEA

2.

Two-armed receptors built by a scaffold as a central element and two arms generally symmetrical and containing several binding sites, have been synthesized (often via a combinatorial approach) and reported to bind different kinds of guests (e.g., anions, cations, small molecules, amino acids, and short peptides). The importance of the central scaffold resides in its ability to: i)

  209

	Figure 2.32. Organocatalysts
	Peptidomimetics (2.111-2.114) were tested as organocatalysts in the conjugate addition reaction
	of several aldehydes to β-nitrostyrene and (E)-2-(furan-2-yl)nitroethene excellent diastereo-(99:1
	dr) and enantioselectivities (up to 98% ee), particularly for catalysts 2.111 (containing L-Pro and

cis-DKP-1) and 2.113 (containing L-Pro and trans-DKP-2), in high yields and under operationally mild conditions. It was shown that the terminal proline residue configuration establishes the absolute configuration of the conjugate addition products: L-Pro gave (2R,3S)-2.115, whereas with D-Pro (2S,3R)-2.116 was obtained. The role of the DKP scaffolds is to tune the degree of selectivity: 2.113 was the best performing catalyst in terms of enantioselectivity, whereas better diastereoselectivity was usually obtained with 2.111. Catalysts 2.

112

and 2.114 parallel the behaviour of 2.111 and 2.113: the more diastereoselective was 2.112 while 2.114 gave better ee values. 209 Scheme 2.11. Conjugate addition reaction of aldehydes to -nitrostyrene

  inhibitors, like -secretase (BACE), an enzyme involving in producing Aβ peptide associated with Alzheimer's disease. For example, bicyclic compounds 2.117 and 2.118 demonstrated their ability in binding to BACE (Figure2.34).219,220

	sufficient occupancy of P1 by methyl substitution of the alkyl chain appeared necessary to reach
	submicromolar BACE-1 inhibition. 222
	developed these inhibitors starting from the x-ray crystallographic data of a ligand-BACE-1
	complex which was reported by Tang and co-workers 221 based on an octapeptide 2.119 that
	showed a great inhibitory activity in nanomolar concentrations. Their aim was to reduce the size of
	OM99-2 while preserving low micromolar activity of compounds. The critical importance of the
	methyl group was demonstrated by the inhibitors 2.120c and 2.120e which were devoid of this
	group and showed a significant loss in activity compared to 2.120b and 2.120d. Therefore,
	219 S. Hanessian, G. Q. Yang, J. M. Rondeau, U. Neumann, C. Betschart, M. Tintelnot-Blomley, J. Med. Chem., 2006,
	49, 4544.

Figure 2.34. Examples of BACE inhibitors

In 2009, researchers in Novartis institutes prepared the macrocyclic inhibitors 2.120a-h by ringclosing metathesis (RCM) followed by aminolysis of the -lactone and hydrogenation. They 220 I. Rojo, J. A. Martin, H. Broughton, D. Timm, J. Erickson, H. C. Yang, J. R.

McCarthy, Bioorg. Med. Chem. Lett., 2006, 16, 191-195. 221

(a) L. Hong, G. Koelsch, X. Lin, S. Wu, S. Terzyan, A. K. Gosh, X. C. Zhang, J.

Tang, Science, 2000, 290, 150; (b)

A. K. Ghosh, G. Bilcer, C. Harwood, R. Kawahama, D. Shin, K. A. Hussain, L. Hong, J. A. Loy, C. Nguyen, G. Koelsch, J. Ermolieff, J. Tang, J.

Med. Chem., 2001, 44, 2865.

.4. Aliphatic and Aromatic Carbocycles

	Figure 2.38. Example of azapeptide peptidomimetic as inhibitor of HIV-1 protease
	Acyclic HIV-1 protease inhibitors were developed using a variety of central linker units, in
	particular, hexadecanoic acid has been used to prepare two peptidomimetics 2.124 and 2.125 Figure 2.40. Aromatic and aliphatic based peptidomimetic examples
	(Figure 2.39) that showed a good inhibitory activity with the IC 50 value of 350 and 180 nM Inhibitors designed as -strand tetrapeptide mimetics, such as 2.126 and 2.129, have the
	respectively. 228 capacity to prevent the Ras processing in a range of ras-transformed tumor cell lines and inhibit
	tumor growth in nude mice. 212,225
	Figure 2.39. HIV-1 protease inhibitors based on acyclic peptidomimetics
	2.2Planar aromatic spacers also have been incorporated into inhibitor design as extended dipeptide
	mimics in, for example, inhibitors of Ras farnesylation. Such inhibitors can slow the development
	of cancers in which oncogenic Ras proteins are active. A series of tetrapeptides CA 1 A 2 X (A:
	aliphatic amino acid, X: Met or Ser) were potent inhibitors of farnesyltransferase (FTase) in vitro,
	but were inactive in cell-based assays. Replacing the central A 1 A 2 residues with rigid hydrophobic
	scaffolds to enforce an extended conformation led to the formation of potent inhibitors that were
	less peptidic in nature. Compounds 2.126, 2.127 (benzene scaffold), 2.128 (1,5-or 1,6-naphtalene
	scaffold), and 2.129 (biphenyl scaffold) are examples of this type of peptidomimetics (Figure
	2.40). 229

Acyclic -strand molecules have typically involved the design and optimization of dipeptide isosteres flanked by peptidic or nonpeptidic appendages. Under this category a useful class of peptidomimetics are azapeptides, in which the α-CH group of one or more amino acid residues in the peptide chain is replaced by a nitrogen atom. Azapeptide-derived inhibitors were originally designed as substrate analogues of papain and cathepsin B, where the bound substrate lies in an extended conformation in the enzyme.

226

Later, azapeptide have been designed as inhibitors of HIV-1 protease, such as 2.123 (Figure 2.38). The α-CH/N replacement forms an azapeptide in a different peptide conformation. 227 225 P. D. Henley, C. P. Waymark, I. Gillies, J. D. Kilburn, J. Chem. Soc., Perkin Trans. I, 2000, 1021-1031. 226 R. Calabretta, C. Giordano, C. Gallina, V. Morea, V. Consalvi, R. Scandurra, Eur. J. Med. Chem., 1995, 30, 931-941. 227 A. Faessler, G. Bold, H.-G. Capraro, R. Cozens, J. Mestan, B. Poncioni, J. Roesel, M. Tintelnot-Blomley, M. Lang, J.

Med. Chem., 1996, 39, 3203-3216.

228 M. J. Bowman, S. Byrne, J.

Chmielewski, Chem. Biol., 2005, 12, 439-444. 229

Y. Qian, A. Vogt, S. M. Sebti, A. D. Hamilton, J.

Med. Chem., 1996, 39, 217-223.

Progressive shortening of the amino acid side chain linked to the aromatic spacer led to other strand mimetics. Carbocycles 2.130 and 2.131 bind in a -strand conformation to Plasmepsin II (an aspartic protease from Plasmodium falciparium) and pancreatic elastase, respectively.

230,231

Figure 2.41. Examples of -strand mimics Some mimetics bear a sulfonamide moiety that orients part of the molecule away from the aromatic spacer and is not part of the -strand mimicry. A crystal structure has demonstrated this fact for the inhibitor 2.132 (Figure 2.42) which bound to caspase-3. 230 O. A. Asojo, S. V. Gulnik, E. Afonina, B. Yu, J. A. Ellman, T. S. Haque, A. M. Silva, J. Mol. Biol., 2003, 327, 173-181. 231 E. Meyer, Protein Data bank, entry 1inc.

  1-42 and it has been shown to interact with the other KLVF) in solid state NMR structure of A 1-42 protofilaments, 244 and in soluble amyloid β-peptide oligomer.245

241 See ref. 44. 242 J. S. Nowick, D. M. Chung, K. Maitra, S. Maitra, K. D. Stigers, Y. Sun, J. Am. Chem. Soc., 2000, 122, 7654. 243 See ref. 237 hydrophobic sequence 16-19 (

  The cleavage of Boc with TFA in CH 2 Cl 2 provided the intermediate 2.146 that was then used for the coupling to diverse amino acids (Scheme 2.13). The synthesis of Val-and Lys-peptidomimetic arms; vi) Boc-Val-OH, HOBt, HBTU, DIPEA, DMF, overnight, 69%; vii) Nɛ-Cbz-Nα-Boc-L-Lys-OH, HOBt, HBTU, DIPEA, DMF, overnight, 70%; viii) = ix) TFA, CH 2 Cl 2 , 1h, r.t., quantitative yield.

	2.3.2. Peptidomimetic Synthesis
	I) Synthesis of 5-amino-2-methoxybenzoic hydrazide derivatives
	The peptidomimetic strand 2.148, bearing the 5-amino-2-methoxybenzoic hydrazide derivative,
	was obtained starting from commercially available 2-methoxybenzoic acid, following procedures
	described in our laboratory. 2-Methoxybenzoic acid was initially nitrated using a mixture of
	ammonium nitrate and sulfuric acid, to give compound 2.142, and successively coupled with tert-
	butylcarbazate affording 2.143 in a good yield. The nitro group of 2.143 was then reduced, by
	catalytic hydrogenation, to afford the N-Boc-protected 5-amino-2-methoxybenzoic hydrazide
	2.144, described in the literature by Nowick. 248 Compound 2.144 was then acetylated to afford
	2.145. Scheme 2.13. Synthesis of key intermediate for the preparation of peptidomimetic strand; i) NH 4 NO 3 , H 2 SO 4 , 80%; ii)
	BocNHNH 2 , HOBt, EDC*HCl, CH 2 Cl 2 , r.t., overnight, 85%; iii) H 2 , Pd/C, MeOH, 95%; iv) Ac 2 O, THF, quantitative
	yield; v) TFA, CH 2 Cl 2 , r.t., quantitative yield.
	244 See ref. 56
	245 L. Yu, R. Edalji, J. E. Harlan, T. F. Holzman, A. P. Lopez, B. Labkovsky, H. Hillen, S. Barghorn, U. Ebert, P. L.

