Patricia Melnyk

Valérie Vingtdeux

Stéphane Burlet

Sabiha Eddarkaoui

Marie-Eve Grosjean

Paul-Emmanuel Larchanché

Guillaume Hochart

Christian Sergheraert

Cecilia Estrella

Mathieu Barrier

Virginie Poix

Pauline Plancq

Cécile Lannoo

Malika Hamdane

André Delacourte

Philippe Verwaerde

Luc Buée

Nicolas Sergeant

Highlight in ACS Chemical Research in Toxicology Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid peptide precursor metabolism

Keywords: Chloroquine, piperazine, Alzheimer's disease, Amyloid Precursor Protein

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aβ) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate processing of APP-CTF and production of Aβ peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyle or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease in Aβ peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aβ production, and is likely to be beneficial for therapeutic applications in AD.

INTRODUCTION

For decades, chloroquine (CQ) has been one of the two most widely used antimalarial drugs with moderate acute toxicity. It is known to cross the BBB. [START_REF] Vodicka | Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice[END_REF] Following a repositioning strategy, CQ and CQ-derived compounds have already been evaluated in several biological applications such as for prion disease, 2-5 HCV [START_REF] Ashfaq | Lysosomotropic agents as HCV entry inhibitors[END_REF][START_REF] Blanchard | Hepatitis C virus entry depends on clathrin-mediated endocytosis[END_REF] and even cancer. [START_REF] Vasquez-Martin | Repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions[END_REF][START_REF] Mahoney | Identification of endoplasmic reticulum stress-inducing agents by antagonizing autophagy : a new potential strategy for identification of anti-cancer therapeutics in b-cell malignancies[END_REF] Different mechanisms have been proposed for these applications. Unfortunately, the toxicity of CQ precludes its use in long-term treatments.

However, CQ-derived compounds such as hydroxychloroquine are administered, for instance, for the treatment of systemic lupus erythematosus [START_REF] Costedoat-Chalumeau | Why all systemic lupus erythematosus patients should be given hydroxychloroquine treatment?[END_REF] or rheumatoid polyarthritis. [START_REF] Suarez-Almazor | Antimalarials for treating rheumatoid arthritis[END_REF] CQ derivatives may therefore maintain the activity with reduced side effects. For that purpose, we previously developed a library of CQ-derivatives among, from which some compounds demonstrated antimalarial properties and reduced cytotoxic effects upon MRC-5 cells (human diploid embryonic lung cell line). [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF][START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF][START_REF] Bonnet | Trypanothione Reductase Inhibition/Trypanocidal Activity Relationships in a 1,4-Bis(3aminopropyl)piperazine Series[END_REF] Based on the recent idea that Alzheimer's disease (AD) could be driven by a prion-like spread of protein aggregates in the brain, [START_REF] Yin | Prion-like Mechanisms in Alzheimer's Disease[END_REF][START_REF] Vingtdeux | Potential contribution of exosomes to the prionlike propagation of lesions in Alzheimer's disease[END_REF] some compounds of this library have also been positively evaluated for their anti-prion properties. [START_REF] Klingenstein | Similar Structure Activity Relationships of Quinoline Derivatives for antiprion and antimalarial effects[END_REF] Recently, we showed that CQ had an indirect modulatory effect on APP metabolism, [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF] the deregulation of which is central to AD pathophysiology. [START_REF] Kummer | Truncated and modified amyloid-beta species[END_REF] The role of CQ in AD has also been controversially emphasized by others in the literature. [START_REF] Yuyama | Chloroquine-induced endocytic pathway abnormalities: Cellular model of GM1 ganglioside-induced Abeta fibrillogenesis in Alzheimer's disease[END_REF][START_REF] Bendiske | Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease?[END_REF] According to the amyloid hypothesis, APP is expected to play a key role in AD. APP metabolism leads to the production and release of amyloid-beta peptides (Aβ), the major component of the amyloid deposits being its 42 aa form. The proteolytic processing of APP brings into play a sequence of cleavages involving either α-or β-secretase at the first step, followed by γ-secretase (Figure 1 [START_REF] Vingtdeux | Potential contribution of exosomes to the prionlike propagation of lesions in Alzheimer's disease[END_REF] ). [START_REF] De Strooper | Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process[END_REF] The α-secretase cleaves APP within the Aβ sequence, producing a soluble fragment (sAPPα) and a carboxy-terminal fragment (CTFα), and inhibits Aβ production. [START_REF] Vingtdeux | Identification and biology of α-secretase[END_REF] Through the amyloidogenic pathway, β-secretase cleaves APP at the first amino acid residue of the Aβ sequence, producing a soluble fragment (sAPPβ) and a transmembrane carboxy-terminal fragment (CTFβ). The APP intracellular domain (AICD) is released by cleavage of CTFβ at the ε-site by γsecretase. [START_REF] Pardossi-Piquard | The physiology of the β-amyloid precursor protein intracellular domain AICD[END_REF] The ε-processed CTFβ stub is subsequently cleaved at γ-sites by γ-secretase to generate Aβ peptides ranging from 46 to 36 amino acids in length, following stepwise successive cleavages along multiple interactive pathways. [START_REF] Takami | Gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment[END_REF][START_REF] Karran | The amyloid cascade hypothesis for Alzheimer's Disease: an appraisal for the development of therapeutics[END_REF][START_REF] Matsumura | Gamma-Secretase Associated with Lipid Rafts: Multiple Interactive Pathways in The Stepwise Processing of Beta-Carboxyl-terminal Fragment[END_REF] Current drug candidates for the treatment of amyloid pathology mainly target β-or γ-secretase, in order to avoid the production of Aβ peptides. Most of them have failed, essentially due to low BBB permeability or severe side effects. [START_REF] Vingtdeux | Identification and biology of α-secretase[END_REF] Given that the mechanism involved in APP processing is now known to be shared by a growing list of type I transmembrane proteins such as Notch, and that β-and γ-secretase have multiple substrates engaged in ubiquitous cellular and tissue functions, their inhibition may have deleterious effects, and more selective inhibition of APP processing by both proteases is a potential alternative. [START_REF] Yan | Targeting the beta secretase BACE1 for Alzheimer's disease therapy[END_REF][START_REF] Hall | Gamma-Secretase modulators: current status and future directions[END_REF] One way to circumvent these side effects of highly potent β-or γ-secretase inhibitors by using dual inhibitors was recently proposed by Strömberg et al. [START_REF] Strömberg | Combining an amyloid-beta (Aβ) cleaving enzyme inhibitor with a γ-secretase modulator results in an additive reduction of Aβ production[END_REF] With regards to γ-secretase, several compounds have been shown (1) to repress or reduce Aβ production, especially Aβ species longer than 40 aa, (2) to maintain AICD release and (3) to spare other γ-secretase substrates. The so-called γ-secretase modulators (GSMs) are small molecules demonstrated to act directly on γ-secretase activity, resulting in a decreased level of long Aβ (1-40 and 1-42) and an increased level of short Aβ peptides (1-37 and 1-38). We previously showed that CQ inhibits Aβ production, whereas levels of other APP metabolites such as APP-CTFs and AICD are maintained and even increased, and γ-secretase cleavage of Notch remains unmodified. [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] Thus CQ can be considered as an indirect γ-secretase modulator since the lysosome is a compartment where Aβ is produced. [START_REF] Tam | The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid[END_REF] Here, we have screened our library of CQ-derivatives in order to identify a family of compounds showing an improved effect on APP processing and lower or limited toxicity. [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF][START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF][START_REF] Bonnet | Trypanothione Reductase Inhibition/Trypanocidal Activity Relationships in a 1,4-Bis(3aminopropyl)piperazine Series[END_REF] Our objective was to identify a compound that can redirect APP metabolism towards the non-amyloidogenic pathway, i.e. that can decrease the level of long Aβ peptides more efficiently than short ones, and increase the level of AICD and sAPPα, with a higher efficiency than CQ or other lysosomotropic compounds. [START_REF] Kummer | Truncated and modified amyloid-beta species[END_REF] For that purpose, we first focused on quinoline compounds derived from CQ as the starting point, and increased the ability of compounds to accumulate in acidic vesicles such as lysosomes by adding a bis(alkylamino)piperazine side chain. Secondly, compounds in which the quinoline moiety was replaced by various heterocyclic or aromatic substructures were studied, with an aliphatic amino chain providing the best efficiency. Finally, we evaluated the importance of the symmetry of the compounds (Figure 2). Here, we identified a series of molecules based on a CQ-derived structure, but with a chemically different structure and improved efficiency. Moreover, among those molecules one had low toxicity and provided an interesting in vivo proofof-concept.

