Eric Le Bourg
email: eric.le-bourg@univ-tlse3.fr

Is lifespan linked with developmental viability in Drosophila melanogaster?

Keywords:

Long-lived lines of flies have been selected by reproduction at older ages and correlated responses observed, and particularly whether development of flies was modified. Using these selected lines or flies subjected to larval crowding of food restriction during development, two studies have computed, among lines, a correlation between viability of eggs and larvae and mean lifespan of adults: mean lifespan was longer when the viability was lower, showing a selection of fittest flies. The present study correlates viability and mean lifespan observed during three decades in a single wild-type strain. Contrary to the previous studies, a slight but not significant positive correlation is observed, showing that lifespan can hardly increase when viability increases. Therefore, it is not clear whether development and lifespan are linked, as hypothesised by some authors.

Introduction

Three centuries ago, [START_REF] Buffon | Histoire naturelle de l'Homme (Natural history of Man)[END_REF] wrote that, among species, duration of development is linked with lifespan. This idea has been confirmed by showing that species are spread on a continuum [START_REF] Stearns | The influence of size and phylogeny on patterns of covariation among lifehistory traits in the mammals[END_REF]) with, on one side, short-lived species with a small body size, a short gestation, maturing quickly, and giving birth at short intervals to many offspring (e.g. mice and rats), and, on the other side, species with the opposite life-history strategy (e.g. elephants or primates).

Regarding, within a species, the relationship between development and ageing, [START_REF] Lints | Genetics[END_REF] reviewed the developmental theory of ageing stating that "some genes affect both traits pleiotropically, or that genes affecting both characters are closely linked". Studies of [START_REF] Lints | Influence of preimaginal environment on fecundity and ageing in Drosophila melanogaster hybrids -III. Developmental speed and life-span[END_REF] making to vary environmental conditions during development of Drosophila melanogaster flies seemed to confirm this statement, showing a negative relationship between growth rate of larvae and lifespan, but [START_REF] Lints | Genetics[END_REF] concluded that Economos andLints (1984, 1986) eventually showed that there was "an optimal growth rate on both sides of which lifespan decreased".

In addition, many studies have selected long-lived lines of flies by reproduction at older ages and observed correlated responses and particularly whether development was modified. Reviewing studies measuring in long-lived and control lines developmental time and viability of flies, i.e. the percentage of eggs reaching adulthood, [START_REF] Wit | Longevity for free? Increased reproduction with limited trade-offs in Drosophila melanogaster selected for increased life span[END_REF] reported that any outcome could be observed. These results could allow to conclude that there is no clear link between development and mean lifespan of flies, but the only study [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] that computed a correlation between the viability and mean lifespan of selected lines seemed to point in another direction (Fig. 1). [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] reported that mean lifespan decreased when viability increased in the 65-90% range, but did not indicate whether they used the longevity of males, females, or of both sexes combined.

In flies subjected to larval crowding or food restriction during development, [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF] also reported a lower mean lifespan, in each sex, when viability increased in the 60-90% range. A high viability means that most of larvae, even the poorly fit ones, reach the adult stage. These flies could have a low lifespan and this could explain the negative relationship between viability and mean lifespan of the lines. As emphasised by Zwann et al. (1991), one can hypothesise that, "if viability is decreased, only the fittest individuals survive to adults (viability selection) and this increase in mean fitness becomes evident in increased adult longevity".

Obviously, these studies do not show that, at the individual level, there is a link between viability and lifespan because viability is measured in groups and not at the individual level. In human beings, the developmental origins of adult health and disease (DOHaD) theory explains that early life events, including in utero, can have major consequences in the adult and even at old age (reviews in [START_REF] Vaiserman | Early life origins of ageing and longevity[END_REF].

The purpose of this paper is to confirm or not the results of [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] and [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF] by correlating viability and mean lifespan. The same wild-type strain was used during three decades and viability was observed for the very rearing groups used to observe lifespan in various experiments, all these groups living in very similar conditions, by controlling the age of parents and larval crowding. Eggs were transferred into vials, making possible to know the viability, developmental duration of flies, and the sex-ratio. In addition, larvae developed without larval crowding on a fresh medium, not used by other flies, and thus with no wastes of previous larvae or flies. For instance, urea, a toxic waste of larvae, which decreases viability and increases development duration [START_REF] Botella | Larval stop, delayed development and survival in overcrowded cultures of Drosophila melanogaster: effect of urea and uric acid[END_REF], increases in crowded cultures [START_REF] Henry | Hormesis-like effect of mild larval crowding on thermotolerance in Drosophila flies[END_REF]. It has been shown that males and females developing in larval crowding conditions that emerged 3 days after the first ones had a lower weight and lifespan, and females had a lower fecundity measured once at young age [START_REF] Lushchak | Larval crowding results in hormesis-like effects on longevity in Drosophila: timing of eclosion as a model[END_REF]: in all experiments reported below flies emerged 9-10 days after egg-laying.

Material and methods

The viability of the wild-type strain Meyzieu, caught at the end of the 1970s near the city of Lyon (France) was observed from 1988 to 2016 at 25°C in nearly 800 weeks (see the whole results in Le Bourg, 2021a). Groups with a very low viability (see Fig. 1 in Le Bourg, 2021a) were however not used to observe lifespan. Flies used in the present article were the control groups of several articles observing the effect of various factors on lifespan (Le [START_REF] Bourg | Hormetic effects of repeated exposures to cold at young age on longevity, aging and resistance to heat or cold shocks in Drosophila melanogaster[END_REF][START_REF] Bourg | Combined effects of suppressing live yeast and of a cold pretreatment on longevity, aging and resistance to several stresses in Drosophila melanogaster[END_REF][START_REF] Bourg | A cold stress applied at various ages can increase resistance to heat and fungal infection in aged Drosophila melanogaster flies[END_REF][START_REF] Bourg | Combined effects of two mild stresses (cold and hypergravity) on longevity, behavioral aging, and resistance to severe stresses in Drosophila melanogaster[END_REF]Bourg, , 2021b;;[START_REF] Bourg | Hypergravity and ageing in Drosophila melanogaster. 2[END_REF][START_REF] Bourg | Food restriction and longevity in Drosophila melanogaster[END_REF][START_REF] Bourg | A mild stress due to hypergravity exposure at youngage increases longevity in Drosophila melanogaster males[END_REF][START_REF] Bourg | Lack of hypergravity-associated longevity extension in Drosophila melanogaster flies overexpressing hsp70[END_REF][START_REF] Bourg | Feeding on frozen live yeast has some deleterious effects in Drosophila melanogaster[END_REF][START_REF] Le Rohellec | Contrasted effects of suppressing live yeast from food on longevity, aging and resistance to several stresses in Drosophila melanogaster[END_REF], and were subjected to the very same rearing conditions (see below). The mean viability in the present study was 74.42% (26 groups, range: 41.37-87.09%).

The mean lifespan of males was 46.09 ± 1.02 days (26 groups, range of means: 35.24-53.76 days), and that of females 52.37 ± 1.27 days (only 25 groups, females being not used in one experiment, range of means: 38.39-65.22 days). The number of harvested eggs in each group was in the 700-2975 range. The number of flies in the longevity experiments was in the 40-130 range, depending on experiments.

In order to obtain the parents of the experimental flies, flies were allowed to lay eggs for one night in a bottle containing the medium (agar, sugar, corn meal and killed yeast) containing a mould inhibitor (para-hydroxymethyl-benzoic acid) and enriched with live yeast at the surface of the medium. This medium was used in all experiments. Flies emerging 9-10 days after egg-laying were transferred to fresh bottles: they are the parents of the experimental flies. Experimental flies were obtained as follows: eggs laid by ca. 5 day-old parents during one night on a Petri dish containing the medium coloured with charcoal were transferred by batches of 25 into 80 ml glass vials. At emergence, flies were transferred in groups of 15 virgin flies of the same sex into 20 ml polystyrene vials. Flies lived at 25°C (LD: 12-12) at the Earth gravity level (1 g) and were transferred twice a week to new vials. To sum up, all groups of flies of the strain used in this article were subjected to the very same conditions. In such conditions, one can expect that viability and lifespan can vary among groups of flies because of random variations, showing a relationship between these traits if it indeed exists. However, a caveat is that, obviously, changes did occur between 1988 and 2016. The stocks of agar, sugar, corn meal, killed yeast were renewed when they were exhausted, and new live yeast was bought very often, as it cannot be used for a long time, even if kept at 4°C. In addition, even if the temperature was always the same, different incubators could be used. Therefore, "very same conditions" should not be understood as meaning that all things were equal for three decades, but simply that care was taken to use similar rearing conditions: temperature, larval density, age of parents of experimental flies, transfers to new vials.

Results and discussion

Figure 2 shows the relationship between viability and lifespan. In each sex, the correlation is positive but not significant (r = 0.371, n = 26 in males, and r = 0.282, n = 25 in females). Pooling the two sexes, as [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF] did, made it became just significant (r = 0.283, p = 0.0440, n = 51). These results thus show hardly a positive correlation between viability and mean lifespan, but in no case that longevity increased when viability decreased, contrary to the results of [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] and [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF]. However, a significant correlation was observed in this last study only if both sexes were pooled, the effect being marginally significant in each sex (Fig. 1). The present results show that viability does not help to predict lifespan of flies able to reach the adult stage. Observing individual durations of development and lifespans could allow to know whether development is linked to lifespan at the individual level, but a previous study of [START_REF] Mayer | Developmental time and adult longevity in two strains of Drosophila melanogaster in a constant low-stress environment[END_REF] only showed a negligible relationship between these traits in both sexes, if any.

One of the strengths of the present study is that viability and lifespan were observed on the same groups and that the rearing conditions were the same for all groups during nearly three decades. One could thus expect that variation of viability and lifespan is linked to random environmental variation and possibly to genetic drift. For instance, larval crowding is not controlled in the stocks of flies kept in bottles for years, if not decades, and this could have an effect on lifespan. [START_REF] Lints | Unexplained variations in life span of the Oregon-R strain of Drosophila melanogaster over a four-year period[END_REF] showed lifespan variations during a 4-year period in a single strain and were unable to explain them. Could the discrepancy between the present results and those of [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] and [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF] be explained by the fact that these authors manipulated developmental conditions, by modifying larval density or food quantity [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF], or age at reproduction [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF]? Indeed, the procedure of the present article is similar to that of [START_REF] Mayer | Developmental time and adult longevity in two strains of Drosophila melanogaster in a constant low-stress environment[END_REF] who, "instead of perturbing the systems of development and aging, … attempted to observe them in a "free running" state by minimizing stresses known to influence the duration of development or the length of imaginal life span".

For the time being, it seems possible to conclude that, when flies develop in controlled conditions, there is no clear relationship between viability and mean lifespan, but these results do not allow concluding that there is not any link between development and ageing, particularly in human beings.

In humans, one of the first studies showing the importance of early-life events was that of [START_REF] Barker | Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales[END_REF] who reported that the 1968-1978 ischaemic heart disease prevalence in various sites of UK, differing by their wealth, was strongly linked to infant mortality in [1921][1922][1923][1924][1925] in the same sites. Because both infant mortality in 1921-1925 and ischaemic heart disease prevalence in the 1970s were higher in lower socio-economic groups, the authors suggested that "the geographical distribution of ischaemic heart disease in England and Wales reflects variations in nutrition in early life". In a subsequent study, [START_REF] Osmond | Early growth and death from cardiovascular disease in women[END_REF] showed that low birth weight is linked to death from cardiovascular disease in both sexes. It is thus not surprising that women suffering from the 1944 Dutch winter famine during their mid-to late gestation gave birth to lowweight babies, and, in addition, that famine during early gestation, even if birth weight was normal, was also associated with cardio-vascular disease at middle age (review in [START_REF] Preston | Developmental origins of health span and life span: a mini-review[END_REF] and death rate in women, but not in men [START_REF] Van Abeelen | Survival effects of prenatal famine exposure[END_REF]. On the whole, early-life events, and particularly foetal nutrition, can have long-lasting adverse consequences in humans (review in [START_REF] Vaiserman | Developmental programming of aging trajectory[END_REF] and results in animal models are not at variance (Tarry-Adkins and Ozanne 2017).

