Caroline Lanthier

Hugo Payan

Irene Liparulo

Bérénice Hatat

Cédric Lecoutey

Marc Since

Audrey Davis

Christian Bergamini

Sylvie Claeysen

Patrick Dallemagne

Maria- Laura Bolognesi

Christophe Rochais

Novel multi target-directed ligands targeting 5-HT 4 receptors with in cellulo antioxidant properties as promising leads in Alzheimer's disease

Keywords: Alzheimer's Disease, 5-HT4 receptor, antioxidant, phenol, MTDL

Highlights

• Conception and synthesis of new MTDLs combining 5-HT4R agonist activity and antioxidant properties

• All compounds showed good %inhibition at 10-6M on 5-HT4R, with 9b and 9g showing Ki < 15 nM on 5-HT4R as well as agonist profile.

• In vitro and in cellulo antioxidant effects were evaluated

• 9b is the best compromise regarding the 5-HT4R activity and antioxidant properties

ABSTRACT

Facing the complexity of Alzheimer's disease (AD), it is now currently admitted that a therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same time Ab protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells.

Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical aminochlorobenzophenone present in numerous 5-HT4R agonists, and diverse antioxidant chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards 5-HT4R and an antioxidant activity in vitro and in cellulo.

Introduction

Alzheimer's disease (AD) is the most common form of dementia and affects more than 50 million people worldwide. 1 More than a century after its discovery, AD is now characterized by synaptic dysfunction and synapse loss correlated with cognitive decline. [START_REF] Forner | Synaptic impairment in Alzheimer's disease: a dysregulated symphony[END_REF] These dysfunction are supposed to be due to the presence of two peptide deposits in and around neuronal cells. The accumulation and aggregation of A peptide is at the origin of the amyloid pathology whereas the neurofibrillary tangles are mainly composed of phosphorylated tau protein. 3 These lesions are generally associated with mitochondria dysfunction, an increase inflammatory response and the presence of oxidative stress. 4 The latter was further particularly linked to the presence of amyloid aggregates which can produce Reactive Oxygen Species (ROS) in the presence of metal ions and which ultimately leads to neuronal cell death. [START_REF] Cheignon | Oxidative stress and the amyloid beta peptide in Alzheimer's disease[END_REF] The control of ROS production is therefore of particular interest in the prevention or the treatment of AD. Despite these progresses, the discovery and development of treatments with disease-modifying effect on AD remain one of the greatest challenges in drug discovery. [START_REF] Cummings | Alzheimer's Disease Drug-Development Pipeline: Few Candidates, Frequent Failures[END_REF] After numerous failures in clinical trials with compounds targeting mainly amyloid [START_REF] Karran | The amyloid cascade hypothesis: are we poised for success or failure?[END_REF] or tau pathologies, [START_REF] Congdon | Tau-targeting therapies for Alzheimer disease[END_REF] it is now currently admitted that a pleiotropic approach is needed for this multifactorial neurodegenerative disease. [START_REF] Frautschy | Why Pleiotropic Interventions Are Needed for Alzheimer's Disease[END_REF] In order to impact several biological targets at the same time, single pleiotropic compounds, known as Multi-Target Directed Ligands (MTDLs), [START_REF] Cavalli | Multi-Target-Directed Ligands to Combat Neurodegenerative Diseases[END_REF][START_REF] Rosini | Multi-Target Design Strategies in the Context of Alzheimer's Disease: Acetylcholinesterase Inhibition and NMDA Receptor Antagonism as the Driving Forces[END_REF][START_REF] Leon | Recent Advances in the Multitarget-Directed Ligands Approach for the Treatment of Alzheimer's Disease[END_REF] have been developed during the last decade. Among them, many examples include compounds able to act on oxidative stress and on another target. 13,[START_REF] Lee | Rational Design of a Structural Framework with Potential Use to Develop Chemical Reagents That Target and Modulate Multiple Facets of Alzheimer's Disease[END_REF][START_REF] Oliveira | Benzoic acidderived nitrones: A new class of potential acetylcholinesterase inhibitors and neuroprotective agents[END_REF] On the other hand, many examples have been described in order to limit amyloid pathology with numerous examples of MTDL acting on -secretase inhibition, [START_REF] Prati | Multitarget Drug Discovery for Alzheimer's Disease: Triazinones as BACE-1 and GSK-3β Inhibitors[END_REF] or inhibiting A aggregation. [START_REF] Lee | Rational Design of a Structural Framework with Potential Use to Develop Chemical Reagents That Target and Modulate Multiple Facets of Alzheimer's Disease[END_REF] In addition to those classical targets, the serotonergic system appears of particular interest, notably via the modulation of 5-HT 4 and 5-HT 6 receptors. [START_REF] Claeysen | Serotonin: A New Hope in Alzheimer's Disease?[END_REF][START_REF] Lalut | Modulating 5-HT 4 and 5-HT 6 Receptors in Alzheimer's Disease Treatment[END_REF] The activation of 5-HT 4 receptors (5-HT 4 R) with partial agonists, such as RS67333, is of particular interest as it can promote the non-amyloidogenic cleavage of Amyloid Protein Precursor (APP) and subsequently decrease the amyloid load in AD transgenic mice models. [START_REF] Cachard-Chastel | 5-HT4 Receptor Agonists Increase sAPPalpha Levels in the Cortex and Hippocampus of Male C57BL/6j Mice[END_REF][START_REF] Cochet | Chronic treatments with a 5-HT4 receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease[END_REF] In this context, we have in the past modulated the structure of RS67333 in order to introduce another target of choice for the treatment of AD such as acetylcholinesterase (AChE) inhibitory activity [START_REF] Lecoutey | Design of Donecopride, a Dual Serotonin Subtype 4 Receptor Agonist/acetylcholinesterase Inhibitor with Potential Interest for Alzheimer's Disease Treatment[END_REF][START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF] or 5-HT 6 R antagonism. [START_REF] Yahiaoui | Synthesis, and Pharmacological Evaluation of Multitarget-Directed Ligands with Both Serotonergic Subtype 4 Receptor (5-HT4R) Partial Agonist and 5-HT6R Antagonist Activities, as Potential Treatment of Alzheimer's Disease[END_REF] This two studies demonstrated that the replacement of the butyl chain of RS67333 by diverse alky moieties has limited impact on both the affinity and the pharmacological profile towards 5-HT 4 R. A similar strategy has been recently followed in order to generate 5-HT 6 R antagonists bearing antioxidant activities. [START_REF] Marcinkowska | Anti-Alzheimer's multitarget-directed ligands with serotonin 5-HT6 antagonist, butyrylcholinesterase inhibitory, and antioxidant activity[END_REF] In this context, we would like to assess the possibility to introduce, on the central piperidine ring of the targeted structures (Figure 1), various chemical moieties known to exhibit antioxidant activities. We postulate that such MTDL would be of particular interest combining, for the first time in a unique compound, antioxidant properties as well as 5-HT 4 R agonism. Several antioxidant scaffolds were chosen for this proof of concept: polyphenol, [START_REF] Spagnuolo | Neuroprotective Role of Natural Polyphenols[END_REF] hydroxycinnamic acid [START_REF] Zhang | A review on the hybrids of hydroxycinnamic acid as multi-targetdirected ligands against Alzheimer's disease[END_REF] and lipoic acid. [START_REF] Bolognesi | Lipoic acid, a lead structure for multi-target-directed drugs for neurodegeneration[END_REF][START_REF] Bolognesi | Toward a rational design of multitargetdirected antioxidants: merging memoquin and lipoic acid molecular frameworks[END_REF] Following this strategy, the two pharmacophores, will be connected through a permanent linker in order to optimize the central distribution of the antioxidant moiety.

Indeed, the presence of the piperidine as a linker appears essential for the 5-HT 4 R activity and the generated compounds should possess physicochemical properties for CNS application. The influence of these substitutions on 5-HT 4 R affinity and the antioxidant activity of the resulting compounds could thus be assessed in vitro and in cellulo.

Results

Chemistry

According to our previous chemical studies, the precursor 7 appears as a central scaffold in the synthesis of RS67333. [START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF][START_REF] Yahiaoui | Synthesis, and Pharmacological Evaluation of Multitarget-Directed Ligands with Both Serotonergic Subtype 4 Receptor (5-HT4R) Partial Agonist and 5-HT6R Antagonist Activities, as Potential Treatment of Alzheimer's Disease[END_REF] Compound 7 was synthesized in a three-step sequence through formation of a β-keto ester 2 by reaction between acid 1, activated in situ with CDI, and potassium 3-ethoxy-3-oxopropanoate. [START_REF] Yang | Discovery and Structure-Activity Relationships of Trisubstituted Pyrimidines/Pyridines as Novel Calcium-Sensing Receptor Antagonists[END_REF][START_REF] Kaspi | Process for the preparation of donepezil[END_REF] Subsequent alkylation with a previously synthesized piperidine chain 6, and followed by reaction with KOH in an EtOH/H 2 O mixture provided a saponification-decarboxylation reaction to afford 7. [START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF] From this precursor 7, different moieties were linked, such as catechol, vanillin or isovanillin widely known for their antioxidant properties. [START_REF] Rice-Evans | Antioxidant Properties of Phenolic Compounds[END_REF][START_REF] Tai | Evaluation of Antioxidant Activity of Vanillin by Using Multiple Antioxidant Assays[END_REF] The first compounds 9a, 9c, 9d, and 9f were obtained by alkylation with the corresponding bromobenzyl derivatives at room temperature. A final debenzylation step was performed with HBr 33% in solution in acetic acid at 0˚C to obtain free alcohols derivatives 9b, 9e, and 9g respectively.

The second goal was to link ferulic and lipoic acid (LA) described as powerful antioxidants in the literature. [START_REF] Zhang | α-Lipoic Acid Improves Behavior by Mitigation of Oxidative Stress, Inflammation, Ferroptosis, and Tauopathy in P301S Tau Transgenic Mice[END_REF][START_REF] Sultana | Ferulic acid ethyl ester protects neurons against amyloid beta-peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity[END_REF][START_REF] Perluigi | In vivo protective effects of ferulic acid ethyl ester against amyloidbeta peptide 1-42-induced oxidative stress[END_REF] In order to link LA and ferulic acid while maintaining the basicity of the nitrogen of the piperidine, an ethylamine linker was added. According to litterature, [START_REF] Yahiaoui | Synthesis, and Pharmacological Evaluation of Multitarget-Directed Ligands with Both Serotonergic Subtype 4 Receptor (5-HT4R) Partial Agonist and 5-HT6R Antagonist Activities, as Potential Treatment of Alzheimer's Disease[END_REF]

In vitro evaluation

As previously mentioned, the main goal of our study was to discover a hit that will have sufficient antioxidant properties, as well as a good affinity toward 5-HT 4 R. All the synthesized compounds, were tested on 5-HT 4 R according to the Grossman et al method. [START_REF] Grossman | Development of a Radioligand Binding Assay for 5-HT4 Receptors in Guinea-Pig and Rat Brain[END_REF] The affinity toward the receptor was evaluated using a displacement assay of the tritiated ligand [ 3 H]-GR113808, a specific and highly potent 5-HT 4 R antagonist used to label specific binding sites in human brain. Those compounds were also developed for their antioxidant properties and preliminary antioxidant in vitro test was also performed. Our attention was focused on two tests, DPPH, and ORAC. [START_REF] Gunesch | Natural antioxidants in hybrids for the treatment of neurodegenerative diseases: a successful strategy?[END_REF] The ORAC Fl assay was conducted in an aqueous medium according to well-described procedure. It evaluates the antioxidant activity via a Hydrogen Atom Transfert (HAT) mechanism. Calculating the AUC of the fluorescence decrease for 1.5 hours, quantitative and kinetic aspects are both taken into account. Here, phenol gifted compounds showed a moderate antioxidant activity compared with a TEAC ranged between Trolox ® (6-Hydroxy-2,5,7,8tetramethylchroman-2-carboxylic Acid), the reference, and quercetin (Table 1), a natural polyphenol antioxidant. The DPPH assay was performed in an organic solvent and enabled the evaluation of antioxidant activity following a Single-Electron-Transfer (SET) mechanism.