RESULTS AND DISCUSSION

The synthetic pathways of chloroquinoline-piperazine derived compounds, heterocyclic and most benzyl-piperazine derived compounds and symmetric compounds have already been detailed, and compounds 1-34, 37, 39, 40, 42-47 have been described. [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF][START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF][START_REF] Bonnet | Trypanothione Reductase Inhibition/Trypanocidal Activity Relationships in a 1,4-Bis(3aminopropyl)piperazine Series[END_REF][START_REF] Melnyk | Use of 1,4-bis(3-aminoalkyl) piperazine derivatives in the treatment of neurodegenerative diseases[END_REF] In addition to previously described mono-or dibenzyl derived compounds, fluorobenzyl or thiazolyl compounds 35, 36 and 41 were easily synthesized by reductive amination according to Scheme 1.

All compounds were tested for their potential interference with APP processing in an SY5Y human neuroblastoma cell line stably expressing the neuronal isoform of human wild-type APP695 (SY5Y-APP wt ), a well-established cellular model for the study of APP metabolism (Figure 3). [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF]33 Thus, to analyze the effect of our compounds on APP metabolism, SY5Y-APP wt were treated or not with 1 and 5 µM of compounds compared to the control condition or to the treatment of SY5Y-APP wt with 1 or 5 µM of CQ for 6 and 24 hours (Figure 3). Small proteolytic fragments of APP catabolism were resolved by 1D Tris-Tricine gels and detected with our well-characterized APP-CterC17 antiserum [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF]33 Identity of APP fragments was previously established by 2D Westernblots. 33 As shown on Figure 3, all APP fragments including APP-CTFs derived from the α-and βcleavages are detected as well as the AICD. The exposure of 5 min enabled to quantify the APP-CTFs whilst following 30 min of exposure AICD signal was quantified. Following treatment with 1 or 5 µM of compound 33, α-and β-CTFs amounts were double when compared to CQ treatment (Figure 3, upper Western-blot). AICD production increased fivefold at 5 µM of compound 33 following 24 hours of treatment (Figure 3, AICD panel). AICD C2 and C10 indicate the concentrations of compound necessary to double (C2) or increase by 10-fold (C10) the quantity of AICDs produced compared with untreated control conditions. These were calculated based on the measure of AICD following a wider range of concentrations.

Cytotoxicity and Aβ1-42 levels, determined for all compounds, were defined as critical parameters and were measured using in vitro assays. All values are reported in Tables 1, 2 and3. Cytotoxicity is expressed as the compound concentration causing 50% cell death (CC50). IC50 indicates the concentration of compound capable of inhibiting secretion of Aβ1-42 by 50% (Tables 123). For the most potent compounds, CTFα, secreted Aβ1-38, Aβ1-40 and sAPPα concentrations were also determined (Table 4). A preliminary ADME (Absorption, Distribution, Metabolism, Excretion) evaluation was performed (Table 5), and finally, the most potent compound was selected for acute in vivo administration by the oral route (Figure 4).

For the reference compound CQ, the AICD C2 and C10 were 1.4 μM and 10.8 μM respectively (Table 1). The AICD C2 value of quinacrine 1.0 μM was comparable to that of CQ, but its high cytotoxicity to SY5Y-APP wt cells precluded the evaluation of other parameters. With the exception of compounds 5 and 8, the AICD C2 values of amide derivatives were comparable to that of CQ (1-5 μM), although AICD C10 values for most compounds were higher than that of CQ (17-40 μM). For aromatic amides, substitution was allowed, but the presence of an electron-donating group was probably unfavorable for activity, although the permeability of cells to these compounds was not measured. In the case of aliphatic amides, tert-butyl amide 6 displayed the same activity as CQ, whereas the others compounds were less effective. The ability of amides to decrease the amount of secreted Aβ1-42 was comparable to that of CQ, but slightly improved in the case of aromatic or bulky hydrophobic substituents.