2.49. The general structure of -hairpins Richardson, L. Miesbauer, L. Solomon, D. Bartley, K. Walter, R. W. Johnson, P. J. Hajduk, E. T. Olejniczak, Biochemistry, 2009, 48, 1870-1877. 246 a) M. D. Smith, L. Cruz, J. Phys. Chem., 2013, 117, 14907-14915. b) K. Hochdörffer, J. März-Berberich, L. Nagel-Steger, M. Epple, W. Meyer-Zaika, A. H. C. Horn, H. Sticht, S. Sinha, G. Bitan, T. Schrader, J. Am. Chem. Soc., 2011, 133, 4348-4358. 247 See ref. 113

The intermediate 2.146 was then successfully coupled to N-Boc-L-Val-OH and N ε -Cbz-N α -Boc-L-Lys-OH using HBTU, HOBt, DIPEA, in dry DMF to afford 2.147a and 2.147b with good yield (69% and 70% respectively). Finally, trifluoroacetic salt 2.148a and 2.148b were obtained in quantitative yield by acidic cleavage with TFA in dry CH 2 Cl 2 (Scheme 2.14). In this way the two peptidomimetic arms are ready to be coupled to our scaffolds.

248 See rif 242. Scheme 2.14.

  4 NO 3 , H 2 SO 4 , 81%; ii) BocNHNH 2 , HBTU, HOBt, DIPEA, DMF, overnight, 95%; iii) Pd/C, H 2 , MeOH, 1h, quantitative yield; iv) benzoic anhydride, THF, 60 o C, 3h, 86%; v) TFA, CH 2 Cl 2 , 1h, r.t., quantitative yield; vi) N ɛ -Cbz-N α -Boc-L-Lys-OH, DMTMM(BF 4 ), NMM, DMF, overnight, 50%. the hydrazine building block 2.165 followed by the coupling reaction with intermediate 2.158 synthesized in a previous step as illustrated in Scheme 2.19.

	Scheme 2.19. The first synthetic pathway; i) H 2 SO 4 , MeOH, 65 o C, reflux, 3h, 96%; ii) NH 2 NH 2 *H 2 O, MeOH, 60 o C,
	quantitative yield; iii) HOBt, HBTU, DIPEA, DMF, overnight
	The commercially available 2-phenylacetic acid was treated with H 2 SO 4 in MeOH to give
	Scheme 2.20. The proposed procedure for the synthesis of another peptidomimetic; iv) TFA, CH 2 Cl 2 , r.t., quantitative
	yield; v) 2-phenylacetic acid, DMTMM(BF 4 ), NMM, DMF, overnight, 89%.
	Scheme 2.18. vii) NaOH (1.2M), MeOH/H 2 O, 30 minutes, quantitative yield; viii) H 2 , Pd/C, MeOH, 1h, r.t.; ix) TFA,
	CH 2 Cl 2 , quantitative yield.

To obtain our target molecule with a non protected hydroxyl, compound 2.162 was treated with NaOH 1.2 M in a mixture of H 2 O/MeOH (1:2.75) giving the desired compound 2.163 in a quantitative yield. As we needed the totally deprotected molecule for the biological test, the Cbz group was removed by hydrogenation and the last protecting group (Boc moiety) was cleaved in an acidic conditions using trifluoroacetic acid in dichloromethane resulting compound 2.157a as our final compound as a TFA salt (Scheme 2.18).

For the synthesis of the second molecular target 2.157b, possessing an OH substituent at the same position of the aromatic ring, the usual path used for the other non-fluorinated peptidomimetics developed in our laboratories was initially applied; this consisted in the preparation of product 2.164 which then underwent reaction with hydrazine hydrate to yield 2.165 as the intermediate for the subsequent coupling reaction. The reaction between intermediates 2.158 and 2.165 using the common coupling reagents HBTU, HOBt led to the formation of product 2.166, but unfortunately due to the presence of a by product, it was not possible to purify this compound. Thus, we decided to change the procedure and to use the intermediate 2.159 that after Boc deprotection underwent coupling to phenylacetic acid directly using DMTMM(BF 4 ), affording 2.166 in a high yield of 89% (Scheme 2.20).

  Synthesis of building blocks; i) CH 3 COCl, CH 2 =CHCH 2 OH, 90%; ii) Et 3 N, Boc 2 O, H 2 O/1,4-dioxane, 96%; iii) CH 3 COCl, CH 3 OH, 82%, iv) Et 3 N, PhCHO, CH 3 OH, then NaBH 4 , 86%.

	Scheme 2.23. Formation of an isopeptide by direct coupling of building blocks 2.168 and 2.170; v) HATU, HOAt,
	DIPEA, DMF, 38%.

Scheme 2.22.

The coupling reaction between protected aspartic acid 2.168 and N-benzylserine 2.170 was then attemted. The reaction performed using Carpino's reagent ATU afforded the unexpected isopeptide 2.172 instead of the dipeptide 2.171 as the sole product, via the selective acylation of the unprotected β-hydroxy group of either (S)-or (R)-N-benzylserine (Scheme 2.23), as demonstrated by spectroscopic methods.

  CH 3 COCl, CH 2 =CHCH 2 OH; ii) Et 3 N, Boc 2 O, 1:1 H 2 O/1,4 dioxane; iii) CH 3 COCl, CH 3 OH; iv) Et 3 N, PhCHO, CH 3 OH, then NaBH 4 ; v) EDC*HCl, DMAP cat. , DCM; vi) TFA, DCM; vii) iPr 2 EtN, iPrOH; viii) PPh 3 , DIAD, H

	Scheme 2.26. i) PPh 3 , DIAD, H 3 N, DCM/Toluene, -20 o C; ii) Me 3 P, Boc-ON, THF, -20 o C-> r.t.; iii) [Pd(PPh 3 ) 4 ], PPh 3 , pyrrolidine, DCM, 0 o C.
	Mitsunobu transformation on compounds 2.174 is a quite sensitive and critical reaction, since
	the activated hydroxyl functionality could undergo the β elimination before reacting with the
	pronucleophile (H 3 N). The C(6) proton can be easily extracted due to its acidity, providing a
	diketopiperazine with an exocyclic double bond (Scheme 2.27).
	Scheme 2.25. i) EDC*HCl, DMAP, CH 2 Cl 2 , 45%.
	Once the coupling reaction and the subsequent ring closure were improved, we concentrated on
	the transformation of the hydroxyl moiety of 2.174 into Boc-protected amino moiety present in
	DKP1 and DKP2. This functional group interconversion was accomplished by a Mitsunobu type
	reaction, followed by reduction and in situ Boc-protection by Staudinger reaction of the obtained
	azides into derivatives 2.176. Deprotection of allyl ester on compounds 2.176 was successfully The next step involved a one-pot Staudinger-Boc protection. The Staudinger reaction, a very
	proceeded via a Pd 0 catalyzed Tsuji-Trost reaction, afforded scaffolds DKP1 and DKP2 (Scheme mild azide reduction, involves the reaction of the azide with a phosphine to generate a phosphazide,
	2.26). which losses N

Scheme 2.27. β elimination competing with the Mitsunobu nucleophilic attack; i) PPh 3 , DIAD, H 3 N.toluene, DCM/Toluene, -20 o C. Monitoring temperature (that should be remains at -20 o C), reaction time and order of reagents addition, it was possible to reduce the amount of 2.177. Reaction was carried out in a mixture of toluene/dichloromethane in a ratio of 2:1 solution, since toluene itself was not sufficient to solubilize diketopiperazines 2.174 at -20 o C. β elimination side reaction is seemed to be particularly competitive in the case of the cis substrate 2.174a. Purification of this product from the byproduct revealed even more complicated, as product 2.175a showed almost the same elution time of the corresponding byproduct 2.177, with various eluents. These are the main reasons why yields are far lower for the cis product. 2 to form an iminophosphorane. Hydrolysis of this intermediate leads to the amine and the very stable phosphine oxide. The mechanism is reported in Scheme 2.28. Scheme 2.30. i) 3 N.tol, DCM/toluene, -20 °C; ix) Me 3 P, Boc-ON, THF, -20 °C -> r.t; x) [Pd(PPh 3 ) 4 ], PPh 3 , pyrrolidine, DCM, 0 °C.

  The acidic Hofmann rearrangement that was utilized in this preparation, converted the amides directly into the corresponding amines in partially aqueous solution, was first developed by Loudon et al.254 The mechanism of the rearrangement has been illustrated in Scheme 2.33.

	Scheme 2.32. Synthesis of the first building block; i) Boc 2 O, Et 3 N, H 2 O/1,4-dioxane, 87%; ii) Ph(OAc) 2 I,
	H 2 O:CH 3 CN:EtOAc, 60%; iii) Cbz-Cl, NaHCO 3 , H 2 O, 67%.

following Hofmann rearrangement carried out in the presence of iodobenzene diacetate in a mixture of EtOAc: H 2 O: MeCN in a ratio of 2:1:2 at 20 o C overnight using thermostatic water bath to maintain the temperature at 20 o C and affording 2.180 in 60% of yield.

  It was thought that by using DCC it can be possible to generate the symmetric anhydride 2.185 which after the coupling reaction with the compound 2.183 provide the dipeptide 2.184. After the purification by flash column chromatography, there were obtained two products successively analysed by NMR spectroscopic. The 1 H NMR analysis surprised us by revealing the first product as the starting material and the second compound, as a cyclized byproduct 2.186 obtained from the activation of carboxylic acid of asparagine and further intramolecular attack of amide group (Scheme 2.36). The first attempt of using symmetric anhydride; i) DCC, CH 2 Cl 2 , ii) CH 2 Cl 2 , overnight.

	Scheme 2.35. i) HATU, HOAt, DIPEA, DMF, overnight or DMTMM(BF 4 ), NMM, DMF, overnight
	In the synthesis of other diketopiperazine scaffolds, we have shown that a good solution to
	perform difficult coupling is the use of symmetric anhydyrides. Thus we planned the synthetic
	route summarized in Scheme 2.33, preparing the symmetric anhydride of the N-(tert-
	butoxycarbonyl)-asparagine 2.179 and coupling it to the other building block 2.183 to form
	dipeptide 2.184. Scheme 2.36.

  t., overnight, 62%; iv) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 57%; v) TFA, CH 2 Cl 2 , 0 °C, 1h, HATU, HOAt, DIPEA, Boc-Gly-OH, DMF, 0 °C to r.t., overnight, 66%; vi) Pd/C, H 2 , MeOH, 5h, then TFA, CH 2 Cl 2 , overnight, 70% over 2 step.