According to recent literature, our initial procedure was completed here by slight modifications to ensure a complete evaluation of our compounds. First calculation of the specific absorbance of DPPH subtracting the absorbance of others components in the solution was realized. The maximum effect was normalized using a high dose of Trolox ® (375 µM) to get the absolute EC 50. [START_REF] Sebaugh | Guidelines for accurate EC50/IC50 estimation -Sebaugh -2011 -Pharmaceutical Statistics[END_REF] The kinetic analysis to reach the plateau of % DPPH RSA at infinite time was determined and EC 50 were calculated using linear or non-linear regression (R²> 0.98) from the dose-effect curves.

In these conditions, Trolox ® and quercetine reached their maximal antioxidant activity very quickly (immediately and few minutes respectively) compared to other active compounds (T 1/2 about 30 min). Their dose-effect curve was a linear (R²>0.99). In our phenol series, when DPPH EC 50 was calculable, EC 50 values obtained after 120 min were very spread from 19.6 to >250 µM, varying as a terminal chemical pattern.

In cellulo evaluation

The pharmacological profile of the selected compounds was assayed towards h5-HT 4 R transiently expressed in COS-7 cells (Figure 2). Three compounds behaved as partial agonists of the receptor with EC 50 of 2.8 ± 1.3 nM, 1.4 ± 0.8 nM and 4.5 ± 2.1 nM for 9b, 9g and 10b, respectively (Figure 4). Compound 10a also behaved as a partial agonist of the 5-HT 4 R, but its irregular profile suggested that it may engage other targets in the COS-7 and no clear pharmacological profile was established. Compound 10b presented an E max of 47 ± 8% that is similar to the one of RS67333, the 5-HT 4 R agonist of reference. Compounds 9b and 9g showed greater efficacy than the reference to induce cAMP production with E max of 72 ± 10% and 57 ± 3%, respectively. The first in vitro antioxidant assays allowed us to select 9b, 9g, 10a-b, to complete their antioxidant evaluation in a cellular context. To define the suitable concentration, the cytotoxicity of 9b, 9g, 10a-b was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF] in a neuroblastoma cell line, SH-SY5Y. As seen on Figure 3, treatment of the cells with compounds 9b, 9g, 10a (0.5 to 10 µM) did not show cytotoxicity. A little decrease of 12% of viability compared to control cells was seen with compound 10b. While at a dose of 50 µM all compounds seem to show a small cytotoxicity with a decreasing of 37%, 40%, 48%, 68% for 9b, 9g, 10a, 10b respectively. Therefore, a single dose of 10 µM was chosen to perform the in cellulo antioxidant assay. The antioxidant activity of selected compounds was evaluated on live SH-SY5Y cells using the fluorogenic probe H 2 DFCDA. The cell-permeant H 2 DCFDA passively diffuses into cells and is retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by ROS, the nonfluorescent H 2 DCF is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) as described by Wang in 1999. [START_REF] Wang | Quantifying Cellular Oxidative Stress by Dichlorofluorescein Assay Using Microplate Reader11Mention of a Trade Name, Proprietary Product, or Specific Equipment Does Not Constitute a Guarantee by the United States Department of Agriculture and Does Not Imply Its Approval to the Exclusion of Other Products That May Be Suitable[END_REF] The level of oxidative stress is directly correlated with the intensity of DCF fluorescence.

To evaluate the antioxidant activity, cells were incubated for 24 hours with the selected compounds, Trolox or vehicle. Then cells were incubated with 10μM H 2 DCFDA probe for 30 minutes. After this time the cells were carefully washed with PBS and the oxidative stress was induced by 30 minutes treatment with tert-butyl hydroxyperoxide (t-BuOOH or TBH). The fluorescence intensity was normalized on protein content (Figure 4).

Discussion

We have demonstrated in our previous study [START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF] that the introduction of a non-susbtituted benzyl group instead of the butyl group present in RS67333 has little impact on affinity towards the 5-HT 4 R and on Acetylcholinesterase inhibition. Based on these results a first series of hydroxyl or methoxy substituted benzylated derivatives was generated. In a previous study, we have demonstrated that the replacement of the butyl substituent of the piperidine of RS67333 by benzyl or methylenecyclohexyl moiety leads to potent AChE inhibitors with good physicochemical properties and in vivo promnesiant activities. [START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF] In order to verify the impact of these modulations on drugability parameters, cLogP were determined. If the protected benzylated compounds 9a, 9d and 9f appear particularly lipophilic, all final compounds are compatible with optimal CNS parameters. [START_REF] Rankovic | CNS Drug Design: Balancing Physicochemical Properties for Optimal Brain Exposure[END_REF] If benzyl piperidine scaffolds are well known pharmacophores for cholinesterase interaction, [START_REF] Bajda | Structure-based search for new inhibitors of cholinesterases Int[END_REF] we have verified that the RS67333 analogs were selective towards butyrylcholinesterase. [START_REF] Rochais | Novel Multi-Target Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities as Potential Agents against Alzheimer's Disease: The Design of Donecopride[END_REF] Docking studies in AChE binding site revealed that the orientation of those ligands was similar to Donepezil and reproduced its interactions. In particular, the benzyl moiety is located at the bottom of the aromatic gorge of AChE and engaged in a hydrophobic interaction with Trp86. We postulate that the introduction of polar or bulky substituents on the benzyl group of our final compounds will be detrimental for their activities. In order to verify this hypothesis, all final compounds 9a-g and 10a-b were evaluated as inhibitors of human AChE. As expected, these modulations were particularly detrimental for the affinity of the ligands towards AChE (See Table S1 in Supporting Informations). Concerning the impact of these modulations on 5-HT 4 R activity, as shown in Table 1, all compounds exhibited good inhibition rates at 10 -6 M.

However, the benzyl protecting group present on the scaffold of compounds 9a, 9d and 9f has detrimental impact on affinity towards 5-HT 4 R receptors probably due to its steric hindrance. On the contrary, smaller substituents lead to enhanced activity. In this assay only 9b, 9g, and 10b exhibited more than 35% of inhibition rate at 10 -8 M allowing us to calculate further data such as IC 50 and Ki. Surprisingly the vanillin derivative 9e showed a decreased potency compared to the isovanillin analog 9g. On the contrary, 9b and 9g are really promising with Ki values under 15 nM. According to our hypothesis, compounds 9b, 9g and 10a behave as partial agonist for the 5-HT 4 R, the first two being able to induce cAMP production with a greater efficiency than the reference RS67333 (Figure 2). All compounds were then engaged in two complementary in vitro antioxidant biological assays.

In the DPPH assay, a SET mechanism test, hydroxylated compounds 9b, 9e, 9g and 10b were the only one to exert antioxidant activity, the catechol 9b being the most potent. In this assay, the lipoic acid derivative 10a did show limited antioxidant activity. In the ORAC evaluation, the antioxidant activities of all compounds were compared to Trolox ® , a widely used antioxidant reference compound. The same compounds 9b, 9e and 9g appeared to be 3-fold more potent than Trolox ® , whereas ferulic derivative 10g demonstrated similar activity than the reference. Both test confirm that the in vitro activity is clearly linked to the introduction of a phenol group as all benzylated compounds are inactive in this assay.

Based on the results we have chosen to test the in cellulo antioxidant activity of these compounds 9b, 9g, 10a and 10b. Compound 9e was not selected because of its lower affinity towards 5-HT 4 R. As already demonstrated for prucalopride, another 5-HT 4 R agonist, we have chosen to evaluate this antioxidant and neuroprotective activities in SH-SY5Y neuroblastoma cell line. [START_REF] Bianco | Prucalopride exerts neuroprotection in human enteric neurons[END_REF] Lipoic acid antioxidant activity is dependent of mitochondrial activity [START_REF] Packer | Alpha-Lipoic Acid as a Biological Antioxidant[END_REF] and therefore cannot be highlighted by an in vitro assay. For that reason, 10a was also selected despite no sign of in vitro antioxidant effect.

A non-toxic dose of 10µM was chosen for the in cellulo antioxidant activity assay, after a general MTT viability assay. Oxidative stress was generated using TBH and the ability of the compounds to limit ROS induction was compared with the well-known antioxidant compound Trolox®. [START_REF] Da Frota | In vitro optimization of retinoic acid-induced neuritogenesis and TH endogenous expression in human SH-SY5Y neuroblastoma cells by the antioxidant Trolox[END_REF] Trolox ® was used at 100 µM and, as expected, exerts a significant reduction of ROS production.

Used at a concentration of 10µM in this assay, compounds 9b, 9g and 10b used at a concentration of 10µM significantly decreased ROS production by at least 50% compared to the control group. However, no activity was obtained in this assay with the lipoic acid substituted compound 10a.

These results are in accordance with the pharmacological profile of the compounds on 5-HT 4 R, towards which no clear profile was established for compound 10a. The three other coupounds demonstrated clear agonism toward the receptor with the catechol substituted compound 9b and the isovanillin substituted compound 9g being interesting candidates to further in vivo evaluation.

Conclusion

This study demonstrates for the time the possibility to generate a series of potent 5-HT 4 R ligands with promising antioxidant activity. Among them compound 9g appears as the best compromise with a Ki of 12.7 nM toward 5-HT 4 R and an antioxidant activity in cellulo. If the balance of activity between the in cellulo or in vitro antioxidant assay (micro molar range) and the in vitro assay for 5-HT 4 R affinity (nanomolar range) might be improved, these series of compound show great promises in more challenging models. In the experiment, the amount of oxidative stress needs to be high enough to be visible with DCF-DA. In the brain, the level of oxidative stress caused by AD is not properly quantified and is lower than the one induced by t-BuOOH. On the base of the obtained data, it seems plausible that, with a suitable pharmacokinetic and a proper Blood Brain Barrier crossing according to its adequate LogP, 9g could have an antioxidant effect at a nanomolar concentration and will be tested in a near future in preclinical animal models of AD.

Experimental section

Chemistry. All commercially available compounds were used without further purification.

Melting points were determined on a Stuart melting point apparatus (SMP50). Analytical thinlayerchromatography (TLC) was performed on silica gel 60 F 254 on aluminium plates (Merck) and visualized with UV light (254 nm). Flash chromatography was conducted on a VWR SPOT II Essential instrument with silica gel 60 (40-63µm). Column size and flow rate followed the manufacturer's recommendations. NMR spectra were recorded at 295K, at 400 or 500 MHz (Bruker Avance III 400/500 MHz) for 1 H NMR and at 100 or 126 MHz for 13

General procedure for deprotection/alkylation of the piperidine moiety A

To a stirred solution of tert-butyl piperidine-1-carboxylate derivative (1.0 eq.) in DCM (20 mL/mmol) was added TFA (2 mL/mmol). The resulting mixture was stirred at room temperature for 1h. Removal of the solvent under vacuum afforded the crude product, which was directly engaged in the next step. The residue obtained (1.0 eq.) was dissolved in DCM or ACN (10 mL/mmol) and bromo-benzyl-alcohol derivatives (1.1-1.5 eq.) and K 2 CO 3 (10.0 eq.) were added.