Overall, secondary and tertiary amines (series Ib and Ic) were more effective than their amide counterparts. However, amines were also more toxic with the exception of the cyclopropyl derivative 15. Because of their cytotoxicity to SY5Y-APP wt cells, as already shown for other cell lines, [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF] the activity of monobenzyl amines 9-11 (except compound 12) and dibenzyl derivatives 17-20 could not be further tested in this assay. The ability of secondary amines to double the amount of AICD fragment was in the same range as for CQ (1-3 μM), although the AICD C10 of these compounds was often higher (except for compound 15). We observed that the activity and cytotoxicity on SY5Y-APP wt cells of amines were highly dependent on their structure. Indeed, steric hindrance might play an important role since the activity of the tert-butyl derivative 14 was comparable to that of CQ, whereas compound 13 was less active. In this series, benzyl or tertbutyl substituents favored the decrease of Aβ1-42 production. Generally, the activity of tertiary amines (series Ic) was higher than that of secondary amines. In tertiary amines, steric hindrance seemed to be important, since substitution with cyclopropylmethyl (compound 23) or isobutyl (compound 24) increased their activity. It was also worth noting the difference between the cyclic amines 25 and 26 in terms of cytotoxicity on SY5Y-APP wt cells, whereas their influence on AICD production remained comparable, even though the Aβ1-42 IC50 was four times higher for the piperidine derivative (1.0 μM for 25 and 4.0 μM for 26).

It is generally accepted that the increased basicity of compounds leads to better uptake by acidic compartments owing to the pH gradient between the cytosol and the compartment. Using CQ as a starting point, we evaluated derivatives with an increased ability to accumulate in acidic vesicles by adding a bis(alkylamino)piperazine side chain. In Series I, compounds 9-13 and 14-19 were chosen to evaluate the influence of a supplementary basic nitrogen, while compounds 1-8 could be compared to CQ. The ability of these compounds to accumulate in acidic cell compartments was estimated by calculating a lysosome accumulation ratio (LAR), similar to the antimalarial accumulation ratio in parasite food vacuoles (VAR). LAR could be determined according to Equation 1, 34 based on a weak base model, proceeding from a derivation of the Henderson-Hasselbach equation in previous work by Hawley et al. 35 As expected, amides 1-8 displayed an LAR similar to that of CQ (LAR = 30 -50.10 3 ) but amines 9-16 and 17-24 showed a 200-fold increase in accumulation (LAR = 56 -60.10 5 ). This seemed to correlate with the comparable activity of amides 1-8 with CQ, whereas tertiary amines showed a greater influence on APP metabolism. Secondary amines, with a higher LAR compared to CQ, also showed a comparable influence on APP metabolism (AICD and Aβ1-42 secretion). It is worth noting that an aliphatic substitution of tertiary amines has little influence on AICD and Aβ1-42 secretion (except compound

22), but increases cytotoxicity by around ten times.

The diisobutylamino side chain was chosen for further studies. We aimed to evaluate the role of the quinoline moiety, as we have previously done for antimalarial activity, [START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF] for a family of analogs in which a common N1,N1-diisobutyl-1,4-bis(3-aminopropyl) piperazine motif was linked to a variety of aromatic entities.

Two series of compounds: heteroarylamines (compounds 27-32, series IIa) and benzylamines (compounds 33-37, series IIb) were selected in order to simultaneously study the influence of the nature of the chemical link on cytotoxicity, the accumulation of AICDs and the secretion of Aβ1-42 (Table 2). With regard to the replacement of the quinoline moiety by a heterocycle, benzimidazole was the most efficient in order to maintain an effect on APP metabolism compared to compound 24. An acridine nucleus was selected for comparison with quinacrine but no results could be obtained because of its cytotoxicity. In the case of the benzoxazole ring (compound 28), the compound decreased AICD level at a concentration of 5.0 μM. Substitution with other heterocycles such as pyrazine (compound 31) led to lower activity, and AICD C10 could not be determined. The influence of the substitution on Aβ1-42 peptide secretion could not be measured in any of these cases. In the case of the pyrimidine and purine derivatives (compounds 30 and 32), no activity at concentrations up to 10 μM could be detected, with regard to either AICD fragment production or Aβ1-42 secretion.

The chloroquinoline nucleus could also be replaced by a benzyl moiety, which induced a slight decrease in the production of AICDs and Aβ1-42 (Table 2). The ability of methoxy 33, chloro 34 or fluoro 35 derivatives to double or increase 10-fold the quantity of AICD fragments generated was in the same range as CQ (C2 = 1 -3 μM and C10 = 10 -16 μM). On the other hand, the nature of the substituent on the phenyl ring modified the quantity of secreted Aβ1-42, resulting in a comparable IC50 for compounds 33 and 34, whereas compound 35 did not induce a decrease in Aβ1-42 secretion at 5 μM. Conversely, compound 37, with a pyridine ring, was found to be slightly less active in terms of AICD production than the phenyl compounds, but no effect could be detected on secreted Aβ1-42 at concentrations below 5 μM. Surprisingly, when the phenyl ring was replaced by a thiazole ring (compound 36), no effect was observed on the secretion of Aβ1-42, and AICD production was shown to decrease at concentrations below 5 μM , in contrast to the other compounds of this series.

Finally, we evaluated the influence of compound symmetry (Table 3) using alkyl-or benzyl-type substituents. Except in the case of derivatives 44, 45 and 46, all the compounds showed higher cytotoxicity on SY5Y-APP wt cells than previous series, making it difficult to evaluate their activity in this cellular model. Commercially available unsubstituted diamine, 38, had no influence on AICD levels or secreted Aβ1-42. With benzyl substituents, at the non-cytotoxic concentration of 5 μM, an increase in Aβ secretion was observed without affecting AICD levels. Surprisingly, pchlorobenzyl derivative 40 behaved differently and was one of the most effective compounds both with regard to the production of AICD (C2 = 0.5 μM) and the secretion of Aβ1-42 (IC50 = 3.1 μM), as seen for Series II. For the dialkyl compounds 45 and 46, a significant increase in Aβ1-42 secretion was measured, with little impact on AICD levels. In contrast, the tetra-isobutyl compound 47 showed a comparable impact to CQ on APP metabolism (C2 = 3.7 μM for 47, 1.4 μM for CQ), but no influence on the secretion of Aβ1-42 at the concentration of 5 μM.