  The yield of each step was high, leading to the formation of a new hairpin 2.204 (Scheme 2.46). The deprotection step; vi) Pd/C, H 2 , MeOH, then TFA, CH 2 Cl 2 , overnight, quantitative yield.

	Scheme 2.46. The synthetic route for the preparation of a new hairpin; ii) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Phe-OH, DMF, 0 °C to r.t., overnight, 87%; iii) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 76%; iv) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, Boc-Leu-OH, DMF, 0 °C to r.t., overnight, 75%; v) TFA, CH 2 Cl 2 , 0 o C, 1h; HATU, HOAt, DIPEA, N ɛ -Cbz-N α -Boc-L-Lys-OH, DMF, 0 °C to r.t.,
	overnight, 75%.

Cleavage of the protectiong groups, namely Cbz by hydrogenation and subsequently Boc with TFA, resulted in the final product 2.205 ready for testing (Scheme 2.47).

Scheme 2.47.

  t., overnight, 75%; v) H 2 , Pd/C, THF/H 2 O; TFA, CH 2 Cl 2 , quantitative yield; v) H 2 , Pd/C, THF:H 2 O, then TFA, CH 2 Cl 2 .

Table 3 .

 3 about the Ar-NH or Ar-CO bonds, occurring in slow exchange on the 1 H NMR time scale. 1. 1 H NMR and 13 C-NMR chemical shifts of 2.193 (1.4 mM) in CD 3 OH at 298 K * Observed at 270.4 K onlyThe conformational analysis of 2.193 was based on 1 H and13 C chemical shifts, vicinal coupling constants and ROEs. The 1 Hα and13 Cα chemical shift deviations (CSD), defined as the differences between experimental chemical shifts and corresponding random coil values, are good descriptors of backbone conformational space for each residue.262 All Val and Ile residues are characterized by

	258 (a) P. G. Vasudev, S. Chatterjee, N. Shamala, P. Balaram, Chem. Rev., 2011, 111, 657; (b) D. Seebach, D. F. Hook,
	A. Glättli, Biopolymers, 2006, 84, 23; (c) F. Fulop, T. A. Martinek, G. K. Tóth, Chem. Soc. Rev., 2006, 35, 323; (d) D. L.

Steer, R. A. Lew, P. Perlmutter, A. I.

Smith, M. I. Aguilar, Curr. Med. Chem., 2002, 9, 811; (e)

R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev., 2001, 101, 3219. 259 D. Seebach, S. Abele, K. Gademann, B. Jaun, Angew. Chem. Int. Ed., 1999, 38, 1595. 260 (a) see ref. 197; (b) M. Marchini, M. Mingozzi, R. Colombo, C. Gennari, M. Durini, U. Piarulli, Tetrahedron, 2010, 66, 9528. 261 See ref. 198.

262 a) D. S. Wishart, C. G. Bigam, A. Holm, R. S. Hodges, B. D. Sykes, J. Biomol. NMR., 1995, 5, 67 ; b) C. M.

Santiveri, E. León, M. Rico, M. A.

Jiménez, Chem. Eur. J., 2008, 14, 488.

Table 3 .3. Temperature

 3 3) exhibit large values (8.8-9.4 Hz range), reflecting  angle values around -120°, as expected for -strand conformations.

	Residue	/T	J±Hz
		(ppb/K)
	Val 2	-6.5	8.8
	Val 3	-6.0	9.2
	Ile 4	-9.4	9.4
	Dapa 5	-4.2	3.8
	Asp 6	-8.2	<2
	Val 7	-8.2	9.4
	NH a	n.d.	bs*
	NH b	-4.5	bs*
	NH c	-5.4	bs*

coefficients and coupling constants for NH protons of 2.193 in methanol * bs (broad signal)

  1 H-1 H TOCSY and ROESY, and 2D 1 H-13 C HSQC and HMBC experiments recorded at 298 K (Tables 3.4 and 3.5).

	Residue	HN (ppm)	  ppm	H  (ppm)	Other protons (ppm)
	Gly 1		3.39
	Val 2		4.26	2.05	 CH 3 0.93
	Val 3	8.06	4.19	2.05	 CH 3 0.90
	Ile 4	8.02	4.10	1.78	CH 2 1.53, 1.15, 1.11; 
					CH 3 0.88;  CH 3 0.85
	Dapa 5	8.35	3.83	4.01, 3.52	CH 2 5.40, 4.10; Bn ~
					(7.33, 7.33, 7.26)
	Asp 6		4.69	2.96, 2.86
	Val 7	8.25	4.40	2.22	CH 3 1.05, 1.03
	Amb				H 3 7.20; H 4 7.86; H 6 8.15;
					OCH 3 3.98

Table 3 .

 3 4. 1 H NMR chemical shifts of 2.217 (0.4 mM) in CD 3 OH at 298 K

  1 H NMR time scale.

	Residue	C  ppm	C  ppm	C  (ppm)	Other carbons (ppm)
	Gly 1	174	44.9
	Val 2	173.5	60.1		 CH 3 19.7, 18.6
	Val 3	173.4	60.1		 CH 3 20.0, 18.8
	Ile 4	174.7	59.4		CH 2 25.8;  CH 3 15.6;   CH 3 10.9
	Dapa 5	168.5	60.3		CH 2 47.9; Bn 129.7, 129.4, 128.8, 136.9
	Asp 6	168.2	52.4		 CO 172.1
	Val 7	171.8	58.7		 CH 3 19.7, 18.5
					C 1 121.5; C 2 156.6;
	Amb	165.2			C 3 113.5; C 4 127.5;
					C 5 131.1; C 6 125.3; OCH 3
					56.9

Table 3 .

 3

	5. 13 C NMR chemical shifts of 2.217 (0.4 mM) in CD 3 OH at 298 K

  .6).

	Residue /T	JHN-	CSD	CSD
		(ppb/K)	HHz	H  ppm	C  ppm
	Val 2	n.d.	n.d.	0.10	-2.1
	Val 3	-8.4	8.7	0.03	-2.1
	Ile 4	-8.1	8.0	-0.13	-1.7
	Dapa 5	-9.2
	Val 7	-5.9	8.5	0.24	-3.5

Table 3 .

 3

6. NMR conformational parameters

  (green curves, t 1/2 is increased by a factor 4 and the final fluorescence is decreased by 85 %), at the ratio of 1:1, it also displays a significant activity shown in fuchsia pink curves (t 1/2 is increased by a factor 1.7 and F is decreased by 40 %). No effect is observed at lower ratio (0.1:1).These results suggest the importance and influence of CF 3 group on the aggregation process of Aβ 1-

	42 .
	Compound 2.212
	Peptide Aβ 1-42 (10 μM)
	2.206 (100 μM)
	Aβ 1-42 + 2.206 (1:10)
	Aβ 1-42 + 2.206 (1:1)
	Compound 2.212 (TFA salt)
	Peptide Aβ 1-42 (10 μM)
	2.207 (100 μM)
	+ LV Aβ 1-42 + 2.207 (1:10) 158 (ratio 10:1) F 12 A-beta 1-42 + LV 158 (ratio 1:1) Aβ 1-42 + 2.207 (1:1)

fibrillation

  1/2 is reduced by a factor of 0.6 and the fluorescence plateau is decreased by 60 %). We suggested that the Aβ 1-42 peptide in the of this compound form oligomeric species which morphologically may be diverse in comparison to the toxic aggregates. Accelerating fibrils formation could be a therapeutic strategy when the candidates are able to induce aggregation into less toxic assemblies.The analogues of compounds 2.216 and 2.217 bearing a lysine amino acid on the peptidomimetic arm were also evaluated. Compound 2.228 has retarded slightly the kinetics of aggregation at the ratio of 1:10 Aβ 1-42 :compound (t 1/2 is increased by a factor of 1.6 and the fluorescence plateau is increased by about 20 %), but at the lower concentrations does not display any activity. On the other hand, compound 2.229 with CF 3 exhibits a more promising inhibitory activity at the ratio of 1:10, Aβ 1-42 :compound (t 1/2 is increased by a factor of 3.5 and the fluorescence plateau is decreased by about 70 %), but represents no effect at lower concentrations.

	60000
	50000 Compound 2.228
	30000
	40000
	25000
	30000											Tampon A Tampon
	20000 20000											Ab 1-42 10 µM A Peptide Aβ 1-42 (10 μM)
	0 15000 10000 10000 40000 0 5000	0 0	5 5	10 15 20 25 30 35 40 45 10 15 20 25 30 35 40	LVP 206 100 µM E 2.216 (100 μM) Tampon A1 Tampon LVP 206 100 µM / Ab 1-42 10 µM (10/1) E LVP 206 10 µM / Ab 1-42 Aβ 1-42 + 2.216 (1:10) Aβ 1-42 10 µM A11 Peptide Aβ 1-42 (10 μM) Aβ 1-42 + 2.216 (1:1) LVP149n 100 µM F 2.228 (100 μM) 1 µM (1/1) E 45 LVP149n 100 µM / Aβ 1-42 Aβ 1-42 + 2.228 (1:10) 10 µM (10/1) F LVP149n 10 µM / Aβ 1-42 Aβ 1-42 + 2.228 (1:1) 10 µM (1/1) F LVP149n 1 µM / Aβ 1-42 10 µM (0,1/1) F Aβ 1-42 + 2.228 (1:0.1)
	35000 Compound 2.229
	30000 30000
	25000
	25000
	20000
	15000 20000											Tampon+Am+DMSO A Tampon
	10000 15000											Ab 1-42 lot 90T 10 µM A Peptide Aβ 1-42 (10 μM) LVP 207 100 µM B 2.217 (100 μM) Tampon A1 Tampon
	0 5000 10000 5000	0	5	10	15	20	25	30	35	40	45	LVP 207 100 µM / ab 1-42 10 µM Aβ 1-42 10 µM A11 Peptide Aβ 1-42 (10 μM) Aβ 1-42 + 2.217 (1:10) (10/1) B LVP 207 10 µM / ab 1-42 10 µM LVP148n 100 µM E 2.229 (100 μM) Aβ 1-42 + 2.217 (1:1) (1/1) B LVP148n 100 µM / Aβ 1-Aβ 1-42 + 2.229 (1:10) 42 10 µM (10/1) E LVP148n 10 µM / Aβ 1-Aβ 1-42 + 2.229 (1:1)
	0	0	5	10	15		20	25	30	35	40	45	10 µM (0,1/1) E 42 10 µM (1/1) E LVP148n 1 µM / Aβ 1-42 Aβ 1-42 + 2.229 (1:0.1)
								Figure 3.17.

presence

Table 3 .