The resulting mixture was stirred at RT or 70°C depending on the product for 2-72h, then concentrated in vacuo. The crude was directly purified by chromatography on silica gel column and concentrated under reduced pressure to afford alkylated compound (isolated yields).

The general procedure to obtain unprotected cathecol B

Appropriate benzylated product (1 eq) was stirred in bromhydric acid 33 % diluted in acetic acid (10 mL.mmol -1 , 0.10M) and DCM (50 mL.mmol -1 , 0.02M) at 0°C for 1 to 4h. The mixture was evaporated in vacuo at rt. Then the crude was directly purified on silica gel column (DCM/MeOH; from 100:0 to 80:20).

Ethyl 3 (4 amino-5-chloro-2-methoxyphenyl)-3-oxopropanoate (2)

To a solution of 4-amino-5-chloro-2-methoxybenzoic acid (5.00 g, 24.8 mmol, 1.0 eq.) in THF (200 mL, 0.12 M) was added CDI (4.42 g, 27.3 mmol, 1.1 eq.) portionwise. The mixture was stirred at rt for 4h. Then, ethyl potassium malonate (5.06 g, 29.7 mmol, 1.2 eq.) and MgCl 2 (2.83 g, 29.7 mmol, 1.2 eq.) were added portionwisely. The resulting mixture was stirred 16h at 40°C. Solvent were evaporated under vacuum. The residue was diluted with water and then extracted with DCM three times. Organics layers were combined and washed with NaHCO 3 sat. solution, then brine, and dried over MgSO 4 . Solvent was evaporated under vaccum to afford a crude product which was purified on silica gel column (Cyclohexane/EtOAc from 100:0 to 60:40). The compound was obtained as a light yellow powder (3.76 g, 60% yield). 1 H NMR (CDCl 3 , 400 MHz) δ 7.92 (s, 1H), 6.23 (s, 1H), 4.55 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.87 (s, 2H), 3.82 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H). 13

(piperidin-4-yl)methanol (4)

To a solution of ethyl isonipecotate (2 mL, 13.0 mmol, 1.0 eq.) in Et 2 O (150 mL) was added portion-wise LiAlH 4 (1.48 g, 39.0 mmol, 3.0 eq.) at 0°C and the reaction mixture was refluxed for 7h. The mixture was then cooled to 0°C and the reaction was then slowly quenched with 1.5 mL of water, 1.5 mL of 15% NaOH solution followed by 4.5 mL of water to afford a granular inorganic precipitate. The white solid was filtered and washed several times with EtOAc.

Evaporation of the filtrate under reduced pressure provided 1.38 g (92% yield) of compound (1) as a colorless oil. 1 H NMR (CDCl 3 , 400 MHz) δ 3.45 (d, J = 5.9 Hz, 2H), 3.08 (m, 2H), 2.59 (dt, J = 12.0 Hz, J = 3.0 Hz, 2H), 1.96 (br s, 1H), 1.71 (m, 2H), 1.60-1.55 (m, 1H), 1.17-1.07 (m, 2H); 13 C NMR (CDCl 3 , 100 MHz) δ 66.9, 45.9 (2C), 38.8, 29.7 (2C); IR (neat, cm -1 )  3369 , 2923, 2855, 1636, 1531, 1425, 1266, 1036. Spectral and analytical data matched with literature. [START_REF] Boyer | Synthesis of ,-Difluoro--amino Esters or gem-Difluoro--lactams as Potential Metallocarboxypeptidase Inhibitors[END_REF]

Tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (5)

To a solution of (1) (1.14 g, 9.96 mmol, 1.0 eq.) in DCM (40 mL) was added di-tertbutyldicarbonate (2.39 g, 10.9 mmol, 1.1 eq.). The solution was stirred for 15h at room temperature. The mixture was then diluted with water and the aqueous layer was extracted several times with DCM. The organic layer was dried over MgSO 4 and concentrated in vacuo.

The crude product was purified by chromatography on silica gel column (cyclohexane/EtOAc, gradient 100:0 to 70:30) to give 1.34 g (62% yield) of compound (2) as white solid. 1 H NMR (CDCl 3 , 400 MHz) δ 4.10 (m, 2H), 3.48 (d, J = 6.1 Hz, 2H) 2.68 (m, 2H), 1.80 (br s, 1H, OH), 1.69 (m, 2H), 1.63 (m, 1H), 1.44 (s, 9H), 1.12 (m, 2H); 13

Tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (6)

To a solution of (2) (2.25 g, 10.5 mmol, 1.0 eq.), imidazole (858 mg, 12.6 mmol, 1.2 eq.) and triphenylphosphine (3.30 g, 12.6 mmol, 1.2 eq.) in dry THF (30 mL) was added drop-wise a solution of iodine (3.20 g, 12.6 mmol, 1.2 eq.) in dry THF (30 mL) at 0°C. The reaction mixture was stirred at room temperature for 14h. The mixture was quenched with a saturated Na 2 S 2 O 3 solution. The aqueous layer was extracted with EtOAc. The organic layer was washed with water, dried over MgSO 4 , and concentrated in vacuo. The crude was several times diluted with cold Et 2 O, and white crystals were filtered. After concentration of filtrate in vacuo,3.13 g of the compound (3) was obtained as a colorless oil (92% yield).

Tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidine-1-carboxylate (7)

To a solution of ethyl 3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropanoate (4) (3.14 g, 11.6 mmol, 1.0 eq.) in DMF (30 mL) and tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (3) (4.14 g, 12.7 mmol, 1.1 eq.) was added K 2 CO 3 (3.19 g, 23.10 mmol, 2 eq.). The mixture was stirred 72h at rt. The reaction mixture was diluted with water, and product was extracted with EtOAc.

Combined organic layers were washed with water and brine and then dried over MgSO 4 .

Evaporation of the solvent gave a crude mixture which was directly dissolved in EtOH (500 mL), H 2 O (100 mL) and KOH (3.23 g, 57.5 mmol, 5.5 eq.) were added. The reaction mixture was refluxed for 3h, EtOH was evaporated. The mixture was diluted with H 2 O and extracted with EtOAc. The organic layer was washed with H 2 O and brine. Evaporation of the solvent provided a crude product, which was purified by column on silica gel, eluting on DCM/EtOAc from 100:0 to 60:40 to give the product 5 as a colorless solid (3.11 g, 67% isolated yield).

Tert-butyl N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-yl}ethyl)carbamate (8)

Following procedure A with tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3oxopropyl]piperidine-1-carboxylate (7) (2.37 g, 5.98 mmol, 1 eq.) and tert-butyl N-(2bromoethyl)carbamate (1.61 g, 7.18 mmol, 1.2 eq.) synthesized following the literature, the reaction was stirred at rt for 16h. After a purification by chromatography on silica gel column (Cyclohexane/EtOAC, gradient 100:0 to 0:100), the compound ( 8) was obtained as a light yellow solid (780 mg, 30% isolated yield).

1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-dibenzyloxyphenyl)methyl]-4piperidyl]propan-1-one (9a)

Following procedure A with tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3oxopropyl]piperidine-1-carboxylate (7) (200 mg, 0.50 mmol, 1 eq.) and 1,2-bis(benzyloxy)-4-(bromomethyl)benzene (230 mg, 0.60 mmol, 1.2 eq.) synthesized following the literature, the reaction was stirred at RT for 10h. After a purification by chromatography on silica gel column From general procedure B with 9d (80 mg, 0.16 mmol, 1.0 eq.) to afford 9e as yellow powder with a yield of 85%. 1 H NMR (MeOD, 400 MHz) δ 7.66 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.91 (dd, J = 8.1, 2.0 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.45 (s, 1H), 4.13 (s, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 3.40 (d, J = 12.6 Hz, 2H), 3.00 -2.79 (m, 4H), 1.96 (d, J = 13.4 Hz, 2H), 1.62 (m, 3H), 1.46 (m, 2H). 13 C NMR (MeOD, 100 MHz ) δ 198.78, 160.3, 150.4, 148.0, 147.9, 131.5, 124.0, 120.

1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-{[3-(benzyloxy)-4hydroxyphenyl]methyl}piperidin-4-yl)propan-1-one (9f)

Following procedure A with 7 (200 mg, 0.05 mmol, 1 eq.) and 2-(benzyloxy)-4-(bromomethyl)-1-methoxybenzene (185 mg, 0.60 mmol, 1.2 eq.), the reaction was stirred at rt for 16h. After a purification by chromatography on silica gel column (DCM/MeOH, gradient 100:0 to 80:20), the compound (9f) was obtained as a light yellow oil (100 mg, 38% isolated yield). 1 H NMR (CDCl 3 , 400 MHz) δ 7.78 (s, 1H), 7.44 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.28 (d, J = 7.2 Hz, 1H), 6.94 (s, 1H), 6.83 (s, 2H), 6.25 (s, 1H), 5.17 (s, 2H), 4.46 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.45 (s, 2H), 2.87 (m, 4H), 1.96 -1.84 (m, 2H), 1.65 (d, J = 10.2 Hz, 2H), 1.62 -1.56 (m, 2H), 1.28 (s, 3H). 13 C NMR (CDCl 3 , 100 MHz) δ 199.0, 159.5, 149.0, 147.9, 147.

1-(4-amino-5-chloro-2-methoxyphenyl)-3-{1-[(3-hydroxy-4methoxyphenyl)methyl]piperidin-4-yl}propan-1-one (9g)

From general procedure B with 9f (80 mg, 0.16 mmol, 1 eq.) to afford acetate salt of 9g as a pale yellow powder with a yield of 70%. 1 H NMR (CDCl 3 , 400 MHz) δ 7.76 (s, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.86 -6.76 (m, 2H), 6.26 (s, 1H), 4.54 (br s, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 3.74 (s, 2H), 3.16 (d, J = 11.5 Hz, 2H), 2.87 (t, J = 7.4 Hz, 2H), 2.27 (t, J = 11.5 Hz, 2H), 2.04 (s, 3H), 1.74 -1.72 (m, 2H), 1.62 -1.60 (m, 2H), 1.54 -1.43 (m, 2H), 1.37 (m, 1H). 13 C NMR (CDCl 3 , 100 MHz) δ 198.5, 176.9, 159.6, 147.9, 147.1, 145.9, 132.

