Marion Gay

Caroline Evrard

Florian Descamps

Pascal Carato

Nicolas Renault

Mathilde Coevoet

Sabiha Eddarkaoui

Catherine Baud

Paul-Emmanuel Larchanché

Luc Buée

Jamal El Bakali

Valérie Vingtdeux

Nicolas Sergeant

Patricia Melnyk

J M Izquierdo

Y Izumi

V Izzo

M Jäättelä

N Jaber

D J Jackson

W T Jackson

T G Jacob

T S Jacques

C Jagannath

A Jain

N R Jana

B K Jang

A Jani

B Janji

P R Jannig

P J Jansson

S Jean

M Jendrach

J H Jeon

N Jessen

E B Jeung

K Jia

L Jia

H Jiang

L Jiang

T Jiang

X Jiang

Y Jiang

A Jiménez

C Jin

H Jin

L Jin

M Jin

S Jin

U K Jinwal

E K Jo

T Johansen

D E Johnson

G V W Johnson

J D Johnson

E Jonasch

C Jones

L A B Joosten

J Jordan

A M Joseph

B Joseph

A M Joubert

D Ju

J Ju

H F Juan

K Juenemann

G Juhász

H S Jung

J U Jung

Y K Jung

H Jungbluth

M J Justice

B Jutten

N O Kaakoush

K Kaarniranta

A Kaasik

T Kabuta

B Kaeffer

K Kågedal

A Kahana

S Kajimura

O Kakhlon

M Kalia

D V Kalvakolanu

Y Kamada

K Kambas

V O Kaminskyy

H H Kampinga

M Kandouz

C Kang

R Kang

T C Kang

T Kanki

T D Kanneganti

H Kanno

A G Kanthasamy

M Kantorow

M Kaparakis-Liaskos

O Kapuy

V Karantza

M R Karim

P Karmakar

A Kaser

S Kaushik

T Kawula

A M Kaynar

P Y Ke

Z J Ke

J H Kehrl

K E Keller

J K Kemper

A K Kenworthy

O Kepp

A Kern

S Kesari

D Kessel

R Ketteler

I Do

C Kettelhut

B Khambu

M M Khan

V K M Khandelwal

S Khare

J G Kiang

A A Kiger

A Kihara

A L Kim

C H Kim

D R Kim

D H Kim

E K Kim

H Y Kim

H R Kim

J S Kim

J H Kim

J H Kim

J C Kim

J H Kim

J H Kim

M.D K W Kim

M M Kim

P K Kim

S W Kim

S Y Kim

Y S Kim

A C Kimchi

T Kimmelman

J S Kimura

K King

V Kirkegaard

L A Kirkin

S Kirshenbaum

Y Kishi

K Kitajima

Y Kitamoto

K Kitaoka

R A Kitazato

W T Kley

M Klimecki

J Klinkenberg

H Klucken

E Knaevelsrud

L Knecht

J L Knuppertz

S Ko

J C Kobayashi

C Koch

U Koechlin-Ramonatxo

Y H Koenig

K Koh

S D Köhler

M Kohlwein

M Koike

E Komatsu

D Kominami

H J Kong

E G Kong

B T Konstantakou

T Kopp

L Korcsmaros

V I Korhonen

N V Korolchuk

Y Koshkina

M I Kou

C Koukourakis

A L Koumenis

T Kovács

W J Kovács

D Kovacs

C Koya

D Kraft

H Krainc

T Kramer

W Kravic-Stevovic

C Krek

C Kretz-Remy

R Kretz-Remy

M Krick

J Krishnamurthy

G Kriston-Vizi

G Kroemer

M C Kroemer

R Kruer

N T Kruger

K Ktistakis

C Kuchitsu

A P Kuhn

A Kumar

D Kumar

R Kumar

S Kumar

M Kumar

H J Kundu

H J Kung

A Kung

S H Kuno

J Kuo

T Kuret

T Kurz

T K Kwok

Y T Kwon

I Kwon

A R La Kyrmizi

A R Spada

La

A phenotypic

Keywords: Alzheimer's disease, Amyloid Precursor Protein, chloroquine, lysosome, autophagy, βsecretase, BACE-1, indirect inhibitor, phenotypic

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

Introduction

Alzheimer's disease (AD), the most frequent form of dementia, is characterized by a slow decline of cognitive and behavioral abilities. Two neuropathological lesions provide the definite diagnosis of AD: neurofibrillary tangles and amyloid deposits [START_REF] Serrano-Pozo | Neuropathological alterations in Alzheimer disease[END_REF]. Neurofibrillary tangles are characterized by intraneuronal accumulation and aggregation of abnormally modified isoforms of the microtubule-associated tau proteins. Parenchymal amyloid deposits are composed of amyloid-β (Aβ) peptides, which derive from its precursor, the so-called amyloid precursor protein (APP) (for a comprehensive review see ref. [START_REF] Strooper | Proteases and Proteolysis in Alzheimer Disease: A Multifactorial View on the Disease Process[END_REF]). Current treatments for AD remain symptomatic and provide only limited benefits to the patients. Therefore, alternative therapeutic options are urgently needed and over the past few years, much effort has been dedicated to the development of disease-modifying therapeutics [START_REF] Cummings | Alzheimer's disease drug development pipeline: 2017, Alzheimer's Dement[END_REF]. One such strategy consists of reducing the levels of toxic species of Aβ peptides by targeting APP processing and more specifically secretases that cleave APP to produce Aβ peptides. Indeed, the metabolism of APP involves the action of several secretases and is sub-cellularly compartmentalized. APP is known to undergo proteolytic processing through two main pathways: the non-amyloidogenic pathway, initiated by α-secretase cleavage and the amyloidogenic pathway initiated by β-secretase cleavage (Figure 1) [START_REF] Andrew | A Greek tragedy: The growing complexity of Alzheimer amyloid precursor protein proteolysis[END_REF][START_REF] Nhan | The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes[END_REF]. Both secretase-mediated steps release soluble ectodomains of APP (sAPPα and sAPPβ) and membrane-bound carboxyl-terminal fragments (APP-CTFs), known as αCTF and βCTF. The latter are further cleaved by γ-secretase to give p3 and Aβ peptides, respectively, along with the APP intracellular domain (AICD).

Redirection of APP processing can be achieved either by blocking the amyloidogenic pathway or by promoting the non-amyloidogenic pathway. Because amyloidogenic processing is initiated by the cleavage of APP by β-secretase (β-site amyloid-precursor-protein-cleaving enzyme 1, BACE-1), this protease has been suggested as an attractive target to lower Aβ levels and the resulting amyloid plaques [START_REF] Vassar | The -Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential[END_REF][START_REF] Yan | Targeting the β secretase BACE1 for Alzheimer's disease therapy[END_REF]. Recently, a mutation in APP (APP-A673T) that protects against the development of AD and age-related cognitive decline has been identified, providing further evidence that reducing β-secretase cleavage of APP may protect against AD [START_REF] Jonsson | A mutation in APP protects against Alzheimer's disease and age-related cognitive decline[END_REF][START_REF] Maloney | Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein[END_REF]. Most efforts regarding β-secretase inhibitors have been focused on active site binders [START_REF] Ghosh | BACE1 (β-secretase) inhibitors for the treatment of Alzheimer's disease[END_REF], and although β-secretase inhibitor drug development has proven challenging, several inhibitors are currently under clinical development [START_REF] Cummings | Alzheimer's disease drug development pipeline: 2017, Alzheimer's Dement[END_REF][START_REF] Vassar | BACE1 inhibitor drugs in clinical trials for Alzheimer's disease[END_REF]. Given that β-secretase has multiple substrates involved in important cellular and tissue functions, its inhibition may produce mechanism-based side effects [START_REF] Yan | Targeting the β secretase BACE1 for Alzheimer's disease therapy[END_REF]. As an alternative, a recent study showed that the anticancer drug Gleevec lowers Aβ levels through indirect inhibition of β-secretase processing of APP [START_REF] Netzer | Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage[END_REF]. Another recent study taking advantage of the compartmentalization of APP processing, showed that β-secretase inhibitors can be designed to specifically inhibit APP processing in the endosomal compartment with no effect on non-amyloid substrates [START_REF] Ben Halima | Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein[END_REF]. Indeed, the endosomal/lysosomal compartments are known to be of importance for APP metabolism, APP degradation as well as for the production of Aβ peptides [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF][START_REF] Hamano | Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression[END_REF][START_REF] Tam | Tyrosine binding protein sites regulate the intracellular trafficking and processing of amyloid precursor protein through a novel lysosome-directed pathway[END_REF]. In this context, the lysosomotropic agent, chloroquine (CQ) has been reported to inhibit Aβ peptide production [START_REF] Melnyk | Chloroquine and Chloroquinoline Derivatives as Models for the Design of Modulators of Amyloid Peptide Precursor Metabolism[END_REF][START_REF] Evrard | Contribution of the endosomal-lysosomal and proteasomal systems in Amyloid-β Precursor Protein derived fragments processing[END_REF]. However, the alkalizing property of CQ also leads to the inhibition of lysosomemediated degradation pathways, resulting in the accumulation of APP-CTFs and AICD [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF][START_REF] Vingtdeux | Intracellular pH regulates amyloid precursor protein intracellular domain accumulation[END_REF][START_REF] González | Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway[END_REF], and in the disruption of the autophagic flux [21]. Autophagy-lysosomal pathway dysfunction has been suggested to play a detrimental role in the pathophysiology of AD [START_REF] Ghavami | Autophagy and apoptosis dysfunction in neurodegenerative disorders[END_REF][START_REF] Correia | Alzheimer's Disease-Related Misfolded Proteins and Dysfunctional Organelles on Autophagy Menu[END_REF][START_REF] Peric | Early etiology of Alzheimer's disease: tipping the balance toward autophagy or endosomal dysfunction?[END_REF]. Therefore, CQ-derived compounds that would maintain the effect on Aβ peptides without disrupting lysosomal functions would be of interest.

We have recently developed two series of compounds derived from either CQ (series A) or amodiaquine (AQ, series B) that demonstrated a strong inhibitory effect on both Aβ1-40 and Aβ1-42 secretions [START_REF] Melnyk | Chloroquine and Chloroquinoline Derivatives as Models for the Design of Modulators of Amyloid Peptide Precursor Metabolism[END_REF][START_REF] Melnyk | Heterocycle compounds and uses thereof for the treatment of diseases involving formation of amyloid plaques and/or where a dysfunction of the APP metabolism occurs[END_REF][START_REF] Melnyk | Use of 1,4-bis(3aminoalkylyl)piperazine derivatives in the treatment of neurodegenerative diseases[END_REF][START_REF] Melnyk | Beta and gamma-carbolines derivatives for the treatment of neurodegenerative diseases[END_REF][START_REF] Barrier | Sulfate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, preparation thereof and use of the same[END_REF]. The lead compound of series A is currently ending phase I clinical trials. However, as observed for CQ, these effects were accompanied by a strong increase in the levels of αCTFs and AICD suggesting an alteration of lysosomal functions. Therefore, based on these earlier works, we sought to develop new series of compounds that would retain the effects on Aβ production without affecting αCTFs and AICD stabilities. To that purpose, in the absence of a defined molecular target of the previously developed compounds, a ligand-based pharmacophore modeling approach [START_REF] Yang | Pharmacophore modeling and applications in drug discovery: Challenges and recent advances[END_REF], coupled with de novo design was implemented.

Herein, we describe a new series of compounds based on a biaryl scaffold decorated with amino side chains. Among these compounds, several were shown to inhibit Aβ peptides secretion and to promote αCTFs and AICD stabilities, as the parent compounds (series A and B). However, the structure activity relationship (SAR) study allowed for the discovery of compounds that kept the effect on Aβ peptides but had no impact on αCTFs and AICD stabilities. Further studies on two structurally very close compounds (30 and 31) with different profile regarding αCTFs and AICD stabilities suggest that an indirect -secretase inhibitory effect can be achieved without the alkalizing property which is associated with the disruption of lysosomal functions.

Results and discussion

Ligand-based pharmacophore modeling

A ligand-based approach was applied to determine a common pharmacophoric model between the two series of compounds developed previously (Figure 2A). Because no model was able to predict compounds with the desired profile (decrease of Aβ peptides secretion with no impact on αCTFs and AICD stabilities), the pharmacophore study took into account the sole inhibitory effect on Aβ production. Hence, a set of 51 compounds of series A [START_REF] Melnyk | Chloroquine and Chloroquinoline Derivatives as Models for the Design of Modulators of Amyloid Peptide Precursor Metabolism[END_REF][START_REF] Barrier | Sulfate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, preparation thereof and use of the same[END_REF] and 35 of series B [START_REF] Melnyk | Heterocycle compounds and uses thereof for the treatment of diseases involving formation of amyloid plaques and/or where a dysfunction of the APP metabolism occurs[END_REF][START_REF] Gay | New phenylaniline derivatives as modulators of amyloid protein precursor metabolism[END_REF] (Data and structures shown in Tables S1 andS2) was chosen regarding the ability of the compounds to inhibit Aβ1-42 secretion (subsequently named anti-Aβ). Compounds were classified into 4 classes: 2 (highly active), 1 (moderately active), 0 (inactive) and ND (not determined). The dominant and common property to all of the molecules is their high positive formal charge, which is comprised between +2 and +4. This ionization state could be ambiguous when predicted pKa get closer to physiological pH 7.4, in particular for both nitrogens of a piperazine group or for nitrogens of benzimidazole and quinoline heterocyclic moieties. In order to increase the possibility of pharmacophore detection on the basis of positively ionized features, the ionization state with the highest formal charge was selected for each molecule. 3D pharmacophore models were scored based on their ability to discriminate between active and inactive compounds (Table S3). The best model, describing four spatial points Positive/Positive/Positive/Aromatic (PPPA), was computed from the 18 molecules with the highest inhibitory activity of Aβ1-42 expression. This 3D pharmacophore model seemed more efficient in comparison with the 3-points PPA pharmacophores. Using the PPPA pharmacophoric model, several substructure were considered. This paper focuses on a biarylbased series of compounds (Figure 2B). This biphenyl scaffold was substituted on the first phenyl ring by N-piperidinylethoxy and 3-(N-piperidinyl)propanamino side chains and a benzylic tertiary amine in ortho position of the second phenyl ring. Structural modifications around this scaffold included, i) the modulation of the nature (piperidine, morpholine, dimethylamine and N-methylpiperazine) and the position (ortho, meta and para) of the benzylic tertiary amine, ii) the length of the linker separating the piperidine moieties from the first phenyl ring (n, m = 2 to 4), iii) replacement of the second phenyl group by a pyrrole, and iv) stepwise removal of the three tertiary amines.