Figure 1. Relationship between viability and mean lifespan in [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] and [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF]. Lifespan is observed, respectively at 25 and 29°C, but the development is done at 25°C in both studies. For the [START_REF] Buck | Extended longevity in Drosophila is consistently associated with a decrease in developmental viability[END_REF] study, lifespan data were determined from Figure 5 and viability ones were extracted from Table 2. For the Zwaan et al.'s (1991) study, data were extracted from Tables 3,4, 5, 7. The correlation is not significant in [START_REF] Zwaan | On the developmental theory of ageing. I. Starvation resistance and longevity in Drosophila melanogaster in relation to pre-adult breeding conditions[END_REF] if sexes are analysed separately, but the authors pooled sexes, which made the correlation being significant (in the figure:

  r = -0.546, p = 0.0057, n = 24; in Zwaan et al. (1991): r = -0.59, p < 0.01). The correlation reported by Buck et al. (2000) is r = -0.791 (r = -0.794 in the figure).

Figure 2 .

 2 Figure 2. Relationship between viability and mean lifespan in the Meyzieu wild-type strain in flies observed during three decades.

Funding details

This research did not receive any specific grant.

Disclosure statement

No conflict of interest.


Manon Garcia

Laurent Hoffer

Raphaël Leblanc

Fatiha Benmansour

Mikael Feracci

Carine Derviaux

Antonio Luis Egea-Jimenez

Philippe Roche

Pascale Zimmermann

Xavier Morelli

Karine Barral
email: karine.barral@inserm.fr

Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread

Keywords: FBDD, fragment-based drug design, PPI, protein-protein interaction, PDB, protein lipophilic Ligand Efficiency, BB, building Homogeneous Time Resolved Fluorescence, LC/MS, liquid chromatography / mass spectroscopy, NMR, nuclear magnetic resonance, rt, room temperature, EDCI, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide, THF, tetrahydrofuran, FA, formic acid, VS, virtual screening, SAR, Structure-activity relationship Fragment-based drug design Small molecule PPI inhibitor Syntenin-Syndecan interaction inhibitors Exosomes biogenesis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Exosomes are lipid-bilayer nanovesicles, released from most cell types, which appear to be important during cancer progression, with a role in metastatic niche formation and tumor immune escape [START_REF] Greening | Exosomes and their roles in immune regulation and cancer[END_REF][START_REF] Wortzel | Exosome-Mediated Metastasis: Communication from a Distance[END_REF]. 'Cancer exosomes' derived from tumor cells are thus gaining considerable attention as oncotargets [START_REF] Rajagopal | The Origin and Functions of Exosomes in Cancer[END_REF]. Syntenin, a 32 kDa PDZ-tandem (PDZ1 and PDZ2) intracellular protein, has a large variety and diversity of interaction partners, including syndecans [START_REF] Grootjans | Syntenin, a PDZ protein that binds syndecan cytoplasmic domains[END_REF]. Syndecans (SDCs) are transmembrane proteins carrying heparin sulfate chains involved in numerous cellular processes, including cell-cell and cell-matrix adhesion, signal transduction, and vesicular trafficking [START_REF] Lambaerts | Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish[END_REF][START_REF] Couchman | Transmembrane signaling proteoglycans[END_REF][START_REF] Christianson | Heparan sulfate proteoglycan as a cell-surface endocytosis receptor[END_REF]. P. Zimmermann and coworkers have identified a molecular pathway, supported by the syndecan heparan sulfate proteoglycans and their adaptor protein syntenin, that binds to the cytosolic tail of syndecans, which regulates the biogenesis and the uptake of a specific subclass of exosome [START_REF] Friand | Syntenin and syndecan in the biogenesis of exosomes[END_REF]. Recent studies established that synteninexosomes might be implicated in the processes of malignant cell invasion and the establishment of niches that promote tumor metastasis [START_REF] Kegelman | Targeting tumor invasion: the roles of MDA-9/Syntenin[END_REF]. Indeed, syntenin expression is upregulated in many cancer cell lines (carcinomas and others cancers) suggesting that it may be an effective target to intervene in metastasis since gain of syntenin expression in tumors cells has been associated with the invasion and the metastatic potential of various solid cancers [START_REF] Das | MDA-9/Syntenin (SDCBP): Novel gene and therapeutic target for cancer metastasis[END_REF].

As knowledge has grown regarding cellular protein interaction networks and their role in numerous cell disorders, including cancer, targeting protein-protein interactions (PPI) has emerged as an encouraging therapeutic approach for the treatment of solid tumors or hematologic malignancies [START_REF] Makley | Expanding the number of 'druggable' targets: non-enzymes and protein-protein interactions[END_REF][START_REF] Cierpicki | Targeting protein-protein interactions in hematologic malignancies: still a challenge or a great opportunity for future therapies?[END_REF][START_REF] Bosc | Fr-PPIChem: An Academic Compound Library Dedicated to Protein-Protein Interactions[END_REF]. Promising results have been obtained with PPI inhibitors such as p53, BclxL or more recently bromodomain inhibitors [START_REF] Bosc | Fr-PPIChem: An Academic Compound Library Dedicated to Protein-Protein Interactions[END_REF][START_REF] Filippakopoulos | Selective inhibition of BET bromodomains[END_REF][START_REF] Ulucan | Druggability of dynamic protein-protein interfaces[END_REF][START_REF] Jung | Targeting BET bromodomains for cancer treatment[END_REF][START_REF] Milhas | Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery[END_REF][START_REF] Faivre | Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer[END_REF]. We therefore aim to apply a Fragment-Based Drug Design (FBDD) strategy for the rapid identification of starting hits and their subsequent optimization into compounds that can selectively disrupt synteninsyndecan PDZ-domain mediated interactions showing known implications in cancer development and dissemination.

In a previous study, Leblanc et al. screened a fragment library, containing 139 potential PDZinhibitory fragments, on syntenin-syndecan-2 complex by HTRF. Five fragments were identified and tested for their selectivity in two other PDZ-mediated interaction complexes implicated in oncology, namely GRASP/Jam-C and Erbin/P0071 complexes [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF]. The most potent and selective fragment, C58 (Figure 1), was confirmed in a secondary dose-response screening to be a reliable inhibitor of syntenin-syndecan interaction with an IC50 of 350 µM (LE = 0.25). Additional experiment on MCF7 breast carcinoma cells established that C58 decreases MCF7 migration and colony formation, alters mammosphere formation and exosomal release [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF]. Taken together, these data clearly indicated that fragment C58 is a selective inhibitor of the syntenin-exosomal pathway.

It should be noted that PDZ domains belong to protein-protein interaction (PPI) networks, and were considered as poorly druggable targets for the development of potent small organic probes. This was confirmed by the screening of large compound libraries in which no potent hits were identified [START_REF] Chen | Highthroughput screen for small molecule inhibitors of Mint1-PDZ domains[END_REF]. However, several studies have reported that small organic compounds were able to disrupt PPIs between PDZ domains and their endogenous protein partners [START_REF] Borg | PDZ Mediated Interactions[END_REF]. Some of these studies also included intensive structure-activity relationship (SAR) studies in which dozens analogs were synthesized and tested. As expected, due to the poor druggable nature of PDZ domains, most non-peptide compounds exhibited moderate inhibition, i.e. IC50 in the 10 micromolar range. One notable exception was recently reported with the development of high affinity compounds (IC50 value below 100 nM) for the Pick1 PDZ domain [START_REF] Lin | Potent PDZ-Domain PICK1 Inhibitors that Modulate Amyloid Beta-Mediated Synaptic Dysfunction[END_REF]. To our knowledge, this is the most potent non-peptide PDZ inhibitor discovered to date. Regarding the syntenin protein, the most potent organic compound reported to date targeted its first PDZ domain and had a Kd value of 21 µM (LE = 0.16) [START_REF] Kegelman | Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin[END_REF]. In this study, we performed SAR analysis and fragment hit optimization aiming to improve the affinity of C58 for the second PDZ domain of Syntenin. X-ray crystallographic structures and molecular modeling were used to guide the fragment optimization process in order to generate new analogues by adding chemical groups that might establish strong interactions with syntenin-PDZ2 domain.

2-Results and discussion

2-1. Fragment expansion and SAR around analogues of C58

A hit identified from screening traditionally require expansion to provide evidence SAR based upon chemical similarity. Therefore, fragment C58 was used as starting point to select the best fragment hit involved in the subsequent optimization process. For this purpose, a rapid fragment hit expansion has been considered by synthetizing nearest analogues around fragment C58 (Figure 1). Figure 1. A. Crystal structure of syntenin PDZ2 domain in complex with compound C58 (PDBID: 6R9H). Compound C58 is depicted in cylinder representation dark green carbon atom.

Nitrogen, oxygen, chlorine and sulfur are colored in blue, red, green and yellow, respectively. Small red spheres represent water molecules around the binding sites. Hydrogen bond interactions are displayed as black dotted lines. Amino acid residues highlighted are those establishing Van der Waals interactions with compound C58. B. 2D structure of hit fragment C58. C. 2D structures of putatively interesting explored modifications around fragment C58.

X-ray crystal structure of C58 bound to syntenin was resolved (PDB code 6R9H) and revealed that C58 is located within the syntenin-PDZ2 domain on the same binding pocket as syndecan-2 peptide as shown in Figure 1A (a superimposition view of binding mode of compounds C58 and C-terminal end peptide of Syndecan is given Figure S1A in Supp. Data). The phenyl ring of fragment C58 exhibits a strong π-stacking with Phe213, its nitrogen atom interacts with the backbone carbonyl of Phe211 through hydrogen bonds and its carboxylic acid forms the expected canonical hydrogen-bonding interactions with backbone amide group of Val209 and Gly210 [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF]. Based on these structural data, we explored four different modifications to improve the affinity of C58 while maintaining its canonical binding mode (Figure 1C).

The main modification involved the terminal hydrophobic moiety of fragment C58. Indeed, PDZ domains are known to recognize C-terminal hydrophobic moieties from their partners. The last residue from syntenin endogenous ligands (syndecan proteins) is an alanine that contains a very small hydrophobic sidechain. Surprisingly, structural data highlighted that PDZ domain from syntenin was also able to bind peptides with large hydrophobic terminal residue (PDB 1W9E). However, binding of the phenylalanine required an induced fit to accommodate the large benzyl group. Thus, 3 different terminal modifications (Ala-like, Val-like and Phelike) were investigated in the SAR studies. A thioether function was also present in the original fragment hit. This sub-optimal moiety, from a medicinal chemistry point of view, was replaced by 1) its similar ether function and 2) with one methylene spacer in the SAR studies. Finally, the influence of the chlorine atom was also assessed by synthesizing the chlorine-free version of each compound.

In a first stage, we aimed to synthesize analogues with a spacer length of two atoms (S-CH2 or O-CH2) between the phenyl ring and the amide bond. A general synthesis of amide derivatives is depicted in Scheme 1. To proceed, we directly condensed the commercial acetyl chloride derivatives (1-4) with the corresponding L-aminoacid methyl ester hydrochloride (Alanine or Valine) in a mixture dichloromethane/trimethylamine at room temperature [START_REF] Elidrisi | Synthesis and NMR elucidation of novel amino acid cavitand derivatives[END_REF]. Methyl ester intermediates 5-12 obtained with good to excellent yields were then hydrolysed with LiOH to give derivatives 13-20 [START_REF] Elidrisi | Pore formation in phospholipid bilayers by amphiphilic cavitands[END_REF].

Scheme 1. Synthesis of analogues 13-20.