The influence on accumulation in acidic compartments in this series did not seem to be critical, since compounds with a comparable theoretical LAR showed widely differing effects on APP metabolism. For instance, with similar LAR values of 15.10 6 , the presence of a p-fluorobenzyl group (compound 35), increased the secretion of Aβ1-42, but did not significantly modify AICD levels compared to a p-methoxy or p-chlorobenzyl group (compounds 33 and 34). In this series, most compounds did not modify the secretion of Aβ1-42 at concentrations below 5 μM. In the case of symmetric compounds (Series 3), except for bis(p-chlorobenzyl) or bis(diisobutyl) compounds 40 and 47, no influence on the quantity of AICD could be measured, but these compounds led to an increase in the secretion of Aβ1-42 at concentrations below 5 μM. Once again, the ability to be accumulated in lysosomes/endosomes could not be considered, since the calculated LARs were comparable (LAR = 15.10 6 ). Thus, LAR values do not seem to be highly relevant or even the only criteria in explaining the relative influence of these compounds on APP metabolism compared to CQ.

Given the interest of quinoline 25, benzimidazole 29 and benzyle 33 compounds demonstrated above, further experiments were designed to evaluate their impact on the metabolism of APP in greater detail. The neurotrophic fragments sAPPα and CTFα 36 , as well as the shorter Aβ1-38 and Aβ1-40 peptides, were quantified (Table 4). The closest analogs of CQ 25 and benzimidazole 29 showed the greatest effects on APP metabolism, with a CTFα C2 value of 0.2 and 0.5 μM respectively and a CTFα C10 close to 10 μM. Substitution with a methoxybenzyle moiety decreased the production of CTFα (C2 = 6.7 μM and C10 = 28.4 μM). Surprisingly, the levels of CTFα and sAPPα seemed uncorrelated for compound 25. At a concentration of 10 μM, which increased the quantity of CTFα produced ten times, sAPPα was only increased by 30%. At this concentration, sAPPα was increased by 78% for benzimidazole 29 and 55% for methoxybenzyle compound 33.

Since the ability of the three compounds 25, 29 and 33 to decrease the secretion of Aβ1-42 was equivalent (IC50 close to 1 μM), benzimidazole 29 appeared more effective with regard to Aβ1-40, with an IC50 of 2.0 μM, compared to 33, with a value of 5.1 μM and 25, with a value of 9.1 μM.

Interestingly, the influence of these compounds on the secretion of the short Aβ1-38 peptide was also very different, as the IC50 was 5.0 μM for compounds 25 and 29, whereas compound 33 seemed to be inactive at concentrations up to 10 μM. These two compounds were also able to inhibit the secretion of Aβ1-38 and Aβ1-40 more effectively. The activity of these three compounds on the metabolism of APP was also confirmed by measuring the secretion of sAPPα, a fragment previously described as being neuroprotective. 36 The mechanism of action of these compounds had to be deciphered. Starting from CQ, the introduction of a polyamino side chain should have led to more efficient accumulation in acidic vacuoles. Interestingly, however, when evaluated as antimalarials, these compounds were shown not to accumulate in parasite food vacuoles. 37 With respect to their influence on the secretion of Aβ peptides of different lengths, the three compounds of interest seemed to have different or complementary mechanisms of action, especially with regard to LAR values. However, a lysosomotropic activity of these compounds cannot be ruled-out. Regarding the possible effect of AICD, whether as a transcriptional gene regulator or a toxic effect, the AICD must reach the cell nucleus. The transcriptional active AICD is supposed to originate from the β-CTF. [START_REF] Belyaev | The Transcriptionally Active Amyloid Precursor Protein (APP) Intracellular Domain Is Preferentially Produced from the 695 Isoform of APP in a beta-Secretasedependent Pathway[END_REF] This mechanism supposes that a γ-secretase cleavage of the β-CTF occurs rapidly along the early endosome / lysosome pathway. AICD can also be produced later in the endosome / lysosome route and being degraded by lysosome proteases [START_REF] Asai | An alternative metabolic pathway of amyloid precursor protein C-terminal fragments via cathepsin B in a human neuroglioma model[END_REF] or secreted in the extracellular space via exosomes [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF] .

Consistent with this mechanism, accumulating evidences suggest that 40% of APP metabolism occurs in the late endosome / lysosome pathway. [START_REF] Tam | The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid[END_REF][START_REF] Vorobyeva | Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation[END_REF] Cyclopamine reduces the Aβ production and redirect part of the APP processing to the lysosome compartment [START_REF] Vorobyeva | Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation[END_REF] giving similar results to that obtain with our small compounds. This effect does not imply a modulation of the gamma-secretase activity. We also tested the in vitro γ-secretase activity as well as the γ-secretase processing of Notch (unpublished data). Both are not modified by our compounds. Interestingly, a nuclear accumulation of AICD was not reported in these studies further supporting the hypothesis that AICD can be produce along the endosome / lysosome pathway giving rise to a transcriptionally active fragment when produced in early endosomal route or alternatively, AICD can also be produced in late endosome compartments such as multivesicular bodies. [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF] Absence of nuclear signal is not due to our APP-CterC17 antibody since our antibody used to visualize the nuclear localization of AICD. [START_REF] Goodger | Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway[END_REF] Taken together, our past and current results strongly suggest that AICD accumulation is related to a modification of the late endosome /lysosome processing of AICD and would therefore explain why a toxic effect of the accumulation of AICD is not herein observed whereas nuclear AICD signaling is reported to be toxic. [START_REF] Ghosal | Alzheimer's disease-like pathological features in transgenic mice expressing the APP intracellular domain[END_REF][START_REF] Ghosal | Aging and exitotxic stress exacerbate neural circuit reorganization in amyloid precursor protein intracellular domain transgenic mice[END_REF][START_REF] Ohkawara | The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis[END_REF] Hence, in sharp contrast to cyclopamine, which is a highly teratogen drug, one of our lead compound has passed the preclinical phase and is currently in clinical phase I. However, further investigation should be done to decipher the precise molecular mechanism of action of our compounds. Some preliminary ADME parameters were evaluated before an in vivo evaluation (Table 5). The three compounds were highly soluble in water (>200 μM at pH 7.4) but showed different logD values, with a very low value of 0.1 for quinoline 25, predicting a low probability of BBB penetration. In contrast, benzimidazole 29 and compound 33 displayed intermediate values of 1.8 and 1.2, respectively. Evaluation of metabolic stability using mouse and human liver microsomes showed high stability for compound 25 (100% of the compound remaining after 1 h) and benzimidazole 29 (79 and 52% of the compound remaining after 1 h). In contrast, compound 33 showed low stability (30% and 27% after 1 h). Compounds 25 and 33 were not evaluated in this study for an in vivo proof-of-concept.