Synthetic chemistry

Compounds 24-37 were obtained as described in Scheme 1. Commercially available benzoxazol-2-one was regioselectively brominated in position 6 (Br2/AcOH), followed by Nalkylation, ring opening under alkaline conditions [START_REF] Carato | Synthesis and binding studies on a new series of arylpiperazino benzoxazol-2-one and benzoxazin-3-one deriv-atives as selective D4 ligands[END_REF]. In this case, dioxane was used instead of MeOH to avoid side products. O-alkylation using NaH as a base gave intermediates 8-14 in moderate to good yields. Subsequent Suzuki-coupling reaction with appropriate boronic acids afforded compounds 15-23 and 37. The last step consisted in a reductive amination, which was performed using two different procedures. Indeed, compounds 24-31 were obtained performing the reaction in DCE and using NaBH(OAc)3 as the reductive agent (procedure A), while compounds 32-36 required the addition of titanium isopropoxide in EtOH and the use of NaBH4 as the reductive agent (procedure B [START_REF] Bhattacharyya | Reductive Alkylations of Dimethylamine Using Titanium(IV) Isopropoxide and Sodium Borohydride: An Efficient, Safe, and Convenient Method for the Synthesis of N,N-Dimethylated Tertiary Amines[END_REF]).

Compounds 39, 41 and 44 were obtained as described in Scheme 2. The first step consisted in a nucleophilic substitution (either O-alkylation or N-alkylation) followed by a Suzuki-coupling reaction to give compounds 39, 40 and 43. Subsequent reductive amination under procedure A afforded final compounds 41 and 44.

Compound 51 was obtained as outlined in Scheme 3. Benzoxazol-2-one was first nitrated regioselectively in position 6 with nitric acid, followed by N-alkylation and ring opening under alkaline conditions to give intermediate 47. Subsequent O-alkylation using K2CO3 as a base, and nitro reduction with ammonium formate gave 49. The latter was subjected to a Clauson-Kaas reaction with 2,5-dimethoxy-3-tetrahydrofurancarboxaldehyde in acetic acid to form pyrrole intermediate 50 [START_REF] Sbardella | Synthesis and in vitro antimycobacterial activity of novel 3-(1H-pyrrol-1-yl)-2-oxazolidinone analogues of PNU-100480[END_REF]. The last step consisted in a reductive amination in DCE and using NaBH(OAc)3 as the reductive agent to give compound 51.

Structure-Activity Relationship studies

Final compounds were evaluated for their ability to modulate APP processing on SY5Y human neuroblastoma cell line stably expressing the neuronal isoform of human wild-type APP 695 (SY5Y-APPwt). Aβ levels (Aβ1-40 and Aβ1-42) after treatment with reference and tested compounds were measured by ELISA (Table 1). Results are expressed as IC50 values which correspond to the concentration of a given compound that inhibits Aβ expression by 50% (either Aβ1-40 or Aβ1-42). APP carboxy-terminal fragments (αCTFs and AICD) levels were assessed by Western-blotting (WB). The effect of reference and tested compounds on αCTFs and AICD are expressed as the intensity of corresponding WB bands. Compounds effect was measured at various concentrations (1, 3, 10 µM, Figure 3) and compared to the effect of CQ at 3µM (Table 1). First, when considering the effect on Aβ levels, regardless of the modulations, all synthesized compounds decreased Aβ levels with IC50 values ranging from 1.2 to 13.7 µM. Indeed, as can be seen from Table 1 and Figures 2A and2B, compounds displaying three tertiary amines (24-30, 32-36 and 51) were all more potent than CQ (3 µM) in increasing AICD and αCTFs levels (up to 6 fold, Table 1). For example, compared to control conditions, compound 30 and 32 (3 µM) increased αCTFs levels by almost 30 and 50-fold, respectively and AICD levels by more than 40 and 30-fold, respectively. On the other hand, replacing one piperidine of compound 30 by a cyclohexyl group [START_REF] Carato | Synthesis and binding studies on a new series of arylpiperazino benzoxazol-2-one and benzoxazin-3-one deriv-atives as selective D4 ligands[END_REF] completely abolished AICD and αCTFs activities as compound 31 had a similar profile as the untreated conditions. This result shows that it is possible to retain the effects on Aβ secretion without affecting αCTFs and AICD stabilities and encouraged us to further refine APP processing profile of compounds 30 and 31.

Effects of compounds 30 and 31 on APP metabolism

Following this primary screening and structure activity relationship analysis, a detailed and comparative analysis of compounds 30 and 31 was performed. First, toxicity assays were performed with both compounds on SY5Y-APPwt cells and in primary neurons. Compounds were tested up to 5 µM and no toxicity was observed (Figure S1). Effects of both compound was then assessed simultaneously on APP metabolism. Full-length APP proteins were resolved and detected with the APP-C17 antiserum as two bands at 110 kDa and 115 kDa. The lowest APP band corresponds to the neosynthesized or immature form of APP whereas the highest band corresponds to the glycosylated, namely the mature form of APP. Both were quantified, and no modification of immature APP levels was observed with either compounds (Figure 4A, D and F). While compound 31 did not modify the expression of mature APP, compound 30 significantly increased mature APP expression at 5 μM by 221% (Figure 4A, C andE). These results were confirmed using primary neuronal cultures in which treatment with compound 30 at 3 and 5 μM induced a significant increase in mature APP of 130% when compared to the control condition (Figure S2A,C). In primary neurons, compound 31 had also no effect on total APP expression (Figure S2A,E).

Full-length APP is further processed by α-, β-and γ-secretases to generate APP-CTFs and AICD. A significant accumulation of APP-CTFs and AICD was observed following treatment with compound 30 (Figure 4B,G,H). This accumulation of APP-CTFs was also observed in primary neurons (Figure S2A andD). Surprisingly, compound 31 did not demonstrate any effect on APP-CTFs or AICD expression in both SY5Y-APPwt cells and primary neuronal cultures (Figure 4B, I, J and Figure S3A,F).

As shown previously (Table 1), compounds 30 and 31 significantly reduced Aβ1-40 and Aβ1-42 production (Figure 4K-N). This reduction of Aβ production may be caused by either an increase of the α-secretase cleavage or a decrease of either the β-or γ-secretase cleavage of APP. Thus, we assessed the secretion of sAPPα and sAPPβ, which are the shedded byproducts of α-and βsecretase, respectively. Both compounds 30 and 31 were found to significantly reduce sAPPβ concentration (Figure 4P andR). On the opposite, sAPPα concentration was significantly increased by compound 30 and to a lesser extend by compound 31 (Figure 4O andQ). Together these results strongly suggest that compounds 30 and 31 reduce Aβ1-40 and Aβ1-42 secretion by repressing β-secretase cleavage of APP while increasing the secretion of sAPPα.

To determine whether compounds 30 and 31 were direct BACE-1 inhibitors, an enzymatic assay monitoring the cleavage of a fluorogenic peptide substrate (Mca-S-E-V-N-L-D-AE-F-R-K(Dnp)-R-R-NH2) was performed [START_REF] Ermolieff | Proteolytic activation of recombinant promemapsin 2 (pro-β-secretase) studied with new fluorogenic substrates[END_REF]. In contrast to reference OM 99-2 (IC50 = 59 nM), under the assay conditions (cf. experimental section), neither compound 30 nor 31 inhibited the cleavage of the fluorogenic peptide even at a concentration of 10 µM (% inhibition < 10%). All together, these data show that compounds 30 and 31 are acting through an indirect mechanism to inhibit β-secretase activity.

Although compounds 30 and 31 derived from the same pharmacophore, only compound 30 increased the expression of mature APP, APP-CTFs and AICD, suggesting that compounds 30 and 31 operated through different mechanisms. CQ is a well-known inhibitor of the autophagic flux and lysosomal degradation pathway, and compounds of series A derive from a CQ library of molecules. Therefore, we assessed whether the activity of compound 30 involved a repression of lysosomal activities that could explain the observed accumulation of mature APP, APP-CTFs and AICD. To address this question, we studied the effect of both compounds 30 and 31 on the autophagic flux. To that purpose, we assessed two markers associated with autophagy: p62 and LC3-I lipidation into LC3-II, which is characterized by the appearance of a second band at 14 kDa under the LC3-I band of 18 kDa. Treatment with compound 30 at 5 μM induced an increase of p62 expression by 476% as compared to the control condition (Figure 5A andB). LC3-I lipidation into LC3-II was also increased by 591% (Figure 5A andD). Similar results were obtained in primary neurons starting at a concentration of 3 μM of compound 30 (Figure S2B, G andH). In sharp contrast, compound 31 showed no effect on these two autophagy markers in either cell systems (Figure 5A, C, E and Figure S3B, I andJ).

As the increase of p62 and LC3 lipidation suggest an inhibition of the autophagic flux, we cotreated the cells with compound 30 and Bafilomycine A1 (BafA1), a well-known inhibitor of the autophagic flux [START_REF] Yamamoto | Bafilomycin A1 Prevents Maturation of Autophagic Vacuoles by Inhibiting Fusion between Autophagosomes and Lysosomes in Rat Hepatoma Cell Line, H-4-II-E Cells[END_REF]. In co-treatment with BafA1, compound 30 demonstrated similar accumulations of p62 and LC3-II as with BafA1 alone (Figure 5F, G andH). Indeed, no additive effect of co-treatments was observed for p62 expression or LC3 lipidation, showing that compound 30 display an inhibitory effect on the autophagic flux. Inhibition of the autophagic flux and accumulation of p62 and LC3 were further confirmed by immunofluorescence in SY5Y-APPwt cells (Figure 5I andJ) and in primary neurons (Figure S2K andL). Treatment with compound 30 at 5 μM displayed a significant accumulation of p62 and LC3 positive vesicles inside these two different cell models. Together, these results demonstrate that compound 30 acts as an inhibitor of the autophagic flux, and thus of lysosomal degradation properties. Importantly, compound 31, which displays a similar activity than compound 30 toward Aβ reduction, does not bear this later property.

Conclusion

This work reports the design, synthesis and in vitro biological evaluation of a biaryl-based series of modulators of APP processing. SAR studies revealed that all compounds were able to decrease Aβ secretion but minor modifications like replacing a piperidine moiety of 30 by a cyclohexyl (compound 31) totally abolished AICD and αCTFs stabilities. The effect of compound 30 and related analogues on AICD and αCTFs stabilities was shown to be linked to an alteration of lysosomal functions. Compound 31, on the other hand, behave as an indirect βsecretase inhibitor with no effect on lysosomes. Importantly, this work shows that it is possible to dissociate the lysosomotropic effect of CQ-derived compounds from their action on Aβ secretion, providing a new profile of indirect β-secretase inhibitors. Although the exact mechanism of action remains to be elucidated, this novel series provides an opportunity for the development of novel -secretase inhibitors potentially useful for the treatment of AD.

Experimental sections

Chemistry

The reaction monitoring was performed by thin layer chromatography (TLC) on Macherey-Nagel Alugram® Sil 60/UV254 (thickness 0.2 mm). TLC were revealed by UV (λ = 254 nm) and/or the appropriate stain (iodine, nynhydrine). Purification of the compounds was carried out by column chromatography (either flash or manual). Manual chromatography was performed using Macherey-Nagel silica gel (0.04-0.063 mm of particule size). Flash chromatography was performed on a Reveleris® Flash Chromatography System using Macherey-Nagel Chromabond flash RS columns. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1 H and 75 MHz for 13 C). Chemical shifts are reported in parts per million (ppm) relative to an internal reference (either tetramethylsilane or residual deuterated solvents 1 H signals). Each signal is reported with its chemical shift (δ), multiplicity (s = singulet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (J in Hz, when applicable) and relative integral. The attributions of protons and carbons were achieved by analysis of 1D and 2D experiments ( 1 H, 13 C, COSY, HQC and HMBC). LC-MS were performed on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionisation and a UV detector (diode array). Elution was performed at a flow rate of 2 mL/min with water-formic acid (pH = 3.8) as eluent A and ACN-formic acid (pH = 3.8) as eluent B, employing a 0.25 min plateau with 0 % B and a linear gradient from 0 % B to 98 % B in 3.25 min, followed by a 0.5 min plateau with 98 % B. Then, column re-equilibration was performed for 1 min. HRMS were recorded on a Hight Resolution Mass Spectrometer (HRMS) Thermo Scientific™ Exactive™.

Analysed compounds were dissolved in methanol and directly injected in the ionisation source ESI, in positive or negative mode according to the analyzed compound, and recorded for one minute. The Xcalibur software was used to determine the elementar composition of main pics of the spectrum. The purity of final compounds was determined by high pressure liquid chromatography (HPLC) using two columns: C18 Interchrom UPTISPHERE and C4 Interchrom UPTISPHERE. The HPLC analysis was carried out on a Shimadzu LC-2010AHT system equipped with a UV detector set at 254 and 215 nm. The compounds were dissolved in 100 µL of buffer B and 900 µL of buffer A. The eluent system used was: buffer A (H2O/TFA, 100:0.1) and buffer B (ACN/H2O/TFA, 80:20:0.1). Retention times (tr) were obtained at a flow rate of 0.2 mL/min for 37 min using a gradient form 100% of buffer A over 1 min, to 100 % buffer B over the next 30 min, to 100% of buffer A over 1 min and 100% of buffer A over 1 min. All tested compounds showed a purity of ≥ 95%. The melting point analyses were performed on Barnstead Electrothermel Melting Point Series IA9200 and were not corrected.

All final compounds were transformed into their hydrochloride salt forms (before testing) using the following procedure: the compound was dissolved in MeOH and 2N HClaq was added dropwise until pH1. The solvent was evaporated and the compound was freeze-dried.