In a second stage, we performed the synthesis of analogues with a spacer length of three atoms (S-CH2-CH2, O-CH2-CH2 or (CH2)3) between the phenyl ring and the amide bond (Scheme 2). Indeed, molecular modeling studies, relying on the X-ray structure of C58 ( 16), suggested that the addition of one carbon spacer in the compound structure could maintain both the PDZ canonical binding mode with the carboxylate group and π-stacking with Phe213, while optimizing the torsion angles from the flexible carbohydrate spacer.

Thus, thiophenol and 4-chlorothiophenol were converted to 3-(phenylthio)propanoic acid 23 and 3-(4-chlorophenylthio)propanoic acid 24 by S-alkylation with 3-chloropropionic acid under alkaline conditions [START_REF] Lamberth | Synthesis and fungicidal activity of N-2-(3-methoxy-4-propargyloxy) phenethyl amides. Part 3: stretched and heterocyclic mandelamide oomyceticides[END_REF][START_REF] Song | Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues[END_REF]. Then, derivatives 23 and 24, as well as commercial propionic acid derivatives 25-27, were condensed with the corresponding L-aminoacid methyl ester hydrochloride (Alanine, Valine or Phenylalanine) in a mixture dichloromethane/trimethylamine at room temperature in the presence of coupling reagent EDCI [START_REF] Matsumoto | Organic reactions mediated by electrochemically generated ArS+[END_REF][START_REF] Liu | 3-Hydrazido and 3-Hydrazono Derivatives of Tenuazonic Acid and their Herbicide Evaluation[END_REF]. As previously, methyl ester intermediates 28-40 obtained with good yields were then hydrolysed with LiOH to give derivatives 41-53 [START_REF] Elidrisi | Pore formation in phospholipid bilayers by amphiphilic cavitands[END_REF]. A primary screening was performed by HTRF with analogues 13-20 and 41-53, at a concentration of 400µM, against Syntenin-Syndecan2 complex (GST-S1FL/SDC2) (Table 1).

None of the analogues 13-20 with a spacer length of two atoms displayed a better affinity than fragment C58 (analogue 16, inhibition of 56%, IC50 of 350 µM). These data revealed the importance of (i) valine as terminal amino acid residue because the corresponding alanine analogue 15 shows an inhibition of 25%, (ii) sulphur atom versus oxygen in the spacer because the corresponding phenoxy analogue 20 exhibits an inhibition of 16%, and (iii) chlorine as para substituent because the corresponding non-substituted analogue 14 displays an inhibition of 20%.

Table 1. Inhibition of Syntenin-Syndecan2 complex interaction in the presence of analogues 13-20 and 41-53 at 400 µM confirmed in counter screening experiment using HTRF assay. To validate and characterize the binding mode of compound 45, we soaked it with syntenin crystals. The X-ray crystal structure of 45 bound to syntenin was resolved (PDB code 6RLC)

and its binding mode into syntenin PDZ2 domain was confirmed with the expected low strain energy in the carbohydrate spacer (Figure 3). The phenyl ring of 45 exhibits a strong π-stacking with Phe213, its nitrogen atom makes a hydrogen-bonding interaction with the backbone carbonyl of Phe211 and its carboxylic acid forms the expected canonical hydrogen-bonding interactions with backbone amide group of Val209 and Gly210 [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF]. Therefore, optimized fragment 45 maintains canonical binding mode and presents a better biological evaluation than the previously identified fragment C58 (a superimposition view of binding mode of compounds C58 and 45 with syntenin is given Figure S1B in Supp. Data). For these reasons, we performed the subsequent growing optimization process starting from fragment 45.

2-2. Optimization of fragment 45 by fragment growing

Based on X-ray crystallographic structure of syntenin in complex with fragment 45, our recently published in silico chemistry driven strategy DOTS was applied to the design of optimized inhibitors [START_REF] Hoffer | Integrated Strategy for Lead Optimization Based on Fragment Growing: The Diversity-Oriented-Target-Focused-Synthesis Approach[END_REF]. The in silico pipeline can be briefly described as follows (see experimental section for details): a focused virtual chemical library was first designed around fragment 45.

The growing strategy consists in exploring the chemical space around this core starting from the logical p-chlorophenyl optimization vector that was naturally included in its structure. Then, the resulting chemical library was virtually screened using the S4MPLE molecular modeling tool [START_REF] Hoffer | Integrated Strategy for Lead Optimization Based on Fragment Growing: The Diversity-Oriented-Target-Focused-Synthesis Approach[END_REF][START_REF] Hoffer | In Silico Fragment-Based Drug Discovery: Setup and Validation of a Fragment-to-Lead Computational Protocol Using S4MPLE[END_REF][START_REF] Hoffer | S4MPLE-Sampler for Multiple Protein-Ligand Entities: Methodology and Rigid-Site Docking Benchmarking[END_REF] with constraints on the shared substructure. Constraints were added in the first stage of the simulation to maintain the original binding mode of the fragment, according to the generic growing paradigm in fragment-based drug discovery. In other words, atoms from fragment 45 were constrained while newly added atoms from virtual synthesis were free to explore putative adjacent sub-pockets without any constraints. The goal was to either catch additional interactions in the vicinity of compound 45 or to extend this fragment towards K214, N215, K250 and D251 residues (see illustration in Figure S2 in Supp. Data), because these residues were known to interact with PIP2 ligand in previously published "Syntenin-PDZdomain/peptide/PIP2" ternary complexes (PDB 4Z33) [START_REF] Egea-Jimenez | Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling[END_REF]. However, it is important to mention that these polar and flexible solvent exposed residues do not define a clear druggable subpocket. This was confirmed by the FTMap web-server [START_REF] Kozakov | The FTMap family of web servers for determining and characterizing ligandbinding hot spots of proteins[END_REF], that identifies binding hot spots of macromolecules using in silico mapping of small organic probes. It has been proven that regions that can bind clusters of multiple probes correlate with known binding hot spots of macromolecules. Using the Syntenin 4Z33 PDB structure as input, the computational mapping identified 2 main regions within the PDZ2-domain that are predicted to be potential binding hot spots. The first concerns the carboxylate binding loop (V209, G210) and the second is located near Phe213. It should be noted that both regions perfectly match the interaction residues with fragment 45 and bound peptide. However, no particular binding hot spots regions were predicted for K214, N215, K250 and D251 residues. The FTMap simulation confirmed that this PIP2-binding area was not a well-defined druggable sub-pocket (see Figure S2 in Supp. Data).

The final selection of compounds was achieved after visual inspection of the top100 best poses with a focus on compounds that were predicted to interact with previously listed residues. In the end, ten compounds were selected using Suzuki-Miyaura coupling reaction (Table 2). At this stage, we focused on the synthesis of biaryl compounds reported in Table 2, and we decided to explore a strategy using a Suzuki-Miyaura coupling reaction that would allow more flexibility in introducing a large diversity of functionalities. To improve the coupling, we set up a general procedure starting with a 4-bromophenyl intermediate (more reactive substrate than 4-chlorophenyl derivatives depicted in Scheme 1) to generate the selected library. As previously described, 4-bromothiophenol was converted to 3-(4-bromophenylthio)propanoic

N N B O O
acid 55 by S-alkylation with 3-chloropropionic acid under alkaline conditions with excellent yield (Figure 4A) [START_REF] Lamberth | Synthesis and fungicidal activity of N-2-(3-methoxy-4-propargyloxy) phenethyl amides. Part 3: stretched and heterocyclic mandelamide oomyceticides[END_REF][START_REF] Song | Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues[END_REF]. Compound 55 was condensed with L-valine methyl ester hydrochloride in the presence of EDCI yielded the desired 4-bromophenyl intermediate 56 [START_REF] Matsumoto | Organic reactions mediated by electrochemically generated ArS+[END_REF][START_REF] Liu | 3-Hydrazido and 3-Hydrazono Derivatives of Tenuazonic Acid and their Herbicide Evaluation[END_REF]. To compare the effect of halogens (Br vs Cl, analogue 45) as para substituent, intermediate 56 was hydrolysed with LiOH to give derivative 57 [START_REF] Elidrisi | Pore formation in phospholipid bilayers by amphiphilic cavitands[END_REF]. Then, we turned to substitution of the bromo group at the C4-position under standard Suzuki-Miyaura conditions in the presence of Pd(PPh3)4 under aqueous basic conditions [START_REF] Matziari | Evaluation of P-1'-diversified phosphinic peptides leads to the development of highly selective inhibitors of MMP-11[END_REF]. After few reaction optimization such as performing the reaction with phenylboronic acid and 56 using different bases (sodium, providing the corresponding carboxylic acid derivatives 58-66 with good to moderate yields (Figure 4A).

potassium
Despite several attempts in different reaction conditions, intermediate 56 failed to react with the commercial pyrazole boronic ester VS10 (see Table 2). The nitrogen-rich heterocycle here could act as a ligand causing side reactions. S1 in Supp. Data.

The newly synthetized derivatives 57-66 were then evaluated for inhibitory activity on Syntenin-Syndecan2 complex through HTRF assay. Compounds 57 and 62 displayed the lowest IC50 values, 47 µM and 7.5 µM respectively (Figure 4B). Analogue 57 corresponds to the brominated version of 45 (Br vs Cl, as para substituent) and analogue 62 contains a phenyl ring substituted in para position with a methyl mesylate instead of chlorine.

Compounds 45, 57 and 62 were investigated using cellular assays. We observed an unusual MCF7 exosomes secretion profile after treatment with compound 62 suggesting a tendency to aggregate (see Figure S3 in Supp. Data). For these reasons, IC50 values for compounds 57 and 62 were also evaluated in presence of 0.01% NP40 detergent to eliminate potential false positive due to aggregation effects in bioassays. IC50 value was not significantly affected by the detergent for compound 57 (51 µM and 47 µM, without and with detergent, respectively).

However, IC50 value for compound 62 increased to 204 µM in the presence of NP40 (Figure 4B). The goal of our growing strategy was to target residues K214, N215, K250, D251 and S252 residues, because they were either residues in the nearby environment of original fragment or residues involved in binding of the PIP2 ligand in 4Z33 ternary complex. However, the growing stage failed to identify any compound with an improved affinity compared to compound 45 (Figure 4B). Since no better compound was identified using the first small growing round and that targeted residues were not predicted to represent some well-defined pocket, the growing approach was stopped at this stage. In the end, compounds 45 and 57 are the two most potent inhibitors in our study.

Subsequently, exosomal fractions released by MCF7 breast cancer cells treated with compounds 45 and 57 (and their corresponding methyl esters 32 and 56 used as controls) were then analysed by western blot, investigating their effect on both syntenin-dependent andindependent exosomal markers (Figure 5A). The amounts of exosomal syntenin were significantly decreased when cells were treated with with 100 µM of compound 45, but not with 100 µM of C58 [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF] or the inactive compound 32

(ester analogue of 45 used as control, Figure 5A & 5B). On the other hand, compound 57 exhibits a tendency to decrease the amounts of exosomal syntenin, but not its negative control 56 (Figure 5A & 5B). Consistent with our previous study [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF], CD63 and the synteninindependent exosomal marker Flotillin-1 remained unaffected regardless of the treatment

(Figure 5A & 5B).
As expected, the different treatments do not alter the cellular levels (Figure 5A & 5B).

3-Conclusion

We established a FBDD process against syntenin-syndecan interactions based on a structureguided optimization strategy. Initial fragment hit C58 identified by HTRF screening was an attractive starting point for inhibitory design. X-ray crystallographic structures and computational docking were used to guide the fragment growing and allowed the synthesis of the first selective syntenin-PDZ2 inhibitors. Investigations into their capacity to control exosomal pathways validate syntenin-PDZ2 as a promising therapeutic target for inhibiting oncogenic processes and could pave the way for effective therapies for primary tumors and metastasis in the future.

4-Experimental section

4-1. Chemistry

All commercial reagents and solvents were purchased from Sigma Aldrich, Fisher Scientific or Fluorochem companies. Furthermore, all dry solvents were obtained via Sigma Aldrich with Sure/Seal™ system and regular solvents were obtained via Sigma Aldrich at technical grade.