Finally, to determine the modulatory effects of our compounds on APP metabolism in vivo, a single dose treatment was carried out in wild-type C57Bl6 mice (Figure 4). Physiologically, AICDs were rarely detected and assumed to be rapidly degraded. After a single administration of compound 29 by the oral route, mice were sacrificed 24 h later and quantification by western blotting highlighted a significant increase in CTFα level. A single dose of compound 29 led to an increase in CTFα in the frontal cortex from a 5 mg/kg dose, and in the hippocampus from a 12.5 mg/kg dose. A 25mg/kg dose was the most efficient and significant, with an increase of CTFα of 29% in the frontal cortex and 20% in the hippocampus. This first in vivo proof-of-concept should be confirmed by further long-term administration studies of compound 29.

CONCLUSION

Previous studies have underlined the interest of CQ and alkaline drugs as AICD enhancers and compounds capable of interacting with the endosome/lysosome pathway, a mechanism that is of growing interest in neurodegenerative diseases including AD. The present study of several series of compounds showed that (1) the chemical structure of CQ could be efficiently modified by adding a bis(alkylamino)piperazine chain, (2) the quinoline nucleus could be replaced by a benzyle or benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allowed the metabolism of APP to be directed toward the decrease of Aβ peptide secretion or the increase of APP-CTF fragments as well as the accumulation of AICDs. Among the compounds evaluated in this study, compounds 25, 29 and 33 showed a better in vitro profile than CQ on the metabolism of APP. Preliminary ADME and in vivo evaluation underlined the possibility that compound 29 could find therapeutic applications in neurodegenerative diseases in which APP metabolism needs to be modulated. Thus benzimidazole 29 is currently under development as a potential anti-Alzheimer drug candidate. From a more fundamental point of view, as the most potent molecules were structurally different from CQ, this raises the question of the mechanism of action as well as the potential specific biological targets of this family of molecules.

METHODS

Chemistry

Compounds 1-34, 37-40 and 42-47 have already been described and were used at a purity of > 95%. [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF][START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF][START_REF] Bonnet | Trypanothione Reductase Inhibition/Trypanocidal Activity Relationships in a 1,4-Bis(3aminopropyl)piperazine Series[END_REF] Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise detailed. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2 mm). Purification of products was carried out by either column chromatography or thick layer chromatography. Column chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). Thick layer chromatography was performed on glass plates coated with Macherey-Nagel Sil/UV254 (thickness 2 mm), from which the pure compounds were extracted with the following solvent system: DCM/MeOH (NH3), 90:10. 1 H and [START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF] C NMR spectra were obtained using a Bruker 300 MHz spectrometer, chemical shifts () were expressed in ppm relative to TMS used as an internal standard. The attributions of the carbons were deduced after 2D experiments had been performed (COSY, HSQC and HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200 W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionization and a UV detector (diode array). The purity of final compounds was verified by two types of high pressure liquid chromatography (HPLC) columns: C18 Deltapak (C18N) and C4 Interchrom UP5WC4-25QS (C4). Analytical HPLC was performed on a Shimadzu system equipped with a UV detector set at 254 nm. Compounds were dissolved in buffer B or MeOH and injected through a 50 µL loop. The following eluent systems were used: buffer A (H2O/TFA, 100:0.05) and buffer B (CH3CN/H2O/TFA, 80:20:0.05). HPLC retention times (HPLC tR) were obtained, at flow rates of 1 mL/min, using the following conditions: for the 10 min method: a gradient run from 100% eluent A for 30 s, then to 100% eluent B for the next 8 min; and for the 40 min method: a gradient run from 100% eluent A for 1 min, then to 100% eluent B for the next 30 min.

(3-{4-[3-(4-Fluorobenzylamino)propyl]piperazin-1-yl}propyl)diisobutylamine (35)

A 3Å molecular sieve (1 g) was added to a solution of {3-[4-(3-aminopropyl)piperazin-1yl]propyl}diisobutylamine [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF] (150 mg, 0.48 mmol) and 4-fluorobenzaldehyde (57 L, 0.53 mmol) in ethanol (6 mL). The reaction medium was stirred at 20°C for 5 h and cooled to 0°C. NaBH4 (45.4 mg, 1.2 mmol) was added portionwise over 15 min, and the reaction medium stirred at 20°C for 12 h. The mixture was filtered over Celite. The solvent was removed under reduced pressure and 20 mL water added to the residue. The product was extracted with dichloromethane, and washed with aqueous HCl 1M. The combined organic fractions were washed with water and dried over sodium sulfate. The solvent was evaporated under reduced pressure. Purification by thick layer chromatography (DCM:MeOH:NH4OH, 90:10:1) was performed and enabled collection of the product as a yellow solid (102 mg, 50%). [START_REF] Vodicka | Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice[END_REF]

Diisobutyl-[3-(4-{3-[(thiazol-2-ylmethyl)amino]propyl}piperazin-1-yl)propyl]amine (36)

A 3Å molecular sieve (1 g) was added to a solution of {3-[4-(3-aminopropyl)piperazin-1yl]propyl}diisobutylamine [START_REF] Ryckebusch | Synthesis and in Vitro and in Vivo Antimalarial Activity of N1-(7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine Derivatives[END_REF] (150 mg, 0.48 mmol) and 2-thiazolecarboxaldehyde (46.4 L, 0.53 mmol) in ethanol (6 mL). The reaction medium was stirred at 20°C for 5 h and cooled to 0°C. NaBH4 (45.4 mg, 1.2 mmol) was added portionwise over 15 min and the reaction medium was stirred at 20°C for 12 h. The mixture was filtered over Celite. The solvent was removed under reduced pressure and 20 mL water added to the residue. The product was extracted with dichloromethane, and washed with aqueous HCl 1M. The combined organic fractions were washed with water and dried over sodium sulfate. The solvent was evaporated under reduced pressure.