6-Bromo

-3H-1,3-benzoxazol-2-one (1)
3H-1,3-Benzoxazol-2-one (5.00 g, 37.00 mmol) was dissolved in acetic acid (50 mL) and bromine (1.9 mL, 37.0 mmol) was added dropwise. The reaction mixture was stirred at 20 °C for 4 h. The reaction mixture was poured onto ice and the precipitate was collected by filtration, washed with water and air-dried to give a pink powder (7.48 g, 34.8 mmol, 94%). Mp 191.6 -192.3 °C. 1 H NMR (300 MHz, DMSO-d6): δ 11.81 (s, 1H), 7.57 (dd, J = 1.9 Hz, J = 0.3 Hz, 1H), 7.30 (dd, J = 8.3 Hz, J = 1.9 Hz, 1H), 7.04 (dd, J = 8.3 Hz, J = 0.3 Hz, 1H). 13

6-Bromo

-3-[3-(piperidin-1-yl)propyl]-1,3-benzoxazol-2-one (3)
Colorless oil (7.76 g, 22.9 mmol, 98%). 1 H NMR (300 MHz, CDCl3): δ 7.36 (d, J = 1.8 Hz, 1H), 7.30 (dd, J = 8.3 Hz, J = 1.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.88 (t, J = 6.7 Hz, 2H); 2.35 -2.28 (m, 6H), 1.98 -1.89 (m, 2H), 1.60 -1.51 (m, 4H), 1.46 -1.38 (m, 2H). 13 C NMR (75 MHz, CDCl3): δ 154. 1, 143.1, 130.8, 126.6, 114.4, 113.5, 109.7, 55.5, 54.5, 40.6, 25.9, 24.8, 24.4. LCMS m/z calc for [M+H] + : 338.1, 340.1, found: 338.9, 340.8.

6-bromo

-3-[4-(piperidin-1-yl)butyl]-1,3-benzoxazol-2-one (4)
To a solution of 1-bromo-4-chlorobutane (2.17 mL, 18.7 mmol), K2CO3 (3.87 g, 28.04 mmol) in ACN (1 mL) was added dropwise at a solution of 6-bromo-3H-1,3-benzoxazol-2-one (1) in ACN (3 mL). The mixture was heated at 80 °C for 12 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was purified by column chromatography (cyclohexane/ethyl acetate), 9:1 (v:v)) to afford 6-bromo-3-(4-chlorobutyl)-1,3-benzoxazol-2one as a white solid (1.2 g, 3.94 mmol). Mp 61.9 -62.4 °C. 1 H NMR (300 MHz, CDCl3): δ 7.39 (d, J = 1.8 Hz, 1H), 7.34 (dd, J = 8.3, 1.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 3.87 (t, J = 6.8 Hz, 2H), 3.60 (t, J = 6.1 Hz, 2H), 2.27 -1.74 (m, 4H). 13 Brown oil (2.16 g, 6.9 mmol, 95%). 1 H NMR (300 MHz, MeOD): δ 6.82 -6.78 (m, 2H), 6.49 (d, J = 8.1 Hz, 1H), 3.15 (t, J = 6.7 Hz, 2H), 2.57 -2.52 (m, 6H), 1.89 -1.80 (m, 2H), 1.69 -1.62 (m, 4H), 1.54 -1.50 (m, 2H). 13 C NMR (75 MHz, MeOD): δ 145.8, 136.7, 122.1, 115.9, 111.5, 107.6, 56.7, 53.9, 41.8, 25.0, 24.7, 23.4. LCMS m/z calc for [M+H] + : 313.1, 315.1, found: 312.9, 314.9.

5-bromo-2-[(4-(piperidin-1-yl)butyl)amino]phenol (7)

Beige solid (246 mg, 0.75 mmol, 50%). Mp 130.3 -130.9 °C. 1 H NMR (300 MHz, DMSO-d6): δ 6.79 -6.73 (m, 2H), 6.40 (d, J = 9.0 Hz, 1H), 3.02 (t, J = 6.5 Hz, 2H), 2.39 -2.22 (m, 6H), 1.59 -1.27 (m, 12H). 13

4-Bromo-N-[2-(piperidin-1-yl)ethyl]-2-[3-(piperidin-1-yl)propoxy]aniline (9)

Brown oil (450 mg, 1.06 mmol, 32 %). 1 [START_REF] Ben Halima | Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein[END_REF] Brown oil (513 mg, 1.17 mmol, 55 %). 1 H NMR (300 MHz, CDCl3): δ 6.96 (dd, J = 8.4, 2.1 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.44 (d, J = 8.4 Hz, 1H), 4.37 (s, 1H), 4.08 (t, J = 5.9 Hz, 2H), 3.12 (s, 2H), 2.76 (t, J = 5.9 Hz, 2H), 2.58 -2.27 (m, 10H), 1.78 -1.36 (m, 16H). 13 The residue was purified by column chromatography (DCM/MeOH(NH3), 9.8 :0.2 (v:v)) to afford 10 as a brown oil (246 mg, 0.56 mmol, 18% over two steps). 1 were added and the reaction was stirred for 30 min and deoxygenated by passing a stream of N2 through it. Pd2dba3 (7 mg, 0.01 mmol) and P(o-tol)3 (22 mg, 0.07 mmol) were added and the mixture was refluxed for 18 h. After cooling to rt, the mixture was poured into water, extracted with ethyl acetate. The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (PE/EtOAc/MeOH(NH3), 10:0:0 to 5.2:4.4: 15) as a brown oil (146 mg, 0.32 mmol, 92%). 1

4-Bromo

-N-[4-(piperidin-1-yl)butyl]-2-[2-(piperidin-1-yl)ethoxy]aniline
0.4 (v/v)) to afford 3'-(2-(piperidin-1-yl)ethoxy)-4'-((3-(piperidin-1- yl)propyl)amino)-[1,1'-biphenyl]-2-carbaldehyde (
3'-(2-(piperidin-1-yl)ethoxy)-4'-((3-(piperidin-1-yl)propyl)amino)-[1,1'-biphenyl]-3- carbaldehyde (16)
Brown oil (600 mg, 1.33 mmol, 96%). 1 H NMR (300 MHz, CDCl3): δ 10.07 (s, 1H), 8.05 (t, J = 1.6 Hz, 1H), 7.81 (m, 1H), 7.75 (dt, J = 7.7 Hz, J = 1.6 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.18 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 6.68 (d, J = 8.2 Hz, 1H), 4.95 (br s, 1H), 4.22 (t, J = 6.2 Hz, 2H), 3.24 (t, J = 6.7 Hz, 2H), 2.83 (t, J = 6.2 Hz, 2H), 2.56 -2.41 (m, 10H), 1.91 -1.87 (m, 2H); 1.67 -1.59 (m, 8H), 1.49 -1.45 (m, 4H). 13 Brown oil (459 mg, 1.05 mmol, 92%). 1 H NMR (300 MHz, CDCl3): δ 10.03 (s, 1H), 7.98 (dd, J = 7.8, 1.4 Hz, 1H), 7.59 (tt, J = 13.1, 6.5 Hz, 1H), 7.45 -7.41 (m, 2H), 6.87 (dd, J = 8.0, 1.9 Hz, 1H), 6.81 (d, J = 1.9 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 5.13 (s, 1H), 4.16 (t, J = 5.8 Hz, 2H), 3.25 (m, 2H), 2.85 (t, J = 5.8 Hz, 2H), 2.66 (t, J = 6.3 Hz, 2H), 2.59 -2.50 (m, 4H), 2.49 -2.40 (m, 4H), 1.67 -1.39 (m, 12H). 13 Brown oil (378 mg, 0.84 mmol, 92%). Brown oil (378 mg, 92%). 1 H NMR (300 MHz, CDCl3): δ 10.04 (s, 1H), 7.98 (dd, J = 7.8, 1.3 Hz, 1H), 7.65 -7.54 (m, 1H), 7.50 -7.37 (m, 2H), 6.86 (dd, J = 8.0, 1.8 Hz, 1H), 6.80 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 5.02 (t, J = 4.9 Hz, 1H), 4.07 (t, J = 6.2 Hz, 2H), 3.25 (dd, J = 11.5, 6.0 Hz, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.57 -2.32 (m, 10H), 1.67 -1.54 (m, 8H), 1.52 -1.41 (m, 4H). 13 2.80 (t, J = 6.1 Hz, 2H), 2.52 -2.30 (m, 14H), 1.90 (quint, J = 6.7 Hz, 2H), 1.68 -1.38 (m, 18H). 13 [START_REF] Barrier | Sulfate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, preparation thereof and use of the same[END_REF] Colorless oil (126 mg, 0.24 mmol, 40%). 1 H NMR (300 MHz, CDCl3): δ 7.53 -7.48 (m, 1H); 7.29 -7.26 (m, 3H), 6.94 -6.89 (m, 2H), 6.64 (d, J = 8.6 Hz, 1H), 4.80 (br s, 1H), 4.14 (t, J = 6.0 Hz, 2H), 3.68 (t, J = 4.4 Hz, 4H); 3.45 (s, 2H); 3.23 (t, J = 6.8 Hz, 2H); 2.79 (t, J = 6.0 Hz, 2H); 2.52 -2.38 (m, 14H), 1.90 (quint, J = 6.8 Hz, 2H), 1.66 -1.56 (m, 8H), 1.50 -1.42 (m, 4H). 13

+ = C33H51N4O. 4-[2-(Morpholin-4-ylmethyl)phenyl]-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1- yl)propyl]aniline

4-(2-[(4-Methylpiperazin-1-yl)methyl]phenyl)-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1yl) propyl]aniline (29)

Colorless oil (213 mg, 0.40 mmol, 54%). 1 H NMR (300 MHz, CDCl3): δ 7.48 -7.45 (m, 1H), 7.27 -7.24 (m, 3H), 6.94 -6.90 (m, 2H), 6.63 (d, J = 8.0 Hz, 1H), 4.13 (t, J = 6.1 Hz, 2H), 3.44 (s, 2H), 3.22 (t, J = 6.8 Hz, 2H), 2.79 (t, J = 6.1 Hz, 2H), 2.50 -2.27 (m, 21H), 1.88 (quint, J = 7.5 Hz, 2H), 1.66 -1.56 (m, 8H), 1.48 -1.42 (m, 4H). 13

4-(2-[(Dimethylamino)methyl]phenyl)-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1yl)propyl] aniline (30)

In the present case 13 equiv. of dimethylamine 2M in THF was used. Compound was obtained as a colorless oil (162 mg, 0.34 mmol, 51%). 1 H NMR (300 MHz, CDCl3): δ 7.51 -7.49 (m, 1H), 7.30 -7.25 (m, 3H), 6.91 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 8.0 Hz, J = 1.8 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 4.74 (br s, 1H), 4.14 (t, J = 6.2 Hz, 2H), 3.39 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.54 -2.40 (m, 10H), 2.18 (s, 6H), 1.93 -1.84 (m, 2H), 1.67 -1.57 (m, 8H), 1.50 -1.42 (m, 4H). 13

2'-((Dimethylamino)methyl)-3-(3-(piperidin-1-yl)propoxy)-N-(3-(piperidin-1-yl)propyl)-[1,1'biphenyl]-4-amine (32)

Brown oil (75 mg, 0.15 mmol, 47%). 1 4H). 13 Brown oil (36 mg, 0.07 mmol, 30%). 1

2'-((Dimethylamino)methyl)-3-(4-(piperidin-1-yl)butoxy)-N-(2-(piperidin-1-yl)ethyl)-[1,1'biphenyl]-4-amine (35)

Brown oil (35 mg, 0.07 mmol, 28%). 1

3-(2-(Piperidin-1-yl)ethoxy)-N-(3-(piperidin-1-yl)propyl)-[1,1'-biphenyl]-4-amine (37)

To a solution of benzeneboronic acid (108 mg, 0.88 mmol) in a mixture of toluene (8 mL) and EtOH (3 mL), K2CO3 (132 mg, 0.96 mmol) and 11 (156 mg, 0.37 mmol) were added. The reaction was stirred for 30 min and deoxygenated by passing a stream of N2 through it. Pd2dba3 (6.73 mg, 0.007 mmol) and P(o-tol)3 (22 mg, 0.073 mmol) were added and the mixture was refluxed overnight. After cooling, the mixture was poured into 1N NaOH and extracted three times with dichloromethane. The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 10:0 to 95:5 (v/v)) to afford a brown oil (110 mg, 0.26 mmol, 71 %). 1

N-(2-(piperidin-1-yl)ethyl)-[1,1'-biphenyl]-4-amine (39)

3-Phenylphenol (1.02 g, 6.02 mmol) was suspended in ACN (50 mL) and K2CO3 (2.49 g, 18.0 mmol) was added. The reaction mixture was stirred at 80 °C for 30 min. Then 1-(2-Chloroethyl)piperidine hydrochloride (1.55 g, 8.42 mmol) was added. The reaction mixture was stirred at 80 °C for 12 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was dissolved in DCM (50 mL) and washed twice with a 1N NaOH solution. The organic layers were combined and dried over MgSO4, filtrated and concentrated.

The residue was purified by flash chromatography (Cyclohexane/AcOEt), 10:0 to 5:5 (v/v)) to afford a yellow oil (690 mg, 2.45 mmol, 41 %). 1

3'-(2-(Piperidin-1-yl)ethoxy)-[1,1'-biphenyl]-2-carbaldehyde (40)

To a solution of 2-formylbenzeneboronic acid (893 mg, 5.95 mmol) in a mixture of toluene (113 mL) and EtOH (45 mL), K2CO3 (891 mg, 6.45 mmol) and 38 (1.4 g, 4.96 mmol) were added. The reaction was stirred for 30 min and deoxygenated by passing a stream of N2 through it. Pd2dba3 (91 mg, 0.02 mmol) and P(o-tol)3 (302 mg, 0.99 mmol) were added and the mixture was refluxed for 2 h. After cooling, the mixture was poured into 1N NaOH and extracted three times with dichloromethane. The combined organic layers were dried over MgSO4 and evaporated to afford an orange oil which was used without further purification (1.27 g, 4.11 mmol, 83 %). 1 H NMR (300 MHz, CDCl3): δ 10.00 (d, J = 0.8 Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.50 -7.34 (m, 3H), 6.99 -6.93 (m, 3H), 4.17 (t, J = 5.9 Hz, 2H), 2.83 (t, J = 5.9 Hz, 2H), 2.56 (m, 4H), 1.65 -1.60 (m, 4H), 1.50 -1.44 (m, 2H). LCMS m/z calc for [M+H] + : 310.1, found: 310.12.