N-(2--{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-yl}ethyl)-5-[(3R)-

1,2-dithiolan-3-yl]pentanamide (10a)

To a stirred solution of 8 (125 mg, 0.28 mmol, 1.1 eq.) in DCM (20 mL/mmol) was added TFA (2 mL/mmol). The resulting mixture was stirred at room temperature for 1h. Removal of the solvent under vacuum afforded the crude product, which was directly engaged in the next step. To a solution of LA (53 mg, 0.26 mmol, 1.0 eq.) in DCM (5 mL) were successively added hydroxybenzotriazole (HOBt) (42 mg, 0.31 mmol, 1.2 eq.) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI) (49 mg, 0.31 mmol, 1.2 eq.), the mixture was stirred at 0˚C in the dark for 30min, then the free amine was added, and the reaction was stirred at rt for 16h. Solvent was evaporated under vaccum then the product was purified on silica gel column with DCM/MeOH/NH 3 (97.5:2.5:0.25), 10a was obtained as white powder with a yield of 44%. 1 H NMR (CDCl 3 , 400 MHz) δ 7.81 (s, 1H), 6.28 (s, 1H), 6.11 (br s, 1H), 4.49 (s, 2H), 3.87 (s, 3H), 3.59 (dq, J = 8.5, 6.5 Hz, 1H), 3.35 (q, J = 5.7 Hz, 2H), 3.24 -3.07 (m, 2H), 2.97 -2.82 (m, 4H), 2.47 (m, 3H), 2.24 -2.17 (m, 2H), 2.01 -1.90 (m, 3H), 1.76 -1.65 (m, 6H), 1.62 (dt, J = 8.5, 6.5 Hz, 2H), 1.57 -1.42 (m, 2H), 1.33 -1.17 (m, 3H). 13 8) (125 mg, 0.28 mmol, 1.1 eq.) and ferulic acid (39 mg, 0.20 mmol , 1.eq.) with EDCI, HOBt, and Et3N. The crude was purified on silica gel column with DCM/MeOH/NH 3 (92.5:7.5:0.75) to afford a bright yellow powder with a yield of 42%. 1

In vitro evaluation

DPPH radical scavenging activity

The antiradical activity of the tested compounds was assessed using a miniaturized DPPH (2,2diphenyl-1-picrylhydrazyl radical) assay. [START_REF] Apak | Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report )[END_REF][START_REF] Schaich | Hurdles and pitfalls in measuring antioxidant efficacy: A critical evaluation of ABTS, DPPH, and ORAC assays[END_REF] The tested compounds were diluted from stock solution in absolute ethanol and incubated in the presence of 100 µM DPPH (Sigma-Aldrich, Saint-Quentin Fallavier, France) solution at room temperature in a 96-wells microplate (Sterilin®, Fisher Scientific SAS, Illkirsh, France) in the dark. The absorbance at 517 nm was then measured every 10 minutes min using Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) and used to calculate the specific absorbance of DPPH in each solution subtracting absorbance of all other components in the solution as following:

ORAC assay

The antioxidant activity of the synthezized compounds was assessed using the Fluorescein Oxygen Radical Absorbance Capacity (ORACFl) assay. [START_REF] Apak | Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report )[END_REF][START_REF] Schaich | Hurdles and pitfalls in measuring antioxidant efficacy: A critical evaluation of ABTS, DPPH, and ORAC assays[END_REF][START_REF] Ou | Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe[END_REF] Trolox® (Sigma-Aldrich, Saint-Quentin Fallavier, France) and other compounds stock solutions were prepared at 10 mM in DMSO and diluted in 10 mM phosphate buffer to obtain working solution at 6 concentration levels. 25 µL of buffer or Trolox® or compounds solution were mixed in a black microplate with 150 µL of fluorescein (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 8 nM and incubated 30 min at 37°C in a multimodal microplate reader (Synergy 2, Biotek, Winooski, VT, USA). Then, 25 µL of AAPH (2,2′-Azobis(2-methylpropionamidine) dihydrochloride, Sigma-Aldrich) solution at 75 mM were added and mixed in each well before the kinetic. Measurements of fluorescence (λex=485/20 nm, λem=520/20 nm) were performed every minute during 2 hours.

Each point was measured in triplicate. The AUC and the Net AUC of the standards and samples were determined using Gen5 Data Analysis Software using equations ( 1) and ( 2) respectively.

-

- ( (1)
) 2
Where F1 is the fluorescence reading at the initiation of the reaction and Fn is the last measurement. The standard curve was obtained by plotting the Net AUC of different compound concentrations against their concentration using GraphPad® Prism statistical software (version 6.01, GraphPad Software, La Jolla, CA, USA). ORAC values of samples were then calculated dividing the slope of standard curve of each compounds by value obtained for Trolox®. ORAC value are given as mean ± standard deviation of three independent experiments.

Affinity as Regards 5-HT 4 Receptors

For competition studies, 2. For some of these compounds, affinity constants were calculated from five-point inhibition curves using the GraphPad Prism 6 software and expressed as Ki ± SD.

In cellulo experiment

Cell lines

SH-SY5Y cells (ATCC, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM, Euroclone, Milan, Italy) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (supplements were purchased from Sigma-Aldrich, St. Louis, MO USA). Cells were grown in a humidified incubator with 95% air and 5% CO2 at 37°C.

Determination of cAMP production

COS-7 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% dialyzed fetal calf serum (dFCS) and antibiotics. Cells were transiently transfected by electroporation with plasmid encoding HA-tagged 5-HT 4 R (100 ng/10 6 cells), then seeded in 96well plates (35,000 cells/well). 24 hours after transfection, cells were exposed to the indicated concentrations of 5-HT 4 R ligands in the presence of 0.1 mM of the phosphodiesterase inhibitor RO201724, at 37°C in 100 µL of HBS (20 mM HEPES; 150 mM NaCl; 4.2 mM KCl; 0.9 mM CaCl 2 ; 0.5 mM MgCl 2 ; 0.1% glucose; 0.1% BSA). After 10 min, cells were then lysed by addition of the same volume of Triton-X100 (0.1%). Quantification of cAMP production was performed by HTRF ® by using the cAMP Dynamic kit (Cisbio Bioassays) according to the manufacturer's instructions.

MTT ASSAY

The effect of 9b, 9g, 10a, 10b on the growth of SH-SY5Y cell line was assessed by MTT-based cell viability test, previously described by Mosmann. [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF] Briefly, cells were seeded in 96-well plates at a density of 5x10 3 cells/well and incubated overnight at 37°C, 5% CO 2 in humidified atmosphere to allow adhesion. After this time, cells were treated for 24 hours with the compounds or vehicle (DMSO) at different concentrations, ranging from 0.5 µM to 50µM, dissolved in complete DMEM.

Then, cells were carefully washed with PBS and treated with 300 μM MTT dissolved in DMEM and incubated for 2 hours at 37°C and 5% CO 2 . Then, the aqueous solution was removed and the intracellular formazan salts were dissolved in DMSO for 15 minutes at room temperature.

Finally, the formazan absorbance from each well was measured at λ= 570 nm, using a microplate reader (Victor2 1420 multilabel counter, Perkin Elmer).

ROS assay

SH-SY5Y cells were seeded in 96-well plates (OptiPlate black, Perkin Elmer) at 5x103 cells/well and incubated overnight at 37°C and 5 % CO 2 to allow adhesion in complete culture medium.

After this time, cells were treated for 24 hours with 10 μM 9f, 9g, 10a, 10b, 100μM Trolox or vehicle (DMSO). After this time, cells were washed with PBS and stained for 30 minutes with 10 μM H2DCFDA probe (2',7'-dichlorodihydrofluorescein diacetate, Thermo Fisher) for ROS detection. Then, cells were carefully washed and treated for 30 minutes with 100 μM TBH (tert-Butyl hydroperoxide solution, Luperox, Sigma-Aldrich) to induce oxidative stress. Afetr this time, cells were washed with PBS and stained for 30 minutes with 10 μM H 2 DCFDA probe (2',7'-dichlorodihydrofluorescein diacetate, Thermo Fisher) for ROS detection.

Finally, cells were washed three times with PBS supplemented with Glucose 5 mM and CaCl 2 1 mM. The DCF fluorescence was measured (λ exc = 485 nm; λ em = 535 nm) with a plate reader (Enspire, PerkinElmer). Data were normalized on protein content determined by Lowry method.

ASSOCIATED CONTENT

Synthesis and spectroscopic characterization of intermediates and analytical analysis of final compounds.

Table S1. Inhibition of hAChE of final derivatives

In vitro tests of AChE biological activity. Inhibitory capacity of compounds on AChE biological activity was evaluated through the use of the spectrometric method of Ellman. [1] Acetyl-iodide and 5,5dithiobis-(2-nitrobenzoic) acid (DTNB) were purchased from Sigma Aldrich. AChE from human erythrocytes (buffered aqueous solution, ≥500 units/mg protein (BCA), Sigma Aldrich) was diluted in 20 mM HEPES buffer pH 8, 0.1% Triton X-100 such as to have enzyme solution with 0.25 unit/mL enzyme activity. In the procedure, 100 µL of 0.3 mM DTNB dissolved in phosphate buffer pH 7.4 were added into the 96 wells plate followed by 50 µL of test compound solution and 50 µL of enzyme (0.05

Figure 1 .

 1 Figure 1. Chemical structure of RS67333 A and targeted structure of the MTDL B.

Scheme 1 . 10 a

 110 Scheme 1. Synthesis of the final derivatives 9-10

Figure 2 .

 2 Figure 2. Determination of the 5-HT 4 R pharmacological profiles for compounds 9b, 9g and 10b, by quantification of cAMP accumulation. The agonism activity of the compounds was determinated with serotonin (5-HT) and the partial agonist RS67333 as references. Data presented are the mean of three independent experiments performed in duplicate.

Figure 3 Figure 3 Figure 4

 334 Figure 3 Figure 3 Cell viability determined by MTT assay. Cells were grown into 96-well plates and treated with 9b, 9g, 10a and 10b for 24 hours at the concentration of 0.5, 1, 10 and 50 µM or vehicle (DMSO). Data are presented as percentage of viable cells in comparison with vehicle treated controls. Error bars indicate ± SEM.

  C NMR in chloroform-d, methanol-d 4 or DMSO-d 6 with chemical shifts (δ) given in parts per million (ppm) relative to TMS as internal standard and recorded. The following abbreviations are used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet. Coupling constants Jare reported in hertz units (Hz). Infrared spectra (IR) were obtained on a PERKIN-ELMER FT-IR spectrometer and are reported in terms of frequency of absorption (cm -1 ) using KBr discs. Highresolution mass spectra (HRMS) were obtained by electrospray (ESI, sampling cone 50V, capillary 0.3kV) on a Xevo G2-XS QTof WATERS mass spectrometer. LC-MS (ESI) analyses were realized with Waters Alliance 2695 as separating module using the following gradients: A (95%)/B (5%) to A (5%)/B (95%) in 4.00min. This ratio was hold during 1.50 min before return to initial conditions in 0.50 min. Initial conditions were then maintained for 2.00 min (A = H 2 O, B = CH 3 CN; each containing HCOOH : 0.1 %; column XBridge BEH C18 2.5 µm/3.0 x 50 mm; flow rate 0.8 mL/min). MS were obtained on a SQ detector by positive ESI. Mass spectrum data are reported as m/z.

1 H

 1 NMR (CDCl 3 , 400 MHz) δ 4.11 (m, 2H), 3.10 (d, J = 5.8 Hz, 2H), 2.68 (m, 2H), 1.83 (m, 2H), 1.64-1.61 (m, 1H), 1.46 (s, 9H), 1.19-1.09 (m, 2H); 13 C NMR (CDCl 3 , 100 MHz) δ 154.6, 79.5, 43.4 (2C), 38.6, 32.5 (2C), 28.4 (3C), 13.5; IR (neat, cm -1 )  2975, 2932, 2850, 1692, 1422 , 1243, 1157, 965. Spectral and analytical data matched with literature.[START_REF] Villalobos | Novel benzisoxazole derivatives as potent and selective inhibitors of acetylcholinesterase[END_REF]

(

  2E)-N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropyl]piperidin-1-yl}ethyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enamide (10b) Following the same procedure as 10a, from N-(2-{4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3oxopropyl]piperidin-1-yl}ethyl)carbamate (

  maximal absorbance of DPPH alone, A_sample^DPPH is the specific absorbance of DPPH in a mix with a tested compound at given concentration, A_min^DPPH is the specific absorbance of DPPH in a mix with a Trolox® from Sigma-Aldrich (Saint-Quentin Fallavier, France) at 375 µM considered as the minimal absorbance. Then, the DPPH radical scavenging activity were calculated according to the following formula: Assays were conduct in triplicate at 10 concentration levels (0, 10, 12.5, 15, 25, 50, 75, 100, 125, 150, 250 µM) of the tested compound. Absorbance calculations were carried out using Microsoft Excel and regressions were performed using GraphPad Prismm 6.01 (La Jolla, CA, USA). The kinetic absorbance data were fitted with a one-phase exponential association model allowing us to calculate the time of half-reaction and the plateau of the maximum effect at each concentration. The EC50 values (concentrations required to obtain 50% scavenging effect) were calculated plotting concentration versus % DPPH RSA at 60, 120, plateau and fitting the data with linear model for Trolox®, one or two-phase exponential association model for other compounds. EC 50 and SD values were the mean of two independent experiments. Trolox® was tested as positive control.