Purification by thick layer chromatography (AcOEt:MeOH:NH4OH, 90:10:2) was performed and enabled collection of the product as a yellow oil (69 mg, 35%). [START_REF] Vodicka | Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice[END_REF]

(4-Fluorobenzyl)-(3-{4-[3-(4-fluorobenzylamino)propyl]piperazin-1-yl}propyl)amine (41)

A 3Å molecular sieve (5 g) was added to a solution of 1,4-bis(3-aminopropyl)piperazine (515 L, 2.50 mmol) and 4-fluorobenzaldehyde (590 L, 5.50 mmol) in ethanol (20 mL). The reaction medium was stirred at 20°C for 5 h and cooled to 0°C. NaBH4 (0.473 g, 12.50 mmol) was added portionwise over 15 min and the reaction medium was stirred at 20°C for 12 h. The mixture was filtered over Celite. The solvent was removed under reduced pressure and 20 mL water added to the residue. The product was extracted with dichloromethane, washed with aqueous HCl 1M. The combined organic fractions were washed with water and dried over sodium sulfate. The solvent was evaporated under reduced pressure. Purification by thick layer chromatography (acetone:NH4OH, 90:10) was performed and enabled collection of the product as a yellow solid (0.18 g, 17%). [START_REF] Vodicka | Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice[END_REF] H NMR (300 MHz, CDCl3) δ 7.21 (dd, 3 J = 8.7 Hz , 4 J = 5.5 Hz, 4H, Haro), 6.93 (m, 4H, Haro), 3.68 (s, 4H, NHCH2), 2.60 (t, 3 J = 6.8 Hz, 4H, NH-CH2), 2.4-2.3 (m, 12H, N-CH2), 1.63 (quint, 3 J = 7.0 Hz, 4H, CH2). [START_REF] Ryckebusch | Synthesis and Antimalarial Evaluation of New 1,4-bis(3aminopropyl)piperazine Derivatives[END_REF]

Cell culture and treatment

The human neuroblastoma cell line SKNSH-SYSY (SY5Y) was cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (PAA), 2 mM L-glutamine (Invitrogen), 1 mM non-essential amino-acids and penicillin/streptomycin (Invitrogen), in a 5% CO2 humidified incubator at 37°C. The human APP695 cDNA was subcloned into eukaryotic expression vector pcDNA3.1 (Invitrogen), allowing for G418 antibiotic selection of stable clones. This APP cDNA was transfected into SY5Y cells using the ethyleneimine polymer ExGen 500 (Euromedex) according to the manufacturer's instructions. SY5Y cells stably expressing the APP695 were selected with Geneticin G418 (Invitrogen) and one clone named SY5Y-APP wt was used here.

For treatment, SY5Y-APP wt cells were plated onto 12-well plates (Falcon) 24 h before drug exposure, and cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum (PAA), 2 mM L-glutamine (Invitrogen), 1 mM non-essential amino acids (Invitrogen), 50 units/mL penicillin/streptomycin (Invitrogen), and 200 µg Geneticin G418 (Invitrogen), under 5% CO2 at 37°C. Cells were exposed to drugs at the indicated concentrations for 24 h. After treatment, the conditioned medium was collected, spun at 200xg to eliminate the cell debris and frozen at -80°C for Aβ1-42, Aβ1-40 and sAPPα quantification. Treated SY5Y-APP wt cells were collected in 50 µl of Laemmli lysis buffer containing protease inhibitors (Complete Mini, Roche Molecular Biochemicals, Meylan, France), sonicated for 5 min and stored at -80°C until use. Total protein quantification of extracted samples was performed by BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol.

Cytotoxicity

SY5Y-APP wt cells cultured as described previously were seeded onto 96-well plates and incubated with the compound at 0.1; 0.3; 1; 3; 10; 30 and 100 µM, or DMSO, diluted in the same culture medium as a control, at 37°C in 5% CO2 for 24h (n=3). Cytotoxicity was determined by using colorimetric MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay-MTS Promega) according to the manufacturer's protocol. Absorbance was read at 490 nm and cytotoxicity was interpreted as the compound concentration causing 50% of cell death (CC50).

Results are shown as a percentage of control conditions which was considered as 100%.

Western blot analysis

Samples were heated at 85°C for 2 min with Reducing Agent (Life Technologies ™) and equal quantities of total proteins (20 µg/lane) were resolved in NuPAGE® Novex® 16% Tris-Tricine precast gels (Life Technologies ™). After electrophoresis, the proteins were transferred onto 0.2 µM PVDF membranes (Life Technologies ™) for 1 h at 20°C using the liquid transfer system (Life Technologies ™). Membranes were blocked with 5% skimmed milk in TNT (15 mM Tris buffer pH 8.4, 140 mM NaCl, 0.05% Tween-20) for 1 h at 20°C. After washing three times, the membrane were incubated with APPCter-C17 rabbit antiserum diluted 1:4,000 in TNT overnight at 4°C. APP-Cter-C17 was raised against the last 17 amino acids of the human APP sequence. [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] To develop the immunoreaction, the blots were incubated with peroxidase-conjugated purified mouse monoclonal anti-goat/sheep IgG (Sigma A 9452, MAb clone GT-34), 1:10,000 in TNT-M, for 1 h at 20°C, and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Membranes were scanned with LAS-4000 Mini Image System. AICD fragment (8 kDa) and CTFα (12 kDa) were detected. Images were obtained with a time exposure from 10 to 320s. Each image was opened with Adobe Photo Shop CS2 (version 9.0.2) computer program, a compose containing all WB bands was created for analysis. Bands quantification was performed by using Image J 1.37v computer program. Each band was transformed in a plot and the area under the curve was calculated. Results were expressed as arbitrary units of optical density. Membranes were then rinsed for 30 min at 20°C and reprobed with a goat polyclonal antibody against α-Actin, GAPDH or Neuron Specific Enolase

Bioavailability properties

Bioavailability-related profile was measured according to Lipinski et al. [START_REF] Lipinski | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF] for aqueous solubility (PBS pH 7.4), and according to Sangster [START_REF] Sangster | Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry[END_REF] for partition coefficient (logD, n-octanol-PBS, pH 7.4) by CEREP (Paris, France).

In vitro metabolic stability (mouse and human liver microsomes)

Stock solutions of compounds were diluted in 100 mM potassium phosphate buffer (KPi, 1 µM final concentrations) pH = 7.4, and test compounds were then incubated for 60 min in an incubator shaker (Eppendorf) at 37°C and 1,400 rpm with regenerating system (NADPH) and microsomal preparation (BD, final concentration 0.3 mg/mL in KPi buffer). Reactions were stopped with cold acetonitrile and Internal Standard (IS) CQ diphosphate was then added for further quantification (based upon Test compound/IS ratio area). Samples were mixed thoroughly and then centrifuged at 13,000 rpm for 10 min. Supernatants were evaporated under vacuum (SpeedVac) at medium drying rate for 2 h and residues reconstituted in water + 0.1 % TFA. Finally, 10 µL volumes were injected into the LCMS system. Microsomal stability was calculated by comparison of area ratio of parent compounds at different times.