Analytical thin layer chromatographiy (TLC) of the reactions was performed on silica gel 60F examined with UV light (λ = 254 nm) and Ninhydrin Spray. Preparative flash column chromatographies were performed using silica gel (Merck) G60 230-240 under compressed air.

The 1 H NMR and 13

4-1-1. General procedure for the synthesis of methyl propanoate and butanoate intermediates

5-12

To a stirred solution of L-aminoacid methyl ester hydrochloride (Alanine or Valine) (4 eq, 4.0 mmol) and triethylamine (5 eq, 5 mmol) in dry dichloromethane was added dropwise the corresponding acetyl chloride derivative (1-4) (1 eq, 1.0 mmol) at 4°C. The mixture was stirred under argon atmosphere at 4°C during 1 h then 1-3h at room temperature. After completion, the resulting mixture was washed twice with 1N HCl solution, once with water, once with brine, dried over Na2SO4, and evaporated in vacuo. Crude was purified by column chromatography eluting with dichloromethane-methanol (100 to 98:2).

4-1-3. General procedure for the synthesis of propanoic acid derivatives 23, 24 and 55

To a stirred solution of commercial thiophenol derivative (1 eq, 5.0 mmol) and sodium hydroxide (2.4 eq, 12.0 mmol) in water, aqueous solution of 3-chloropropionic acid was added dropwise (1.7 eq, 8.5 mmol). The mixture was refluxed during 2-16 h. After completion, 1N

HCl solution was added dropwise at 4°C until pH 1, to afford a crude precipitate which was filtered and dried. The crude residue does not require further purification. Hz, 1H), 2.93 (dd, J = 13.7 Hz and J = 9.2 Hz, 1H), 2.58 (t, J = 6.0 Hz, 2H); 13 (td, J = 7.2 Hz and 1.6 Hz, 2H), 2.07 (m, 1H), 0.67 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H). [M+H] + and isotopic peak 376.1; white solid. Yield = 72%.

4-1-3-1. 3-(phenylthio)propanoic acid 23

4-1-5. General procedure for Suzuki synthesis of valine derivatives 58-66

To a solution of compound 56 (1eq, 0.2 mmol), boronic acid or ester derivative (1.5 eq, 0.3 mmol) and Pd[P(Ph3)]4 (5 mol%) in a mixture toluene/ethanol (2/0.5 mL) was added an aqueous solution of Na2CO3 (2 eq, 0.4 mmol, 0.5 mL). The resulting mixture was refluxed under argon atmosphere during 18 h, cooled and then concentrated. To the resulting residue was added dropwise 1N HCl solution 4°C until pH 1, the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed once with water, once with brine, dried over Na2SO4, and evaporated under vacuum. Crude was purified by column chromatography eluting with dichloromethane-methanol (98:2 to 80:20).

4-1-5-1. (2S)-3-methyl-2-(3-{[4-(3-sulfamoylphenyl)phenyl]sulfanyl}propanamido)butanoic

acid 58 1

4-1-5-2.(2S)-3-methyl-2-[3-({4-[4-(piperidine-1sulfonyl)phenyl]phenyl}sulfanyl)propanamido]butanoic acid

4-2. HTRF GST-S1FL/SDC2 assay 4-2-1. Production of GST-syntenin full length for HTRF experiments

Competent E.coli (ER2566 strain) cells were transformed with the human syntenin1 pGEX-5X expression vector (GE Healthcare). Expression of N-terminally GST-tagged syntenin-1 full length was induced overnight at 30 °C and by the addition of 0.4mM IPTG (Fisher Scientific).

Protein was purified using GSTrap4B columns 28-4017-45 (GE Healthcare).

4-2-2. HTRF screen

HTRF assays were performed in white 96Well Small Volume™HiBase Polystyrene Microplates (Greiner) with a total working volume of 100μL, as described previously [START_REF] Milhas | Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery[END_REF].

Briefly, compounds were firstly dispensed into the wells at 400µM for the primary screen or with serial DMSO dilutions for IC50 measurement assays. Primary screening assays have been performed in monoplicate while IC50 measurements were performed in triplicates. All HTRF reagents were purchased from CisBio Bioassays and reconstituted according to the supplier protocols. The plates were spin for 3 min at 500g and then incubated at 4°C for 16 h. The HTRF signals were recorded on a POLAR star Omega plate reader (BMG Labtech) with an excitation filter at 337 nm and fluorescence wavelength measurement at 620 and 665 nm, an integration delay of 60μs and an integration time of 400μs. Results were analyzed with a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)]*10 4 . Percentage of inhibition was calculated using the following equation: % inhibition = [(compound signal)-(min signal)]/[(max signal)-(min signal)]*100, where 'max signal' is the signal ratio with the compound vehicle alone (DMSO) and 'min signal' the signal ratio without protein B. For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y=100/(1+((X/IC50)^Hill slope)).

4-3. Molecular modeling 4-3-1. Binding site preparation

MOE version 2016 (Chemical Computing Group Inc., Montreal, QC, Canada) was used to prepare the binding sites starting from the X-ray structure of the syntenin PDZ domain in complex with compound 45 (6RLC). All residues, with at least one atom within 14 Å radius from reference fragment were selected to define the binding site. A large binding site was defined because the docking engine (S4MPLE) relies on a FF-based energy function and the goal is to explore adjacent sub-pockets using a growing strategy. Two different binding site files were prepared because both potential orientations of the N215 sidechain (flip of terminal amide) were considered.

4-3-2. Design of virtual library

The creation of the virtual focused library relied on the procedure described in the DOTS methodology [START_REF] Hoffer | Integrated Strategy for Lead Optimization Based on Fragment Growing: The Diversity-Oriented-Target-Focused-Synthesis Approach[END_REF]. Briefly, a focused library was generated starting from an activated substructure (here, fragment 45 that contains an aryl halide reactive function), a collection of commercially available BBs and a set of in silico encoded medicinal chemistry relevant chemical reactions. This chemistry-driven strategy is expected to design accessible compounds with high reaction yields. The single Suzuki rule (#31 according to the reference study [START_REF] Hartenfeller | A Collection of Robust Organic Synthesis Reactions for In Silico Molecule Design[END_REF] was enabled for the design stage. A raw focused library of 1865 compounds was automatically designed. Then, several fully automated post-processing stages were applied to this raw focused library to extract a subset of duplicate-free compounds with a focus on structures with Then, yuck filters, which were used to design the clean subset of the ZINC database (http://zinc.docking.org), were employed to reject compounds with undesired functional groups. Cxcalc and Jcsearch programs from ChemAxon were respectively used to compute descriptors and detect undesired substructures. The last post-processing stage involved various computations which are required for the subsequent virtual screening: major microspecies, partial charges, force-field atomic types, and single 3D-conformer were computed for each compound. A maximum common substructure (MCS) algorithm was also used to flag atoms that were already present in the fragment 45. These atoms were constrained in the subsequent virtual screening stage (see below). The mapping of force-field atomic types was done using programs from AmberTools (http://ambermd.org), while all the other steps were performed using in house tools relying on the ChemAxon Java API. In the end, the virtual focused chemical library contained 402 compounds.

4-3-3. Virtual screening

The virtual screening was performed on both prepared binding sites as described in the DOTS methodology [PMID 29883107]. More precisely, the final subset of compounds was screened using the S4MPLE conformational tool with constraints on the original location of the fragment 45. Unflagged atoms, corresponding to newly added atoms from virtual synthesis, were free to explore adjacent sub-pockets without any constraints. The sampling stage mainly consisted in three independent simulations of 400 generations with a population of 30 individuals. All saved poses were merged into one file before switching to the post-processing stage that involved the minimization of all non-redundant poses while unlocking all ligand atoms. The energy used to rank all screened compounds was equal to the best energy of the complex minus the energy of the best conformer of the free ligand. The latter is obtained by performing three independent simulations with the same parameters on the ligand alone. The goal is to compute a potential energy difference between bound and free forms to estimate the interaction energy while considering the strain energy of the ligand. The cDNA encoding mCherry-syntenin was derived from the eGFP-syntenin cDNA construct, by restriction-ligation. The expression vector for Ce-RAB5(Q79L) was received from W.

Annaert (K.U. Leuven, Belgium). The CAY10594 inhibitor (used at 10 μM) was purchased from Santa Cruz (sc-223874) [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF].

4-4-2. Cancer cell viability

The effects of the compounds C58 and SyntOFF were tested both on MCF-7 and MCF-7

SyntKO cells. Briefly, the cells were treated with increasing concentrations of the compounds for 48hours and early/late apoptosis was measured by using the FITC Annexin V apoptosis detection kit with 7-AAD (Biolegend, San Diego, CA) according to the manufacturer. Annexin

V positive but 7-AAD negative (early apoptotic cells) and Annexin V positive and 7-AAD positive (late stage apoptosis) was determined by using FACS LSRII flow cytometer (BD Biosciences) and data were analyzed with Flowjo software (Tree Star).

4-4-3. Exosomes and total cell lysates

For comparative analyses, exosome-enriched fractions were collected from equivalent amounts of culture medium, conditioned by equivalent amounts of cells. MCF-7 cells were treated with indicated compounds or with DMSO 0.2% as control, in medium containing exosome-depleted FCS (10%). After 16hours, cell media were collected and exosomes were isolated by three sequential centrifugation steps at 4 °C: 10 min at 500×g, to remove cells; 30 min at 10,000×g, to remove cell debris; and 1h30min at 100,000×g, to pellet exosomes (exosome-enriched fraction), followed by one wash with 1400µL of PBS1X (100,000×g, 1h), to remove soluble serum and secreted proteins. Exosomal pellets were then re-suspended in 100µL of PBS1X.

The lysates from corresponding cultures were cleared by centrifugation at 1500rpm for 5 min and then resuspended in lysis buffer (TrisHCL pH 7.4 30mM, NaCl 150mM, 1% NP40 (IGEPAL), 1µg/ml aprotinin, 1µg/ml leupeptin). Although only little variations were observed from sample to sample, exosomal amounts loaded for the western blot were normalized according to the number of parent cells from where exosomes were secreted. Table 1. Inhibition of Syntenin-Syndecan2 complex interaction in the presence of analogues 13-20 and 41-53 at 400 µM confirmed in counter screening experiment using HTRF assay.

List of captions for Figures, Schemes and Tables

Table 2. 2D structure of selected compounds by the virtual growing strategy. Building blocks used to build the compounds are depicted with reactive groups (boronic acid or ester) leading to aromatic-carbon bonds highlighted in red.

Scheme 2 .

 2 Scheme 2. Synthesis of analogues 41-53 and their ester derivatives 28-40.

Figure 2 .

 2 Figure 2. Selectivity profiles and IC50 of compounds C58 (red) and 45 (blue) using HTRF assay without and with 0.01% NP40.

Figure 3 .

 3 Figure 3. Crystal structure of syntenin PDZ2 domain in complex with compound 45 (PDBID: 6RLC). Compound 45 is displayed in cylinder representation orange carbon atom. Nitrogen, oxygen, chlorine and sulfur are colored in blue, red, green and yellow, respectively. Small red spheres represent water molecules around the binding sites. Hydrogen bond interactions are depicted as black dotted lines. Amino acid residues indicated are those establishing Van der Waals interactions with compound 45.

Figure 4 .

 4 Figure 4. A. General scheme for synthesis of derivatives 57 and 58-66 via Suzuki-Miyaura coupling. B. Structure, IC50 and LE values of analogues 57-66 on Syntenin-Syndecan2 complex HTRF assay. (a) in parenthesis IC50 and LE values with NP40 0.01%. Additional physicochemical efficiency metrics are shown TableS1in Supp. Data.

Figure 5 .