 3 the aggregation but only at a high ratio (t 1/2 is decreased by a factor of 0.75) and the final fluorescence is decreased by 23 %) while the trans analog, 2.207 delays the aggregation at a high (1:10) and a lower (1:1) A 1-42 :2.207 ratios with a t 1/2 increased respectively by a factor 4.17 and 1.71 and a final fluorescence plateau decreased by 85 and 43 %. However, this truncated molecule remains less active than the compound containing the tetratpeptide arm, KLVF, 2.205. is increased by a factor of 3.5 and the fluorescence plateau is decreased by about 70 %) but similarly to the compound without CF 3 represents no effect at lower concentrations. summary of the activity of compounds tested by ThT is reported in a table below. 7. A summary of compounds with their activity in different concentration of Aβ 1-42 /compound morphology and in the quantity of aggregates formed in presence of 2.217. After 5 h of aggregation, whereas the sample of Aβ 1-42 alone had almost aggregated into fibrils still containing some spherical prefibrillar aggregates (not shown), the sample incubated with 2.217 showed a more dense network of fibers which are substantially thicker. At 29 h, once the maximum aggregation was reached, a dense network of fibers with the classical morphology, is observed for Aβ 1-42 alone.

	1-42 (10 μM)
	2.157a (100 μM)
	Aβ 1-42 + 2.157a (1:10)
	Aβ 1-42 + 2.157a (1:1)

accelerates Although, the analogue of 2.228 which bears valine amino acid on peptidomimetic arm, 2.216 does not show significant activity on the aggregation process (just at the ratio of 1:10 Aβ 1-42 :compound a fiddling decrease on the fluorescence plateau was noted about -30 % associated with an increase of the t 1/2 by a factor of 1.6), more interesting result obtained with compound 2.217 (CF 3 analog of 2.216), which promotes the aggregation of Aβ 1-42 since its t 1/2 was reduced by 0.54. However, the final fluorescence was slightly decreased (-57%) indicating that the quantity and/or the morphology of the fibers were different from those of the control sample. The precursor of this molecule, 2.213 which bears the tripeptide Val-Val-Ile instead of the tetrapeptide Gly-Val-Val-Ile, did not show activity even at a high ratio A 1-42 :compound 1:10. This demonstrates that the simultaneous presence of the trans-DKP scaffold and of two arms tetrapeptide and 5trifluoroacetamido-2-methoxybenzhydrazide-based peptidomimetic, are required for the molecules to accelerate Aβ 1-42 fibrillation.

A

  Since its original development in 1981 by Jorgensson et al., CE (capillary electrophoresis) has been undergoing ups and downs. The development of fully automated systems and further CE techniques, such as micellar electrokinetic chromatography (MEKC), capillary isotachophoresis (ITP), capillary isoelectric focusing (IEF), and capillary gel electrophoresis (CGE) in the early 1990s helped to spread the use of the CE technology. 279 Unlike the other chromatography systems that have different mobile phase, like pressurized gas for the gas chromatography (GC) or high pressure pumped in high performance chromatography (HPLC) or solvent in paper chromatography, the electro osmotic flow (EOF) is a mobile phase pump in CE. The electrical potential maintained across the CE's capillary tube by the electrical circuit of the a) capillary, b) buffer, c) reservoirs, d) electrodes, and e) power supply sets up some pretty interesting conditions that make the buffer solution flow from one buffer reservoir to the other, just as if it were being pumped. So everything injected into the buffer flows with the EOF. The separation wouldn't occur unless the analytes flow towards the detector at different speeds due to their different electrophoretic mobilities. The collection and analysis of CE data has many characteristics in common with other chromatography-like analyses. Many analysts have utilized data systems developed for HPLC and GC systems. Like HPLC, capillary electrophoresis can use UV absorption detection.

	In 2004, a first CE method was proposed to separate different soluble oligomers of Aβ 1-42

  Alzheimer's disease (AD), the most common form of dementia, is a degenerative disorder of the brain that leads to memory loss. In 2013, there were 35 million persons afflicted with AD worldwide, a number expected to double by 2050.Currently, there are no disease-modifying therapies approved for AD. A number of promising targets and therapeutic strategies for the treatment of AD are under active investigation. At present, it is too early to determine if one strategy, and which strategy, will work best. Inhibition of Aβ production, assembly, and toxicity has become a primary focus for contemporary drug development programs. Among the small molecules found in the literature, some peptide derivatives have shown interesting inhibition activities on Aβ 1-42 aggregation. Yet peptide-based drug discovery could be a serious option for addressing new therapeutic challenging candidates and the number of approved peptides is dramatically increasing, in particular because they often offer greater efficacy, selectivity, specificity and a reduced risk of unforeseen side-reactions compared to small organic molecules.

	Absorbance at 190 nm (mAU)
	Spike	New species
		+ spikes
	ES
	CHAPTER 4
	ES′
		6 h
	LS	4 h
	Absorbance at 190 nm (mAU)	0 h
	Time (min)
	Figure 3.27. Electrophoretic profile of Aβ 1-42 peptide (100 µM) obtained immediately (0 h), 4 h and 6 h after sample
	reconstitution (t 0 ) in the presence of compound 2.217 at Aβ 1-42 : compound ratio of 1:1
	ES CONCLUSIONS
	ES′
		6 h
		4 h
		0 h
	Time (min)

  dramatically the early oligomerization process. Cellular evaluation on the effect of Aβ 1-42 toxicity on human neuroblastoma cells of few representative and promissing cis and trans DKP molecules is currently under investigation.

1-42 aggregation kinetics. EC evaluation confirmed that molecules 2.193, 2.205, 2.217 are able to modulate also

  t. overnight. Volatiles were removed under reduced pressure and the residue was diluted with EtOAc and consecutively washed with 1 M KHSO 4 (2×) or citric acid (10% solution), aqueous NaHCO 3 (2×) and brine (1×), dried over Na 2 SO 4 and the solvent evaporated under reduced pressure to afford the crude product.

	Synthesis procedures
	2-Methoxy-5-Nitrobenzoic acid (2.142)
	To an ice-cold suspension of 2-methoxybenzoic acid (3 g, 19.72 mmol) in concentrated aqueous
	H 2 SO 4 (36 mL) was added dropwise NH 4 NO 3 (1.74 g, 21.69 mmol, 1.1 eq). The reaction mixture
	was stirred at 0 °C for 30 min then at room temperature for 3 h. Ice cold water (150 mL) was added
	and the resulting precipitate was filtered and the pale solid was washed with water (200 mL) and
	dried (over P 2 O 5 in a desiccator) to give 2.142 as a white solid (3.1 g, 80%). R f : 0.1 (EtOAc/
	cyclohexane 80/20); mp: 149 -150 °C (litt. = 161-162 °C); 1 H NMR (200 MHz, CD 3 OD) δ 8.64 (d,
	J = 2.9 Hz, 1H), 8.40 (dd, J = 9.3, 2.9 Hz, 1H), 7.31 (d, J = 9.3 Hz, 1H), 4.02 (s, 3H); 13 C NMR
	(50 MHz, CD 3 OD) δ 165.

Hydrazinecarboxylic Acid tert-butyl ester (2.143)

	To a solution of 2.142 (3.05 g, 15.47 mmol) in dry CH 2 Cl 2 (70 mL), was added tert-
	butyloxycarbazide (2.25 g, 17.02 mmol, 1.1 eq), DIPEA (8.1 mL; 46.41 mmol, 3 eq), EDC•HCl
	(3.26 g; 17.02 mmol, 1.1 eq) and HOBt (2.3 g; 17.02 mmol, 1.1 eq). The reaction mixture was
	stirred under argon overnight. CH 2 Cl 2 was evaporated under reduced pressure and the residue was

dissolved in EtOAc (200 mL). The organic phase was washed with 10% aqueous citric acid (50 mL), water (100 mL), 10% aqueous K 2 CO 3 (50 mL), and brine (25 mL). The organic phase was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The resulting crude brown (1-[N'-(

5-Acetylamino-2-methoxybenzoyl) hydrazinocarbonyl]-2-methyl-propyl) carbamic acid tert-butyl ester (2.147a)

  Compound 2.145 (500 mg, 1.54 mmol) was deprotected according to general procedure A. The corresponding trifluoroacetate salt and Boc-L-Val-OH (369.3 mg, 1.7 mmol, 1.1 eq) were dissolved in DMF(15 mL). DIPEA (3.4 mL, 19.25 mmol, 12.5 eq), HBTU (645 mg, 1.7 mmol, 1.1 eq) and HOBt (229.7 mg, 1.7 mmol, 1.1 eq) were successively added to the reaction flask. The reaction mixture was stirred under argon at room temperature for 24 h. Then DMF was evaporated under reduced pressure and the residue was dissolved in EtOAc (200 mL), washed with 10 % aqueous citric acid (50 mL), 10 % aqueous K 2 CO 3 (50 mL). The organic phase was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (EtOAc/cyclohexane 80:20). The solid obtained was recrystallized from EtOAc/petroleum ether. Product 2.147a was obtained as a white solid (453 mg, 69 %). mp: 136-140 o C; 1 H NMR (400 MHz, CDCl 3 ) δ 12.15(d, J=7.0 Hz, 1H), 11.42 (d, J=7.0 Hz, 1H), 8.82 (s, 1H), 8.47 (d, J=7.0 Hz, 1H), 8.11 (s, 1H), 7.02 (d, J=7.0 Hz, 1H), 5.60 (d, J=8.7 Hz, 1H), 4.79 (m,1H), 4.08 (s, 3H), 2.19 (s, 3H), 2.02 (m, 1H), 1.48 (s, 9H), 0.88 (m, 6H); 13 C NMR (101 MHz, CDCl 3 ) δ 169.