N,N-Dimethyl-1-(3'-(2-(piperidin-1-yl)ethoxy)-[1,1'-biphenyl]-2-yl)methanamine (41)

To a solution of 40 (925 mg, 2.99 mmol) in DCE (37 mL), dimethylamine 2.0 M in THF (15 mL, 29.9 mmol), NaBH(OAc)3 (950 mg, 4.48 mmol) and acetic acid (0.26 mL, 4.48 mmol) were added and the mixture was stirred at rt for 12 h. The reaction was quenched by pouring the mixture into saturated NaHCO3 solution (25 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 10:0 to 97:3 (v/v)) to afford a brown oil (266 mg, 0.79 mmol, 26 %). 1 H NMR (300 MHz, MeOD): δ 7.51 (dd, J = 7.4, 1.6 Hz, 1H), 7.39 -7.27 (m, 3H), 7.25 -7.19 (m, 1H), 6.95 (m, 1H), 6.92 -6.84 (m, 2H), 4.17 (t, J = 5.7 Hz, 2H), 3.45 (s, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.57 (d, J = 5.2 Hz, 4H), 2.10 (s, 6H), 1.64 (m, 4H), 1.50 (m, 2H). 13

4-Bromo-N-(2-(piperidin-1-yl)ethyl)aniline (42)

To solution of 4-bromoaniline (1.34 mL, 12 mmol) in DMF (50 mL), K2CO3 (3.2 g, 23 mmol), NaI (8.7 g, 58 mmol) and 3-chloropropylpiperidine hydrochloride (3.4 g, 18 mmol) were added.

The reaction mixture was stirred at 100 °C for 24 h. DMF was removed under reduced pressure and the residue was taken up in 20 mL of water. The aqueous layer was extracted with DCM (3 x 60 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by column chromatography (EtOAc/MeOH, 8.5:1.5 (v:v)) to afford a brown oil (779 mg, 2.76 mmol, 23 %). 1 H NMR (300 MHz, CDCl3): δ 7.23 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 8.8 Hz, 1H), 3.15 (t, J = 6.2 Hz, 2H), 2.67 -2.41 (m, 6H), 1.97 -1.76 (m, 2H), 1.77 -1.44 (m, 6H). 13 To a solution of 2-formylbenzeneboronic acid (791 mg, 5.27 mmol) in a mixture of toluene (35 mL) and EtOH (17 mL), K2CO3 (724 mg, 5.24 mmol) and 42 (779 mg, 2.62 mmol) were added.

The reaction was stirred for 30 min and deoxygenated by passing a stream of N2 through it.

Pd2dba3 (133 mg, 0.055 mmol) and P(o-tol)3 (395 mg, 0.48 mmol) were added and the mixture was refluxed for 96 h. After cooling, the mixture was poured into water and extracted with dichloromethane. The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by semi-preparative chromatography (eluent H2O formic, ACN formic, 100 :0 to 70:30 (v/v)) to afford orange oil (341 mg, 1.05 mmol, 40%). 1

2'-((Dimethylamino)methyl)-N-(3-(piperidin-1-yl)propyl)-[1,1'-biphenyl]-4-amine (44)

To a solution of 43 (156 mg, 0.42 mmol) and dimethylamine hydrochloride (69 mg, 0.85 mmol) in EtOH (1 mL) was added triethylamine (0.12 mL, 0.85 mmol) and titanium isopropoxide (0.25 mL, 0.85 mmol). The mixture was stirred at rt for 12 h. NaBH4 (32 mg, 0.85 mmol) was added and the resulting mixture was further stirred for 2 h. The reaction was quenched by pouring the mixture into aqueous ammonia (2.5 mL) and extracted with DCM (3 x 10 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 10:0 to 98:2 (v/v)) to afford brown oil (119 mg, 0.34 mmol, 40%). 1 H NMR (300 MHz, CDCl3): δ 7.60 -7.45 (m, 1H), 7.37 -7.23 (m, 3H), 7.20 (d, J = 8.6 Hz, 2H), 6.65 (d, J = 8.6 Hz, 2H), 5.08 (s, 1H), 3.41 (s, 2H), 3.24 (t, J = 6.3 Hz, 2H), 2.47 (dd, J = 14.0, 7.5 Hz, 6H), 2.18 (s, 6H), 1.96 -1.74 (m, 2H), 1.74 -1.57 (m, 4H), 1.49 (d, J = 5.1 Hz, 2H). 13 6-Nitro-3H-1,3-benzoxazol-2-one (45) 70% Nitric acid (50 mL, 0.80 mol) was cooled to 0 °C and 3H-1,3-benzoxazol-2-one (5.00 g, 37 mmol) was added. The reaction mixture was stirred at rt for 4 h and then poured in ice. The resulting precipitate was collected by filtration, washed with water and dried to give a pink solid (5.80 g, 32.5 mmol, 87 %). Mp > 250 °C. 1 H NMR (300 MHz, DMSO-d6): δ 12.41 (s, 1H), 8.20 (d, J = 2.2 Hz, 1H), 8.13 (dd, J = 8.6 Hz, J = 2.2 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H). 13

5-Nitro-2-([3-(piperidin-1-yl)propyl]amino)phenol (47)

To a solution of 6-nitro-3-[3-(piperidin-1-yl)propyl]-1,3-benzoxazol-2-one (46) (1.0 g, 3.28 mmol) in dioxane (5 mL), aqueous 2.5N NaOH (26 mL, 65.5 mmol) was added. The mixture was stirred at 20 °C for 48 h. After cooling to 0 °C, a 1N HCl solution was added to reach pH 8. After 30 min of stirring, the mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 9.5:0.5 (v/v)) to give compound 47 as an orange solid (715 mg, 2.56 mmol, 78%). Mp: 152.3 -153.0 °C. 1 H NMR (300 MHz, CDCl3): δ 7.69 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 8.8 Hz, 1H), 3.23 (t, J = 6.5 Hz, 2H); 2.67 -2.57 (m, 6H), 1.95 -1.91 (m, 2H), 1.71 -1.67 (m, 4H), 1.53 -1.48 (m, 2H). 13 72%). 1 H NMR (300 MHz, CDCl3): δ 7.86 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H), 7.62 (d, J = 2.4 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H), 6.19 (br t, J = 4.9 Hz, 1H), 4.17 (t, J = 6.1 Hz, 2H), 3.28 (m, 2H), 2.77 (t, J = 5.8 Hz, 2H), 2.48 (m, 4H), 2.45 -2.33 (m, 6H), 1.85 -1.82 (m, 2H), 1.65 -1.54 (m, 8H), 1.49 -1.39 (m, 4H). 13 The organic layer was dried over MgSO4 and evaporated. The residue was purified by semipreparative chromatography (eluent H2O formic, ACN formic, 9:1 to 1:9 (v/v)). to afford compound 51 as a yellow oil (28 mg, 0.06 mmol, 31%). molecules. Among the 86 molecules of the entire dataset, pharmacophore detection was computed from a reduced set of molecules including only highly active (HA) compounds or from a larger set including also moderately active (HMA) compounds for anti-Aβ1-42 activities.

Pharmacophore evaluation. Learned only from active molecules, 3D pharmacophore models were evaluated by their efficiency to discriminate active and inactive compounds. Briefly, the robustness of pharmacophore models was evaluated by screening all the conformers of 86 reference molecules against 3D pharmacophore models with Align-it program. The matching molecules were ranked according to a Tanimoto fitting value between pharmacophore features and concerned atoms of fitted molecules whereas for each non-matched molecule was conferred an arbitrary high RMSD value. ROC scores depict the area under the curve of the plot true positive = f(false positives) according to ranking (Table S4).

Biological evaluations

Antibodies. Primary antibodies used in this study for western-blot analysis included a wellcharacterized homemade rabbit antiserum against the last 17 amino acids of APP, named APP-Cter-C17 (1/5000) [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF][START_REF] Vingtdeux | Intracellular pH regulates amyloid precursor protein intracellular domain accumulation[END_REF][START_REF] Sergeant | Progressive decrease of amyloid precursor protein carboxy terminal fragments (APP-CTFs), associated with tau pathology stages, in Alzheimer's disease[END_REF]. Anti-β-actin (1/10 000) was obtained from Sigma. Anti-LC3B

(1/2000) and anti-p62/SQSTM1 (1/2000) antibodies were purchased from Cell Signaling Technology. Secondary antibodies (peroxidase-labelled goat anti-rabbit IgG, 1/5000 or peroxidase-labelled horse anti-mouse IgG, 1/50000) were obtained from Vector Laboratories.

Cell culture and treatment. The human neuroblastoma cell line SY5Y-APP 695WT was maintained in Dulbecco's modified Eagle medium (DMEM, high glucose, pyruvate -GIBCO, Life Technologies) supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 1 mM non-essential amino acids and penicillin/streptomycin (GIBCO, Life Technologies) at 37 °C in a 5% CO2 humidified incubator [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF]. For compounds treatment, a 10 mM stock solution was diluted in freshly supplemented DMEM medium to obtain the precise final concentration of drug. Bafilomycin A1 (BafA1), the vacuolar 362 type H+ ATPase inhibitor, was purchased from Merck Millipore and was used at a final dilution of 100 nM. Cells were plated at a density of 5.10 5 cells per well into 12-well plates and cultured with 1 mL supplemented DMEM cell medium for 24 h before drug exposure. The following day, cell medium was replaced with fresh medium containing drugs diluted at the indicated concentrations. Cells were treated for 24 h.

At the end of treatments, the cell medium was collected and kept at -80 °C until use, cells were rinsed once with PBS and extracted in 100 µL of Laemmli buffer (10 mM Tris, 20% glycerol and 2% Sodium dodecyl sulfate) using a cell-scraper. The cell lysate was further sonicated (30 pulses of 0.5 s, 60 Hz) for 5 min. Total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo scientific) according to the manufacturer's instructions.

Samples were stored at -80 °C until analysis.

Primary neuronal cultures and drug treatment. Primary neuron cultures were obtained from C57BL6 wild-type mice as described previously [START_REF] Domise | AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo[END_REF]. Briefly, females were sacrificed at 18. according to the manufacturer's instructions. SY5Y-APP 695WT cells were plated into a 96-well plate at 2*10 4 cells per well with 100 µL of DMEM supplemented medium. Cells were treated as described above and LDH measurement was performed at the end of treatment. Primary neuronal cultures were plated into a 48-well plate at a density of about 1.5x10 5 cells per well with Neurobasal supplemented medium. Cells were cultured and treated as described above.

LDH measurement was performed at the end of treatment. Quantification of secreted Aβ1-40/1-42 and sAPPα/sAPPβ. Conditioned media of SY5Y-APP 695WT collected at the end of treatments were centrifuged at 1000g for 5 min to eliminate cells debris. Aβ1-40 and Aβ1-42 peptide concentrations in pg/mL were determined using amyloidbeta 40 and 42 Human ELISA kits (Invitrogen) according to the manufacturer's instructions after 1:10 and 1:2 dilution of supernatants in ELISA buffer, respectively. sAPPα and sAPPβ concentrations in ng/mL were determined using the sAPPα/sAPPβ kit (Meso Scale Diagnostics, MSD ® ) according to the manufacturer's instructions.

Western Blot

Immunofluorescence. SY5Y-APP 695WT cells were plated at 3x10 5 cells per well on poly-Dlysine coated glass coverslip with 1 mL of DMEM supplemented medium. Primary neurons were plated at 2.5x10 5 cells par well with 1 mL of Neurobasal supplemented medium.

Treatment with compound 30 at µM was performed following the same protocols described above. At the end of treatments, cells were fixed with 4% paraformaldehyde in PBS during 15 min at rt and rinsed 3 times 5 min with PBS. Cells were then permeabilized using 0.25% Triton X-100 during 15 min at rt and rinsed with PBS 3 times for 5 min before blocking in 1% BSA in PBS for 1 h at rt. After 3 washes with PBS, cells were incubated with primary antibodies directed against LC3-B (1/200) and p62 (1/200) at 4 °C overnight in a PBS solution containing 1% BSA and 0.02% Triton X-100. The following day, coveslips were rinsed with PBS and incubated with anti-IgG secondary antibodies coupled to Alexa Fluor 568 (1/500) (Invitrogen).

Nuclei were visualized using DAPI (Thermo Scientific). Images were acquired on a Zeiss Axio Imager Z2 microscope (Carl Zeiss, Germany) equipped with a Hamamatsu ORCA-Flash4.0 digital camera (Hamamatsu Photonics, Japan) and ApoTome.2 system (Carl Zeiss, Germany).

For Apotome image acquisition, the Axio Imager was used with optical plan-apochromat 63x/1.4 oil M27.