 5 Figure 5. MCF-7 cells were treated with vehicle (Ctrl) or the indicated compound (32, 45, 56 & 57; 100µM) in medium containing exosome-depleted FCS (10%) for 16h. A. Exosomes and corresponding total cell lysates were analyzed by western blot, tracing different markers, as indicated. B. Histograms represent mean signal intensities (±SEM) in exosomes, relative to controls (Ctrl). Data were obtained from N independent experiments, as indicated. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with a Tukey posttest.

4- 1

 1 -2-4. (S)-2-(2-(4-chlorophenylthio)acetamido)-3-methylbutanoic acid 16 1 H NMR (DMSO-d 6 , 300 MHz): δ = 12.04 (bs, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 3.96 (dd, J = 8.5 Hz and 5.5 Hz, 1H), 3.64 (d, J = 14.5 Hz, 1H), 3.53 (d, J = 14.5 Hz, 1H), 1.98-1.70 (m, 1H), 0.85-0.53 (m, 6H).

4- 1 - 4 -

 14 12. (S)-methyl 2-(3-(4-chlorophenoxy)propanamido)-3-phenyl-propanoate39 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.55 (d, J = 7.8 Hz, 1H), 7.38-7.22 (m, 7H), 6.91 (d, J = 9.0 Hz, 2H), 4.53 (m, 1H), 4.11 (t, J = 6.1 Hz, 2H), 3.63 (s, 3H), 3.06 (dd, J = 13.7 Hz J = 5.5

4- 1 - 4 -

 14 14. (S)-methyl 2-(3-(4-bromophenylthio)propanamido)-3-methylbutanoate 561 H NMR (CDCl3, 250 MHz): δ = 7.34 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 5.98 (bd, J = 8.6 Hz, 1H), 4.50 (dd, J = 8.8 Hz and 4.6 Hz, 1H), 3.66 (s, 3H), 3.12 (t, J = 7.2 Hz, 2H), 2.45

59 1 H

 1 NMR (DMSO-d 6 , 400 MHz): δ = 8.02 (bd, J = 8.4 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 4.16 (dd, J = 8.6 Hz and 5.7 Hz, 1H), 3.48-3.39 (m, 2H), 3.22 (t, J = 7.2 Hz, 2H), 2.92 (t, J = 5.4 Hz, 4H), 2.10-1.99 (m, 1H), 1.61-1.50 (m, 4H), 1.42-1.34 (m, 2H), 0.88 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H).

4- 4 . Cellular evaluation 4 - 4 - 1 .

 4441 Cell culture and reagents MCF-7 cell lines were purchased from the American Type culture collection (Manassas, VA).Cells were grown in DMEM-F12 (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Eurobio) at 37°C under 5% CO2. Syntenin CRISPR/Cas9 knockout (SyntKO) MCF-7 cells were generated following the procedures as previously described[START_REF] Imjeti | Syntenin mediates SRC function in exosomal cell-to-cell communication[END_REF]. Briefly, SDCBP-specific gRNA oligos sgRNASDCBP1 targeting exon1 (F: CACCgCTATCCCTCACGATGGAAGT; R: AAACACTTCCATCGTGAGGGATAGc) were cloned into the pX458 two-in-one CRISPR targeting vector (Addgene, Cambridge, MA), individually transfected into MCF7 cells and sorted for GFP expression after 48h. Single cell clones were grown and screened by western blotting for syntenin expression. One individual clone has been used in the in vitro experiments.

Figure 1 .

 1 Figure 1. A. Crystal structure of syntenin PDZ2 domain in complex with compound C58 (PDBID: 6R9H). Compound C58 is depicted in cylinder representation dark green carbon atom. Nitrogen, oxygen, chlorine and sulfur are colored in blue, red, green and yellow, respectively. Small red spheres represent water molecules around the binding sites. Hydrogen bond interactions are displayed as black dotted lines. Amino acid residues highlighted are those establishing Van der Waals interactions with compound C58. B. 2D structure of hit fragment C58. C. 2D structures of putatively interesting explored modifications around fragment C58.

Figure 2 .

 2 Figure 2. Selectivity profiles and IC50 of compounds C58 (red) and 45 (blue) using HTRF assay without and with 0.01% NP40.

Figure 3 .

 3 Figure 3. Crystal structure of syntenin PDZ2 domain in complex with compound 45 (PDBID: 6RLC). Compound 45 is displayed in cylinder representation orange carbon atom. Nitrogen, oxygen, chlorine and sulfur are colored in blue, red, green and yellow, respectively. Small red spheres represent water molecules around the binding sites. Hydrogen bond interactions are depicted as black dotted lines. Amino acid residues indicated are those establishing Van der Waals interactions with compound 45.

Figure 4 .

 4 Figure 4. A. General scheme for synthesis of derivatives 57 and 58-66 via Suzuki-Miyaura coupling. B. Structure, IC50 and LE values of analogues 57-66 on Syntenin-Syndecan2 complex HTRF assay. (a) in parenthesis IC50 and LE values with NP40 0.01%. Additional physicochemical efficiency metrics are shown TableS1in Supp. Data.

Figure 5 .Scheme 1 .Scheme 2 .

 512 Figure 5. MCF-7 cells were treated with vehicle (Ctrl) or the indicated compound (32, 45, 56 & 57; 100µM) in medium containing exosome-depleted FCS (10%) for 16h. A. Exosomes and corresponding total cell lysates were analyzed by western blot, tracing different markers, as indicated. B. Histograms represent mean signal intensities (±SEM) in exosomes, relative to controls (Ctrl). Data were obtained from N independent experiments, as indicated. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with a Tukey posttest.

Table 2 .

 2 2D structure of selected compounds by the virtual growing strategy. Building blocks used to build the compounds are depicted with reactive groups (boronic acid or ester) leading to aromatic-carbon bonds highlighted in red.

	VS

ID Substructure VS ID Substructure VS01 VS06 VS02 VS07 VS03 VS08 VS04 VS09 VS05 VS10

VS04-07 and VS-09 as well as boronic esters VS01, VS03 and VS08 in the

	or cesium carbonate) in various solvents such toluene, THF, 1,4-dioxane or ethanol,
	we were able to conclude that use of Pd(PPh3)4 (5 mol%) and aqueous Na2CO3 in a mixture
	toluene/ethanol (4/1) at reflux temperature achieved optimum yield (reactions monitored by
	LC/MS analysis). Furthermore, under these conditions, we noticed that the cross-coupled
	product is hydrolyzed to the corresponding carboxylic acid derivative, probably due to methyl
	ester sensitivity to basic hydrolysis. Therefore, intermediate 56 reacted readily with boronic
	acids VS02, presence of Pd(PPh3)4 and K2CO3 in refluxing aqueous toluene/ethanol mixture for 18h

  3H).13 C NMR (DMSO-d 6 , 101 MHz): δ = 172.19, 168.21, 157.19, 129.63, 125.24, 116.86, 67.07, 57.65, 52.25, 30.27, 19.42, 18.70; LC/MS (ESI): 298.5 [M-H] -, 300.1 [M+H] + ; colorless oil. Yield = 93%. Hz, 3H). 13 C NMR (DMSO-d 6 , 75 MHz): δ = 173.20, 168.52, 136.62, 129.34, 128.27, 126.18, 57.82, 36.37, 30.47, 19.47, 18.23; LC/MS (ESI): 266.6 [M-H] -, 268.3 [M+H] + ; beige solid.

	4-1-1-1. (S)-methyl 2-(2-(phenylthio)acetamido)propanoate 5
	1 H NMR (CDCl3, 250 MHz,): δ = 7.29-7.09 (m, 6H), 4.48 (p, J = 7.2 Hz, 1H), 3.63 (s, 3H), 3.56 (d, J = 0.9 Hz, 2H), 1.25 (d, J = 7.2 Hz, 3H). LC/MS (ESI): 252.5 [M-H] -, 254.4 [M+H] + ; light brown oil. Yield = 92%. 4-1-1-2. (S)-methyl 3-methyl-2-(2-(phenylthio)acetamido)butanoate 6 1 H NMR (DMSO-d 6 , 400 MHz): δ = 8.39 (bd, J = 8.3 Hz, 1H), 7.39-7.35 (m, 2H), 7.34-7.26 (m, 2H), 7.23-7.15 (m, 1H), 4.49 (dd, J = 8.3 Hz and 6.2 Hz, 1H), 3.80 (d, J = 14.5 Hz, 1H), 4-1-1-4. (S)-methyl 2-(2-(4-chlorophenylthio)acetamido)-3-methylbutanoate 8 1 H NMR (DMSO-d 6 , 400 MHz): δ = 7.21 (bs, 4H), 7.09 (bd, J = 8.4 Hz, 1H), 4.43 (dd, J = 8.9 Hz and 4.8 Hz, 1H), 3.65 (d, J = 16.8 Hz, 1H), 3.63 (s, 3H), 3.55 (d, J = 16.8 Hz, 1H), 2.11-1 H NMR (DMSO-d 6 , 400 MHz): δ = 8.50 (bd, J = 7.3 Hz, 1H), 7.34-7.26 (m, 2H), 7.00-6.94 (m, 3H), 4.54 (d, J = 14.8 Hz, 1H), 4.50 (d, J = 14.8 Hz, 1H), 4.39 (p, J = 7.3 Hz, 1H), 3.64 (s, 3H), 1.33 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 101 MHz): δ = 173.18, 168.13, 158.21, 129.91, 121.64, 115.19, 67.08, 52.40, 47.76, 17.32; LC/MS (ESI): 236.6 [M-H] -, 238.5 [M+H] + ; colorless oil. Yield = 74%. = 9.1 Hz, 2H), 4.50 (m, 1H), 4.40 (s, 2H), 3.69 (s, 3H), 1.38 (d, J = 7.2 Hz, 3H). LC/MS (ESI): 270.5 [M-H] -, 272.2 [M+H] + ; colorless oil. Yield = 89%. 4-1-1-8. (S)-methyl 2-(2-(4-chlorophenoxy)acetamido)-3-methylbutanoate 12 1 H NMR (DMSO-d 6 , 400 MHz): δ = 8.33 (bd, J = 8.2 Hz, 1H), 7.34 (d, J = 9.1 Hz, 2H), 6.96 4-1-2. General procedure for the synthesis of amino acid derivatives 13-20, 41-53 and 57. To a stirred solution of ester intermediate derivative (5-12, 28-40 or 56) (1eq, 0.5 mmol) in THF at 0°C, aqueous solution of LiOH (10 eq, 5.0 mmol) was added dropwise. The resulting mixture was stirred 1 h at room temperature. After completion, THF was removed in vacuo. 4-1-2-2. (S)-3-methyl-2-(2-(phenylthio)acetamido)butanoic acid 14 1 H NMR (DMSO-d 6 , 300 MHz): δ = 12.66 (bs, 1H), 8.19 (d, J = 8.6 Hz, 1H), 7.39-7.35 (m, Yield = 88%. 4-1-2-3. (S)-2-(2-(4-chlorophenylthio)acetaamido)propamoic acid 15 1 H NMR (DMSO-d 6 , 300 MHz): δ = 12.56 (bs, 1H), 8.43 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 4.20 (p, J = 7.3 Hz, 1H), 3.69 (s, 3H), 1.25 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 75 MHz): δ = 174.24, 167.85, 135.75, 130.96, 130.20, 129.22, 1.99 (m, 1H), 0.74 (d, J = 6.8 Hz, 3H), 0.73 (d, J = 6.9 Hz, 3H). 4-1-1-5. (S)-methyl 2-(2-phenoxyacetamido)propanoate 9 48.28, 36.59, 17.70; LC/MS (ESI): 272.5 [M-H]

3.70 (d, J = 14.5 Hz, 1H), 3.63 (s, 3H), 2.02 (m, 1H), 0.85 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H).

13

C NMR (DMSO-d 6 , 101 MHz): δ = 172.

24, 168.76, 136.54, 129.36, 128.30, 126.25, 58

.01, 52.

21, 36.23, 30.48, 19.33, 18.

47; LC/MS (ESI): 280.5 [M-H] -, 282.4 [M+H] + ; light yellow oil. Yield = 98%.