  ). Product 2.147b was obtained as a white solid (3.75 g, 70 %). R f : 0.40 (AcOEt 100%); mp: 136-138 °C; 1 H NMR (400 MHz, CDCl 3 ) δ 11.9 (s, 1H),11.3 (d, J=7.0 Hz, 1H), 8.78 (s, 1H), 8.44 (d, J=9.0 Hz, 1H), 7.9 (s, 1H), 7.28 (m, 5H), 6.97 (d, J=9.0 Hz, 1H), 5.64(d, J=7.5 Hz, 1H), 5.01 (s, 2H), 4.92 (m, 1H), 4.76 (s, 1H), 4.02 (s, 3H), 3.04 (m, 2H), 2.20 (s, 3H), 1.81 (m, 1H), 1.74 (m, 1H), 1.46 (s, 9H), 1.35-1.46 (m, 4H);13 C NMR (101 MHz, CDCl 3 ) δ 169.0, 166.3,

  ; HRMS (ESI) m/z calcd for [C 29 H 39 N 5 O 8 Na] + : 608.2696 [M+Na] + ; found: 608.2697; Anal. Calcd for C 29 H 39 N 5 O 8 : C 59.47, H 6.71,

  ; MS (ESI Positive) m/z 400 [M+23] + ; Anal. Calcd for C 15 H 18 F 3 N 3 O 5 + 0.15 H 2 O: C 47.43, H 4.87,

  ). Product 2.151a was obtained as a white solid (501 mg, 80%). R f : 0.4 (EtOAc/Cyclohexane 1:1); mp: 228-230 o C; 1 H NMR (300 MHz, CDCl 3 ) δ 11.86(d, J= 9

Hz, 1H, NH)

,

11.40

(d, J= 6 Hz, 1H, NH), 9.48 (s, 1H, NH), 8.39 (s, 2H), 8.35 (d, 1H)

, 7.11 (d, J= 9 Hz, 1H), 5.52 (d, J= 9 Hz, 1H), 4.72 (t, J= 6 Hz, 1H), 4.12 (s, 3H), 1.44 (s, 9H), 0.96 (t, J= 3 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 166

  The resulting precipitate was recrystallized from EtOAc/cyclohexane to give compound 2.154 as a white solid (3.73 g, 98%). R f : 0.7 (AcOEt/ MeOH 80:20); mp: 124 °C (litt 140-141 °C); 1 H NMR (300 MHz, DMSO) δ 8.41 (bs, NH, 1H), 7.80 (s, NH, 1H), 7.34 (s, NH, 1H), 4.04 (bs, NH 2 , 2H), 1.38 (s, 9H); 13 C NMR (75 MHz, DMSO) δ 159.9, 155.9, 78.7, 28.1; MS (ESI Positive) m/z 213 [M+23] + ; Anal. Calcd for C 6 H 14N 4 O 3 : C 37.89, H 7.42, N 29.46; found C 38.03, H 7.39, N 29.53; IR ν max : 3252, 2974, 1730, 1651, 1229, 1173.

tert-Butyl-2-[2-(Phenoxycarbonyl) Hydrazinecarbonyl] Hydrazinecarboxylate (2.155)

  g, 60%) as a white solid. R f : 0.4 (EtOAc/CH 3 OH 80:20); mp: 140-142 °C; 1 H NMR (300 MHz, DMSO) δ 9.45 (s, NH, 1H), 8.56 (s, NH, 1H), 8.37 (s, NH, 1H), 8.27 (s, NH, 1H), 7.40 (m, 2H), 7.22 (m, 1H), 7.12 (m, 2H), 1.40 (s, 9H); 13 C NMR (75 MHz, DMSO) δ 157.

  .1; MS (ESI Positive MeOH) m/z 333 [M+Na] + ; Anal. Calcd for C 13 H 18N 4 O 5 : C 49.88, H 5.91, N 17.90; found C 50.29, H 5.52, N 17.87; IR ν max : 3301, 1663, 1521, 1238, 1158. carbazate (411 mg, 3.11 mmol, 1.1 eq) and the flask was left at 0 o C for 30 minutes and at r.t.overnight. The solvent was evaporated and the crude was purified directly by silica flash column chromatography (cyclohexane/EtOAc 8:2 to 7:3) to obtain product 2.159 as a light-yellow powder (848 mg, 95%). R f : 0.5 (EtOAc/MeOH 8:2); mp: 185-187 o C; 1 H NMR (300 MHz, DMSO) δ 10.36 (s, 1H), 8.99 (s, 1H), 8.30 (s, 1H), 8.01 (dd, J=11.3, 2.0Hz, 1H), 1.43 (s, 9H);13 C NMR (75 MHz, DMSO) δ 162.9, 155.3, 151.9 (J = 243.7Hz), 144.2 (J = 17.2Hz), 138.1, 122.0, 120.1, 119.1 (J = 20.2Hz), 79.3, 28.0; 19 F NMR (200 MHz, DMSO) δ -127.76 (s); HRMS Calc for C 12 H 14 FN 3 O 6 [M+Na] + (338.0764): found 338.0765; IR ν max

	(S)-tert-Butyl 2-[2-(1-Amino-3-Methyl-1-Oxobutan-2 yl) Hydrazinecarbonyl]
	Hydrazinecarboxylate (2.156)

  ; HRMS (ESI) m/z calcd for [C 23 H 33 N 2 O 8 ] + : 465.22314 [M+H] + ; found: 465.22326; Anal. Calcd for C 23 H 32 N 2 O 8

  , 1.46 (s, 9H); MS (ESI) m/z calcd: 227.2134 [M+Na] + ; found 227.2656; IR ν max

  ; HRMS Calcd for C 48 H 73 N 12 O 11 [M+H] + (993.5522): found 993.5533; Anal. Calcd for C 52 H 74 F 6 N 12 O 15 .2H 2 O: C 49.67, H 6.27,

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H NMR (300 MHz, DMSO)13 C NMR (75 MHz, DMSO)

H NMR (400 MHz, CD 3 OD)13 C NMR (101 MHz, CD 3 OD)

H NMR (400 MHz, DMSO)13 C NMR (101 MHz, DMSO)

H NMR (400 MHz, CD 3 OD) 13 C NMR (101 MHz, CD 3 OD)

H NMR (400 MHz, DMSO) 13 C NMR (101 MHz, DMSO)

C NMR (101 MHz, DMSO)1 H NMR (400 MHz, CD 3 OD)

C NMR (101 MHz, CD 3 OD)1 H NMR (400 MHz, CD 3 OD)

C NMR (101 MHz, CD 3 OD)

ACKNOWLEDGEMENTS

Firstly, I would like to thank my supervisors: Prof. Umberto Piarulli and Prof. Sandrine Ongeri for giving me the opportunity to participate in this project. Thank you for making

Scheme 2.28. Mechanism of the Staudinger reaction

The intermediate iminophosphorane reacted directly with 2-(tert-butoxycarbonyloxyimino)-2phenylacetonitrile (Boc-ON), present in the reaction medium, affording the desired Boc-protected amine in very good yield. Finally the DKP scaffold allyl esters 2.176 (Scheme 2.29), were deallylated in the presence of a catalytic amount of tetrakis (triphenylphosphine) [Pd(PPh 3 ) 4 ] and pyrrolidine, i.e. a nucleophile acting as an allyl scavenger to give the amino acid derivatives DKP1&DKP2 in good yield. The mechanism of the deprotection of allyl ester was reported in Scheme 2.29. The diketopiperazine scaffolds obtained were then used to couple to our -strands.

Scheme 2.29. Mechanism of allyl deprotection Such methodology is of special interest for peptide synthesis because the deprotection conditions are usually mild enough to be compatible with the presence of acid labile t-Bu and Boc protections. 253 A scheme of the whole synthetic route to DKP1 & DKP2 is reported below (Scheme 2.30). Med. Chem., 1999, 42, 5197-5211. Scheme 2.37. Synthesis procedure of dipeptide 2.187; i) DCC, CH 2 Cl 2 , 2h, r.t.; ii) CH 2 Cl 2 .

The dipeptide obtained in this way was then deprotected in acidic condition (TFA/DCM) and the subsequent ring closure in a basic condition lead to the formation of new protected diketopiperazine scaffold 2.178 in a quantitative yield.

Evaluation

Abstract

In this chapter, diverse methods of evaluation for the synthesized compounds will be described. The conformational search by NMR analysis and molecular modelling was performed for the one representative hairpin mimic, proving the expected structure. In order to evaluate the capability of the synthesized compounds to modulate the aggregation kinetics of Aβ1-42 peptide, Thioflavin-T fluorescence assay was carried out and the compounds exhibiting interesting results were studied in details using transmission electron microscopy (TEM) and capillary electrophoresis.

From the ThT fluorescence spectroscopy evaluation we can conclude that …

Cis-DKP compounds showed their ability to inhibit A 1-42 aggregation. The more significant inhibitory activities with cis-DKP hairpins bearing the GVVI sequence was obtained with 2.193 that at the ratio of 1:10 (Aβ 1-42 :compound) the Aβ fibrillation is delayed by increasing the t 1/2 by a factor of 2 with no effect on the fluorescence plateau. No effect was observed at lower ratio. The tetrapeptide sequence GVVI was a good choice because it improved the binding and the inhibitory activity of the hairpins.

The KLVF sequence was also incorporated in the hairpin structure instead of GVVI as the hydrophobic KLVFF sequence in the central region of A peptide (residues 16-20) appears to be important for stabilizing intra-and intermolecular interactions involved in aggregation and amyloid for-mation. 275 Small peptides mimicking this hydrophobic sequence were demonstrated to modulate the kinetics of aggregation. 276,277 The sequence KLVFFAE (16-22) has been also introduced in macrocyclic -sheet mimics by Nowick et al. 278 that showed significant inhibitory activity on A 1-42 aggregation at a A 1-42 :macrocycle ratio of 1:0.5 and on protection against A 1-42 toxicity towards PC-12 cells at a A 1-42 :macrocycle ratio of 1:5.