β-secretase in vitro activity assay. These experiments were performed on human recombinant BACE-1 protein at Eurofins Cerep (Celle-Lévescault, France), with minor variations to the experimental protocol described by Ermolieff, J. et al. [START_REF] Ermolieff | Proteolytic activation of recombinant promemapsin 2 (pro-β-secretase) studied with new fluorogenic substrates[END_REF]. The cleavage of fluorescent Mca-

S-E-V-N-L-D-AE-F-R-K(Dnp)-R-R-NH2 substrate to Mca-S-E-V-N-L-NH2

While compound 39 (

 39 Aβ1-40: IC50 = 9.1 µM and Aβ1-42: IC50 = 13.7 µM) displayed similar activity to CQ (Aβ1-40: IC50 = 7.0 µM and Aβ1-42: IC50 = 12.7 µM), all other evaluated compounds were found to be more active (IC50 ranging from 1.2 to 5.2 µM). The different modulations applied, including the modification of the position of the piperidine borne by the second phenyl group (compare 24, 26 and 27), the replacement of the piperidine by other tertiary amines (compare 24 vs 25 and 26-29) and the modification of the length of both piperidine alkyl chains on the other side of the molecule (30 vs 32-36) had little impact on Aβ levels. Finally, replacing one of the phenyl rings of 30 (Aβ1-40: IC50 = 2.8 µM and Aβ1-42: IC50 = 3.4 µM) with a pyrrole (compound 51) led to a two-fold decrease in activity (Aβ1-40: IC50 = 5.0 µM and Aβ1-42: IC50 = 6.5 µM). The main factor that appears to influence this activity is the number of tertiary amines. Indeed, 41 (two tertiary amines, Aβ1-40: IC50 = 1.5 µM and Aβ1-42: IC50 = 1.2 µM) was 10-fold more active than 39 (one tertiary amine, Aβ1-40: IC50 = 9.1 µM and Aβ1-42: IC50 = 13.7 µM), while adding an extra tertiary amine did not improve activity (e.g. compare 41, Aβ1-40: IC50 = 1.5 µM and Aβ1-42: IC50 = 1.2 µM and 33, Aβ1-40: IC50 = 1.4 µM and Aβ1-42: IC50 = 1.2 µM). Hence, with regard to Aβ levels, two basic centers are required in this series to get an efficient inhibitory activity. Interestingly, compounds displaying one or two tertiary amines had little (41 and 44) or no impact (31 and 39) on AICD and αCTFs stabilities while compounds with three tertiary amines significantly promote AICD and αCTFs stabilities.

  C NMR (75 MHz, DMSO-d6): δ 154.5, 144.5, 130.3, 126.9, 113.5, 113.2, 111.7. LCMS m/z calc for [M-H] + : 211.9, 213.9, found: 211.8, 213.8. General procedure for the synthesis of compounds 2 and 3. 6-bromo-3H-1,3-benzoxazol-2one 1 (5.00 g, 23.36 mmol) was suspended in ACN (150 mL) and K2CO3 (9.69 g, 70.09 mmol) was added. The reaction mixture was stirred at 80 °C for 30 min. 1-(2-Chloroethyl)piperidine hydrochloride (4.30 g, 23.36 mmol) or 3-chloropropylpiperidine hydrochloride (5.5 g, 28 mmol) was added and the reaction mixture was stirred at 80 °C for another 12 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 9.8:0.2 (v/v)) to afford compounds 2 and 3. 6-Bromo-3-[2-(piperidin-1-yl)ethyl]-1,3-benzoxazol-2-one (2) Beige solid (7.58 g, 23.1 mmol, 99 %). Mp 85.1-85.8 °C. 1 H NMR (300 MHz, CDCl3): δ 7.36 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.3, 1.8 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 3.90 (t, J = 6.6 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 2.51 -2.38 (m, 4H), 1.60 -1.35 (m, 6H). 13 C NMR (75 MHz, CDCl3): δ 154.1, 143.1, 130.7, 126.6, 114.4, 113.5, 109.9, 56.0, 54.7, 40.3, 26.0, 24.2. LCMS m/z calc for [M+H] + : 325.1, 327.1 found: 325.1, 327.1.

1 .

 1 C NMR (75 MHz, CDCl3): δ 154.1, 143.1, 130.2, 126.9, 114.8, 113.8, 109.2, 77.5, 77.0, 76.6, 44.0, 41.6, 29.2, 25.0. LCMS m/z calc for [M+ACN+H] + : 345.6, 347.6, found: 345.1, 347.1. 6-Bromo-3-(4-chlorobutyl)-1,3-benzoxazol-2-one (1.2 g, 3.94 mmol) was dissolved in piperidine (19 mL) and refluxed for 12 h. The mixture was concentrated in vacuo and the residue was dissolved in 1N NaOH (20 mL) and extracted with DCM (3 x 60 mL). The combined organic layers were dried over MgSO4, filtrated and concentrated in vacuo. The residue was purified by column chromatography (DCM/MeOH(NH3), 9.5 :0.5 (v:v)) to afford a beige powder (1.07 g, 3.0 mmol, 16% over two steps). Mp 100.2 -101.0 °C. 1 H NMR (300 MHz, CDCl3): δ 7.37 (d, J = 1.8 Hz, 1H), 7.32 (dd, J = 8.3, 1.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 3.83 (t, J = 7.2 Hz, 2H), 2.38 -2.27 (m, 6H), 1.86 -1.70 (m, 2H), 1.66 -1.42 (m, 8H). 13 C NMR (75 MHz, CDCl3): δ 154.0, 143.1, 130.4, 126.7, 114.6, 113.6, 109.4, 58.5, 58.4, 54.6, 42.3, 42.2, 30.0, 25.8, 24.4, 23.9. LCMS m/z calc for [M+H] + : 353.1, 355.1, found: 353.1, 355.General procedure for the synthesis of compounds 5-7 is exemplified by the protocol used for the synthesis of 5. To a solution of 6-bromo-3-[2-(piperidin-1-yl)ethyl]-1,3-benzoxazol-2one (2) (4.0 g, 12.3 mmol) in dioxane (19 mL), aqueous 2.5N NaOH (98.4 mL, 246 mmol) was added. The mixture was stirred at 20 °C for 12 h. After cooling to 0 °C, a 1N HCl solution was added to reach pH 8. After 30 min of stirring, the mixture was extracted with DCM (3 x 100 mL). The combined organic layers were dried over MgSO4 and evaporated to afford 5-bromo-2-((2-(piperidin-1-yl)ethyl)amino)phenol 5 as a beige solid (3.1 g, 10.4 mmol, 84%) that was used without further purification. Mp 126.8 -127.5 °C. 1 H NMR (300 MHz, CDCl3): δ 6.95 (d, J = 2.1 Hz, 1H), 6.87 (dd, J = 8.3, 2.1 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 3.25 -3.15 (t, J = 5.7 Hz, 2H), 2.60 -2.31 (m, 6H), 1.75 -1.64 (m, 4H), 1.56 -1.45 (m, 2H). 13 C NMR (75 MHz, CDCl3): δ 151.1, 135.8, 122.7, 120.9, 119.8, 113.8, 57.3, 54.3, 44.7, 25.0, 23.9. LCMS m/z calc for [M+H] + : 299.1, 301.1, found: 299.1, 301.1. 5-Bromo-2-([3-(piperidin-1-yl)propyl]amino)phenol[START_REF] Vassar | The -Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential[END_REF]

  C NMR (75 MHz, DMSO-d6): δ 145.2, 137.1, 121.9, 115.7, 110.6, 105.4, 58.0, 53.9, 42.5, 40.3, 40.1, 39.8, 39.5, 39.2, 39, 38.7, 26.3, 25.4, 24.0, 23.8. LCMS m/z calc for [M+H] + : 327.1, 329.1 found: 327.2, 329.2. General procedure for the synthesis of compounds 8, 9, 11-14 is exemplified by the protocol used for the synthesis of 8. To a solution of 5-bromo-2-([2-(piperidin-1yl)ethyl]amino)phenol (5) (550 mg, 1.84 mmol) in DMF (23 mL), at 0 °C, NaH (147 mg, 3.68 mmol) was added. After stirring the mixture for 30 min, 1-(2-chloroethyl)piperidine hydrochloride (338 mg, 1.84 mmol) was added at 0 °C. The reaction mixture was stirred for another 12 h at rt. The mixture was diluted with 20 mL of water and extracted with DCM (3 x 60 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by column chromatography (DCM/MeOH(NH3), 9.5:0.5 (v:v)) to afford 4-bromo-2-(2-(piperidin-1-yl)ethoxy)-N-(2-(piperidin-1-yl)ethyl)aniline (8) as a brown oil (587 mg, 1.43 mmol, 78 %).1 H NMR (300 MHz, CDCl3): δ 6.96 (dd, J = 8.4, 1.9 Hz, 1H), 6.86 (d, J = 1.9 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 4.91 (s, 1H), 4.10 (t, J = 5.8 Hz, 2H), 3.22 -3.05 (m, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.68 -2.34 (m, 10H), 1.71 -1.52 (m, 8H), 1.51 -1.39 (m, 4H).13 C NMR (75 MHz, CDCl3): δ 146.9, 138.0, 124.0, 114.1, 110.8, 107.3, 66.7, 58.0, 57.3, 54.9, 54.4, 40.3, 26.1, 26.0, 24.5, 24.2. LCMS m/z calc for [M+H] + : 410.4, 412.4 found: 410.2, 412.2.

2 4-

 2 Bromo-2-(3-(piperidin-1-yl)propoxy)-N-(3-(piperidin-1-yl)propyl)aniline (12) Brown oil (940 mg, 2.1 mmol, 56 %). 1 H NMR (300 MHz, CDCl3): δ 6.94 (dd, J = 8.4, 2.1 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.44 (d, J = 8.4 Hz, 1H), 4.55 (s, 1H), 4.01 (t, J = 6.4 Hz, 2H), 3.22 -3.03 (m, 2H), 2.55 -2.20 (m, 12H), 2.07 -1.95 (m, 2H), 1.90 -1.76 (m, 2H), 1.70 -1.55 (m, 8H), 1.50 -1.40 (m, 4H).

4 -

 4 Bromo-2-(4-chlorobutoxy)-N-(2-(piperidin-1-yl)ethyl)aniline (10) To a solution of 5-bromo-2-([2-(piperidin-1-yl)ethyl]amino)phenol (5) (1 g, 3.34 mmol) in DMF (42 mL), at 0 °C, NaH (334 mg, 8.36 mmol) was added. After stirring the mixture for 30 min, 1-bromo-4-chlorobutane (0.43 ml, 3.34 mmol) in DMF (5 ml) was added dropwise at 0 °C. The reaction mixture was stirred for another 12 h at rt. The mixture was diluted with 40 mL of water and extracted with DCM (3 x 60 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by column chromatography (DCM/MeOH(NH3), 9.8 :0.2 (v:v)) to afford 4-bromo-2-(4-chlorobutoxy)-N-(2-(piperidin-1yl)ethyl)aniline as a colorless oil (450 mg, 1.15 mmol). 1 H NMR (300 MHz, CDCl3): δ 6.97 (dd, J = 8.3, 2.1 Hz, 1H), 6.84 (s, 1H), 6.44 (dd, J = 8.3, 1.5 Hz, 1H), 4.88 (s, 1H), 4.06 -3.97 (m, 2H), 3.70 -3.58 (m, 2H), 3.21 -3.07 (m, 2H), 2.61 (t, J = 5.2 Hz, 2H), 2.48 -2.35 (m, 4H), 2.12 -1.94 (m, 4H), 1.68 -1.44 (m, 6H). 13 C NMR (75 MHz, CDCl3): δ 146.8, 137.9, 123.9, 113.6, 110.9, 107.4, 67.4, 57.1, 54.3, 44.7, 40.2, 29.4, 26.7, 26.2, 24.5. LCMS m/z calc for [M+H] + : 389.1, 391.1, 393.1, found: 389.1, 391.1, 393.2. 4-Bromo-2-(4-chlorobutoxy)-N-(2-(piperidin-1-yl)ethyl)aniline (438 mg, 1.12 mmol) was dissolved in piperidine (11 mL) and was refluxed for 12 h. The mixture was concentrated in vacuo and the residue was dissolved in 1N NaOH (20 mL) and extracted with DCM (3 x 60 mL). The combined organic layers were dried over MgSO4, filtrated and concentrated in vacuo.

3 .

 3 Generalprocedure for the synthesis of compounds 15-23 is exemplified by the protocol used for the synthesis of 15. 2-Formylbenzeneboronic acid (63 mg, 0.42 mmol) was dissolved in a mixture of toluene (8 mL) and EtOH (3.2 mL). K2CO3 (62 mg, 0.45 mmol) and 4-bromo-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]aniline (11) (150 mg, 0.35 mmol)

  3'-(2-(piperidin-1-yl)ethoxy)-4'-((3-(piperidin-1-yl)propyl)amino)-[1,1'-biphenyl]-4carbaldehyde (17) Brown oil (285 mg, 0.63 mmol, 88%). 1 H NMR (300 MHz, CDCl3): δ 10.01 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.23 (dd, J = 8.4 Hz, J = 2.1, 1H), 7.10 (d, J = 2.1 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 5.05 (br s, 1H), 4.23 (t, J = 6.3 Hz, 2H), 3.27 (t, J = 6.6 Hz, 2H), 2.86 (t, J = 6.0 Hz, 2H), 2.61 -2.39 (m, 10H), 1.93 (m, 2H), 1.75 -1.58 (m, 8H), 1.53 -1.43 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 191.9, 147.6, 146.2, 139.6, 134.0, 133.3, 127.0, 126.4, 121.1, 110.0, 66.5, 57.9, 57.2, 54.8, 42.1, 25.8, 25.5, 24.1. LCMS m/z calc for [M+H] + : 450.3, found: 450.2. 3'-(2-(piperidin-1-yl)ethoxy)-4'-((2-(piperidin-1-yl)ethyl)amino)-[1,1'-biphenyl]-2carbaldehyde (18)

  4'-((2-(piperidin-1-yl)ethyl)amino)-3'-(3-(piperidin-1-yl)propoxy)-[1,1'-biphenyl]-2carbaldehyde[START_REF] Vingtdeux | Intracellular pH regulates amyloid precursor protein intracellular domain accumulation[END_REF]

3'-( 4 -

 4 (piperidin-1-yl)butoxy)-4'-((2-(piperidin-1-yl)ethyl)amino)-[1,1'-biphenyl]-2carbaldehyde (20)Brown oil (150 mg, 0.32 mmol, 71%).1 H NMR (300 MHz, CDCl3): δ 10.04 (s, 1H), 7.98 (dd, J = 7.8, 1.3 Hz, 1H), 7.65 -7.56 (m, 1H), 7.49 -7.38 (m, 2H), 6.86 (dd, J = 8.0, 1.8 Hz, 1H), 6.78 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 5.01 (d, J = 4.8 Hz, 1H), 4.04 (t, J = 6.2 Hz, 2H), 3.31 -3.18 (m, J = 11.6, 4.6 Hz, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.54 -2.24 (m, 10H), 1.94 -1.79 (m, 4H), 1.79 -1.53 (m, 8H), 1.53 -1.37 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 193.