4-1-1-3. (S)-methyl 2-(2-(4-chlorophenylthio)acetaamido)propamoate 7

1 H NMR (CDCl3, 400 MHz): δ = 8.58 (bd, J = 7.0 Hz, 1H), 7.38 (bs, 4H), 4.27 (p, J = 7.2 Hz, 1H), 3.69 (s, 2H), 3.61 (s, 3H), 1.26 (d, J = 7.3 Hz, 3H).

13

C NMR (CDCl3, 101 MHz): δ = 173.21, 168.07, 135.65, 131.02, 130.22, 129.25, 52.37, 48.30, 36.46, 17.42; LC/MS (ESI): 286.5 [M-H] -, 288.1 [M+H] + ; white solid. Yield = 69%. 13 C NMR (DMSO-d 6 , 101 MHz): δ = 170.87, 166.40, 131.98, 131.94, 128.90, 128.38, 56.33, 51.18, 36.68, 30.15, 17.79, 16.53; LC/MS (ESI): 314.6 [M-H] -, 316.2 [M+H] + ; colorless oil. Yield = 90%.

4-1-1-6. (S)-methyl 3-methyl-2-(2-phenoxyacetamido)butanoate

10 1 H NMR (DMSO-d 6 , 400 MHz): δ = 8.28 (bd, J = 8.3 Hz, 1H), 7.35-7.25 (m, 2H), 6.95 (m, 3H), 4.62 (d, J = 14.7 Hz, 1H), 4.58 (d, J = 14.7 Hz, 1H), 4.43 (dd, J = 8.3 Hz and 6.4 Hz, 1H), 3.65 (s, 3H), 2.16-1.98 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H). 13 C NMR (DMSO-d 6 , 101 MHz): δ = 172.22, 168.50, 158.27, 129.90, 121.54, 115.03, 66.80, 57.60, 52.25, 30.30, 19.42, 18.67; LC/MS (ESI): 264.7 [M-H] -, 266.5 [M+H] + ; colorless oil. Yield = 63%.

4-1-1-7. (S)-methyl 2-(2-(4-chlorophenoxy)acetamido)propanoate 11

1 H NMR (CDCl3, 250 MHz): δ = 7.20 (d, J = 9.1 Hz, 2H), 6.99 (bd, J = 6.7 Hz, 1H), 6.80 (d, J (d, J = 9.1 Hz, 2H), 4.64 (d, J = 14.7 Hz, 1H), 4.59 (d, J = 14.7 Hz, 1H), 4.23 (dd, J = 8.2 Hz and 6.4 Hz, 1H), 3.65 (s, 3H), 2.14-1.98 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, Then, 1N HCl solution was added dropwise until pH 2-3 and the aqueous layer was extracted with ethyl acetate. The organic layer was washed once with water, once with brine, dried over Na2SO4, and evaporated in vacuo to afford either a crude residue which does not requires further purification or a crude which was purified by column chromatography eluting with dichloromethane-methanol (95:5).

4-1-2-1. (S)-2-(2-(phenylthio)acetamido)propanoic acid

13 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.69 (bs, 1H), 8.49 (d, J = 7.2 Hz, 1H), 7.42-7.26 (m, 4H), 7.19 (t, J = 7.0 Hz, 1H), 4.24 (p, J = 7.1 Hz, 1H), 3.72 (s, 2H), 1.28 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 173.88, 167.63, 136.15, 128.86, 127.82, 125.73, 47.76, 36.02, 17.19; LC/MS (ESI): 238.6 [M-H] -, 240.4 [M+H] + ; beige solid. Yield = 77%. 2H), 7.3-7.25 (m, 2H), 7.22-7.14 (m, 1H), 4.16 (dd, J = 8.6 Hz and 5.5 Hz, 1H), 3.81 (d, J = 14.5 Hz, 1H), 3.71 (d, J = 14.5 Hz, 1H), 2.03 (m, 1H), 0.84 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.4 -, 274.1 [M+H] + ; white solid. Yield = 78%.

  H NMR (DMSO-d 6 , 300 MHz): δ = 12.68 (bs, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.35-7.24 (m, 2H), 7.00-6.91 (m, 3H), 4.59 (s, 2H), 4.22 (dd, J = 8.5 Hz and 5.7 Hz, 1H), 2.10 (m, 1H), 0.88 , 254.3 [M+H] + ; HRMS (TOF, ESI+) cald for C12H16NO3S [M+H] + 254.0851, found 254.0851; white powder. Yield = 93%. J = 8.6 Hz and J = 4.4 Hz, 1H), 4.23 (dd, J = 8.5 and J = 5.9 Hz, 1H), 3.19 (t, J = 7.2 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.16-2.01 (m, 1H), 0.93 (bd, J = 5.7 Hz, 6H); 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.75, 170.32, 136.16, 128.97, 128.56, 125.78, 57.28, 34.97, 29.88, 28.67, 19.04, 18.05 ; LC/MS (ESI): 280.6 [M-H] -, 282.3 [M+H] + ; HRMS (TOF, ESI+) cald for C14H20NO3S [M+H] + 282.1164, found 282.1165; white solid. Yield = 96%. H NMR (DMSO-d 6 , 300 MHz): δ = 12.51 (bs, 2H), 8.04 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 9.2

	4-1-2-6. (S)-3-methyl-2-(2-phenoxyacetamido)butanoic acid 18 4-1-2-19. (S)-2-(3-(4-chlorophenoxy)propanamido)-3-methylbutanoic acid 51 4-1-2-22. (S)-2-(3-(4-bromophenylthio)propanamido)-3-methylbutanoic acid 57
	4-1-2-13. (S)-2-(3-(4-chlorophenylthio)propanamido)-3-methylbutanoic acid 45 4-1-2-16. (S)-3-methyl-2-(3-(phenoxypropanamido)butanoic acid 48 1 H NMR (CDCl3, 250 MHz): δ = 7.16 (d, J = 7.6 Hz, 2H), 6.77 (d, J = 7.6 Hz, 2H), 6.40 (d, J 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.67 (bs, 2H), 8.16 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 8.6
	1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.70 (bs, 1H), 8.19 (d, J = 8.6 Hz, 1H), 7.38-7.20 (m, = 8.5 Hz, 1H), 4.55 (dd, J = 8.6 Hz and J = 4.7 Hz, 1H), 4.17 (t, J = 5.6 Hz, 2H), 2.67 (t, J = Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 4.21 (dd, J = 8.6 Hz and 5.8 Hz, 1H), 3.20 (t, J = 7.1 Hz,
	(d, J = 6.7 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H). 13 C NMR (DMSO-d 6 , 75 MHz): δ = 173.11, 4-1-2-10. (S)-3-methyl-2-(3-phenylthio)propanamido)butanoic acid 42 Hz, 2H), 7.34 (d, J = 9.3 Hz, 2H), 4.16 (dd, J = 8.4 Hz and 6.0 Hz, 1H), 3.14 (t, J = 7.1 Hz, 2H), 7.00-6.84 (m, 3H), 4.21 (m, 1H), 4.18 (t, J = 5.8 Hz, 2H), 2.82-2.58 (m, 2H), 2.16-1.99 5.7 Hz, 2H), 2.19 (m, 1H), 0.94-0.85 (m, 6H) ; 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.81, 2H), 2.59 (t, J = 7.0 Hz, 2H), 2.07 (m, 1H), 0.92 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H).
	168.25, 158.26, 129.91, 121.57, 115.08, 66.95, 57.33, 30.38, 19.54, 18.37; LC/MS (ESI): 250.7 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.67 (bs, 1H), 8.17 (d, J = 8.6 Hz, 1H), 7.39 (bd, J = 4.5 2H), 2.54 (t, J = 7.0 Hz, 2H), 2.03 (m, 1H), 0.87 (bd, J = 6.6 Hz, 6H) ; 13 C NMR (DMSO-d 6 , (m, 1H), 0.91 (d, J = 6.7 Hz, 6H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 13 C NMR (63 MHz, 169.68, 157.34, 129.15, 124.34, 116.37, 64.66, 57.17, 35.07, 29.91, 19.03, 17.99 ; 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.96, 170.16, 135.71, 131.74, 129.96, 118.46, 57.04,
	= 96%. 4-1-2-5. (S)-2-(2-phenoxyacetamido)propanoic acid 17 [M-H] -, 252.4 [M+H] + ; white solid. Yield = 45%. 4-1-2-7. (S)-2-(2-(4-chlorophenoxy)acetamido)propanoic acid 19 75 MHz) δ 173.41, 170.71, 135.69, 130.86, 130.42, 129.40, 57.62, 34.99, 30.34, 28.91, 19.58, 18.48. LC/MS (ESI): 314.6 [M-H] -, 316.2 [M+H] + ; HRMS (TOF, ESI+) cald for 4-1-2-14. (S)-2-(3-(4-cholorophenylthio)propanamido)-3-phenylpropamoic acid 46 66, 169.87, 137.60, 135.18, 130.58, 130.19, 129.05, 128.88, 128.06, 126.30, 53.38, 36.98, 34.85, 28.59 ; LC/MS (ESI): 362.6 [M-H] -, 364.1 [M+H] + ; HRMS (TOF, ESI+) cald for C18H19NO3SCl [M+H] + 364.0774, found 364.0776; white solid. Yield = 97%. 4-1-2-15. (S)-2-(3-(phenoxypropanamido)propanoic acid 47 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.63 (bs, 1H), 8.35 (d, J = 7.3 Hz, 1H), 7.38-7.17 (m, 2H), 7.02-6.84 (m, 3H), 4.27 (p, J = 5.8 Hz, 1H), 4.18 (td, J = 6.2 Hz and 2.8 Hz, 2H), 2.62 DMSO) δ = 172.82, 169.80, 158.49, 129.39, 120.55, 114.60, 64.16, 57.18, 35.23, 29.93, 19.04, 18.00; LC/MS (ESI): 264.4 [M-H] -, 266.3 [M+H] + ; HRMS (TOF, ESI+) cald for C14H20NO4 4-1-2-17. (S)-2-(3-(phenoxypropanamido)-3-phenylpropanoic acid 49 73, 169.45, 158.42, 137.61, 129.37, 129.06, 128.06, 126.30, 120.56, 114.60, 63.97, 53.39, 36.92, 35.26; LC/MS (ESI): 312.5 [M-H] -, 314.2 [M+H] + ; HRMS (TOF, ESI+) cald for C18H20N2O4 [M+H] + 314.1387, found 314.1390; colorless oil. Yield = 75%. 4-1-2-18. (S)-2-(3-(4-chlorophenoxy)propanamido)propanoic acid 50 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.07 (d, J = 7.2 Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H), 6.76 (d, LC/MS (ESI): 298,5 [M-H] -, 300,1 [M+H] + ; HRMS (TOF, ESI+) cald for C14H19NO4Cl [M+H] + 300.1003, found 300.1003; white solid. Yield = 74%. 4-1-2-20. (S)-2-(3-(4-chlorophenoxy)propanamido)-3-phenylpropanoic acid 52 124.35, 116.38, 64.49, 53.40, 36.91, 35.11; LC/MS (ESI): 346,6 [M-H] -, 348,3 [M+H] + ; HRMS (TOF, ESI+) cald for C18H19NO4Cl [M+H] + 348.1003, found 348.1006; white solid. Yield = 94%. 4-1-2-21. (S)-3-methyl-2-(4-(phenylbutanamido)butanoic acid 53 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.65 (bs, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.36-7.26 (m, 2H), 7.28-7.18 (m, 3H), 4.20 (dd, J = 8.1 Hz and J = 6.0 Hz, 1H), 2.61 (t, J = 7.5 Hz, 2H), 2.25 34.30, 29.80, 28.05, 19.07, 17.92; LC/MS (ESI): 358.8 [M-H] -and isotopic peak 360.8, 359.8 [M+H] + and isotopic peak 361.9; HRMS (TOF, ESI+) cald for C14H19NO3SBr [M+H] + Hz, 4H), 7.26 (dd, 4-1-2-12. (S)-2-(3-(4-cholorophenylthio)propanamido)propamoic acid 44 C14H19NO3SCl [M+H] + 316.0774, found 316.0776; white solid. Yield = 89%. [M+H] + 266.1387, found 266.1395; colorless oil. Yield = 76%. 360.0269, found 360.0272; white solid. Yield = 96%.
	1 H NMR (DMSO-d 6 , 300 MHz): δ = 12.62 (bs, 1H), 8.27 (d, J = 7.4 Hz, 1H), 7.34-7.26 (m, 4-1-2-9. (S)-2-(3-phenylthio)propanamido)propamoic acid 41 1 H NMR (CDCl3, 250 MHz): δ = 7.28-7.14 (m, 4H), 6.17 (bd, J = 7.0 Hz, 1H), 4.51 (p J = 7.1 (td, J = 6.2 Hz and 1.2 Hz, 2H), 1.30 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = J = 9.0 Hz, 2H), 4.08-3.92 (m, 3H), 4.00-2.40 (t, J = 6.1 Hz, 2H), 1.07 (d, J = 7.3 Hz, 3H); 13 C (t, J = 7.3 Hz, 2H), 2.11 (m, 1H), 1.92-1.74 (m, 2H), 0.93 (bd, J = 6.7 Hz, 6H) ; 13 C NMR
	2H), 7.02-6.93 (m, 3H), 4.48 (s, 2H), 4.31 (p, J = 7.3 Hz, 1H), 1.31 (d, J = 7.3 Hz, 3H). 13 C 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.59 (bs, 1H), 8.31 (d, J = 7.2 Hz, 1H), 7.39 (bd, J = 4.3 Hz, 1H), 3.12 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 1.38 (d, J = 7.2 Hz, 3H) ; 13 C NMR 13 C NMR (DMSO-d 6 , 63 MHz) δ = 173.92, 169.26, 158.48, 130.20, 129.39, 120.57, 116.00, NMR (DMSO-d 6 , 63 MHz): δ = 174.19, 169.01, 157.36, 129.15, 124.36, 116.44, 64.56, 47.79, (DMSO-d 6 ,63 MHz): δ = δ 173.01, 172.18, 141.87, 128.24, 128.19, 125.64, 57.20, 34.66, 34.60,
	NMR (DMSO-d 6 , 75 MHz): δ = 174.21, 167.88, 158.23, 129.90, 121.63, 115.22, 67.17, 47.72, Hz, 4H), 7.26 (dd, J = 8.7 Hz and J = 4.3 Hz, 1H), 4.25 (p, J = 7.3 Hz, 1H), 3.18 (t, J = 7.3 Hz, (CDCl3, 63 MHz): δ = 207.45, 170.05, 133.02, 131.95, 130.52, 128.30, 47.29, 35.12, 28.86, 114.65, 110.85, 64.01, 47.52, 35.18, 17.35; LC/MS (ESI): 236.6 [M-H] -, 238.7 [M+H] + ; HRMS 35.10, 17.55 ; LC/MS (ESI): 270.5 [M-H] -, 272.3 [M+H] + ; HRMS (TOF, ESI+) cald for 29.72, 27.14, 19.10, 18.10; LC/MS (ESI): 262.6 [M-H] -, 264.4[M+H] + ; HRMS (TOF, ESI+)
	(TOF, ESI+) cald for C12H16NO4 [M+H] + 238.1074, found 238.1079; colorless oil. Yield = C12H15NO4Cl [M+H] + 272.0690, found 272.0689; white solid. Yield = 52%. cald for C15H22NO3 [M+H] + 264.1594, found 264.1590; white solid. Yield = 92%.
	90%.