The presence of a free amine at the N-terminus of the hairpin increased significantly the activity of all the molecules towards the aggregation of A 1-42 , compared to the activity of the protected compounds; in particular, the protected hairpin 2.198 precursor of 2.199, and the protected hairpin 2.204 precursor of 2.205, suggesting that ionic interactions are likely to be established between the terminal amino group and acidic residues of the amyloid peptide (likely the asp 23 which is crucial for the hairpin structure of Aβ oligomeric structure). Both protected derivatives 2.198 and 2. 204 tended to self-aggregate at high concentration at 100 μM and 10 μM) and to be organized in structures that bind the ThT dye already in the control experiments.

To confirm that the whole molecule of the hairpin was essential to modulate A 1-42 aggregation, synthetic intermediates were also evaluated by ThT fluorescence assays. As expected, the 5acetamido or 5-trifluoroacetamido-2-methoxybenzhydrazide-based peptidomimetic arms 2.148a and 2.152a as well as the truncated molecules containing solely the 5-acetamido or 5trifluoroacetamido-2-methoxybenzhydrazide-based peptidomimetic arm linked to cis DKP, 2.188 and 2.200 displayed no significant activity. Surprisingly, truncated molecule containing solely the 5-trifluoroacetamido-2-methoxybenzhydrazide-based peptidomimetic arm, 2.207 (trans DKP) displayed a significant activity on the aggregtaion process. Indeed, the cis analog, 2.200 slightly 275 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, 1996, 35, 13914-13921. 276 L. O. Tjenberg, J. Näslund, F. Lindqvist, A. R. Karlström, J. Thyberg, L. Terenius, C. Nordstedt, J. Biol. Chem., 1996, 271, 8545-8548. 277 T. Takahashi, H. Mihara, Acc. Chem. Res., 2008, 41, 1309-1318. 278 P.-N. Cheng, C. Liu, M. Zhao, D. Eisenberg, J. S. Nowick, Nature Chemistry, 2012, 4, 927-933.

Transmission electron microscopy

Transmission electron microscopy uses high energy electrons (up to 300 kV accelerating voltage) which are accelerated to nearly the speed of light. The electron beam behaves like a wavefront with wavelength about a million times shorter than light waves. When an electron beam passes through a thin-section specimen of a material, electrons are scattered. A sophisticated system of electromagnetic lenses focuses the scattered electrons into an image or a diffraction pattern, or a nano-analytical spectrum, depending on the mode of operation. You can see objects to the order of a few angstrom (10 -10 m). For instance, it is possible to study small details in the cell or different materials down to near atomic levels. The possibility for high magnifications has made the TEM a valuable tool in both medical, biological and materials research.

In order to evaluate the activity of synthesized compounds the Thioflavin-T fluorescence assay was first employed but the reduction or the increase of the final fluorescence intensity of Aβ 1-42 induced by the peptidomimetics should not necessarily be interpreted quantitatively in terms of the amount of fibrillar material formed (changes in the binding constant or the quantum yield of the dye and binding of the dye to aggregated synthetic molecules might also affect the fluorescence intensity). Thus, it is important to assay any potential inhibitor or accelerator by an independent method. Transmission electron microscopy (TEM) analyses were performed on the 3 of most interesting compounds 2.193, 2.217 and 2.205 that showed the most significant effects on Aβ 1-42 aggregation during the ThT-fluorescence assays.

To complement the fluorescence assay on 2.193, transmission electron microscopy (TEM) analyses was performed validating that this product delays Aβ 1-42 fibrillation (Figure 3.21). Images were recorded at 15 h of fibrillation kinetics, corresponding to half-aggregation. Major differences were observed in quantity of aggregates formed in presence of 2.193. After 15 h of aggregation, the sample of Aβ 1-42 control mainly contained a network of globular prefibrillar aggregates and some fibers. In the Aβ sample incubated with 2.193, aggregates of the same morphology were observed but the network is significantly less dense.

General procedure A for deprotection reactions:

To a solution of the N-Boc-protected amino acid or peptide in CH 2 Cl 2 (0.13 M) was added a half volume of TFA and the reaction was stirred at r.t. for 1-3 h. The solvent was evaporated, toluene (2×) was added followed by evaporation, and then ether was added and evaporated to afford the corresponding TFA salt.

General procedure B for coupling reactions:

The N-protected amino or peptide acid (3 equiv) was dissolved in DMF (0.1 M) under a nitrogen atmosphere and the solution was cooled in an ice bath. HOAt (3 equiv), HATU (3 equiv)

and DIPEA (5 equiv) were then added. The solution was stirred at 0 °C for 1 h and then the solution of the TFA salt in DMF was added. The reaction was stirred at 0 ºC for 30 minutes to 1 h and at r. 13 C NMR (100 MHz, CDCl 3 , 50ºC) δ 175. 1, 168.1, 164.9, 157.0, 135.4, 129.4, 128.8, 128.6, 81.4, 59.5, 52.4, 47.3, 40.9, 40.6, 28.7 : 3382, 3325, 3227, 1715, 1659, 1647, 1272, 1162, 1125.

(S)-4-amino-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoic acid (2.179)

Triethylamine (16 mL, 113.5 mmol, 3 eq) was added dropwise to a stirred solution of Boc 2 O (9.9 g, 45.4 mmol, 1.2 eq) and L-asparagine(5 g, 37.8 mmol) in H 2 O/1,4-dioxane (100 mL, 1:1 v/v).

After 18 h the solution was extracted with petroleum ether (2x50 mL) and the aqueous phase was cooled on ice and carefully acidified to pH 3 by slow addition of 1 M KHSO 4 solution. The urethane was then extracted into EtOAc (3x 50 mL) and the combined organic phases were washed with brine (2x25 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give product 2.179 as a white solid (6.12 g, 70%). 1 H NMR (400 MHz, DMSO) δ 12.51 (s, 1H), δ 7.29 (s, 1H), δ 6.88 (s, 1H), δ 6.84 (d, 1H), δ 4.20 (m, 1H)

Nα-Boc-L-diaminopropionic acid (2.180)

A slurry of Boc-L-asparagine 2.179 (500 mg, 2.15 mmol) in ethyl acetate (4 mL), acetonitrile (4 mL), and water (2 mL) and iodosobenzene diacetate (837.4 mg, 2.6 mml, 1.2 eq) was sooled and stirred at 16 o C for 30 min. The temperature was raised to 20 o C and the reaction was stirred until Compound 2.188 (0.172 mmol, 1 equiv) was deprotected according to general procedure A. The resulting TFA salt was then coupled to Boc-Ile-OH (0.26 mmol, 3 eq) according to the general procedure B affording 2.189 (54 mg, 78%) as a white solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 95:5). R f : 0.2 (CH 2 Cl 2 /CH 3 OH: 94:6); 1 H NMR (400 MHz, CD 3 OD) δ 7.98 (d, J=2.5 Hz, 1H) ,7.83 (dd,J=9.0,2.5 Hz,1H),5H),7.11 (d,J=9.0 Hz,1H),5.35 (d,J=15.1 Hz,1H),4.54 (d,J=8.2 Hz,1H),4.42 (t,J=4.2 Hz,1H),4.28 (d,J=7.9 Hz,1H),2H),3.94 (s,3H),3.81 (dd,J=7.0,4.1 Hz,2H),3.15 (dd,J=16.3,4.2 Hz,1H),2.90 (dd,J=16.3,4.2 Hz,1H),1H),2.11 (s,3H),1H),(m, 1H), 1.37 (s, 9H), 1.12 (d, J=6.8 Hz, 3H), 1.06 (d, 3H, J=6.8 Hz), 0.92-0.88 (m, 6H); 13 C NMR (100 MHz, CD 3 OD) δ 174. 9, 172.1, 171.5, 171.4, 168.2, 167.3, 166.2, 157.8, 155.6, 136.8, 133.4, 129.9, 129.2, 129.0, 126.8, 124.3, 121.3, 113.3, 80.7, 60.1, 58.7, 58.4, 56.8, 53.2, 40.7, 39.1, 38.8, 32.0, 28.7, 25.9, 23.5, 19.6, 19.1, 16.2, 11.6 : 3282, 2958, 2931, 1652, 1534, 1493, 1247, 1159, 737.

Compound 2.190

Compound 2.189 (0.056 mmol, 1 eq) was deprotected according to general procedure A. The resulting TFA salt was coupled to Boc-Val (0.17 mmol, 3 eq) according to the general procedure B affording 2.190 (42 mg, 83%) as a white solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 95:5). R f : 0.15 (CH 2 Cl 2 /CH 3 OH: 94:6); mp 182-185 °C; 1 H NMR (400 MHz, CD 3 OD) δ 8.05 (d, J=2.5 Hz, 1H), 7.89 (dd, J=9.0, 2.5 Hz, 1H), 7.38-7.26 (m, 5H) 6, 174.2, 171.6, 171.1, 170.4, 168.4, 167.0, 164.2, 158.3, 155.6, 136.7, 133.7, 130.0, 129.2, 129.1, 127.0, 124.4, 120.9, 113.4, 80.4, 61.6, 58.7, 58.0, 57.8, 57.0, 53.1, 47.7, 40.3, 38.3, 38.2, 32.8, 31.8, 28.7, 28.0, 26.1, 23.6, 19.9, 19.7, 19.3, 18.8, 16.2, 11.5 : 3314, 2946, 2833, 1653, 1541, 1495, 1249, 1165, 1025.