  3'-(3-(piperidin-1-yl)propoxy)-4'-((3-(piperidin-1-yl)propyl)amino)-[1,1'-biphenyl]-2carbaldehyde (21)Brown oil (573 mg, 1.24 mmol, 80%).1 H NMR (300 MHz, CDCl3): δ 10.02 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.49 -7.36 (m, 2H),[START_REF] Vassar | The -Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential[END_REF].85 (dd, J = 8.0, 1.7 Hz, 1H), 6.80 (s, 1H),[START_REF] Vassar | The -Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential[END_REF].67 (d, J = 8.0 Hz, 1H), 4.79 (s, 1H), 4.07 (t, J = 6.4 Hz, 2H), 3.25 (d, J = 6.2 Hz, 2H), 2.58 -2.33 (m, 12H), 2.07 -1.99 (m, 2H), 1.96 -1.79 (m, 2H), 1.61 (m, 8H), 1.51 -1.41 (m, 4H).13 C NMR (75 MHz, CDCl3): δ 193.3, 146.8, 145.9, 138.7, 133.8, 133.3, 130.7, 127.5, 125.1, 123.8, 112.3, 109.2, 67.0, 57.4, 56.0, 54.8, 54.7, 42.4, 26.9, 26.5, 25.9, 24.5. LCMS m/z calc for [M+H] + :464.3, found 464.4.4'-((4-(piperidin-1-yl)butyl)amino)-3'-(2-(piperidin-1-yl)ethoxy)-[1,1'-biphenyl]-2carbaldehyde (22)Brown oil (439 mg, 0.94 mmol, 97%).1 H NMR (300 MHz, CDCl3): δ 10.02 (s, 1H), 7.98 (dd, J = 7.8, 1.5 Hz, 1H), 7.59 (td, J = 7.5, 1.5 Hz, 1H), 7.45 (dd, J = 8.0, 1.2 Hz,1H), 7.40 (d, J = 7.5 Hz, 1H), 6.86 (dd, J = 8.0, 1.9 Hz, 1H), 6.82 (d, J = 1.9 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 4.61 (s, 1H), 4.14 (t, J = 5.9 Hz, 2H), 3.21 (d, J = 6.1 Hz, 2H), 2.80 (t, J = 6.0 Hz, 2H), 2.63 -2.26 (m, 10H), 1.77 -1.38 (m, 16H).13 C NMR (75 MHz, CDCl3): δ 193.2, 146.6, 145.7, 139.0, 133.8, 133.3, 130.6, 127.5, 126.7, 125.0, 124.2, 113.1, 109.2, 66.6, 59.1, 58.0, 55.0, 54.6, 43.4, 27.5, 26.0, 24.6, 24.5, 24.2. LCMS m/z calc for [M+H] + : 464.65, found 464.45 3'-(2-cyclohexylethoxy)-4'-((3-(piperidin-1-yl)propyl)amino)-[1,1'-biphenyl]-3-carbaldehyde (23)Brown oil (748 mg, 1.67 mmol, 80%).1 H NMR (300 MHz, CDCl3): δ 10.03 (s, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.60 -7.57 (m, 1H), 7.48 -7.41 (m, 2H), 6.86 -6.66 (m, 3H), 4.77 (br s, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.26 (m, 2H), 2.48 (m, 6H), 2.46 -0.97 (m, 21H).13 C NMR (75 MHz, CDCl3): δ 193.3, 146.8, 146.0, 138.6, 133.8, 133.3, 130.6, 127.5, 126.6, 125.2, 123.6, 112.0, 109.1, 66.5, 57.4, 54.7, 42.4, 36.7, 34.8, 33.3, 26.4, 26.2, 25.7, 24.3. LCMS m/z calc for [M+H] + : 449.3, found 449.4.General procedure for the synthesis of compounds 24-30 is exemplified by the protocol used for the synthesis of 24. 3'-(2-(piperidin-1-yl)ethoxy)-4'-((3-(piperidin-1yl)propyl)amino)-[1,1'-biphenyl]-4-carbaldehyde 17 (290 mg, 0.65 mmol) was dissolved in DCE (12 mL). Piperidine (96 µL, 0.97 mmol) was added. After 2 h at rt, NaBH(OAc)3 (206 mg, 0.97 mmol) and acetic acid (56 µL, 0.97 mmol) were added. After 16 h at rt, saturated NaHCO3 solution was added. The reaction mixture was stirred for another hour. DCM was added and the layers were separated. The organic layer was washed three times with saturated NaHCO3 solution. The organic layer was dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 10:0 to 9:1 (v/v)) to afford 2-[2-(Piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]-4-[4-(piperidin-1-ylmethyl)phenyl]aniline 24 as a colorless oil (260 mg, 0.50 mmol, 77%).1 H NMR (300 MHz, CDCl3): δ 7.51 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.13 (dd, J = 8.1 Hz, J = 1.9 Hz, 1H), 7.04 (d, J = 1.9 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.50 (s, 2H), 3.22 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H), 2.54 -2.34 (m, 14H), 1.93 -1.84 (m, 2H), 1.66 -1.38 (m, 18H).13 C NMR (75 MHz, CDCl3): δ 146.2, 140.2, 138.3, 136.1, 129.6, 129.1, 126.0, 120.2, 110.1, 66.6, 63.6, 58.1, 57.5, 55.1, 54.5, 42.5, 26.6, 26.0, 24.4. LCMS m/z calc for [M+H] + : 519.4, found: 519.3. HR-MS: m/z Calculated: 519.4057, found: 519.4060 [M+H] += C33H51N4O. 4-(4-[(Dimethylamino)methyl]phenyl)-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1yl)propyl] aniline[START_REF] Melnyk | Heterocycle compounds and uses thereof for the treatment of diseases involving formation of amyloid plaques and/or where a dysfunction of the APP metabolism occurs[END_REF] In the present case dimethylamine 2 M in THF was used and the product was obtained as a colorless oil (130 mg, 0.27 mmol, 61%).1 H NMR (300 MHz, CDCl3): δ 7.49 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.12 (dd, J = 8.2 Hz, J = 2.1 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 4.20 (t, J = 6.1 Hz, 2H), 3.45 (s, 2H), 3.23 (t, J = 6.8 Hz, 2H), 2.82 (t, J = 6.1 Hz, 2H), 2.55 -2.41 (m, 10H), 2.25 (s, 3H), 1.90 -1.85 (m, 2H), 1.66 -1.58 (m, 8H), 1.51 -1.44 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 146.2, 140.4, 138.3, 136.4, 129.5, 129.0, 126.4, 120.2, 110.0, 66.4, 64.1, 58.1, 57.4, 54.8, 45.3, 42.5, 26.4, 25.8, 24.3. LCMS m/z calc for [M+H] + : 479.3, found: 479.4. HR-MS: m/z Calculated: 479.3744, found: 479.3716 [M+H] + = C30H47N4O. 2-[2-(1-piperidyl)ethoxy]-4-[3-(1-piperidylmethyl)phenyl]-N-[3-(1-piperidyl)propyl]aniline (26) Brown oil (17 mg, 0.03 mmol, 5%). 1 H NMR (300 MHz, CDCl3): δ 7.49 -7.47 (m, 1H), 7.45 -7.43 (m, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.24 -7.20 (m, 1H), 7.5 (dd, J = 8.1 Hz, J = 1.8 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 4.22 (t, J = 6.0 Hz, 2H), 3.54 (s, 2H), 3.23 (t, J = 6.9 Hz, 2H), 2.83 (t, J = 6.7 Hz, 2H), 2.6 -2.4 (m, 14H); 1.89 (quint, J = 6.7 Hz, 2H), 1.70 -1.50 (m, 12H), 1.50 -1.40 (m, 6H). 13 C NMR (75 MHz, CDCl3): δ 146.2, 141.4, 138.4, 129.2, 128.4, 127.3, 127.0, 125.0, 120.3, 110.3, 110.0, 66.6, 64.0, 58.1, 57.4, 55.1, 54.7, 54.5, 42.5, 26.5, 26.0, 25.9, 24.4, 24.2. LCMS m/z calc for [M+H + ]: 519.4, Found: 519.3. HR-MS: m/z Calculated: 519.3985, found: 519.4060 [M+H + ] = C33H51N4O. 2-[2-(Piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]-4-[2-(piperidin-1ylmethyl)phenyl]aniline (27) Colorless oil (114 mg, 0.22 mmol, 66%). 1 H NMR (300 MHz, CDCl3): δ 7.54 -7.51 (m, 1H), 7.29 -7.25 (m, 3H), 6.93 (d, J = 1.8 Hz, 1H), 6.89 (dd, J = 8.0 Hz, J = 1.8 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 4.98 (br s, 1H), 4.15 (t, J = 6.1 Hz, 2H), 3.42 (s, 2H), 3.23 (t, J = 6.8 Hz, 2H),

  General procedure for the synthesis of compounds 31-36 is exemplified by the protocol used for the synthesis of 31. To a solution of 3'-(2-cyclohexylethoxy)-4'-((3-(piperidin-1yl)propyl)amino)-[1,1'-biphenyl]-3-carbaldehyde 23 (300 mg, 0.67 mmol) and dimethylamine hydrochloride (110 mg, 1.34 mmol ) in EtOH (2 mL) was added triethylamine (186 µL, 1.34 mmol) and titanium isopropoxide (400 µL, 1.34 mmol). The mixture was stirred for 12 h at rt. NaBH4 (51 mg, 1.34 mmol) was then added and the mixture was further stirred for 2 hours. The reaction was quenched by pouring the mixture into aqueous ammonia (2.5 mL) and extracted with DCM (3 x 10 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 10:0 to 9:1 (v/v)) to afford 3-(2-cyclohexylethoxy)-2'-((dimethylamino)methyl)-N-(3-(piperidin-1-yl)propyl)-[1,1'biphenyl]-4-amine as a colorless oil (157 mg, 0.33 mmol, 49%). 1 H NMR (300 MHz, CDCl3): δ 7.53 -7.49 (m, 1H), 7.33 -7.28 (m, 3H), 6.91 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 8.0 Hz, J = 1.8 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 4.64 (br s, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.40 (s, 2H), 3.24 (t, J = 6.4 Hz, 2H), 2.49 -2.40 (m, 6H), 2.18 (s, 6H), 1.94 -1.85 (m, 2H), 1.80 -1.60 (m, 11H), 1.54 -1.42 (m, 3H), 1.33 -1.10 (m, 3H), 1.06 -0.92 (m, 2H).

  2'-((Dimethylamino)methyl)-3-(2-(piperidin-1-yl)ethoxy)-N-(2-(piperidin-1-yl)ethyl)-[1,1'biphenyl]-4-amine (33) Brown oil (25 mg, 0.05 mmol, 30%). 1 H NMR (300 MHz, CDCl3): δ 7.51 (dd, J = 4.7, 2.3 Hz, 1H), 7.33 -7.24 (m, 3H), 6.91 (d, J = 1.8 Hz, 1H), 6.87 (dd, J = 8.0, 1.8 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 4.96 (s, 1H), 4.15 (t, J = 5.8 Hz, 2H), 3.40 (s, 2H), 3.25 (t, J = 6.1 Hz, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.73 -2.39 (m, 10H), 2.19 (s, 6H), 1.66 -1.56 (m, 8H), 1.46 (d, J = 5.1 Hz,

  2'-((Dimethylamino)methyl)-N-(2-(piperidin-1-yl)ethyl)-3-(3-(piperidin-1-yl)propoxy)-[1,1'biphenyl] -4-amine[START_REF] Ermolieff | Proteolytic activation of recombinant promemapsin 2 (pro-β-secretase) studied with new fluorogenic substrates[END_REF]

  2'-((Dimethylamino)methyl)-N-(4-(piperidin-1-yl)butyl)-3-(2-(piperidin-1-yl)ethoxy)-[1,1'biphenyl]-4-amine (36) Brown oil (75 mg, 0.15 mmol, 47%). 1 H NMR (300 MHz, CDCl3): δ 7.54 -7.47 (m, 1H), 7.35 -7.24 (m, 3H), 6.91 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 8.0, 1.8 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 4.58 (s, 1H), 4.05 (t, J = 6.5 Hz, 2H), 3.39 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H), 2.59 -2.34 (m, 12H), 2.19 (s, 6H), 2.09 -1.97 (m, 2H), 1.95 -1.82 (m, 2H), 1.69 -1.53 (m, 8H), 1.51 -1.39 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 145.5, 143.0, 137.4, 136.4, 130.2, 129.9, 129.6, 126.6, 126.4, 122.4, 112.4, 109.2, 66.9, 61.1, 57.6, 56.2, 54.8, 54.7, 45.4, 42.6, 27.0, 26.7, 26.0, 24.5. LCMS m/z calc for [M+H] + : 493.4, found: 493.4. HR-MS: m/z Calculated: 493.3901, found: 493.3906 [M+H] + = C31H49N4O.

[

  M+H] + = C27H40N3O. 1-(2-(3-Bromophenoxy)ethyl)piperidine (38) 3-Bromophenol (1.63 g, 9.42 mmol) was suspended in ACN (50 mL) and K2CO3 (3.91 g, 28.3 mmol) was added. The reaction mixture was stirred at 80 °C for 30 min. Then 1-(2-Chloroethyl)piperidine hydrochloride (2.43 g, 13.2 mmol) was added. The reaction mixture was stirred at 80 °C for 12 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was dissolved in DCM (50 mL) and washed twice with a 1N NaOH solution. The organic layers were combined and dried over MgSO4, filtrated and concentrated in vacuo to afford 38 (2.45 g, 8.57 mmol, 91%). The product was used without further purification in the next step. 1 H NMR (300 MHz, CDCl3): δ 7.14 (d, J = 8.1 Hz, 1H), 7.08 -7.04 (m, 2H), 6.83 (ddd, J = 8.1, 2.5, 1.4 Hz, 1H), 4.08 (t, J = 6.1 Hz, 2H), 2.75 (t, J = 6.1, 2H), 1.65 -1.53 (m, 6H), 1.49 -1.39 (m, 2H). LCMS m/z calc for [M+H] + : 286.1, 288.1 found: 286.0, 288.0

  C NMR (75 MHz, CDCl3): δ 147.7, 131.9, 114.1, 108.4, 57.7, 54.5, 43.4, 29.7, 25.5, 24. LCMS m/z calc for [M+H] + : 283.1, 285.1 found: 283.1, 285.1 4'-((3-(piperidin-1-yl)propyl)amino)-[1,1'-biphenyl]-2-carbaldehyde (43)

  H NMR (300 MHz, CDCl3): δ 10.02 (d, J = 0.7 Hz, 1H), 8.57 (s, 1H), 7.98 (dd, J = 7.7, 1.0 Hz, 1H), 7.64 -7.55 (m, 1H), 7.47 -7.35 (m, 2H), 7.22 -7.14 (m, 2H), 6.71 (d, J = 8.6 Hz, 2H), 3.34 -3.23 (m, 2H), 3.11 -2.99 (m, 6H), 2.13 (dd, J = 13.9, 6.5 Hz, 2H), 1.97 -1.84 (m, 4H), 1.72 -1.53 (m, 2H).13 C NMR (75 MHz, CDCl3): δ 193.2, 168.3, 148.2, 146.4, 133.6, 133.4, 131.3, 130.6, 127.5, 126.7, 126, 112.3, 55.1, 53.1, 40.9, 23.8, 23.1, 22.6. LCMS m/z calc for [M+H] + : 323.2, found 323.2.

  C NMR (300 MHz, DMSO-d6): δ 154.7, 143.2, 142.5, 137.2, 121.2, 109.8, 105.8. LCMS m/z calc for [M-H] + : 179.0, found: 179.0. 6-Nitro-3-[3-(piperidin-1-yl)propyl]-1,3-benzoxazol-2-one (46) 6-Nitro-3H-1,3-benzoxazol-2-one (45) (6.30 g, 35 mmol) was suspended in ACN (150 mL) and K2CO3 (6.91 g, 50 mmol) was added. The reaction mixture was stirred at 80 °C for 30 min. 3-Chloropropylpiperidine hydrochloride (3.96 g, 20 mmol) was added and the reaction mixture was stirred at 80 °C for another 5 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 9.8:0.2 (v/v)) to give compound 46 as a beige solid (5.98 g, 19.6 mmol, 98%). Mp 81.1 -81.7 °C. 1 H NMR (300 MHz, CDCl3): δ 8.21 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 2.38 -2.28 (m, 6H), 2.02 -1.98 (m, 2H), 1.56 -1.53 (m, 4H), 1.45 -1.41 (m, 2H). 13 C NMR (75 MHz, CDCl3): δ 154.1, 143.0, 141.9, 137.3, 120.7, 108.0, 106.1, 55.4, 54.4, 41.1, 25.9, 24.4, 24.3. LCMS m/z calc for [M+H] + : 306.2, found: 306.2.

  C NMR (75 MHz, CDCl3): δ 145.4, 144.3, 136.7, 118.5, 108.8, 106.4, 56.3, 54.1, 41.2, 25.1, 24.5, 23.5. LCMS m/z calc for [M+H] + : 280.2, found: 280.1. 4-Nitro-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]aniline (48) 5-Nitro-2-([3-(piperidin-1-yl)propyl]amino)phenol (47) (200 mg, 0.72 mmol) was suspended in ACN (6 mL) and K2CO3 (300 mg, 2.15 mmol) was added. The reaction mixture was stirred at rt for 10 min. 1-(2-Chloroethyl)piperidine hydrochloride (160 mg, 0.86 mmol) was then added and the reaction mixture was stirred at 60 °C for another 5 h. The inorganics were removed by filtration and the solvent was evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 9.8:0.2 (v/v)) to give a brown oil (200 mg, 0.52 mmol,

  C NMR (75 MHz, CDCl3): δ 145.1, 144.3, 136.3, 120.3, 106.48, 106.3, 66.4, 57.6, 57.2, 54.8, 54.5, 42.0, 25.8, 25.7, 24.2. LCMS m/z calc for [M+H] + : 391.3, found: 391.1. 2-[2-(Piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]benzene-1,4-diamine (49) 4-Nitro-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]aniline (48) (100 mg, 0.255 mmol) was dissolved in MeOH (5 mL). Pd/C 10 % (20 mg, 7 mol%) was added and the reaction mixture was stirred at 40 °C for 30 min. The catalyst was filtered through a celite pad and the filtrate was evaporated to give 49 as a purple oil which was used in the next step without further purification. 1 H NMR (300 MHz, CDCl3): δ 6.43 (d, J = 8.2 Hz, 1H), 6.30 -6.20 (m, 2H), 4.03 (t, J = 6.3 Hz, 2H), 3.63 (br s, 3H), 3.06 (t, J = 7.1 Hz), 2.74 (t, J = 6.3 Hz, 2H), 2.47 -2.44 (m, 4H), 2.42 -2.31 (m, 6H), 1.78 -1.73 (m, 2H), 1.62 -1.52 (m, 8H), 1.47 -1.35 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 147.2, 137.3, 131.8, 111.8, 108.0, 111.4, 66.3, 58.0, 57.5, 55.0, 54.7, 43.5, 26.7, 26.0, 24.3. LCMS m/z calc for [M+H] + : 361.3, found: 361.1. 1-(3-[2-(Piperidin-1-yl)ethoxy]-4-([3-(piperidin-1-yl)propyl]amino)phenyl)-1H-pyrrole-3carbaldehyde (50) 2-[2-(Piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]benzene-1,4-diamine (49) was dissolved in acetic acid (5 mL).

4 -

 4 (3-[(Dimethylamino)methyl]-1H-pyrrol-1-yl)-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl) propyl]aniline (51) To a solution of 1-(3-[2-(piperidin-1-yl)ethoxy]-4-([3-(piperidin-1-yl)propyl]amino)phenyl)-1H-pyrrole-3-carbaldehyde (50) (85 mg, 0.19 mmol) in DCE (2 mL), dimethylamine in THF (2M, 144 µL, 0.29 mmol) was added. After 1 h 30 of stirring at rt, NaBH(OAc)3 (61 mg, 0.29 mmol) and acetic acid (17 µL, 0.29 mmol) were added. After 5 h at rt, saturated NaHCO3 solution was added. The reaction mixture was stirred for another 1 h. DCM was added and the layers were separated. The organic layer was washed twice with saturated NaHCO3 solution.

5 days

 5 of gestation to collect forebrains of foetuses. Dissection was performed under a microscope in ice-cold Hank's balanced salt solution containing 0.5% w/v D-glucose and 25 mM Hepes buffer. Cells were dissociated mechanically in dissection medium containing 0.01% w/v papain, 0.1% w/v dispase II, and 0.01% w/v DNase and incubated at 37 °C twice for 10 min. Cells were then centrifuged at 220g for 5 min at 4 °C, resuspended in Neurobasal medium supplemented with 2% B27, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM Glutamax (Invitrogen), filtered through a 40 μm cell strainer, counted, and plated on poly-L-ornithine-and laminin-coated 12-well plates at a density of 5*10 5 cells per well. New medium was added at a ratio of 1:3 of starting volume every 3 days until the end of experiments. Drug treatments with 30 and 31 at 1, 3 or 5 µM were performed directly in the conditioned cell media for 24 h at 16 days in vitro (DIV).Cytotoxycity assays. Determination of cell toxicity was assessed by measurement of lactate dehydrogenase (LDH) release (CytoTox 96® Non-Radioactive Cytotoxicity Assay -Promega)

  Analysis. Cell protein lysates were prepared for western-blot analysis by diluting the sample with 1 volume of NuPAGE® lithium dodecyl sulfate (LDS) 2X sample buffer supplemented with 20% NuPAGE® sample reducing agents (Invitrogen). Samples were heated 10 min at 100 °C. 10 µg of total proteins per well were loaded onto precast 4-12% Criterion XT Bis-Tris polyacrylamide gels (Bio-Rad) and electrophoresis was achieved after applying a tension of 150 V during 90 min using a Criterion electrophoresis Cell with the NuPAGE® MOPS SDS running buffer (1X). Proteins were transferred to a nitrocellulose membrane of 0.4 µM pore size (G&E Healthcare) using the Criterion blotting system and applying a tension of 100 V for 45 min. To resolve proteins of low molecular weights such as carboxy-terminal fragments of APP, 12% Criterion XT Bis-Tris polyacrylamide gels (Bio-Rad) were used and electrophoresis was performed during 70 min at 150 V in a NuPAGE® MES SDS running buffer (1X). Proteins were transferred to a nitrocellulose membrane of 0.2 µm pore size (G&E Healthcare) at 100 V for 40 min. Molecular weights calibration was achieved using molecular weight markers (Novex and Magic Marks, Life Technologies). Protein transfer and quality were determined by a reversible Ponceau Red coloration (0.2% Xylidine Ponceau Red and 3% Trichloroacetic acid). Membranes were then blocked in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 (v/v) (TBS-T) and 5% (w/v) of skimmed milk or 5% (w/v) of bovine serum albumin depending on the antibody during 1 h. Membrane was rinsed three-times 10 min in TBS-T before incubation with the primary antibody overnight at 4 °C. Membrane was rinsed 3 times 10 min with TBS-T and then incubated with the secondary antibody for 45 min at room temperature (RT). The immunoreactive complexes were revealed using the ECL TM Western Blotting Detection Reagents (G&E Healthcare) and image acquisitions were performed with the Amersham Imager 600 (G&E Healthcare). Quantifications of protein expression levels were performed with ImageJ Software (NIH).

Figure 2 .BFigure 3 .Figure 4 .Figure 5 .Scheme 1 a 37 a

 2345137 Figure 2. Design of biaryl series based on series A and B. A. Structure of compound A1 and B1, representative compounds of series A and B, respectively. B. Ligand-based approach leading to biaryl series.

  H NMR (300 MHz, CDCl3): δ 6.95 (dd, J = 8.4, 2.1 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 4.81 (t, J = 5.0 Hz, 1H), 4.01 (t, J = 6.2

	Hz, 2H), 3.14 (m, 2H), 2.69 -2.25 (m, 12H), 2.11 -1.91 (m, 2H), 1.73 -1.31 (m, 12H). 13 C
	NMR (75 MHz, CDCl3): δ 147.0, 137.8, 123.7, 113.7, 110.8, 107.5, 67.1, 57.5, 57.2, 56.1, 54.7,
	40.3, 26.8, 26.1, 25.9, 24.4. LCMS m/z calc for [M+H] + : 424.2, 426.2 found: 424.3, 426.2.
	4-Bromo-2-[2-(piperidin-1-yl)ethoxy]-N-[3-(piperidin-1-yl)propyl]aniline (11)
	Brown oil (2.30 g, 5.42 mmol, 79 %). 1 H NMR (300 MHz, CDCl3): δ 6.95 (dd, J = 8.4 Hz, J =
	2.2 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 4.73 (br s, 1H), 4.09 (t, J = 6.2
	Hz, 2H), 3.13 (t, J = 6.7 Hz, 2H), 2.76 (t, J = 6.1 Hz, 2H), 2.51 -2.36 (m, 10H), 1.88 -1.79
	(m, 2H), 1.64 -1.57 (m, 8H), 1.49-1.41 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 146.2, 138.1,
	124.1, 114.4, 110.7, 107.2, 66.6, 57.9, 57.4, 54.8, 42.5, 26.3, 26.0, 24.3. LCMS m/z calc for
	[M+H]

+ : 424.2, 426.2, found: 424.3, 426.

  J = 6.6 Hz, 2H), 2.47 -2.43 (m, 6H), 1.90 -1.60 (m, 13H), 1.50 -1.44 (m, 3H), 1.25 -1.22 (m, 3H), 1.04 -0.97 (m, 2H).13 C NMR (75 MHz, CDCl3): δ 146.9, 137.9, 123.5, 113.5, 110.5, 107.4, 66.6, 57.3, 54.7, 42.5, 36.5, 34.8, 33.3, 26.5, 26.2, 26.1, 25.7, 24.3. LCMS m/z calc for [M+H] + : 423.2, 425.2, found: 423.2, 425.0.

	C
	NMR (75 MHz, CDCl3): δ 146.5, 138, 124.2, 114.6, 110.7, 107.1, 66.8, 59.1, 57.9, 55, 54.6,
	43.5, 27.4, 26.0, 24.5, 24.2. LCMS m/z calc for [M+H] + : 438.2, 440.2, found 438.3, 440.3.

4-Bromo-2-(2-cyclohexylethoxy)-N-(3-(piperidin-1-yl)propyl)aniline (14)

Brown oil (80 mg, 0.19 mmol, 59 %).

1

H NMR (300 MHz, CDCl3): δ 6.96 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 6.83 (d, J = 2.1 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 3.99 (t, J = 6.75 Hz, 2H),

3.13 (t,

  C NMR (75 MHz, CDCl3): δ 193.2, 146.7, 146.1, 139, 133.8, 133.3, 130.6, 127.5, 126.7, 125.2, 124.1, 109.4, 66.6, 58.1, 57.4, 55, 54.4, 40.2, 26.1, 26, 24.5, 24.2. LCMS m/z calc for [M+H] + : 436.3, found 436.3.

  3, 146.8, 146.2, 138.8, 133.8, 133.3, 130.7, 127.5, 126.7, 125.2, 123.7, 112.2, 109.4, 68.2, 59.1, 57.3, 54.6, 54.4, 40.3, 26.2, 25.9, 24.5, 23.5. LCMS m/z calc for [M+H] + : 464.3, found 464.4.

  H NMR (300 MHz, CDCl3): δ 7.54 -7.47 (m, 1H), 7.35 -7.24 (m, 3H), 6.91 (d, J = 1.8 Hz, 1H), 6.85 (dd, J = 8.1, 1.8 Hz, 1H), 6.64 (d, J = 8.1 Hz,

	1H), 4.58 (s, 1H), 4.05 (t, J = 6.5 Hz, 2H), 3.39 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H), 2.59 -2.34
	(m, 12H), 2.19 (s, 6H), 2.09 -1.97 (m, 2H), 1.95 -1.82 (m, 2H), 1.69 -1.53 (m, 8H), 1.51 -
	1.39 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 145.5, 143.0, 137.4, 136.4, 130.2, 129.9, 129.6,
	126.6, 126.4, 122.4, 112.4, 109.2, 66.9, 61.1, 57.6, 56.2, 54.8, 54.7, 45.4, 42.6, 27.0, 26.7, 26.0,
	24.5. LCMS m/z calc for [M+H] + : 493.4, found: 493.4. HR-MS: m/z Calculated: 493.3901,
	found: 493.3906 [M+H] + = C31H49N4O.

  H NMR (300 MHz, CDCl3): δ 7.61 -7.41 (m, 1H), 7.40 -7.19 (m, 3H), 6.90 (d, J = 1.6 Hz, 1H), 6.86 (dd, J = 8.0, 1.6 Hz, 1H), 6.63 (d, J = 8.0 Hz,

	1H), 4.86 (s, 1H), 4.16 -3.91 (m, 2H), 3.36 (s, 2H), 3.32 -3.16 (m, 2H), 2.66 (t, J = 6.3 Hz,
	2H), 2.39 (dd, J = 19.5, 12.2 Hz, 10H), 2.19 (s, 6H), 1.96 -1.33 (m, 16H). 13 C NMR (75 MHz,
	CDCl3): δ 145.7, 143.0, 137.5, 136.4, 130.2, 129.9, 129.5, 126.6, 126.4, 122.3, 112.3, 109.3,
	68.0, 61.2, 59.2, 57.6, 54.6, 54.5, 45.4, 40.5, 27.7, 26.2, 26.0, 24.5, 24.5, 23.6. LCMS m/z calc
	for [M+H] + : 493.4, found 493.5. HR-MS: m/z Calculated: 493.3901, found: 493.3906 [M+H] +
	= C31H49N4O.

  8H), 1.55 -1.38 (m, 4H).13 C NMR (75 MHz, CD2Cl2): δ 148.3, 143.4, 140.7, 130.5, 128.0, 127.8, 122.0, 112.2, 111.6, 68.6, 60.0, 59.1, 56.9, 56.6, 44.2, 28.2, 28, 27.8, 26.3, 26.2. LCMS m/z calc 422.3 for [M+H] + : 422.3 , found:. HR-MS: m/z Calculated: 422.3093, found: 422.3156

	(m,
	H NMR (300 MHz, CD2Cl2): δ
	7.61 -7.53 (m, 2H), 7.44 -7.36 (m, 2H), 7.30 -7.22 (m, 1H), 7.15 (dd, J = 8.2, 2.0 Hz, 1H),
	7.09 (d, J = 2.0 Hz, 1H), 6.68 (d, J = 8.2 Hz, 1H), 4.94 (s, 1H), 4.18 (t, J = 6.0 Hz, 2H), 3.24 (t,
	J = 6.7 Hz, 2H), 2.78 (t, J = 6.0 Hz, 2H), 2.60 -2.31 (m, 10H), 1.91 -1.75 (m, 2H), 1.70 -1.55

  , 127.4, 127.2, 119.7, 113.7, 113.3, 66.0, 58.0, 55.1, 26, 24.2. LCMS m/z calc for [M+H] + : 282.2 found: 282.1. HR-MS: m/z Calculated: 282.1939, found: 282.1846 [M+H] + = C19H24NO.

	H NMR (300 MHz, CDCl3): δ 7.64 -7.56 (m,
	2H), 7.44 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.22 -7.14 (m, 2H), 6.91 (ddd, J = 8.2,
	2.5, 0.8 Hz, 1H), 4.18 (t, J = 6.1 Hz, 2H), 2.82 (t, J = 6.1 Hz, 2H), 2.61 -2.46 (m, 4H), 1.72 -
	1.57 (m, 4H), 1.53 -1.41 (m, 2H). 13 C NMR (75 MHz, CD2Cl2): δ 159.2, 142.7, 141.1, 129.7,
	128.7

  C NMR (75 MHz, CDCl3): δ 158.4, 142.9, 142.2, 136.3, 129.8, 128.8, 127.3, 126.6, 122.1, 122.1, 115.9, 113.1, 66, 30.9, 58, 55.1, 45.4, 26, 24.2. LCMS m/z calc for [M+H] + : 339.2, found: 339.2. HR-MS: m/z Calculated: 339.2358, found: 339.2422 [M+H] + = C22H31N2O.

  C NMR (75 MHz, CDCl3): δ 147.8, 142.7, 136.5, 130.5, 130.1, 130, 129.7, 126.6, 126.5, 112.0, 61.0, 58.3, 54.7, 45.4, 43.9, 26.2, 25.8, 24.5. LCMS m/z calc for [M+H] + : 352.3, found 352.3. HR-MS: m/z Calculated: 352.2747, found: 352.2743 [M+H] + = C23H34N3.

  2,5-Dimethoxy-3-formyl-2,3,4,5-tetrahydrofuran (380 μL, 2.71 mmol) was added and the reaction mixture was heated at 90 °C for 2 h. The solvent was then evaporated and the residue taken up in saturated NaHCO3 solution (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO4 and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH3), 9.8:0.2 (v/v)) to afford 50 as a brown oil (410 mg, 0.93 mmol, 43 %).1 H NMR (300 MHz, CDCl3): δ

	9.81 (s, 1H), 7.54 (dd, J = 2.1 Hz, J = 1.7 Hz, 1H), 6.95 (dd, J = 2.9 Hz, J = 2.2 Hz, 1H), 6.89
	(dd, J = 8.2 Hz, J = 2.3 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 6.74 (dd, J = 2.8 Hz, J = 1.8 Hz, 1H),
	6.59 (d, J = 8.4 Hz, 1H), 4.91 (br s, 1H), 4.15 (t, J = 6.1 Hz, 2H), 3.21 (t, J = 6.6 Hz, 2H), 2.80
	(t, J = 6.2 Hz, 2H), 2.59 -2.35 (m, 10H), 1.84 -1.89 (m, 2H), 1.69 -1.53 (m, 8H), 1.53 -1.38
	(m, 4H). 13 C NMR (75 MHz, CDCl3): δ 185.5, 146.3, 138.5, 129.0, 127.7, 127.3, 123.0, 114.8,
	109.3, 108.9, 105.8, 66.8, 57.9, 57.4, 55.0, 54.7, 42.5, 26.2, 25.9, 24.2. LCMS m/z calc for
	[M+H] + : 439.3, found: 439.2.

  An in-house dataset including 51 compounds from series A and 35 from series B was used for the computation of pharmacophore models for inhibition of Aβ1-42 secretion (TableS1, FigureS1). Sketching, 3D conversion and conformer generation of the 86 molecules was performed with ChemAxon (Marvin 14.8.11, 2014, http://www.chemaxon.com).

	1 H NMR (300 MHz, CDCl3): δ 6.91 -6.85 (m, 3H), 6.82 (d, J = 2.3 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H), 6.23 (dd, J = 2.0 Hz, J = 2.1 Hz, 1H), 4.68 (br s, 1H), 4.15 (t, J = 6.2 Hz, 2H), 3.40 (s, 2H), 3.19 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.53 -2.41 (m, 10H), 2.29 (s, 6H), 1.87 -1.84 (m, 2H), 1.65 -1.58 (m, 8H), 1.49 -1.42 (m, 4H). 13 C NMR (75 MHz, CDCl3): δ 146.3, 137.1, 130.9, 121.4, 119.5, 113.9, 110.5, 109.7, 105.5, 66.7, 57.9, 57.5, 56.5, 54.9; 45.0, 42.7, 26.5, 26.0, 24.4. LCMS m/z calc for [M+H] + : 468.4, found: 468.3. HR-MS: m/z Calculated: 468.3697, found: 468.3696 [M+H] + = C28H46ON5O. 4.2 Computational methods set using Align-It program [36] onto a linux workstation for pharmacophore perception of anti-Aβ1-42 compounds. This program takes advantage of detection of positively charged atoms and allows to customize the nature of pharmacophore features by specifying the argument funcGroup CHARGE. In this, hydrogen bond donor features were not taken into account in order to avoid the redundant and non-informative features because of overlapping with positive charge features. We chose a 3-features (Positive/Positive/Aromatic) or a more restrictive 4-features (Positive/Positive/Positive/ Aromatic) layout for generation of pharmacophores Training chemical dataset. Pharmacophore detection. Common pharmacophore features were generated from training models. B_1 and B_10 were chosen as templates for 3D flexible superimposition of other

  For statistical analysis, the non-parametric Mann-Whitney test was selected using GraphPad Prism 6 (GraphPad Software) and statistical significance was set at *p<0.05, **p<0.001, ***p<0.001 and ****p<0,0001. All data are reported as mean  SEM.

	Statistical Analysis. ASSOCIATED CONTENT
	Supporting Information. Supplementary tables S1-S3, figures S1 & S2, 1 H and 13 C NMR
	spectroscopic data and HRMS spectra for all final compounds
	Corresponding authors:
	Dr. Jamal El Bakali
	Inserm UMR-S1172 -Onco and Neurochemistry team, School of Pharmacy, 3 rue du Pr.
	Laguesse -BP 83 -59006 Lille cedex -France -Tel +33 (0)3 20 96 49 66.
	was monitored
	after 60 min incubation at rt in the presence or absence of test molecules. Compounds 30 and
	31 were evaluated at concentrations of 1 and 10 µM. Both compounds were found totally
	inactive (% inhibition < 10%). Under the assay conditions, reference OM 99-2 displayed an
	IC50 value of 59 nM.

e-mail : jamal.el-bakali@univ-lille.fr

Dr. Valerie Vingtdeux

Inserm UMR-S1172 -Alzheimer & Tauopathies team -Jean-Pierre Aubert Research Centrerue M. Polonowski -59045 Lille cedex -France -Tel + 33(0)3 20 29 88 91 e-mail : valerie.vingtdeux@inserm.fr

Table 1 . Effects of compounds 24-37, 39, 41, 44 and 51 on Aβ1-40 and Aβ1-42 secretion

 1 Compound concentration inhibiting 50% of Aβ1-40 or Aβ1-42 peptide secretion in SY5Y cells. IC50 values are expressed as mean ± SD of at least two experiments performed in triplicate. b Mean values calculated on the basis of at least three independent experiments with less than 10% deviation. c CTFα increase compared to chloroquine ([CTF]compound/[CTF]CQ) at 3 µM. AICD increase compared to chloroquine ([AICD]compound/[AICD]CQ) at 3 µM

	Cpd X	n m		Aβ1-40 IC50 (µM) a	Aβ1-42 IC50 (µM) a	αCTF vs CQ (3µM) b,c	AICD vs CQ (3µM) b,d
	CQ				7.0	12.7	1	1
	A1				1.7 ± 0.2 2.1 ± 0.5	1.3	1.8
	B1				5.1 ± 0.1 11.2 ± 4.2	2.1	2.5
	24	N	3 2	p-CH2-piperidine	2.1 ± 0.6 3.3 ± 2.1	2.6	2.5
	25	N	3 2	p-CH2-N(Me)2	1.5 ± 0.1 1.7 ± 0.4	2.1	2.5
	26	N	3 2	m-CH2-piperidine	4.3 ± 0.7 4.8 ± 0.6	3.3	2.6
	27	N	3 2	o-CH2-piperidine	2.6 ± 0.1 4.1 ± 1.3	4.3	2.7
	28	N	3 2	o-CH2-morpholine	2.8 ± 1.2 4.2 ± 1.5	3.1	1.7
	29	N	3 2 o-CH2-N-methylpiperazine 2.6 ± 0.2 2.7 ± 0.8	5.1	3.7
	30	N	3 2	o-CH2-N(Me)2	2.8 ± 1.2 3.4 ± 1.9	6.4	3.6
	31	CH 3 2	o-CH2-N(Me)2	2.1 ± 0.3 2.8 ± 0.1	0.1	0.1
	32	N	3 3	o-CH2-N(Me)2	2.1 ± 0.3 2.0 ± 0.1	4.5	5.1
	33	N	2 2	o-CH2-N(Me)2	1.4 ± 0.5 1.2 ± 0.6	5.0	4.8
	34	N	2 3	o-CH2-N(Me)2	2.0 ± 0.2 1.8 ± 0.7	4.5	3.4
	35	N	2 4	o-CH2-N(Me)2	1.6 ± 0.1 1.6 ± 0.3	4.4	4.8
	36	N	4 2	o-CH2-N(Me)2	1.7 ± 0.3 1.6 ± 0.8	4.2	3.0
	37	N	3 2	/	5.2 ± 0.1 4.0 ± 0.4	2.4	2.1
	39				9.1 ± 0.6 13.7 ± 3.3	0.1	0.2
	41				1.5 ± 0.3 1.2 ± 0.3	0.6	0.7
	44				3.9 ± 1.0 3.6 ± 0.1	0.9	0.8
	51				5.0 ± 0.9 6.5 ± 2.0	3.6	2.0

a d

ACKNOWLEDGMENT

We expressed our thanks to C. Mésangeau, N. Deguine, F. Copper and L. Barber for their contribution in organic synthesis, F. Sauvé, A. Trichies and R. Dufossez for their help with the in vitro experiments. We thank the animal core facility (animal facilities of Université de Lille-Inserm) of "Plateformes en Biologie Santé de Lille" as well as C. Degraeve, M. Besegher-Dumoulin, J. Devassine, R. Dehaynin and D. Taillieu for animal care.

ABBREVIATIONS

AD, Alzheimer's disease; AICD, APP intracellular domain; APP, amyloid precursor protein; CTF, carboxy-terminal fragment; CQ, chloroquine.

Funding Sources

This work was supported by Inserm and the University of Lille. Grants were obtained from ANR (VIDALZ), Laboratory of Excellence Labex DISTALZ and France Alzheimer. Marion Gay, Caroline Evrard and Florian Descamps are the recipient of fellowships from the University of Lille 2. The 300 MHz NMR facilities were funded by the Région Nord Pas-de-Calais (France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER).

Notes

The authors declare no competing financial interest.