The LCMS system for the microsomal stability assay consisted of an Orbitrap Exactive instrument (Thermo) equipped with an electrospray ionization source used in positive mode (M+H + ). The apparatus was managed with Xcalibur software. Tune parameters were set as: sheet gas flow rate at 70 L/min, aux gas flow rate at 20 L/min, spray voltage at 3.00 kV, capillary temperature at 275°C, capillary voltage at 95 V, tube lens voltage at 165 V and skimmer voltage at 36 V. Tray temperature was set at 4°C and oven temperature at 30°C. The analytical column was a C18 Hypersil Gold Thermo 50 x 2 mm, 1.9 µm (Thermo). The mobile phase consisted of water + 0.05% TFA (A) and acetonitrile + 0.05% TFA (B). The linear gradient elution program was as follows: 0-100% of B for 3.5 min, followed by an isocratic hold at 100% B for 1 min and 2 min of reequilibration with 100% A for a total run of 6 min at a flow rate of 400 µL/min. Due to the basicity of polyamines, good sensitivity was achieved using TFA in the mobile phase.

In vivo acute treatment

Statistics

Statistical analysis was performed with Graph Pad Prism® software computer program. Analysis was performed using one-way ANOVA (F value), followed by a Fisher test. p<0.05, 0.01 and 0.001 were considered statistically significant (respectively indicated *, ** and ***).

  H NMR (300 MHz, CDCl3) δ 7.20 (dd, 3 J = 8.7 Hz, 4 JF = 5.5 Hz, 2H, Haro), 6.93 (m, 3 J = 3 JF = 8.7 Hz, 2H, Haro), 3.67 (s, 2H, NHCH2), 2.59 (t, 3 J = 6.8 Hz, 2H, NH-CH2), 2.5-2.2 (m, 14H, N-CH2), 1.96 (d, 3 J = 7.2 Hz, 4H, 2 N-CH2), 1.68 (m, 2H, 2 CH), 1.5-1.4 (m, 4H, 2 CH2), 0.78 (d, 3 J = 6.6 Hz, 12H, 4 CH3). 13 C NMR (75 MHz, CD3OD) δ 129.8 (2 CHaro), 115.1 (2 CHaro), 64.1 (2 NCH2), 57.2 (NCH2), 57.0 (2 NCH2), 53.5 (NHCH2), 53.4 (2 NCH2), 48.3 (NHCH2), 26.7 (2 CH), 24.6 (2 CH2), 21.1 (4 CH3). LCMS (ESI + ): Calc. for [M+H] + : 421.36; Found: 421.47. HPLC (C4, 35 min): tR 6.7 min, PHPLC 99%; HPLC (C18, 35 min): tR 3.8 min, PHPLC > 99%.

  H NMR (300 MHz, CDCl3) δ 7.66 (d, 3 J = 3.4 Hz ,1H, Haro), 7.20 (d, 3 J = 3.4 Hz, 1H, Haro), 4.07 (s, 2H, NHCH2), 2.70 (t, 3 J = 6.7 Hz, 2H, NH-CH2), 2.5-2.3 (m, 14H, N-CH2), 1.97 (d, 3 J = 7.2 Hz, 4H, 2 N-CH2), 1.7-1.5 (m, 6H, 2 CH, 2 CH2), 0.79 (d, 3 J = 6.5 Hz, 12H, 4 CH3). 13 C NMR (75 MHz, CD3OD) δ 142.6 (CHaro), 118.9 (CHaro), 64.1 (2 NCH2), 57.0 (2 NCH2), 53.4 (3 NHCH2), 51.2 (NHCH2), 48.5 (NHCH2), 27.1 (CH2), 26.7 (2 CH), 24.6 (CH2), 21.1 (4 CH3). LCMS (ESI + ): Calc. for [M+H] + : 410.32; Found: 410.40. HPLC (C4, 40 min): tR 3.8 min, PHPLC 99%; HPLC (C18, 35 min): tR 3.8 min, PHPLC 99%.

  C NMR (75 MHz, CD3OD) δ 129.8 (CHaro), 115.5 (CHaro), 57.2 (NCH2), 53.4 (2 NCH2), 53.3 (NHCH2), 48.3 (NHCH2), 26.8 (CH2). LCMS (ESI + ): Calc. for [M+H] + : 417.27; Found: 417.04. HPLC (C4, 40 min): tR 5.9 min, PHPLC > 99%; HPLC (C18, 35 min): tR 3.2 min, PHPLC 95%.

  (1:1000; Santa Cruz Biotechnology). In vitro results are shown as the concentrations of compound able to double (C2) or multiply 10-fold (C10) the quantity of AICD or CFTα quantified by Image J. Secreted Aβ1-38, Aβ1-40, Aβ1-42 and sAPPα quantification Conditioned medium was used to determine the secreted Aβ1-38, Aβ1-40 and Aβ1-42 concentrations, using the Human Aβ (1-40) and Aβ (1-38) Assay Kits (IBL) and the INNOTEST TM beta-Amyloid (1-42) ELISA Kit (Innogenetics). For sAPPα concentrations, the human sAPPα (highly sensitive) Assay Kit (IBL) was used according to the manufacturer's instructions. Each sample was loaded in duplicate onto a 96 well plate. Experiments were done in triplicate. Results expressed in ng/ml were compared to control conditions arbitrarily given an average value of 100%. Results are presented as IC50, the concentration able to decrease to 50% the basal quantity of secreted Aβ peptide 1-38, 1-40 and 1-42.

Female 4 -

 4 month-old C57Bl6 mice were treated p.o. (gavage) with either carrier (water) or compound 29 as hydrochloride at 1; 3; 6; 12.5 and 25 mg/kg (minimum number of animals per group = 6). After 24 h, mice were sacrificed and the brain immediately removed to dissect the frontal cortex and hippocampus. Tissues were stored at -80°C until western blot analysis. All experiments were carried out in accordance with the European Communities Council Directive of November 24th, 1986 (86/609/EEC) and the experimental protocols were approved by the local institutional animal research committee (Project number: CEEA-35014, agreement number 59-350208).

Figure 1 .

 1 Figure 1. Amyloid Precursor Protein (APP) metabolism (adapted from Vingtdeux et al.16)

Figure 2 .Figure 3 :

 23 Figure 2. Chloroquine (CQ) and piperazine-derived compounds (Series I, II and III)

Scheme 1 .