Compound 2.191

Compound 2.190 (0.028 mmol, 1 equiv) was deprotected according to general procedure A. The resulting TFA salt was coupled to Boc-Val (0.11 mmol, 3 equiv) according to the general procedure B affording 2.191 (21 mg, 74%) as a white solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 95:5). R f : 0.2 (CH 2 Cl 2 /CH 3 OH: 94:6); 1 H NMR (400 MHz, CD 3 OD) δ 8.10 (d, J=2. 5 Hz, 1H), 8.02 (dd, J=9.1, 2.5 Hz, 1H), 7.36-7.28 (m, 5H), 7.16 (d, J=9.1 Hz, 1H), 5.41 (d, J=15.2 Hz, 1H), 4.98 (d, J=8.5 Hz, 1H), 4.76-4.71 (m, 1H), 4.39-4.42 (m, 2H), 4.13 (d, J=6.2 Hz, 1H), 4.08 (d, J=4.4 Hz, 1H), 4.03-3.97 (m, 4H), 3.92-3.88 (m, 1H), 3.77-3.69 (m, 2H), 3.30-3.28 (m, 1H), 2.88 (dd, J=17.2, 4.6 Hz, 1H), 2.11 (s, 3H)

6H), 0.93-0.83 (m, 18H); 13 C NMR (100 MHz, CD 3 OD) δ 174.2, 174.0, 173.5, 171.5, 171.1, 169.1, 168.4, 167.0, 162.5, 158.0, 155.5, 136.6, 133.8, 130.0, 129.2, 129.0, 127.2, 124.5, 124.4, 120.2, 113.5, 80.4, 61.1, 59.7, 58.6, 57.7, 57.2, 55.9, 52.9, 47.5, 43.8, 40.2, 38.3, 37.9, 33.0, 32.5, 31.9, 28.7, 26.2, 23.7, 19.9, 19.8, 19.6, 19.3, 19.0, 18.3, 17.3, 16.2, 13.1, 11.6

Compound 2.192

Compound 2.191 (0.028 mmol, 1 eq) was deprotected according to general procedure A.

The resulting TFA salt was coupled to Boc-Gly (0.11 mmol, 3 equiv) according to the general procedure B affording 2.192 (23 mg, 77%) as a light yellow solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 96:4 to 93:7). R f : 0.2 (CH 2 Cl 2 /CH 3 OH: 94:6); 1 H NMR (400 MHz, CD 3 OD) δ 8. 29-8.24 (m, 2H), 7.35-7.28 (m, 5H), 7.17 (d, J=9.1 Hz, 1H), 5.36 (d, J=15.2 Hz, 1H), 5.01 (d, J=8.9 Hz, 1H), 4.57 (d, J=7.3 Hz, 1H), 4.44-4.37 (m, 1H), 4.12 (d, J=4.1 Hz, 1H), 4.07-4.02 (m, 4H), 3.97-3.84 (m, 3H), 3.81-3.69 (m, 3H), 3.36-Compound 2.194 Compound 2.148b (0.084 mmol, 1.1 eq) that was obtained according to general procedure A, was coupled to DKP-1 (30 mg, 0.076 mmol, 1 eq) according to general procedure C, affording 2.194 (16 13 C NMR (100 MHz, CD 3 OD) δ 171.0, 170.7, 170. 1, 166.4, 164.5, 154.2, 135.6, 132.0, 128.5, 128.0, 127.8, 127.5, 127.3, 125.4, 122.9, 119.6, 112.0, 79.4, 65.9, 59.1, 55.5, 54.4, 52.6, 52.1, 47.3, 46.8, 40.8, 40.6, 40.1, 30.9, 29.0, 27.4, 22.6, 22.1, 11.7 N 12.28 Found C 56.63, H 6.54, N 11.80; IR ν max : 3304, 2932, 1643, 1531, 1484, 1251, 1168, 735, 669.

Compound 2.195

Compound 2.194 (0.05 mmol, 1 eq) was deprotected according to general procedure A.

The resulting TFA salt was then coupled to Boc-Ile-OH (0.15 mmol, 3 eq) according to the general procedure B affording 2. 195 13 C NMR (100 MHz, CD 3 OD) δ 174.3, 174.0, 173.6, 171.5, 171.1, 170.0, 168.4, 167.1, 164.9, 162.9, 158.8, 157.9, 155.6, 138.4, 136.7, 133.8, 130-128.7, 127.1, 124.4, 120.3, 113.4, 80.4, 67.3, 61.2, 59.9, 58.6, 58.0, 57.1, 53.0, 52.3, 47.7, 41.6, 40.3, 38.6, 38.3, 34.2, 34.0, 32.3, 32.4, 30.7, 28.7, 26.1, 24.0, 23.7, 19.9, 19.8, 19.1, 18.5, 16.2, 11.5

Compound 2.198

Compound 2.197 (0.022 mmol, 1 eq) was deprotected according to general procedure A.

The resulting TFA salt was coupled to Boc-Gly-OH (0.08 mmol, 3 eq) according to the general procedure B affording 2.198 (18 mg, 66%) as a white solid after purification by 6, 171.4, 171.0, 170.9, 169.3, 169.0, 168.4, 168.3, 166.2, 165.3, 162.9, 158.8, 158.5, 154.7, 154.6, 137.5, 136.7, 129.4, 129.2, 129.0, 128.6, 128.3, 128.1, 127.9, 127.4, 126.3, 125.7, 125.4, 123.6, 121.5, 112.8, 62.2, 62.1, 58.3, 57.8, 56.3, 53.6, 53.5, 51.9, 51.7, 51.3, 48.0, 46.8, 41.9, 40.6, 40.4, 39.2, 38.5, 38.4, 37.9, 32.5, 31.0, 30.7, 30.6, 29.4, 26.5, 24.1, 23.1, 21.5, 21.0, 19.2, 19.1, 18.5, 18.1, 16.7, 12.4

Compound 2.206

Compound 2.148a (0.64 mmol, 1.1 eq) that was obtained according to general procedure A, was coupled to DKP-2 (230 mg, 0.58 mmol, 1 eq) according to general procedure C, affording 2.206 (260 mg, 54%) as a white solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 96:4). 3, 170.6, 170.4, 170.0, 167.4, 166.9, 164.3, 156.5, 154.2, 135.7, 132.0, 128.4, 128.0, 127.4, 125.3, 122.9, 119.7, 112.0, 79.1, 59.2, 57.3, 55.5, 54.3, 50.9, 48.0, 46.8, 42.3, 38.7, 36.6, 30.1, 27.4, 24.3, 22.2, 18.3, 17.1, 14.4, 9.9 N 12.32; IR ν max : 3271, 2917, 1731, 1653, 1541, 1522, 1457, 1250, 1177.

Compound 2.209

Compound 2.207 (0.26 mmol, 1 equiv) was deprotected according to general procedure A. The resulting TFA salt was then coupled to Boc-Ile-OH (0.78 mmol, 3 eq) according to the general procedure B affording 2.209 (219 mg, 98%) as a light yellow solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 94:6). R f : 0.35 (CH 2 Cl 2 /CH 3 OH: 94:6); mp 79-81 o C; 1 H NMR (400 MHz, CD 3 OD) δ 8.17 3, 173.3, 170.6, 170.5, 167.4, 166.9, 164.0, 163.4, 156.4, 155.3, 135.7, 129.6, 128.4, 127.9, 127.4, 126.1, 123.9, 120.2, 112.2, 79.2, 59.2, 57.3, 55.5, 54.3, 50.8, 47.9, 46.7, 38.7, 37.3, 27.4, 27.2, 24.3, 24.1, 17.9, 14.3, 9.9 13 C NMR (100 MHz, CD 3 OD) δ 173.2, 173. 1, 170.9, 170.4, 170.1, 167.4, 166.7, 164.4, 156.6, 154.2, 135.7, 132.0, 128.4, 128.0, 127.4, 125.4, 122.8, 119.7, 111.9, 79.1, 60.1, 58.9, 57.7, 57.4, 55.5, 54.3, 50.9, 47.9, 46.5, 42.3, 38.4, 36.5, 36.4, 30.3, 27.3, 24.4, 22.1, 18.7, 18.3, 17.4, 17.2, 14.3, 11.8, 9.7 : 3295, 2965, 1640, 1541, 1495, 1297, 1168, 844.

Compound 2.211

Compound 2.209 (0.25 mmol, 1 equiv) was deprotected according to general procedure A. The resulting TFA salt was then coupled to Boc-Val-OH (0.75 mmol, 3 eq) according to the general procedure B affording 2. 211 172.9, 172.2, 171.7, 170.7, 170.5, 164.0, 163.4, 156.5, 155.4, 135.7, 128.4, 127.9, 127.4, 126.2, 123.9, 120.1, 112.2, 79.1, 60.3, 58.9, 58.4, 57.2, 55.6, 54.4, 52.8, 50.9, 48.0, 42.3, 37.4, 35.5, 34.5, 27.2, 24.5, 24.2, 18.3, 17.2, 15.8, 14.1, 11.7, 9.7 : 3282, 2964, 1688, 1633, 1497, 1468, 1207, 1174, 1018, 846.