13

C NMR (DMSO-d

6

, 75 MHz): δ = 172.63, 167.91, 135.26, 130.39, 129.58, 128.71, 57.31, 35.85, 29.92, 18.96, 17.74; LC/MS (ESI): 300.1 [M-H] -, 302.1 [M+H] + ; HRMS (TOF, ESI-) cald for C13H15NO3SCl [M-H] -300.0467, found 360.0462; LC/MS (ESI): 300.1 [M-H] -, 302.1 [M+H] + ; white solid. Yield 17.61; LC/MS (ESI): 222.8 [M-H] -, 224.4 [M+H] + ; white solid. Yield = 78%.

1 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.72 (bs, 1H), 8.40 (d, J = 7.3 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 4.42 (s, 2H), 4.32 (p, J = 7.0 Hz, 1H), 1.34 (d, J = 7.2 Hz, 3H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 171.33, 164.71, 154.09, 126.68, 122.38, 113.99, 64.34, 44.79, 14.56; LC/MS (ESI): 256.5 [M-H] -, 258.3 [M+H] + ; white solid. Yield = 95%.

4-1-2-8. (S)-2-(2-(4-chlorophenoxy)acetamido)-3-methylbutanoic acid 20

1 H NMR (CDCl3, 300 MHz): δ = 7.21 (d, J = 9.0 Hz, 2H), 6.92 (bd, J = 8.8 Hz, 1H), 6.82 (d, J = 8.9 Hz, 2H), 4.23 (dd, J = 8.8 Hz and 4.7 Hz, 1H), 4.47 (s, 1H), 2.27-2.13 (m, 1H), 0.91 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H).

13

C NMR (CDCl3, 101 MHz): δ = 174.28, 167.45, 154.65, 128.71, 126.37, 115.14, 66.53, 55.61, 29.97, 17.99, 16.54; LC/MS (ESI): 284.5 [M-H] - , 285.9 [M+H] + ; white solid. Yield = 85%. 2H), 2.50 (t, J = 7.3 Hz, 2H), 1.30 (d, J = 7.3 Hz, 3H); 13 C NMR (DMSO-d 6 , 101 MHz): δ = 174.59, 170.34, 136.45, 129.55, 128.64, 126.20, 47.94, 35.18, 28.44, 17.69 ; LC/MS (ESI):

252.6 [M-H] -4-1-2-11. (S)-3-phenyl-2-(3-phenylthio)propanamido)propamoic acid 43 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.72 (bs, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.31-7.10 (m, 10H), 4.37 (td, J = 9.2 Hz and J = 5.0 Hz, 1H), 3.04-2.93 (m, 3H), 2.78 (dd, J = 13.7 Hz and J = 9.4 Hz, 1H), 2.34 (t, J = 7.3 Hz, 2H) ; 13 C NMR (DMSO-d

6 , 63 MHz): δ = 172.67, 169.98, 137.62, 136.04, 129.05, 128.97, 128.53, 128.05, 126.29, 125.79, 53.39, 36.96, 35.04, 28.46 ; LC/MS (ESI): 328.5 [M-H] -, 330.3 [M+H] + ; HRMS (TOF, ESI+) cald for C18H20NO3S [M+H] + 330.1164, found 330.1165; white solid. Yield = 49%. 17.02 ; LC/MS (ESI): 286.5 [M-H] -, 288.1 [M+H] + ; HRMS (TOF, ESI+) cald for C12H15NO3SCl [M+H] + 288.0461, found 288.0461; white solid. Yield = 85%.

1 1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.82 (bs, 1H), 8.34 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.33-7.23 (m, 5H), 4.48 (m, 1H), 3.09 (m, 3H), 2.88 (dd, J = 8.4 Hz and 6.0 Hz, 1H), 2.44 (t, J = 7.2 Hz, 2H) ; 13 C NMR (DMSO-d 6 , 63MHz): δ = 172.1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.83 (bs, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.36-7.18 (m, 7H), 7.08-6.83 (m, 3H), 4.48 (m, 1H), 4.10 (t, J = 6.2 Hz, 2H), 3.09 (dd, J = 13.8 Hz and 4.9 Hz, 1H), 2.90 (dd, J = 13.8 Hz and 9.3 Hz, 1H), 2.58 (t, J = 6.1 Hz, 2H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 13 C NMR (DMSO-d 6 , 63 MHz) δ = 172.1 H NMR (DMSO-d 6 , 250 MHz): δ = 12.85 (bs, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.40-7.26 (m, 7H), 6.94 (d, J = 9.0 Hz, 2H), 4.51 (m, 1H), 4.13 (t, J = 6.0 Hz, 2H), 3.12 (dd, J = 13.7 Hz and J = 4.8 Hz, 1H), 2.93 (dd, J = 13.7 Hz and J = 9.3 Hz, 1H), 2.60 (t, J = 6.0 Hz, 2H) ; 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.72, 169.33, 157.28, 137.61, 129.13, 129.05, 128.06, 126.30,

  H NMR (DMSO-d 6 , 400 MHz): δ = 8.24 (bd, J = 8.2 Hz, 1H), 7.33 (d, J = 4.3 Hz, 4H), 7.24-7.17 (m, 1H), 4.27 (dd, J = 8.2 Hz and 6.4 Hz, 1H), 3.63 (s, 3H), 3.13 (t, J = 7.2 Hz, 2H), 2.46 (t, J = 7.3 Hz, 2H), 2.07-1.90 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H).13 C 6 , 75 MHz): δ = 172.47, 170.86, 135.62, 130.89, 130.45, 129.41, 57.87, 52.06, 34.91, 30.39, 28.83, 19.40, 18.72; LC/MS (ESI): 328.63 [M-H] -, 330.1 [M+H] + ; white solid. Yield = 62%. H NMR (DMSO-d 6 , 250 MHz): δ = 8.55 (bd, J = 7.8 Hz, 1H), 7.35-7.20 (m, 7H), 6.99-6.84 (m, 3H), 4.53 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.63 (s, 3H), 3.12-2.86 (m, 2H), 2.56 (t, J = 6.3

	4-1-3-3. 3-(4-bromophenylthio)propanoic acid 55 4-1-4-9. (S)-methyl 2-(3-(phenoxypropanamido)-3-phenyl-propanoate 36
	1 H NMR (CDCl3, 250 MHz): δ 7.35 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 3.07 (t, J =
	7.2 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H). 13 C NMR (CDCl3, 63 MHz): δ 178.34, 134.72, 132.79,
	132.51, 121.44, 34.68, 29.49. LC/MS (ESI): 259.3 [M-H] -and isotopic peak 361.4; white solid. NMR (DMSO-d 6 , 101 MHz): δ = 172.54, 170.99, 136.47, 129.55, 128.70, 126.22, 57.86, 52.10, 4-1-4-6. (S)-methyl 2-(3-(4-chlorophenylthio)propanamido)-3-phenylpropanoate 33 Hz, 2H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 171.86, 169.63, 158.41, 137.20, 129.38, 129.00,
	Yield = 97%. 35.03, 30.38, 29.32, 28.62, 19.43, 18.73; LC/MS (ESI): 294.6 [M-H] -, 296.1 [M+H] + ; colorless 1 H NMR (DMSO-d 6 , 300 MHz): δ = 8.38 (bd, J = 7.8 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.31(d, 128.16, 126.45, 120.58, 114.59, 63.90, 53.51, 51.70, 36.85, 35.18; LC/MS (ESI): 328.3
	oil. Yield = 55%. J = 8.5 Hz, 2H), 7.28-7.17 (m, 5H), 4.48 (m, 1H), 3.60 (s, 3H), 3.09-2.96 (m, 3H), 2.88 (dd, J [M+H] + ; colorless oil. Yield = 91%.
	4-1-4. General procedure for the synthesis of amino acid derivatives 28-40 and 56 = 13.7 Hz and 9.2 Hz, 1H), 2.40 (t, J = 7.2 Hz, 2H); 13 C NMR (DMSO-d 6 , 75 MHz): δ =
	To a stirred solution of the corresponding propionic acid derivative (23-27 or 55) (1 eq, 1.0 4-1-4-3. (S)-methyl 3-phenyl-2-(3-(phenylthio)propanamido)propanoate 30 172.38, 170.54, 137.59, 135.52, 130.89, 130.42, 129.52, 129.41, 128.68, 126.99, 54.00, 52.26, 4-1-4-10. (S)-methyl 2-(3-(4-chlorophenoxy)propanamido)propamoate 37
	37.23, 34.97, 28.64; LC/MS (ESI): 376.5 [M-H] 97, 169.45,
	158.46, 129.39, 120.58, 114.62, 63.93, 51.69, 47.54, 35.11, 17.05; LC/MS (ESI): 252.5
	[M+H] + ; colorless oil. Yield = 67%.
	J = 9.0 Hz, 2H), 4.02 (dd, J = 8.3 Hz and J = 6.5 Hz, 1H), 3.95 (t, J = 6.1 Hz, 2H), 3.43 (s, 3H),
	1 H NMR (CDCl3, 250 MHz): δ 7.39-7.07 (m, 5H), 3.09 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 7.3 Hz, 3 Hz, 4H), 7.25-49, 170.38, 4-1-4-8. (S)-methyl 3-methyl-2-(3-(phenoxypropanamido)butanoate 35 2.56-2.33 (m, 2H), 1.82 (m, 1H), 0.68 (d, J = 6.7 Hz, 3H), 0.65 (d, J = 6.7 Hz, 3H); 13 C NMR
	2H). LC/MS (ESI): 181.6 [M-H] -, 183.6 [M+H] + ; white powder. Yield = 94%. 7.14 (m, 1H), 4.27 (p, J = 7.2 Hz, 1H), 3.62 (s, 3H), 3.13 (t, J = 7.3 Hz, 2H), 2.45 (t, J = 7.3 135.57, 130.90, 130.44, 129.42, 52.26, 48.01, 34.97, 28.63, 17.46; LC/MS (ESI): 301.1 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.35 (bd, J = 8.3 Hz, 1H), 7.35-7.24 (m, 2H), 7.00-6.88 (DMSO-d 6 , 63 MHz): δ = 171.92, 169.83, 157.33, 129.17, 124.36, 116.37, 64.59, 57.40, 51.49,
	Hz, 2H), 1.26 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 75 MHz): δ 173.52, 170.50, 136.41, [M+H] + ; white solid. Yield = 41%. (m, 3H), 4.26 (dd, J = 8.2 Hz and 6.5 Hz, 1H), 4.18 (t, J = 6.1 Hz, 2H), 3.66 (s, 3H), 2.78-2.59 34.98, 29.95, 18.87, 18.19; LC/MS (ESI): 314.2 [M+H] + ; colorless oil. Yield = 68%.
	4-1-3-2. 3-(4-chlorophenylthio)propanoic acid 24 129.54, 128.76, 126.24, 52.26, 48.02, 35.16, 28.51, 17.45; LC/MS (ESI): 266.5 [M-H] -, 268.4 (m, 2H), 2.06 (m, 1H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H). 13 C NMR (DMSO-
	1 H NMR (CDCl3, 250 MHz): δ 7.35-7.08 (m, 4H), 3.07 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.2 Hz, [M+H] + ; colorless oil. Yield = 45%. 4-1-4-5. (S)-methyl 2-(3-(4-chlorophenylthio)propanamido)-3-methylbutanoate 32 d 6 , 63 MHz): δ = 13 C NMR (63 MHz, DMSO) δ 171.95, 169.96, 158.47, 129.39, 120.56, 114.58,
	2H). 13 C NMR (CDCl3, 63 MHz): δ 175.58, 132.74, 132.16, 130.80, 128.18, 33.13, 28.39. 1 H NMR (DMSO-d 6 , 300 MHz): δ = 8.19 (bd, J = 8.1 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 7.35 64.08, 57.40, 51.48, 35.14, 29.96, 22.50, 18.87, 18.20, 15.61; LC/MS (ESI): 280.4 [M+H] + ;
	colorless oil. Yield = 82%.

LC/MS (ESI): 215.7 [M-H] -; white powder. Yield = 75%. mmol) and L-aminoacid methyl ester hydrochloride (Alanine, Valine or Phenylalanine) (1 eq, 1.0 mmol) in dry dichloromethane, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (2 eq, 2.0 mmol) and triethylamine (2 eq, 2 mmol) were added. The mixture was stirred under argon atmosphere during 5-16 h at room temperature. After completion, the resulting mixture was washed twice with 1N HCl solution, once with water, once with brine, dried over Na2SO4, and evaporated in vacuo. Crude was purified by column chromatography eluting with dichloromethane-methanol (100 to 98:2).

4-1-4-1. (S)-methyl 2-(3-(phenylthio)propanamido)propanoate 28

1 H NMR (DMSO-d 6 , 300 MHz): δ = 8.32 (bd, J = 6.8 Hz, 1H), 7.33 (d, J = 4.

4-1-4-2. (S)-methyl 3-methyl-2-(3-(phenylthio)propanamido)butanoate 29

1 1 H NMR (DMSO-d 6 , 300 MHz): δ = 8.38 (bd, J = 7.7 Hz, 1H), 7.35-7.18 (m, 10H), 4.49 (m, 1H), 3.60 (s, 3H), 3.06-2.99 (m, 3H), 2.88 (dd, J = 13.7 Hz and 9.1 Hz, 1H), 2.41 (t, J = 7.3 Hz, 2H). 13 C NMR (DMSO-d 6 , 75 MHz): δ = 172.41, 170.65, 137.62, 136.37, 129.53, 128.73, 128.67, 126.98, 126.23, 54.01, 52.26, 37.22, 35.16, 28.51; LC/MS (ESI): 342.5 [M-H] -, 344.2 [M+H] + ; white solid. Yield = 71%.

4-1-4-4. (S)-methyl 2-(3-(4-chlorophenylthio)propanamido)propamoate 31

1 H NMR (DMSO-d 6 , 300 MHz): δ = 8.32 (bd, J = 6.8 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 9.0 Hz, 2H), 4.26 (p, J = 7.3 Hz, 1H), 3.62 (s, 3H), 3.13 (t, J = 7.2 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 1.25 (d, J = 7.3 Hz, 3H).

13

C NMR (DMSO-d 6 , 75 MHz): δ = 173.

(d, J = 9.0 Hz, 2H), 4.26-4.12 (m, 1H), 3.63 (s, 3H), 3.14 (t, J = 7.1 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.00 (m, 1H), 0.87 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H).

13

C NMR (DMSO-d -, 378.1 [M+H] + ; white powder. Yield = 54%.

4-1-4-7. (S)-methyl 2-(3-(phenoxypropanamido)propanoate

34 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.49 (bd, J = 7.0 Hz, 1H), 7.36-7.25 (m, 2H), 6.99-6.90 (m, 3H), 4.33 (p, J = 7.3 Hz, 1H), 4.19 (td, J = 6.1 Hz and 2.2 Hz, 2H), 3.65 (s, 3H), 2.62 (t, J = 6.2 Hz, 2H), 1.31 (d, J = 7.3 Hz, 3H). 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.

1 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.49 (d, J = 7.0 Hz, 1H), 7.35 (d, J = 9.1 Hz, 2H), 6.97 (d, J = 9.1 Hz, 2H), 4.32 (p, J = 7.2 Hz, 1H), 4.18 (td, J = 6.1 Hz and J = 2.3 Hz, 2H), 3.64 (s, 3H), 2.61 (t, J = 6.1 Hz, 2H), 1.30 (d, J = 7.3 Hz, 3H); 13 C NMR (DMSO-d 6 , 63 MHz): δ = 172.95, 169.32, 157.33, 129.16, 124.38, 116.42, 64.46, 51.70, 47.54, 34.96, 17.05; LC/MS (ESI): 286.2 [M+H] + ; white powder. Yield = 73%. 4-1-4-11. (S)-methyl 2-(3-(4-chlorophenoxy)propanamido)-3-methylbutanoate 38 1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.12 (d, J = 8.3 Hz, 1H), 7.11 (d, J = 9.0 Hz, 2H), 6.72 (d,

  C NMR (DMSO-d 6 , 63 MHz): δ = 171.84, 169.50, 157.27, 137.18, 129.15, 128.99, 128.16, 126.46, 124.37, 116.37, 64.43, 53.50, 51.71, 36.84, 35.03; LC/MS (ESI): 362.2 [M+H] + ; %; white powder. NMR (DMSO-d 6 ,63 MHz): δ = 172.30, 172.12, 141.81, 128.24, 128.20, 125.66, 57.42, 51.41, 34.61, 34.44, 29.77, 27.02, 18.92, 18.30 ; LC/MS (ESI): 278.4 [M+H]

	Yield = 77%.
	4-1-4-13. (S)-methyl 3-methyl-2-(4-(phenylbutanamido)butanoate 40
	1 H NMR (DMSO-d 6 , 250 MHz): δ = 8.15 (d, J = 8.0 Hz, 1H), 7.36-7.27 (m, 2H), 7.24-7.17 (m,
	3H), 4.19 (dd, J = 8.0 Hz and J = 6.5 Hz, 1H), 3.65 (s, 3H), 2.59 (t, J = 7.6 Hz, 2H), 2.21 (t, J
	= 7.3 Hz, 2H), 2.04 (m, 1H), 1.88-1.75 (m, 2H), 0.91 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.7 Hz,
	3H) ; 13 C

+ ; colorless oil. Yield = 35%.

  Bond acceptors ≤ 10, H-Bond donors ≤ 5, rotational bond count ≤ 14, and formal charge count ≤ 2) were used to automatically extract a subset of compounds with reasonable properties.

	reasonable physiochemical properties. More precisely, a structural check was performed with
	StructureChecker from ChemAxon (http://www.chemaxon.com) to eliminate compounds with
	any structural warning. Duplicates and compounds that contain at least one undesired element
	(other than C, H, N, O, P, S, F, Cl, Br or I) were also discarded. Usual threshold values for
	common physico-chemical descriptors (molecular weight ≤ 550, cLogP ≤ 5.5, tPSA ≤ 150 Ų,
	H-

Acknowledgements

This work was supported by grants from the National Research Agency (ANR, Investissements d'Avenir, A * MIDEX project ANR-11-IDEX-0001-02), the Institut National du Cancer (INCa, subvention 2013-105), the Belgian Foundation against cancer (STK, FA/2014/294), the French National Research Agency (ANR-18-CE13-0017, Project SynTEV), the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 747025, the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 with the use of the AFMB laboratory Xray facility. The authors acknowledge the European Synchrotron Radiation Facility (ESRF) as well as the SOLEIL synchrotron (Proxima 1 and Proxima 2 beamlines) for provision of synchrotron radiation facilities. M. Garcia was the recipient of La Ligue, French Foundation for Cancer Research. R. Leblanc was the recipient of the Fondation ARC, French Foundation for Cancer Research.

4-4-4. Western blots

The proteins were heat-denaturated in Laemmli sample buffer, fractionated in 12.5% or 15% gels by SDS-PAGE and electro-transferred to nitrocellulose membrane. Membranes were stained with Ponceau red and immunoblotted with the indicated primary antibodies for: CD9

(1/5000), CD63 (1/5000), CD81 (1/5000) and ADAM10 (1/1000) antibodies provided by E.

Rubinstein (Université Paris-Sud UMRS_935, Villejuif); syntenin (Homemade; 1/3000), Tubulin (Sigma-Aldrich; 1/10000), ALIX (Homemade, 1/500), HSP70 (Santa Cruz Biotechnology; 1/500), Flotillin-1 (BD Biosciences; 1/1000), EGFR (Cell Signaling; 1/1000), EpCAM (Santa Cruz Biotechnology; 1/200), Fibronectin (BD Biosciences; 1/5000), Src (Cell signaling; 1/1000), TSP1 (Lab Vision; 1/100), SDC1 intracellular domain (Homemade, 1/1000), SDC4 intracellular domain (Abnova; 1/2000), and HRP-conjugated secondary antibodies (Mouse or Rabbit, Thermofisher scientific; 1/10000). Signals were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) [START_REF] Leblanc | Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loadifing with syndecan and EpCAM cargo[END_REF].