  5 µg of proteins (5-HT 4 B membrane preparations, HTS110M, Eurofins. Eurofins's 5-HT 4 B membrane preparations are crude membrane preparations made by use of their proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression.) were incubated in duplicate at 25 °C for 60 min in the absence or the presence of 10-6 or 10-8M of each compound together with 0.2 nM [ 3 H]-GR 113808 (NET 1152, Perkin Elmer) in 25 mM Tris buffer (pH 7.4, 25°C). At the end of the incubation, homogenates were filtered through Whatman GF/C filters (Alpha Biotech) presoaked with 0.5% polyethylenimine using a Brandel cell harvester. Filters were subsequently washed three times with 1 mL of ice-cold 25 mM Tris buffer (pH 7.4, 4 °C). Non-specific binding was evaluated in parallel, in the presence of 30 µM serotonin.

Table 1 .

 1 Binding to h5-HT 4 R and in vitro antioxidant activities of final derivatives

							h5-HT 4 R			DPPH	ORAC
	Cmpd	R 1	R 2	R 3	cLogP*	% inhibition at 10 -8 M	% inhibition at 10 -6 M	Ki (nM)	IC 50 (nM)	EC 50 at (µM) 120 min	(µM compo und / µM Trolox® )
	9a	O-Bn	O-Bn	-	6.78	3%	89%	-	-	>250	< 0.2
	9b	OH	OH	-	3.48	76%	100%	5.9 ± 0.7	7.5± 0.7	19.6 ± 0.8	3.40 ± 0.02
	9c	O-CH 2 -O	-	3.84	22%	100%	-	-	>250	< 0.2
	9d	O-CH 3	O-Bn	-	5.55	0%	97%	-	-	>250	< 0.2
	9e	O-CH 3	OH	-	3.92	7%	95%	-	-	180.3 ± 25.5	3.88 ± 0.55
	9f	O-Bn	O-CH 3	-	5.62	7%	88%	-	-	>250	< 0.2
	9g	OH	O-CH 3	-	3.94	64%	100%	12.7 ± 2.1	14.5 ± 5.8	85.5 ± 2.0	3.41 ± 0.59
	10a	-	-	Lipoic acid	4.98	26%	99%	-	-	>250	0.76 ± 0.11
	10b	-	-	Ferulic acid	3.65	39%	100%	101 ± 8.0	101 ± 3.6	45.5 ± 2.0	1.47 ± 0.25
	Serotonin	-	-	-	-	0%	65%	246± 31	323 ± 25	-	-
	Trolox	-	-	-	-	-	-	-	-	22.9 ± 0.9	1
	Quercetin	-	-	-	-	-	-	-	-	9.3 ± 1.5	6.50 ± 0.73

* calculated logP with ALOGPS 2.1.

  HRMS/ESI: m/z calcd. for C 22 H 34 ClN 3 O 4 [M+H] + 440.2316, found 440.2324;

	1 H NMR (CDCl 3 , 400 MHz) δ 7.71 (s, 1H), 6.20 (s, 1H), 4.98 (br s, 1H, NH), 4.49 (s, 2H, NH 2 ),
	3.77 (s, 3H), 3.14 (t, J = 6.0 Hz, 2H), 2.86 -2.72 (m, 4H), 2.35 (t, J = 6.0 Hz, 2H), 1.92 -1.76
	(m, 2H), 1.67 -1.54 (m, 2H), 1.56 -1.44 (m, 2H), 1.38 (s, 9H), 1.24 -1.08 (m, 3H). 13 C NMR
	(CDCl 3 , 100.0 MHz) δ 199.0, 159.4, 156.0, 147.7, 132.2, 118.9, 111.2, 97.5, 79.1, 57.5, 55.6,
	53.8 (2C), 40.9, 37.4, 35.6, 32.2 (2C), 31.3, 28.5 (3C); MS m/z [M+H] + 441.8; IR (neat, cm -1 ) 
	3486, 3376, 3325, 2980, 2925, 2855, 1690, 1625, 1573, 1467, 1367, 1311, 1175, 1216, 1114, 969,
	610; Mp 119°C;

  5, 116.3, 115.1, 114.0, 110.3, 96.7, 60.5, 55.1, 54.8, 51.9 (2C), 39.9, 33.3, 30.2, 29.1 (2C); MS m/z [M+H] + 432.8; MP: 120.5˚C; IR (neat, cm -1 )  3360, 3220, 2932, 2855, 1620, 1586, 1420, 1254, 1216, 1177, 1136, 1084, 735; HRMS/ESI: m/z calcd. for C 23 H 30 ClN 2 O 4 + [M+H] + 433.1894,

	found 433.1894;

  7, 137.1, 132.1, 129.3, 128.5 (2C), 127.8, 127.5 (2C), 122.3, 118.7, 115.5, 111.4, 111.2, 97.5, 70.9, 62.7, 56.0, 55.7, 53.4 (2C), 40.8, 35.2, 31.7 (2C), 31.1; MS m/z [M+H] + 523.6; IR (neat, cm -1 )  3360, 2923, 2834, 2788, 1618, 1587, 1463, 1450, 1414, 1261, 1216, 1177, 735; HRMS/ESI: m/z calcd. for C 30 H 36 ClN 2 O 4 [M+H] + 523.2364, found 523.2365;

  2, 125.78, 122.2, 118.6, 116.8, 111.3, HRMS/ESI: m/z calcd. for C 23 H 31 ClN 2 O 4 + [M+H] + 433.1894, found 433.1894;

	110.7, 97.5, 61.0, 55.9, 55.7, 52.1 (2C), 40.4, 34.2, 30.5, 30.1 (2C), 22.9; MS m/z [M+H] + 432.8;
	MP: 80.8˚C; IR (neat, cm -1 )  3360, 3220, 2932, 2855, 1620, 1586, 1420, 1254, 1216, 1177,
	1136, 1084, 735;

  C NMR (CDCl 3 , 100 MHz) δ 198.9, 172.7, 159.5, 147.7, 132.2, 118.9, 111.3, 97.6, 56.87, 56.5, 55.7, 53.7 (2C), 40.9, 40.3, 38.5, 36.5, 36.1, 35.6, 34.7, 32.3, 31.2 (2C), 28.9, 25.5;.MS m/z [M+H] + 527.8; MP: 122.5˚C; IR (neat, cm -1 )  3217, 2926, 2844, 1642, 1586, 1462, 1419, 1316, 1254, 1250, 1170; HRMS/ESI: m/z calcd. for C 23 H 39 ClN 3 O 3 S 2 [M+H] + 528.2121, found 528.2118;

  J = 2.0 Hz, 1H),6.80(d, J = 8.2 Hz, 1H), 6.37 (br s, 1H), 6.23 -6.07 (m, 2H), 4.40 (br s, 2H), 3.84 (s, 3H), 3.77 (s, 3H), 3.46 -3.41(m, 2H), 2.91 (d, J = 11.0 Hz, 2H), 2.86 -2.82 (m, 2H), 2.51 (t, J = 5.8 Hz, 2H), 2.45 -2.43 (m, 1H), 1.98 -1.96 (m, 3H), 1.67 (d, J = 9.2 Hz, 2H), 1.57 -1.51 (m, 2H), 1.27 (s, 2H). 13 C NMR (CDCl 3 , 100 MHz) δ 198.8, 166.2, 159.5, 147.7,

	H NMR (CDCl 3 , 400 MHz) δ 7.72 (s, 1H), 7.43 (d, J = 15.5 Hz, 1H), 6.96 (dd, J = 8.2, 2.0 Hz, 1H), 53.8 (2C), 40.7, 36.0, 35.3, 31.8 (2C), 31.0; MS m/z [M+H] + 515.77; MP: 85˚C; IR (neat, cm -1 )  3435, 2938, 2837, 1647, 1616, 1585,1511, 1459, 1419, 1264, 1214, 1156; HRMS/ESI: m/z calcd. 6.90 (d, 147.4, 146.8, 140.7, 132.2, 127.4, 122.3, 118.9, 118.3, 114.8, 111.3, 109.5, 97.5, 57.1, 55.9, 55.7, for C 27 H 35 ClN 3 O 5 [M+H]+ 516.2265, found 516.2264.

1± 2.1 1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[(3,4-dihydroxyphenyl)methyl]-4-piperidyl]propan-1- one (9b)

						hAchE
	Cmpd	R 1	R 2	R 3	cLogP*	% inhibition at	IC50 (nM)
						10 -6 M
	9a	O-Bn	O-Bn	-	6.78	32%	NT
	9b	OH	OH	-	3.48	70%	137.9± 5.2
	9c	O-CH2-O	-	3.84	41%	NT
	9d	O-CH3	O-Bn	-	5.55	48%	NT
	9e	O-CH3	OH	-	3.92	67%	535 ± 49
	9f	O-Bn	O-CH3	-	5.62	8%	NT
	9g	OH	O-CH3	-	3.94	11%	NT
	10a	-	-	Lipoic acid	4.98	15%	NT
	10b	-	-	Ferulic acid	3.65	23%	NT
	Donepezil	-	-	-	-	97%	12.

[1]

Ellman, G. L.; Courtney, K. D.; Andres Jr., V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88-95.

1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-(1,3-benzodioxol-5-ylmethyl)-4-piperidyl]propan-1-one (9c)

La partie de l'image avec l'ID de relation rId28 n'a pas été trouvé dans le fichier.

Acknowledgment

This work was supported by funding from the Fondation Plan Alzheimer (AAP2015 Project TRIAD 016). The authors gratefully acknowledge the Conseil Régional de Normandie, as well as the European Community (FEDER) for their contribution to the CERMN's analytical platform. European COST action CA15135 (Multi-Target Paradigm for Innovative Ligand Identification in the Drug Discovery Process, MuTaLig) supports this article with the funding of a Short Term Scientific Mission attributed to CL. ABBREVIATIONS AD, Alzheimer's disease; DCF, dichlorofluorescein; DPPH, 2,2-diphenyl-1-picrylhydrazyl; HAT, Hydrogen Atom Transfert; LA, Lipoic Acid; MTDL, multi-target directed ligands; ROS, Reactive Oxygen Species; SET, Single-Electron-Transfer; TBH, tert-butyl hydroxyperoxide; TEAC, Trolox Equivalent Antioxidant Capacity.

AUTHOR INFORMATION

Corresponding Author

*E-Mail: christophe.rochais@unicaen.fr; Tel.: +33-2-31-56-68-13. Fax +33-2-31-56-68-03.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Novel multi target-directed ligands targeting 5-HT 4 receptors with in cellulo antioxidant properties as promising leads in Alzheimer's disease Caroline Lanthier, 1 Hugo Payan, [START_REF] Forner | Synaptic impairment in Alzheimer's disease: a dysregulated symphony[END_REF] Irene Liparulo, 3 Bérénice Hatat, 1,[START_REF] Forner | Synaptic impairment in Alzheimer's disease: a dysregulated symphony[END_REF] Cédric Lecoutey, 1 Marc Since, 1 Audrey Davis, 1 Christian Bergamini, 3 Sylvie Claeysen, [START_REF] Forner | Synaptic impairment in Alzheimer's disease: a dysregulated symphony[END_REF] Patrick Dallemagne, 1 Maria-Laura Bolognesi, 3

Contents of Supplementary Information

Inhibition properties of AChE for compound 9a-g and 10a-b p2

Analytical spectrum for compound 9a-g and 10a-b p4

U final). After 5 min of preincubation at 25 °C, the reaction was then initiated by the injection of 50 µL of 10 mM acetylthiocholine iodide solution. The hydrolysis of acetylthiocholine was monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine, at a wavelength of 412 nm using a 96-well microplate plate reader (TECAN Infinite M200, Lyon, France). Test compounds were dissolved in analytical grade DMSO. Donepezil was used as a reference standard. The rate of absorbance increase at 412 nm was followed every minute for 10 min. Assays were performed with a blank containing all components except acetylthiocholine, in order to account for non-enzymatic reaction. The reaction slopes were compared and the percent inhibition due to the presence of test compounds was calculated by the following expression: 100 -(vi/v0x100) where vi is the rate calculated in the presence of inhibitor and v0 is the enzyme activity.

First screening of AChE activity was carried out at a 10 -6 or 10 -5 M concentration of compounds under study. For the compounds with significant inhibition (≥ 50%), IC50 values were determined graphically by plotting the % inhibition versus the logarithm of six inhibitor concentrations in the assay solution using the Origin software.


Jean-Pierre Malval

Marion Cranney

Sylvain Achelle

Huriye Akdas-Kiliç

Jean-Luc Fillaut

Françoise Nolwenn Cabon

Robin-Le Guen

Olivier Soppera

Yann Molard

Françoise Robin-Le Guen

Porosity-driven large amplitude dynamics for nitroaromatic sensing with fluorescent films of alternating D-π-A molecules

come

The detection of nitroaromatic explosives constitutes a strategic concern in the context of homeland security and environment protection. A large panel of analytical techniques going from expensive and sophisticated instrumentations to low-cost portable detectors is currently available for this purpose. Of particular interest, fluorescent vapor-phase sensors have been extensively developed [1][2][3][4][5][6] in concomitance with the emergence of a huge variety of fluorescent sensing materials such as conjugated organic/inorganic polymers 2,7 , dendrimers [8][9][10] , molecular imprinted polymers 11 , supramolecular 3D-architectures 12 . Small fluorescent molecules 1 are also attractive since they constitute relevant building block motifs for self-assembly thin-film sensors. In this domain, one challenging issue relies in controlling at molecular scale the directionality of structural interactions 13,14 to optimize the macromolecular film porosity which drives the sensor time response with the maintain of a reasonable fluorescence emission in solid state which guarantees the sensor sensitivity.

In line with this objective, we present herein the DNT sensing performances of fluorescent thin-films made of alternated triphenylamine-pyrimidine chromophores (Scheme 1) whose V-shaped π-conjugated structure is initially configured to promote cofacial self-packing. The structural introduction of a long flexible alkyl chain into a judicious position of the chromophore backbone hardly affects the electronic and the photophysical properties but drastically impacts the DNT-vapor sensing dynamics. E-Mails: jean-pierre.malval@uha.fr and sylvain.achelle@univ-rennes1.fr ). This band corresponds to a strongly allowed S0→S1 transition implying a long range charge delocalization from the external triphenyl amine moieties (donors) to the pyrimidine core (acceptor) 15,16 . In liquid medium, these chromophores are strongly emissive with fluorescence quantum yields (Φf) higher than 0.6 whatever the solvent polarity (Table S2). The radiative rate constant (kr) remains also invariant indicating that the fluorescence deactivation process occurs via a relaxed S1 state with the same electronic configuration in both cases. In addition, the fluorescence bands undergo a strong red shift (∆νfluo) by about 4300 cm -1 on going from toluene to acetonitrile. Such solvatochromic effects is in line with the enhancement of the S1 state dipole moment with respect to that of S0 one. This high emissivity associated with a strong charge transfer at S1 state clearly indicates the occurrence the radiative deactivation from an intramolecular charge transfer ( 1 ICT) state with a typical planar geometry. In thin films, the dense molecular organization in solid state combined to the inherent heterogeneous environment around the emitting species open up new radiationless routes such aggregation-caused quenching 17,18 which competes with the 1 ICT state radiative deactivation. As a consequence, the fluorescence time decays of the films become non exponential as compared to those measured in liquid solution (see Figure S1). The averaged emission lifetimes in solid state are divided by a factor ~ 2 for both compounds with corresponding fluorescence quantum yields decreasing to values close to 0.2 (Table S1). In parallel, both absorption and fluorescence spectra are redshifted on going from toluene to spin-cast film (see Figure 1). In order to probe the polarity environment around the emitting species in solid state, the maximum fluorescence wavenumbers (νf MAX ) in thin-films have been reported on the solvatochromatic plots for each chromophore as shown in Figure S2. These solvatochromic plots are obtained by correlating νfluo MAX of the dyes diluted in various solvents with the corresponding Lippert-Mataga polarity function 19, 20 (∆f). According to this methodology, it can be inferred that the emitting species in thin films experience a surrounding environment with an estimated ∆f of about 0.27 and 0.23 for MON and MOL respectively. Such a significant difference in the polarity function clearly denotes a more polar environment for MON molecules as compared to MOL ones which should be ascribed to a more compact molecular organization for MON film with a subsequent enhancement of dipolar interactions. Indirectly this structural effect should preclude a change in film porosity on going from MON to MOL. It is clear that the long alkyl side chain of MOL contributes to enlarge the intermolecular spacing and thereby promotes a larger free volume within the thin-film. In order to illustrate such a structural effect, the self-assembly properties of the chromophores have been investigated by scanning tunneling microscopy (STM) in 1-phenyloctane on highly oriented pyrolytic graphite (HOPG) surfaces. Figure 2 depicts STM topographic images of self-assembled layers of MON and MOL at the solid-liquid interface. assume that the intermolecular interactions governing the self-assembly of MOL molecules result from a combination of π-π stacking interactions between superimposed triphenylamine groups as well as Van der Waals interactions between the partially interdigitated alkyl chains. These latter intermolecular repulsive interactions will strongly influence the macromolecular porosity in thinfilm configuration with subsequent effects on the time detection of DNT vapors as will be developed hereafter. In liquid medium, the presence of DNT leads to a dynamic quenching of the chromophores emission. Both fluorescence intensities and emission lifetimes decrease upon addition of DNT leading to Stern-Volmer constants of ca. 48 and 44 M -1 for MON and MOL respectively in acetonitrile (see Figure S3).

This quenching process typically proceeds through a photoinduced electron transfer (PeT) from the singlet excited state of the chromophore (donor) to DNT (acceptor). Consecutively, a back electron (BeT) should regenerate the chromophore to ground state. However, it is possible to observe an effective photo oxidation of the dyes in presence of a large excess of DNT. Figure S4A shows the evolution of the absorption spectrum of an irradiated ACN solution of MOL in presence of 100 eq. of DNT. While the absorption spectrum of MOL without DNT is relatively invariant upon irradiation (Figure S4B), the presence of DNT clearly triggers important spectral changes. The main absorption band of MOL collapses in concomitance with the growth of a new band in 500-650 nm range. This band corresponds to the pyrimidine protonated form of MOL since it can be neutralized by addition of a potassium hydroxide ACN solution. This acid form corresponds in fact to a by-product consecutive to the production of protons due to the dimerization of MOL radical cations (MOL •+ ). Indeed, it is well established that the one-electron oxidation of partially substituted triphenylamine derivatives generates unstable radical cations which undergo dimerization in the para position to the nitrogen centers [21][22][23] . In order to confirm the occurrence of these sequencing reactions, cyclic voltammetry coupled with spectroelectrochemical measurements have been performed. First, it should be noted that both MOL and MON present similar oxidation potentials whose values are gathered in Table S3. The cyclic voltammogram of MOL which is displayed in Figure 3B shows a first oxidation wave at 0.88 V vs. SCE. This irreversible wave which is only observed during the first forward scan corresponds to the oxidation of MOL to MOL •+ . The second oxidation wave at higher potential (Eox 2 ~ 1.09 V vs. SCE) should be ascribed to the oxidation of the follow-up product (i.e. dimer). Figure 3A depicts the spectroelectrochemical spectrum of MOL in ACN and its time dependent absorption changes upon applying a constant potential corresponding to the first oxidation reaction. Interestingly, the spectral changes are comparable to those previously observed for the photo oxidation of MOL in presence of DNT with the difference that an additional band is growing in the 600-900 nm range. This band should presumably correspond to the dimer product which is generated in larger yield as compared to that of the photo oxidation reaction with DNT. In Figure 3C, the time-dependent absorption changes of MOL at 420 nm have been correlated to those of the products absorbing at 520 nm and 700 nm respectively. Whereas a first-order kinetics reaction is derived for the production reaction of the acid form at 520 nm, a secondorder kinetics is clearly observed for the long-wavelength absorbing product which is consistent with a dimer production. Hence in liquid medium, the presence of DNT promotes a dynamic fluorescence quenching and, in a less extent, a photoinduced oxidation of the chromophores. In solid-state, the response of the molecule films exposed to DNT vapors mainly corresponds to an oxidative fluorescence quenching. To illustrate this effect Figure S5 displays several epifluorescence images of MOL thin-film before and after exposure to DNT vapors. As a reference experiment, the image (a) shows the fluorescence of a film which was excited at 365 nm during 5 min under N2 stream. No photobleaching was observed demonstrating the film photostability. However, when the same film is irradiated with introduction of DNT vapors, a strong decrease of the film epifluorescence is observed after 15 s excitation as shown in image (b). The corresponding absorption band of MOL (see Figure S5C) clearly collapses which confirms the photo oxidation process. In this latter case, it should be emphasized that we did not detect the absorption bands of the by-products presumably because their subsequent generation which is diffusion-controlled is strongly hampered in solid state. Hence, it is clear that the analyte detection for both chromophores occurs through a DNT- Whereas the fluorescence signal of MOL film decreases rapidly with a short quenching half-time of 13 s (see also Figure S6), the time-dependence for the fluorescence quenching of MON film is much more slower with a halftime (t1/2 ~ 430 s) more than one order of magnitude longer than that measured for MOL. In addition, the quenching efficiency after 30s DNT exposure is about 75 % for MOL whereas it hardly reaches a value of 10 % for MON film. Such a striking difference in the sensing time response should be confidently ascribed to the distinctive porosities within each film. Moreover, increasing the thickness of MOL film does not drastically affect its DNT sensing dynamics as compared to that observed for MON (see Figure S7). We clearly evidence the porogen role of the long alkyl side chain of MOL which appears as an efficient intermolecular spacer in solid state. This alkyl chain is thereby responsible for a lower density film which facilitates DNT diffusion as well as the analyte mass uptake. Reciprocally, it is worth stressing that the very slow sensing dynamics observed for MON film in presence of DNT vapors illustrates the fact that this π-conjugated material is perfectly configured to promote very dense sandwich-type supramolecular assemblies. Here, we show that a relatively simple structural change at the molecular scale which leaves invariant the electronic properties of the πconjugated chromophore has a drastically impact on the sensing time response performances in thin solid film. In addition, similar sensing effects can be observed for other nitroaromatic analytes such 2,6 dinitrotoluene (2,6-DNT) as shown in Figure S8. Similar time-sensing responses as those measured for DNT can be derived which nicely generalizes our approach. In summary, we have developed a strategy to drastically accelerate the sensing dynamics for the detection of nitroaromatics on the basis of light-emitting molecule films. The key feature consists in incorporating a long flexible alkyl chain in the backbone of a D-π-A alternated chromophore which is intrinsically configured to promote cofacial sandwich-type self-packing. The resulting antagonistic intermolecular interactions lead to a significant change of the film porosity with a subsequent faster DNT sensing whose detection dynamics is enhanced by more than one order of magnitude.

Notes and references

Scheme 1 .

 1 Scheme 1. Molecular structures of the chromophores.

Fig. 1

 1 Fig.1 Normalized absorption and fluorescence spectra of compounds in toluene and in spin-cast film (absorption: triangles and fluorescence: circles).

Fig. 2

 2 Fig.2 Topographic STM images at 1-phenyloctane-HOPG interface of molecular self-assembled layers. MON: A. (89.6 nm x 83.3 nm; -1.0 V); B. (4 nm x 17.6 nm, -1.0 V); C. Possible self-assembly model (up: side view, down: front view). MOL: D. (89.6 nm x 83.3 nm; -1.5 V); E. (8.8 nm x 7.6 nm, -1.5 V); F. Possible self-assembly model. Two distinctive self-organizations are observed. In the case of MON, the molecules self-assemble in compact domains composed of parallel rows with an oblique lattice. Using self-correlation measurements, we identify its mean unitcell parameters a =0.42 ± 0.02nm, b = 1.77 ± 0.04nm and α = 88 ± 2°, leading to an area of 0.74 nm² per molecule. These values indicate that MON molecules are in edge-on conformation with their long axis parallel to the substrate, probably slightly tilted from the direction perpendicular to the surface. On the basis of the model shown in Figure 2C, π-π stacking implying the entire aromatic structure of MON constitutes the main interaction that governs the observed self-organization. In the case of MOL, the molecules also self-assemble in domains composed of parallel lines with an oblique lattice. By contrast to MON, MOL molecules are lying flat on the substrate with the pyrimidine cores completely exposed. The parameters of the unit-cell are c = 1.55 ± 0.02nm, d = 2.95 ± 0.02nm and β = 83 ± 2° which leads to an area of 2.27 nm 2 per molecule. It is noteworthy that this area is ~ 3 times larger than that measured for MON. Based on the model shown in Figure 2F, we may

  A c c e p t e d m a n u s c r i p t

Fig. 3 A

 3 Fig.3 A. Time-dependent changes of the absorption spectrum of MOL upon the first oxidation reaction at constant potential of 0.85 V / SCE. B. CVs of MOL oxidation in ACN (0.18 mM MOL, 0.1 M TBAPF6, 100 mV s -1 ). C. Log-log plots of ∆A420 nm vs. ∆A520 nm and ∆A420 nm vs. ∆A700 nm (∆Aλi are in absolute values).

A

  c c e p t e d m a n u s c r i p t triggered oxidative fluorescence sensing. However, depending on the nature of the chromophore, the real-time sensing dynamics are strikingly different.

Figure 4

 4 displays the kinetics for fluorescence quenching of MON and MOL films exposed to low vapor pressure of DNT (~ 60 ppb). Both films have similar thicknesses of about 40 ± 5 Å.

Fig. 4

 4 Fig.4 Time-dependent fluorescence intensity changes of MON and MOL films with similar thicknesses (i.e. 40 ± 5 Å) exposed to a 300 mL min -1 N2 flow with DNT at a vapor pressure of ~ 60 ppb. Halftime for fluorescence quenching is also indicated for each film.


PhD Ioana A Cristea
email: ioanacristea@psychology.ro

MD, PhD Florian Naudet
email: floriannaudet@gmail.com

US Food and Drug Administration approval of esketamine and brexanolone

In March 2019, The US Food and Drug Administration (FDA) approved two new antidepressantsesketamine for treatment-resistant depression (TRD) [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] and brexanolone for postpartum depression (PPD) [START_REF]Briefing Information for the November 2, 2018 Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] . Both had "Breakthrough" designation, an expedited review process for drugs "intended to treat a serious condition and preliminary clinical evidence indicates that the drug may demonstrate substantial improvement over available therapy on a clinically significant endpoint(s)" [START_REF]Breakthrough Therapy[END_REF] . Though new interventions are welcome for these debilitating disorders, we are concerned these approvals share common critical features, and could further lower the bar in the evaluation of treatments for mental disorders.

Both drugs were approved for medically supervised administration only, with a boxed warning due to serious adverse effects (Table ). These include sedation, dissociation and suicidal ideation and behaviors for esketamine [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] and loss of consciousness and syncope for brexanolone [START_REF]Briefing Information for the November 2, 2018 Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] .

Moreover, although the risk of misuse for esketamine is unknown, the abuse potential of the related molecule, ketamine, is well-documented, albeit less common than for other frequently abused hallucinogens like ecstasy or LSD 1 .

Brexanolone's administration through a continuous 60 hours intravenous drip 2 and the requirement of a minimum inpatient stay of 2 1/2 days represent considerable hindrances for mothers, due to the danger of disrupting breastfeeding, caregiving and early attachment. Many healthcare facilities do not have psychiatric mother-baby units, making hospitalization equivalent to mother-infant separation. Patients for whom these conditions are unacceptable might grapple with anxiety and guilt over the loss of a potential cure. Had it not been for the treatment administration, few PPD patients in the pivotal trials would have required inpatient care [START_REF] Meltzer-Brody | Brexanolone injection in postpartum depression: two multicentre, double-blind, randomised, placebo-controlled, phase 3 trials[END_REF] .

Effective treatments are often accompanied by serious adverse effects, which many patients are willing to withstand as trade-off for expected benefits. Still, a balance needs to be A c c e p t e d m a n u s c r i p t struck between benefits and harms. For esketamine and brexanolone, the degree of medicalization and risk of serious adverse effects need to be counterbalanced by strong efficacy results. Yet results from the pivotal trials were modest at best (Table ). For esketamine [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] , one short-term trial demonstrated significant benefits over placebo, while two others found no difference. A dose-response relationship suggested in a phase 2 study could not be confirmed [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] . In a first for the FDA's Division of Psychiatry Products, a maintenance trial showing esketamine's continued antidepressant response counted towards the requirement of two positive trials [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] .

Furthermore, for one trial, the FDA reviewer described data integrity issues 1 , such as an "unusual response curve shift" at posttest, "discrepancies between the locked datasets" and "reported protocol violations" (p.32). No efficacy results were reported for the 24-week follow-up of the short-term trials [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] . Brexanolone showed a large effect compared to placebo at 60 hours postinfusion 2 in a phase 2 trial. Two short-term phase 3 trials showed considerably smaller effects at the same timepoint, and, in one, differences had largely dissipated at 30 days (the longest followup). [START_REF]Briefing Information for the November 2, 2018 Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] Perhaps as a consequence of the drugs' rapid onset of action [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF][START_REF] Meltzer-Brody | Brexanolone injection in postpartum depression: two multicentre, double-blind, randomised, placebo-controlled, phase 3 trials[END_REF] , the timing of outcome assessment drifted to shorter durations, ranging from hours (60 hours in the pivotal brexanolone trials 2 ) to days (28 days in the esketamine trials [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] ), even for follow-up (e.g., 30 days for brexanolone trials) [START_REF]Briefing Information for the November 2, 2018 Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] . Furthermore, we are wary of the notion that multifactorial, protracted or poorly defined conditions like TRD or PPD could be both rapidly and enduringly resolved. Treatment-resistant depression is marred by diagnostic ambiguity regarding the nature and number of failed treatments, as well as by significant heterogeneity [START_REF] Conway | Toward an Evidence-Based, Operational Definition of Treatment-Resistant Depression: When Enough Is Enough[END_REF] . The concept itself misleadingly implies developing sensitivity to a highly effective treatment (e.g., "antibiotic resistance"), though most antidepressants show modest benefits over placebo [START_REF] Cipriani | Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis[END_REF] and thus may not be effective to begin with A c c e p t e d m a n u s c r i p t for many patients. Beyond semantics, TRD is a chronic and disabling form of depression 5 , as is PPD, which can result in negative long-term effects for both woman and child [START_REF] Curry | Interventions to Prevent Perinatal Depression: US Preventive Services Task Force Recommendation Statement[END_REF] . Moreover, PPD was linked to a constellation of risk factors, including history of physical and sexual abuse, lack of social and financial support, or medical complications like gestational diabetes [START_REF] Curry | Interventions to Prevent Perinatal Depression: US Preventive Services Task Force Recommendation Statement[END_REF] .

Therefore, for both disorders, benefits measured on a symptom scale at 60 hours or 28 days can only be a surrogate of long-term functional outcomes. For esketamine, longer-term effects were assessed in a maintenance study [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] , a design long criticized for inflating true treatment effects [START_REF] Kopec | Randomized discontinuation trials: utility and efficiency[END_REF] . In this 500-days study, esketamine was superior to placebo for time to relapse in stable remitters (Table ). Yet most of the drug-placebo separation occurred early (2-4 weeks postrandomization), leading the FDA to question whether "functional unblinding" partially impacted results [START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] .

Both drugs were compared to placebo and not the relevant "standard of care". For new treatments, particularly those with risk of serious harms, head-to-head trials against existent treatments are crucial. Superiority on patient-relevant outcomes -symptoms, but also quality of life and functioning -needs to be shown to justify widespread use. For example, psychological interventions, notably cognitive behavioral therapy and interpersonal therapy, are effective for both treating [START_REF] Stephens | Effectiveness of Psychological Interventions for Postnatal Depression in Primary Care: A Meta-Analysis[END_REF] and preventing PPD [START_REF] Curry | Interventions to Prevent Perinatal Depression: US Preventive Services Task Force Recommendation Statement[END_REF] , with benefits that persist at 6-months follow-up [START_REF] Stephens | Effectiveness of Psychological Interventions for Postnatal Depression in Primary Care: A Meta-Analysis[END_REF] .

Psychotherapies showed moderate effects for chronically depressed patients [START_REF] Van Bronswijk | Effectiveness of psychotherapy for treatment-resistant depression: a meta-analysis and meta-regression[END_REF] , several of whom would have presumably met at least one TRD definition [START_REF] Conway | Toward an Evidence-Based, Operational Definition of Treatment-Resistant Depression: When Enough Is Enough[END_REF] . Though more invasive, physical therapies, such as electroconvulsive therapy, also showed effectiveness for TRD [START_REF] Milev | Canadian Network for Mood and Anxiety Treatments (CANMAT) 2016 Clinical Guidelines for the Management of Adults with Major Depressive Disorder: Section 4. Neurostimulation Treatments[END_REF] .

Since 2012, the FDA approved approximately 50 therapeutics with Breakthrough designation, most commonly for cancer [START_REF] Puthumana | Clinical Trial Evidence Supporting FDA Approval of Drugs Granted Breakthrough Therapy DesignationClinical Trial Evidence Supporting FDA Approval of Drugs Granted Breakthrough Therapy DesignationLetters[END_REF] . The designation is frequently disconnected from the low-quality evidence supporting subsequent approvals [START_REF] Puthumana | Clinical Trial Evidence Supporting FDA Approval of Drugs Granted Breakthrough Therapy DesignationClinical Trial Evidence Supporting FDA Approval of Drugs Granted Breakthrough Therapy DesignationLetters[END_REF] . We fear that the arguably already low A c c e p t e d m a n u s c r i p t bar for antidepressants, where some previously approved drugs had similar numbers of failed and positive trials [START_REF] Turner | Selective Publication of Antidepressant Trials and Its Influence on Apparent Efficacy[END_REF] , will be lowered further. The FDA should hold future antidepressants to higher evidentiary standards before granting approval and the European Medicines Agency should carefully consider whether the existing evidence for brexanolone and esketamine is sufficient to warrant approval. 2) Versus placebo / Mean difference (least square) for scales / Hazard ratio for time-to-event outcomes [START_REF]Breakthrough Therapy[END_REF] As described in the FDA DSaRM; Events; % exposures in drug arm [START_REF] Meltzer-Brody | Brexanolone injection in postpartum depression: two multicentre, double-blind, randomised, placebo-controlled, phase 3 trials[END_REF] Two Phase 2 trials (Study 2003/SYNAPSE and SUI2001) were not included due uncertainty as to whether they were considered pivotal for the FDA assessment. [START_REF] Conway | Toward an Evidence-Based, Operational Definition of Treatment-Resistant Depression: When Enough Is Enough[END_REF] As required by the study's hierarchical testing procedure the 56-mg dose should not have been formally analysed unless the 84-mg dose showed superiority to placebo [START_REF] Cipriani | Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis[END_REF] p-values were reported one-sided in the FDA PDAC for esketamine (i.e., compared to p= 0.025) and were transformed to two-sided (i.e., compared to p= 0.05) for comparability. When two-sided p values were given for the results posted on clinicaltrials.gov, these were used [START_REF] Curry | Interventions to Prevent Perinatal Depression: US Preventive Services Task Force Recommendation Statement[END_REF] p-value was not reported and calculated with the Altman-Bland (2011) method.

A c c e

  p t e d m a n u s c r i p t

Table .

 . Efficacy and safety data from pivotal trials[START_REF] Naudet | Joint Meeting of the Psychopharmacologic Drugs Advisory Committee (PDAC) and the Drug Safety and Risk Management Advisory Committee (DSaRM)[END_REF] Note. MADRS, Montgomery-Asberg Depression Rating Scale; HAM-D, Hamilton Depression Rating Scale 1) Data sources are FDA Briefing documents for Psychopharmacologic Drugs Advisory Committee (PDAC) and Drug Safety and Risk Management Advisory Committee Meeting (DSaRM) for NDA 211243 esketamine (https://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/PsychopharmacologicDrugsAdvisoryCommittee/UCM630970.pdf) and NDA 211371 brexanolone (https://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/PsychopharmacologicDrugsAdvisoryCommittee/UCM624643.pdf). All efficacy results were cross-checked against results posted on clinicaltrials.gov, with the exception of brexanolone study 547-PPD-202A, where no results were posted.

	Brexanolone
	547-PPD-202A		Phase 2	90	HAM-D	60	-12.2	0.008	30	-11.9	0.004 7) Loss of
	Study Esketamine 4) TRD3001 (TRANSFORM-1) 5) TRD3002 (TRANSFORM-2) 547-PPD-202B 547-PPD-202C	NCT (Clinicaltrials.gov) NCT02417064 NCT02418585 NCT02942004 NCT02942017	Trial type Phase 3 Short-term Phase 3 Short term Phase 3 Phase 3	Dose 56 mg 84 mg 56-84 mg μg/kg/h 60 μg/kg/h 90 μg/kg/h μg/kg/h 90	Primary outcome Primary outcome Time frame MADRS 4 weeks MADRS 4 weeks MADRS 4 weeks m hours HAM-D 60 hours hours a n u s c r i p t Longest follow-up Effect size 2) p-value Time frame Effect size 2) p-value -4.1 (-7.7 to -0.5) 0.026 6) 24 weeks Only safety data -3.2 (-6.9 to 0.5) 0.088 6) 24 weeks Only safety data -4 (-7.3 to -0.6) 0.020 6) 24 weeks Only safety data (-20.8 to -3.7) days (-19.9 to -3.9) -5.5 (-8.8 to -2.2) 0.001 30 days -5.6 (-9.5 to -1.8) 0.004 -3.7 (-6.9 to -0.5) 0.025 30 days -3.8 (-7.6 to -0.0) 0.048 4.5 to -0.5) days to 3.1) HAM-D 60 -2.5 (-0.016 30 0.5 (-2 0.67	Adverse events 3) (serious & of special interest) Depression (6; 2.6%) Suicidal ideation or behavior (4; 1.7%) Headache (1; 0.4%) Severe sedation (6; 2.6%) Dissociation (151; 65%) Road traffic accident/death (1; 0.9%) Cerebal hemorrhage (1; 0.9%) Severe sedation (1; 0.9%) consciousness, syncope, pre-syncope (6; 4%) Suicidal ideation and intentional overdose (1; 0.7%)
											Dissociation (80;
	TRD3005 (TRANSFORM-3) TRD3002 (SUSTAIN-1) A c c e p t e d NCT02422186 Phase 3 Short-term 28-84 mg MADRS NCT02493868 Phase 3 Maintenance NR Time to relapse in stable remitters	4 weeks 500 days	-3.6 (-7.2 to 0.07) 0.49 (0.3 to 0.8)	0.058 6) 24 weeks 0.003	Only safety data	70%) Depression (1; 1.4%) Dizziness/Fall/Hip fracture (1; 1.4%) Suicidal ideation or behavior (1; 1.4%) Blood pressure increased (1; 1.4%) Dissociation (57; 79%)

Authors' contributions

IAC and FN conceived, drafted and revised the manuscript.

Declaration of interest

IAC and FN have no conflicts of interest to disclose.

A c c e p t e d m a n u s c r i p t


O A Smolyanskaya
email: smolyanskaya@corp.ifmo.ru

Q Cassar

V Kravtsenyuk

E L Odlyanitskiy

Patrick Mounaix

Jean-Paul Guillet

K I Zaytsev

V Petrov

O V Kravtsenyuk

J.-P Guillet

N V Petrov

Interaction of Terahertz Radiation with Bio-Like Objects: Theoretical and Numerical Modelling, Real Objects and Phantom Experiments

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I. INTRODUCTION

New biomedical devices require test objects to check their performance and periodic calibration to monitor the system efficiency over the time. A phantom is a bio-like object mimicking the intrinsic real-tissue properties that serves as a test object. A variety of phantoms are commercially available. Phantoms are already used in spectroscopy within various spectral range. However, currently, the THz frequency range suffers from a lack of investigations in this direction. Only the first steps have been performed [START_REF] Liakhov | Fabrication and characterization of biotissuemimicking phantoms in the THz frequency range[END_REF]. In the present work, we investigate interaction of submillimeter radiation with the breastmimicking skin-mimicking phantoms. Results for breastmimicking phantom were compared with numerical models based on a double-Debye model of the dielectric permittivity for different content of fat, fibrous and cancerous tissue. The wave front propagation using the angular spectrum representation was used to simulate the theoretical response of breast-mimicking phantoms.

II. MODEL OF WAVEFRONT PROPAGATION

Electromagnetic fields can be represented in various ways. The angular spectrum representation is a numerical technique describing optical fields in a homogeneous media [START_REF] Petrov | Application of terahertz pulse time-domain holography for phase imaging[END_REF]. Optical fields are described as a superposition of plane waves and evanescent waves. A useful approach to describe optical field diffraction is to conduct the Fourier analysis at a given plane so that the different Fourier components of the field distribution are identified as plane waves propagating away from that plane in different directions. Thus taking less computation time for numerical reconstruction. This technique is preferable for the analysis of light diffraction by histological slides [START_REF] Smolyanskaya | Glycerol dehydration of native and diabetic animal tissues studied by THz-TDS and NMR methods[END_REF]. Angular spectrum representation consists of the following stages: (i) the representation of the field through the angular spectrum of 2D waves (Eq.1); (ii) multiplication by the transfer function that contains a complex refractive index of the object (Eq.2); (iii) the back transition from plane waves to the wave field in the calculated z-plane (Eq.3). is the spatial and frequency distribution of the complex index of refraction.

III. MATERIALS AND METHODS

A. Sample preparation

We have used three-component phantoms made of fat, protein and water of different content. Vegetable oil (10-75%), soya (13-75%) and water (0-70%) were homogenized to generate an emulsion. Then, the phantoms were deposited into vacuum packages.

B. THz-Spectrometer

The commercially available TPS 4000 (Teraview Ltd, UK) spectrometer working in reflection mode was used. The spectral range of the system is from 0.06 to 4.50 THz. The entire system was under an air-dried dome to limit the interaction of water molecules with generated pulses. Samples were mounted on a sapphire substrate during measurement. The sapphire cut was chosen not to exhibit a birefringence that would disrupt results of measurements [START_REF] Cassar | Mounaix Pilot study of freshly excised breast tissue response in the 300 -600 GHz range[END_REF]. Each sample was measured 5 times, independently, i.e. the sample was removed and replaced again into system.

C. Numerical modelling

A double-Debye model of the dielectric permittivity was used to compare optical properties of the threecomponent phantoms and some biological tissue (adipose, fibrous and cancerous) taken from our previous paper [START_REF] Fitzgerald | Use of finite difference time domain simulations and Debye theory for modelling the terahertz reflection response of normal and tumour breast tissue[END_REF].

IV. RESULTS AND DISCUSSION

The propagation dynamics of the pulsed THz radiation through a tissue sample was numerically simulated in the temporal and spectral domains. The index of refraction, n re , absorption coefficient, , as well as real, re , and imaginary, im , part of dielectric permittivity was calculated from the experimentally obtained data. We have obtained the time-domain spatial evolution of the THz pulse. Agreement between measured and simulated THz waveforms is confirmed by the comparison was shown. The spectral dependences of the absorption coefficient of the three-component phantoms consisting of fat, protein and water are depicted. It is shown that the spectral window, the absorption coefficient of phantoms containing water exceeds the absorption coefficient of phantoms free of water. This is due to the fact, that water molecules, which strongly absorb THz radiations, are replaced by low absorbing components (fat and protein). Our results were compared with simulated signals of adipose, fibrous and cancerous tissues taken from our previous paper [START_REF] Fitzgerald | Use of finite difference time domain simulations and Debye theory for modelling the terahertz reflection response of normal and tumour breast tissue[END_REF].

V. CONCLUSION

To overcome the lack of knowledge on tissuemimicking phantoms within the submillimeter spectral range, we conducted preliminary studies on skin-and breast tissue-mimicking samples. Simulations performed for skin-mimicking phantoms have match the experimental spectral data. Thus demonstrating advantages of the technique used in this study. The spectral dependences of absorption coefficient extracted from measurements for breast-mimicking phantom are clearly differentiated into two distinct groups. One for phantoms containing water and those being water-free. Such a discrimination is also shown via the double-Debye model of the dielectric permittivity. Thus, finding of a proper composition of tissue phantom is possible by an appropriate matching of model and experimental data, in particular for prediction of impact on THz radiation probing depth of tissue reversible dehydration using hyperosmotic optical clearing agents [START_REF] Smolyanskaya | Glycerol dehydration of native and diabetic animal tissues studied by THz-TDS and NMR methods[END_REF]. Deeper investigations are however still required to fulfill a set of phantoms that could be used to perform tissue measurement within the THz-range. Such a set would be profitable to design new medical THz-devices without human biopsy.

ACKNOWLEDGMENTS

The reported study was funded by RFBR and CNRS -51-16002, and by the Government of the Russian Federation (Grant 08-08).