Compound 2.214

Compound 2.212 (0.056 mmol, 1 equiv) was deprotected according to general procedure A. The resulting TFA salt was then coupled to Boc-Gly-OH (0.16 mmol, 3 eq) according to the general procedure B affording 2.214 (40 mg, 67%) as a white solid after purification by flash chromatography (CH 2 Cl 2 /CH 3 OH: 93:7). R f : 0. (d, J=8 Hz, 1H), 7.34-7.26 (m, 5H), 7.11 46.4, 40.1, 38.9, 36.5, 36.4, 31.0, 30.9, 30.1, 24.1, 19.3, 19.2, 19.1, 18.5, 18.2, 17.9, 15.2, 10.7 1, 168.0, 167.3, 164.3, 157.5, 156.9, 154.3, 137.1, 135.9, 132.1, 128.5, 128.1, 127.8, 127.5, 127.4, 125.5, 123.0, 119.6, 112.1, 79.4, 65.9, 60.1, 55.6, 52.1, 51.2, 47.1, 40.1, 37.3, 31.0, 29.0, 27.4, 22.6, 22.3 6, 172.5, 172.0, 168.8, 168.2, 165.3, 158.8, 157.8, 156.8, 156.5 (CF 3 ), 138.4, 137.1, 131.0, 129.8, 129.4, 129.2, 128.9, 128.8, 128.7, 127.7, 125.4, 121.2, 118.8, 113.6, 80.6, 67.3, 60.6, 60.5, 58.8, 57.0, 55.8, 53.3, 52.9, 52.8, 52.3, 48.4, 43.7, 41.4, 40.2, 38.8, 38.2, 38.0, 32.5, 30.7, 30.4, 28.8, 26.4, 25.7, 23.9, 17.9, 15.9, 11.4 7, 171.3, 171.1, 170.7, 170.0, 169.3, 169.2, 168.0, 166.5, 166.0, 165.9, 163.2, 163.1, 156.1, 155.4, 152.7, 137.4, 137.3, 137.2, 136.5, 132.8, 132.7, 128.5-127.2, 123.6, 121.6, 121.4, 112.7, 112.5, 78.0, 65.2, 65.0, 60.1, 60.0, 58.7, 57.6, 57.4, 56.9, 56.8, 56.4, 56.2, 53.7, 51.2, 51.0, 50.5, 50.3, 46.8, 46.5, 40.4, 38.9, 36.8, 36.6, 32.0, 31.9, 30.7, 30.5, 30.2, 30.1, 29.1, 28.2, 24.2, 23.8, 22.5, 20.7, 19.2, 18.3, 18.2, 15.3, 15.2, 10.8 9, 170.8, 170.5, 170.1, 169.3, 166.6, 166.0, 165.8, 163.2, 154.6, 136.5, 129.5, 129.4, 128.6, 127.8, 125.6, 123.5, 121.6, 117.3, 114.5, 112.9, 112.8, 58.8, 58.2, 57.6, 56.8, 56.3, 50.9, 50.4, 49.4, 46.6, 43.8, 40.2, 39.1, 38.8, 36.5, 31.6, 31.0, 30.1, 29.1, 26.7, 25.5, 24.2, 22.4, 19.3, 19.1, 18.6, 18.5, 17.9, 15.3, 10.8 4, 172.9, 171.9, 170.8, 170.4, 167.4, 166.9, 163.9, 155.3, 136.8, 135.7, 129.7, 128.7, 128.4, 128.0, 127.9, 127.4, 126.3, 126.2, 123.9, 120.1, 112.2, 79.2, 59.0, 58.8, 57.4, 55.6, 54.7, 53.2, 50.9, 40.9, 40.1, 38.8, 37.0, 36.7, 30.7, 30.0, 27.3, 24.4, 22.1, 20.4, 18.3, 17

SPPS (Solid Phase Peptide Synthesis)

The SPPS was accomplished using the semi-automatic synthesizer Biotage® Initiator™ assisted by microwaves; the Fmoc strategy and the Rink Amide MHBA Resin (100-200 mesh; loading: 1.1 mmol/g) were used. Each coupling step consisted in i) activation of the Fmoc-protected amino acid, ii) addition of the activated amino acid to the resin at the synthesizer to effect the coupling reaction and the following steps of capping, deprotection and washing.

•

Solutions and solvents

In order to execute the programs set, the synthesizer required two solutions and two solvents:

), methanol and DMF. Methanol was used only as washing solvent, DMF was used for washes and reactions (coupling, capping and deprotection).

• Resin preparation

The resin was weighted in a 10 mL vial and processed with the swelling task and the swelling-Fmoc deprotection task (see 7.2.5.1 and 7.2.5.2). At the end of these operations the resin was ready for the SPPS.

• Activation of Fmoc-AA-OH and its addition to the resin

To a solution of the desired Fmoc-AA-OH (0.4 mmol, 4.0 eq with respect to the resin -0.1 mmol, 1.0 eq) in DMF (3.5 mL) DIC (63 μL, 0.4 mmol, 4.0 eq), HOAt (54.4 mg, 0.4 mmol, 4.0 eq) and DIPEA (139 μL, 0.8 mmol, 8.0 eq) were added at 0 °C, under stirring and inert atmosphere.

After 25 min, the reaction mixture was added to the resin at the synthesizer and a cycle of coupling-capping-deprotection-washing was effected. At the end of this cycle it is possible to add another residue or effect a capping-washing cycle if the peptide sequence was completed.

• SPPS "tasks"

The programs "Tasks" used in the SPPS instrument are reported here. Each task can be modified in every parameter and it is performed under mechanical stirring at 1000 rpm.

• Swelling task DMF (3.0 mL) was added to the resin: the swelling was accomplished at r.t. in 30 min. At the end of the swelling step, the vial was empty.

• Swelling-Fmoc deprotection task DMF (3.5 mL) was added to the resin and the swelling was performed at r.t. for 15 min. Then two deprotection steps were carried out adding the solution 25% piperidine in DMF (3.0 mL for each step) to the beads: the reaction was performed at r.t. under inert atmosphere for 5 min and 15 min for the first andthe second deprotection step respectively. The resin was washed 8 times with DMF (3 mL × 30 s for every wash). At the end of each step, the vial was empty.

• Coupling-capping-Fmoc deprotection task

The activated Fmoc-AA was added to the beads and the coupling reaction assisted by microwaves started was carried out at 70 °C under inert atmosphere for 12 min. The beads were washed six times with DMF (2.5 mL × 20 s for every wash). The solution 25% Ac 2 O in DMF (3 mL) was added to the resin: the capping step was performed at r.t. under inert atmosphere for 15 min. The beads were washed six times with DMF (2.5 mL × 20 s for every wash). Two deprotection steps were carried out adding the solution 25% piperidine in DMF (3.0 mL for each step) to the beads: the reaction was performed at r.t. under inert atmosphere for 5 min and 7 min for the first and the second deprotection step respectively. The beads were washed twice with DMF, MeOH, DMF, MeOH and DMF (3.0 mL × 20 s for every wash; 3.0 mL × 20 s for the last washes in MeOH and DMF). At the end of each step, the vial was empty.

• Coupling-capping task

The activated Fmoc-AA was added to the beads and the coupling reaction assisted by microwaves started was carried out at 70 °C under inert atmosphere for 12 min. The beads were washed six times with DMF (2.5 mL × 20 s for every wash). The solution 25% Ac 2 O in DMF (3 mL) was added to the resin: the capping step was performed at r.t. under inert atmosphere for 15 min. The beads were washed six times with DMF (2.5 mL × 20 s for every wash). The beads were washed twice with DMF, MeOH, DMF, MeOH (3.0 mL × 20 s for every wash). Eventually, the beads were washed eight times with DMF (3.0 mL × 30 s). At the end of each step, the vial was empty.

• Capping task

The solution 25% Ac 2 O in DMF (3 mL) was added to the resin: the capping step was performed at r.t. under inert atmosphere for 15 min. The beads were washed six times with DMF (2.5 mL × 20 s for every wash). The beads were washed twice with DMF, MeOH, DMF (3.0 mL × 20 s for every wash). Eventually, the beads were washed twice with MeOH (3.0 mL × 30 s) and eight times with DMF (3.0 mL × 30 s). At the end of each step, the vial was empty.

Cleavage cocktail preparation

Under stirring and inert atmosphere, in a round bottom flask the cleavage cocktail was prepared adding TIS (1%V/V), EDT (2.5%V/V), H 2 O (2.5%V/V) and TFA (94%V/V) in a ratio of 3.0 mL of cleavage cocktail per 0.1 mmol of resin.

• Cleavage reaction

The cleavage procedure was formed by three cleavage cycles. The on resin-peptide, stored in a vial, was swollen first with DMF (3.5 mL), then with DCM (3.5 mL). The resin was put under inert atmosphere and the cleavage cocktail (3.0 mL per 0.1 mmol of resin to treat) was added: the reaction was carried out at r.t. under mechanical stirring and inert atmosphere. After 1 h, the liquid was filtered off under nitrogen flow and collected in a round bottom flask, where neat TFA (1.0 mL) used for washing the beads was collected too: the filtered mixture was concentrated. Another aliquot of cleavage cocktail was added to the beads and the same procedure described above was accomplished: the cleavage was completed after three cycles. All the filtered, combined fractions were concentrated and poured in cold Et 2 O was added to provoke peptide precipitation. The ether in excess was eliminated with a syringe and concentrated: this step had to be repeated at least five times. The white solid so obtained was purified with RP-HPLC and freeze-dried.

Compound 2.235

The tetrapeptide GVVI was accomplished on 0.22 mmol of resin (200 mg). The relative spps scheme was reported below. 9, 170.8, 166.1, 58.6, 57.8, 56.9, 36.9, 31.4, 30.5, 24.6, 19.6, 19.5, 18.9, 18.3, 15.8, 11.3 1, 171.8, 170.8, 168.6, 158.3, 138.1, 129.5, 128.4, 126.6, 58.2, 53.9, 52.1, 51.6, 40.9, 38.9, 38.1, 31.0, 30.9, 26.9, 24.5, 23.5, 22.0, 21.5, 21.3, 19.5, 18.4

Molecular Modelling Experimental part

Molecular mechanics studies were carried out using MacroModel from Schrödinger software suite. 287 An arbitrary initial conformation of 2.193 was energy-minimized using conjugate gradient method 288 with OPLS_2005 force field 289 and GBSA as an implicit water solvation model. 290 Convergence criterion was set to 0.05 kJ•mol -1 •Å -1 on energy gradient.

An unconstrained conformational search was then performed starting from this structure and using MCMM (Monte Carlo Multiple Minima) method 291 with the same force field, solvation model and convergence criterion. 100,000 conformations were generated, energy-minimized and deduplicated, resulting in 1583 unique conformations within a sliding energy window of 21 kJ/mol (~5 kcal/mol). Clustering of these results using centroid linkage method with 0.66 Å merge distance threshold (as suggested by Kelley penalty function) 292 yielded 69 representative conformations. Conformations exhibiting the maximum number of hydrogen bonds participating in the structure of a hairpin (3 out of the 4 possible) were selected, and the most extended one (based on the radius of gyration with the benzyl group excluded) within this group was retained.

This conformation was subjected to a 1 µs-long molecular dynamics at 310 K using the same force field and solvation model as above. Geometry optimization at the B3LYP/6-31G* level 293 with water PCM solvation model 294 using Gaussian 295 was performed on the first frame where simultaneous formation of the 4 key hydrogen bonds occurred. 3 J(H N -H α ) coupling constants were calculated according to the Karplus equation 296 from dihedral angles averaged over the trajectory.

Biological Evaluation Experimental Part

Thioflavin T was obtained from Sigma. Aβ 1-42 was purchased from American Peptide (batch 1207090T). 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 NaCl buffer (pH 7.4). Stock solutions of the tested compounds were dissolved in DMSO with the final concentration kept constant at 0.5% (v/v). APPENDIX 1 H AND 13 C SPECTRA