 1 Scheme 1. Synthesis of benzyl derivatives 35, 36 and 41.

  values calculated on the basis of at least three independent experiments with less than 10% deviation; nd: not determined; a Compound concentration causing 50% of cell death after 24 h treatment; b Concentration doubling the quantity of AICD; c Concentration increasing the quantity of AICDs generated 10-fold; d Concentration decreasing the secretion of Aβ by 50%; e increase in the quantity of Aβ at 5 μM.

  [START_REF] Vingtdeux | Potential contribution of exosomes to the prionlike propagation of lesions in Alzheimer's disease[END_REF]

				N H	N		R1	Series I
		N H	N		N		N	R2	1-8 : R1=H, R2=COAr, COR
							9-16: R1=H, R2= benzyl, alkyl
	Cl	N	Cl	N			17-24: R1=R2= benzyl, alkyl 25-26: NR1R2= pyrrolinyl, piperidinyl
		CQ
							Series II
			R3
				N H	N		27-32: R3=heteroaryl
					N	N	33-37: R3=benzyl
			R4	N	N	R4	Series III
				R5	N	N	R5	38-46: R4=H, R5=H, benzyl, alkyl
							47: R4=R5= iBu

Table 1 .

 1 In vitro evaluation of compounds from Series I on SY5Y-APP WT cellsMean values calculated on the basis of at least three independent experiments with less than 10% deviation; nd: not determined; a compound concentration causing 50% of cell death after 24 h treatment; b Concentration doubling the quantity of AICD; c Concentration increasing the quantity of AICDs generated 10-fold; d Concentration decreasing the secretion of Aβ by 50%.

		Ref	SY5Y AICD AICD	Aβ1-42	ref	SY5Y AICD AICD	Aβ1-42	Ref	SY5Y AICD AICD	Aβ1-42
			CC50		C2		C10		IC50		CC50		C2		C10		IC50	CC50	C2	C10	IC50
			(μM) a		(μM) b	(μM) c	(μM) d		(μM) a		(μM) b	(μM) c	(μM) d	(μM) a	(μM) b	(μM) c	(μM) d
	CQ		30		1.4		10.8	12.7
	Quinacrine		5		1.0		>5		>5
	R				Series Ia							Series Ib			Series Ic
			N H	N	N	N H	R			N H	N	N	N H	R	N H	N	N	N	R	R
			Cl	N			O				Cl	N					Cl	N
	phenyl	1	57		2.0		16.9	10.3	9	<5						17	1.0
	4-methoxyphenyl	2	64		1.3		36.0	10.8	10	1.0						18	1.0
	4-chlorophenyl	3	17		2.1		13.9	9.6	11	1.0						19	1.0
	4-nitrophenyl	4	17		1.3		>17	7.3	12	17		1.0		>17	7.1	20	3.0
	methyl	5	>100		16.8		>100	>20	13	48		1.4		19.3	>10	21	18	0.8	5.8	11.3
	t-butyl	6	33		2.1		9.0		11.3	14	27		0.8		10.1	8.7	22	65	0.9	>60	18.4
	cyclopropyl	7	75		5.1		39.5	>20	15	60		1.0		6.7		16.4	23	6.5	0.6	3.0	>6
	i-propyl	8	> 30		10.1		21.6	15.0	16	10		3.7		>10	>10	24	5.3	0.4	2.7	1.0
	pyrrolidinyl																25	60	1.2	5.4	1.0
	piperidinyl																26	8.5	0.9	6.3	4.0

Table 2 .

 2 In vitro evaluation of compounds of Series II on SY5Y cellsMean values calculated on the basis of at least three independent experiments with less than 10% deviation; nd: not determined; a compound concentration causing 50% of cell death after 24 h treatment; b Concentration doubling the quantity of AICD; c Concentration increasing the quantity of AICDs generated 10-fold; d Concentration decreasing the secretion of Aβ by 50%; e decrease in the quantity of AICDs; f increase in the quantity of Aβ at 5 μM.

		Ref	SY5Y	AICD	AICD	Aβ1-42		Ref	SY5Y		AICD	AICD	Aβ1-42
			CC50	C2		C10	IC50			CC50		C2	C10	IC50
			(μM) a	(μM) b	(μM) c	(μM) d			(μM) a		(μM) b	(μM) c	(μM) d
	CQ		30		1.4		10.8	12.7
	R				Series IIa			R				Series IIb
			R	N H	N	N	N				R	H N	N	N	N
	7-chloro-4-	24	5.3		0.4		2.7	1.0	4-	33	82		1.5	10.5	1.0
	quinolinyl								methoxyphenyl
	6-chloro-2-	27	<5		>5		nd	>5	4-chlorophenyl	34	30		1.3	16	2.4
	methoxyacridin-
	9-yl
	2-benzoxazolyl	28	>100	e		nd	>10	4-fluorophenyl	35	>30		2	9.5	f
	2-	29	30		0.8		3.0	1.5	2-thiazolyl	36	nd		e	e	>10
	benzimidazolyl
	2-pyrimidinyl	30	30		>10		nd	>10	4-pyridinyl	37	>100		3	5	>10
	2-pyrazinyl	31	30		4.1		nd	>10
	6-purinyl	32	>60		>10		nd	>10

Table 3 .

 3 In vitro evaluation of compounds of Series III on SY5Y cells

	Ref	SY5Y	AICD	AICD	Aβ1-42	Ref	SY5Y	AICD	AICD	Aβ1-42
		CC50
		(μM) a

Table 5 .

 5 Preliminary ADME results

	Ref	Aqueous	logD pH 7.4	Metabolic	Metabolic
		solubility PBS pH		stability	stability
		7.4
		(μM)		mLM a	hLM b
	25	227	0.1	100%	100%
	29	278	1.8	79%	52%
	33	200	1.2	30%	27%
	Mean values calculated on the basis of two independent experiments with less than 10% deviation; a mLM: mouse liver
	microsomes; b hLM: human liver microsomes

Reactants: (i) ArCHO, NaBH4, EtOH, rt

This work was supported by the University of Lille 2, CNRS and Inserm. Grants were obtained from FEDER, OSEO and ANR.

ACKNOWLEDGMENT

We express our thanks to Emmanuelle Boll and Alexandre Barras for their contributions to organic synthesis and analysis, and to Nathalie Duhal and Dr Mostafa Kouach, CUMA, for help with the LCMS experiment.

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).

SUPPORTING INFORMATION

NMR spectra of compounds 35, 36 and 41 are available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS