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# A Novel in vivo Anti-amnesic Agent, Specially Designed to Express Both Acetylcholinesterase (AChE) Inhibitory, Serotonergic Subtype 4 Receptor (5-HT4R) Agonist and Serotonergic Subtype 6 Receptor (5-HT6R) Inverse Agonist Activities, With a Potential Interest Against Alzheimer's Disease

Bérénice Hatat, Samir Yahiaoui, Cédric Lecoutey, Audrey Davis, Thomas

Freret, Michel Boulouard, Sylvie Claeysen, Cédric Lecoutey, Patrick

Dallemagne

► To cite this version:

Bérénice Hatat, Samir Yahiaoui, Cédric Lecoutey, Audrey Davis, Thomas Freret, et al.. A Novel in vivo Anti-amnesic Agent, Specially Designed to Express Both Acetylcholinesterase (AChE) Inhibitory, Serotonergic Subtype 4 Receptor (5-HT4R) Agonist and Serotonergic Subtype 6 Receptor (5-HT6R) Inverse Agonist Activities, With a Potential Interest Against Alzheimer's Disease. Frontiers in Aging Neuroscience, 2019, 11, pp.148. 10.3389/fnagi.2019.00148 . hal-02399864

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# A Novel in vivo Anti-amnesic Agent, Specially Designed to Express Both Acetylcholinesterase (AChE) Inhibitory, Serotonergic Subtype 4 Receptor (5-HT₄R) Agonist and Serotonergic Subtype 6 Receptor (5-HT₆R) Inverse Agonist Activities, With a Potential Interest Against Alzheimer's Disease

OPEN ACCESS

**Edited by:**
Rommy Von Bernhardt,
Pontifical Catholic University of Chile,
Chile

**Reviewed by:**
Romain Lefebvre,
Ghent University, Belgium
Joachim Neumann,
Institut für Pharmakologie und
Toxikologie, Germany

**\*Correspondence:**
Christophe Rochais
christophe.rochais@unicaen.fr
Patrick Dallemagne
patrick.dallemagne@unicaen.fr

**Received:** 11 April 2019
**Accepted:** 05 June 2019
**Published:** 19 June 2019

**Citation:**
Hatat B, Yahiaoui S, Lecoutey C, Davis A, Freret T, Boulouard M, Claeysen S, Rochais C and Dallemagne P (2019) A Novel in vivo Anti-amnesic Agent, Specially Designed to Express Both Acetylcholinesterase (AChE) Inhibitory, Serotonergic Subtype 4 Receptor (5-HT₄R) Agonist and Serotonergic Subtype 6 Receptor (5-HT₆R) Inverse Agonist Activities, With a Potential Interest Against Alzheimer's Disease. *Front. Aging Neurosci.* 11:148. doi: 10.3389/fnagi.2019.00148

Bérénice Hatat<fnref n="1" />,²†, Samir Yahiaoui<fnref n="1" />†, Cédric Lecoutey<fnref n="1" />, Audrey Davis<fnref n="1" />, Thomas Freret³, Michel Boulouard³, Sylvie Claeysen², Christophe Rochais<fnref n="1" />* and Patrick Dallemagne<fnref n="1" />*

This work describes the conception, synthesis, in vitro and in vivo biological evaluation of novel Multi-Target Directed Ligands (MTDL) able to both activate 5-HT₄ receptors, block 5-HT₆ receptors and inhibit acetylcholinesterase activity (AChE), in order to exert a synergistic anti-amnesic effect, potentially useful in the treatment of Alzheimer's disease (AD). Indeed, both activation of 5-HT₄ and blockage of 5-HT₆ receptors led to an enhanced acetylcholine release, suggesting it could lead to efficiently restoring the cholinergic neurotransmission deficit observed in AD. Furthermore, 5-HT₄ receptor agonists are able to promote the non-amyloidogenic cleavage of the amyloid precursor protein (APP) and to favor the production of the neurotrophic protein sAPPα. Finally, we identified a pleiotropic compound, [1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-(3-methylbenzyl)piperidin-4-yl)propan-1-one fumaric acid salt (10)], which displayed in vivo an anti-amnesic effect in a model of scopolamine-induced deficit of working memory at a dose of 0.3 mg/kg.

**Keywords:** Alzheimer's disease, acetylcholinesterase, 5-HT₄ receptors, 5-HT₆ receptors, MTDL

<footnote n="1">¹These authors have contributed equally to this work</footnote>

<footnote n="1">¹Normandie Université, UNICAEN, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), Caen, France, ²IGF, University of Montpellier, CNRS, INSERM, Montpellier, France, ³Normandie Université, UNICAEN, INSERM, U1075, GIP CYCERON, COMETE, Caen, France</footnote>

<footnote>**Abbreviations:** AD, Alzheimer's disease; ACh, acetylcholine; AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; DPZ, donepezil; MTDL, multi-target directed ligands.</footnote>
<figure><img src="image_3.png" /></figure>

**GRAPHICAL ABSTRACT** | Design of a MTDL able *in vitro* to both inactivate 5-HT6R and AChE and to activate 5-HT4R and finally displaying *in vivo* antiamnesiant effect.

## INTRODUCTION

The pathogenesis of Alzheimer's Disease (AD) is complex and related to the abnormality and dysfunction of multi-systems. Thus, to be potentially more effective, a treatment might consider more than a single target such as acetylcholinesterase (AChE) the main focus of marketed AD drugs.

Within this framework, the design of some pleiotropic ligands known as Multi-Target Directed Ligands (MTDL; Cavalli et al., 2008) appears as a promising approach to tackle the complex origin of the disease as demonstrated recently for several G Protein-Coupled Receptors (GPCRs) and enzymes (Dolles and Decker, 2017; Dolles et al., 2018). We recently described compound 1 (donecopride), which is currently a novel preclinical drug candidate exhibiting both an *in vitro* dual-binding site AChE inhibitory activity and a serotonergic subtype 4 receptor (5-HT4R) agonist effect leading to *in vivo* procognitive and anti-amnesic effects in mice (Figure 1; Lecoutey et al., 2014; Rochais et al., 2015). Indeed, 5-HT4R agonists are able to promote the “non-amyloidogenic” cleavage of the amyloid precursor protein (APP) by α-secretase, inducing the decrease in amyloid-β peptide (Aβ) production in primary neurons (Lezoualc'h, 2007; Russo et al., 2009), the release of soluble and neuroprotective sAPPα protein (Cho and Hu, 2007), and the *in vivo* improvement of memory in rodents (Lelong et al., 2003; Nirogi et al., 2018).

5-HT₄R is a GPCR. Interestingly, another serotonin GPCR, the serotonergic subtype 6 receptor (5-HT₆R) appears as a valuable target to treat cognitive impairments in the field

of neurodegenerative disorders, notably AD (Karila et al., 2015). In fact, its blockade confers to 5-HT₆R antagonists procognitive effects (Benhamú et al., 2014). Among these 5-HT₆R antagonists, 2 (idalopirdine) was studied in phase 3 of clinical trials (Wilkinson et al., 2014), and 3 (landipirdine), a dual antagonist of the 5-HT₆ and 5-HT₂A receptors is currently under investigation in the field of Parkinson Disease (Figure 1; Ellis and Fell, 2017).

The procognitive activity of 5-HT₆R antagonists is probably mediated by modulation of neurotransmitters' release. Indeed, 5-HT₄R activation enhances the liberation of acetylcholine (Kilbinger and Wolf, 1992; Consolo et al., 1994), dopamine (Steward et al., 1996; Lucas et al., 2001) and serotonin (Ge and Barnes, 1996), while blockade of 5-HT₆R enhances the liberation of acetylcholine (Shirazi-Southall et al., 2002; Riemer et al., 2003; Hirst et al., 2006; Marcos et al., 2006; Zhang et al., 2007) and glutamate (Dawson et al., 2000, 2001). Consequently, 5-HT₆R antagonists can improve cognition since they limit the activation of the mTOR pathway (de Bruin and Kruse, 2015). The acute administration of 5-HT₄R agonists or 5-HT₆R antagonists leads to procognitive effects. However, recently we demonstrated that chronic 5-HT₄R activation or chronic 5-HT₆R blockade, also improved memory performances in object recognition test in mice (Quiedeville et al., 2015). These ligands are active at lower doses than those needed for an acute effect and furthermore, seem to be devoid of side effects in these conditions. We thus considered that co-modulation of these two receptors could represent a valuable strategy against memory deficits in AD (Claeysen et al., 2015; Lalut et al., 2017).
Therefore, we recently reported the synthesis and the *in vivo* procognitive effect displayed by the dual compound **4** with *in vitro* 5-HT₄R agonist and 5-HT₆R antagonist effects (**Figure 2**; Yahiaoui et al., 2016). Based on the evidence that 5-HT₄R, 5-HT₆R and AChE are all valuable therapeutic targets in AD treatment, the present work aimed at designing, starting from our dual compound **4** and the benzyl analog of donecopride **5a** (Rochais et al., 2015), novel MTDLs associating 5-HT₄R agonist, 5-HT₆R antagonist and AChE inhibitory balanced activities (**Figure 2**).

# EXPERIMENTAL SECTION

## Chemistry

### General Materials and Methods

All chemical reagents and solvents were purchased from commercial sources and used without further purification. Melting points were determined on a STUART SMP50 melting point apparatus. ¹H, ¹³C and ¹⁹F NMR spectra were recorded on a BRUKER AVANCE III 400MHz with chemical shifts expressed in parts per million (in chloroform-*d*, methanol-*d*₄ or DMSO-*d*₆) downfield from TMS as an internal standard

and coupling in Hertz. IR spectra were recorded on a Perkin-Elmer BX FT-IR apparatus using KBr pellets. High resolution mass spectra (HRMS) were obtained by electrospray on a BrukermaXis. The purities of all tested compounds were analyzed by LC-MS, with the purity all being higher than 95%. Analyses were performed using a Waters Alliance 2695 as separating module (column XBridge C18 2.5 µM/4.6 × 50 mM) using the following gradients: A (95%)/B (5%) to A (5%)/B (95%) in 4.00 min. This ratio was held during 1.50 min before return to initial conditions in 0.50 min. Initial conditions were then maintained for 2.00 min (A = H₂O, B = CH₃CN; each containing HCOOH: 0.1%). MS were obtained on a SQ detector by positive ESI.

### Procedures for the Deprotection and N-alkylation of tert-butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxo-propyl]piperidine-1-carboxylate (8)

Procedure A: to a solution of compound **8** in DCM (5 mL) was added TFA (1 mL) dropwise. The reaction mixture was stirred at room temperature for 1 h and then concentrated under reduced pressure. Evaporation of the solvent provided a light-yellow oil, which was thereafter dissolved in 1,4-dioxane (30 mL). To the resulting solution, 20 equivalent of K₂CO₃

<figure><img src="image_4.png" /><figcaption>**FIGURE 1** | Structure of donecopride, idalopirdine and landipirdine.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**FIGURE 2** | Structure of compounds **4** and **5a**.</figcaption></figure>
were added and thereafter 1.2 equivalent of commercially available benzylbromide derivative was added. The reaction mixture was then stirred at reflux until the full consumption of the starting material. The mixture was then concentrated in vacuo, diluted with water and extracted twice with EtOAc. The combined organic phases were washed with brine, dried over MgSO₄, filtrated and concentrated under pressure. The crude product was thereafter purified by silica column flash chromatography using the appropriate eluting system.

Procedure B: to a solution of compound **8** in DCM (5 mL/mmol) was added TFA (1 mL/mmol) dropwise. The reaction mixture was stirred at room temperature for 15 min and then concentrated under reduced pressure. Evaporation of the solvent provided a light-yellow oil, which was thereafter dissolved in DMF (10 mL/mmol). To the resulting solution, 10 equivalent of K₂CO₃ were added and thereafter 1.2 equivalent of commercially available benzylbromide derivative was added. The reaction mixture was then stirred at 110°C until the full consumption of the starting material. The mixture was then concentrated in vacuo, diluted with EtOAc and washed twice with brine. The organic layer was dried over MgSO₄, filtrated and concentrated under pressure. The crude product was thereafter purified by silica column flash chromatography using the appropriate eluting system.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-benzyl)piperidin-4-yl)propan-1-one (5a; Rochais et al., 2015)

Following procedure A. Pale yellow oil (23%); ¹H NMR (400 MHz, CDCl₃) δ 7.71 (s, 1H), 7.19 (m, 5H), 6.18 (s, 1H), 4.39 (s, 2H), 3.76 (s, 3H), 3.45 (s, 2H), 2.81 (m, 4H), 1.90 (br t, *J* = 10.2 Hz, 2H), 1.61 (br d, *J* = 9.2 Hz, 2H), 1.52 (q, *J* = 6.9 Hz, 2H), 1.22 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.2, 159.5, 147.7, 137.7, 132.3, 129.5 (2C), 128.2 (2C), 127.1, 118.9, 111.3, 97.5, 63.3, 55.6, 53.7 (2C), 40.8, 35.4, 31.9 (2C), 31.4; HRMS (m/z) calcd for C₂₂H₂₈ClN₂O₂ [M + H]⁺ 387.183382, found 387.183120.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-chlorobenzyl)piperidin-4-yl)propan-1-one (5b)

Following procedure B. Pale yellow solid (33%); mp 125.4°C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.48 (dd, *J* = 7.7, 1.6 Hz, 1H), 7.32 (dd, *J* = 7.8, 1.4 Hz, 1H), 7.22 (dt, *J* = 7.5, 1.4 Hz, 1H), 7.16 (dt, *J* = 7.7, 1.8 Hz, 1H), 6.25 (s, 1H), 4.47 (s, 2H), 3.83 (s, 3H), 3.59 (s, 2H), 2.92-2.88 (m, 4H), 2.08-2.03 (m, 2H), 1.69-1.67 (m, 2H), 1.62-1.57 (m, 2H), 1.30-1.23 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.6, 147.8, 136.5, 134.3, 132.3, 130.8, 129.4, 128.0, 126.7, 119.1, 111.4, 97.7, 59.7, 55.7, 54.1 (2C), 41.1, 35.7, 32.5 (2C), 31.5; MS (m/z) [M + H]⁺ 421.61-423.63; HRMS (m/z) calcd for C₂₂H₂₇Cl₂N₂O₂ [M + H]⁺ 421.1450, found 421.1462.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-bromobenzyl)piperidin-4-yl)propan-1-one (5c)

Following procedure B. Pale brown solid (43%); mp 118.1°C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.51 (dd, *J* = 7.9,

1.1 Hz, 1H), 7.48 (dd, *J* = 7.3, 1.5 Hz, 1H), 7.26 (dt, *J* = 7.5, 1.1 Hz, 1H), 7.08 (dt, *J* = 7.8, 1.7 Hz, 1H), 6.25 (s, 1H), 4.49 (s, 2H), 3.82 (s, 3H), 3.56 (s, 2H), 2.92-2.88 (m, 4H), 2.09-2.04 (m, 2H), 1.69-1.67 (m, 2H), 1.62-1.57 (m, 2H), 1.28-1.25 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.6, 147.8, 138.2, 132.7, 132.3, 130.8, 128.3, 127.3, 124.7, 119.0, 111.3, 97.6, 62.2, 55.7, 54.1 (2C), 41.1, 35.7, 32.5 (2C), 31.4; MS (m/z) [M + H]⁺ 465.59-467.59-469.59; HRMS (m/z) calcd for C₂₂H₂₇BrClN₂O₂ [M + H]⁺ 465.0944, found 465.0945.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-fluorobenzyl)piperidin-4-yl)propan-1-one (5d)

Following procedure B. Pale yellow solid (56%); mp 115°C; ¹H NMR (399.75 MHz, CDCl₃) δ 7.77 (s, 1H), 7.36 (td, *J* = 7.6, 1.8 Hz, 1H), 7.22 (m, 1H), 7.10 (td, *J* = 7.6, 1.2 Hz, 1H), 7.01 (m, 1H), 6.24 (s, 1H), 4.47 (s, 2H), 3.82 (s, 3H), 3.56 (s, 2H), 2.88 (m, 4H), 2.00 (br t, *J* = 10.7 Hz, 2H), 1.67 (br d, *J* = 9.3 Hz, 2H), 1.58 (m, 2H), 1.27 (m, 3H); ¹³C NMR (100.53 MHz, CDCl₃) δ 199.1, 161.2 (d, *J* = 244.5 Hz), 159.5, 147.7, 132.2, 131.7 (d, *J* = 4.6 Hz), 128.6 (d, *J* = 8.0 Hz), 125.0 (d, *J* = 14.7 Hz), 123.8 (d, *J* = 3.4 Hz), 118.9, 115.2 (d, *J* = 22.5 Hz), 111.2, 97.5, 63.3, 55.6 (2C), 53.6 (2C), 40.9, 35.5, 32.3, 31.3, 29.7; ¹⁹F NMR (376.10 MHz, CDCl₃) δ -117.75 (s); MS (m/z) [M + H]⁺ 405.56-407.55; HRMS (m/z) calcd for C₂₂H₂₇ClFN₂O₂ [M + H]⁺ 405.1745, found 405.1747.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-iodobenzyl)piperidin-4-yl)propan-1-one (5e)

Following procedure B. Pale yellow solid (44%); mp 106°C; ¹H NMR (400 MHz, CDCl₃) δ 7.81 (dd, *J* = 7.8, 1.1 Hz, 1H), 7.78 (s, 1H), 7.44 (d, *J* = 7.4 Hz, 1H), 7.30 (dt, *J* = 7.4, 1.1 Hz, 1H), 6.92 (dt, *J* = 7.6, 1.7 Hz, 1H), 6.25 (s, 1H), 4.48 (s, 2H), 3.83 (s, 3H), 3.50 (s, 2H), 2.92-2.88 (m, 4H), 2.11-2.06 (m, 2H), 1.69-1.67 (m, 2H), 1.62-1.57 (m, 2H), 1.31-1.25 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.2, 159.6, 147.8, 141.0, 139.4, 132.3, 130.4, 128.7, 128.1, 119.0, 111.3, 100.7, 97.7, 67.0, 55.8, 54.0 (2C), 41.1, 35.7, 32.4 (2C), 31.4; MS (m/z) [M + H]⁺ 513.59-515.61; HRMS (m/z) calcd for C₂₂H₂₇ClFN₂O₂ [M + H]⁺ 513.0806, found 513.0824.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-methylbenzyl)piperidin-4-yl)propan-1-one (5f)

Following procedure B. Yellow-pale solid (45%); mp 119°C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.37 (m, 1H), 7.13 (m, 1H), 6.25 (s, 1H), 4.44 (br s, 2H), 3.84 (s, 3H), 3.41 (s, 2H), 2.91-2.83 (m, 4H), 2.35 (s, 3H), 1.95 (br t, *J* = 11.6 Hz, 2H), 1.67-1.53 (m, 4H), 1.28-1.18 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.5, 137.4, 137.1, 132.2, 130.1, 129.7, 126.8, 125.4, 119.0, 111.2, 97.6, 61.1, 55.6, 54.1, 41.0, 35.8, 32.5, 31.4, 19.3; MS (m/z) [M + H]⁺ 401.65-403.64; HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₂ [M + H]⁺ 401.1996, found 401.1994.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-benzyloxybenzyl)piperidin-4-yl)propan-1-one (5g)

Following procedure B. Yellow oil (16%); ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.46-7.44 (m, 2H), 7.40-7.37 (m, 3H),
7.32 (m, 1H), 7.21 (dt, *J* = 7.9, 1.6 Hz, 1H), 6.96–6.91 (m, 2H), 6.24 (s, 1H), 5.08 (s, 2H), 4.47 (s, 2H), 3.82 (s, 3H), 3.62 (s, 2H), 2.95–2.87 (m, 4H), 2.05–2.00 (m, 2H), 1.68–1.66 (m, 2H), 1.61–1.56 (m, 2H), 1.31–1.26 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.6, 157.1, 147.8, 137.5, 132.3, 130.9, 128.6 (2C), 128.0, 127.8, 127.3 (3C), 120.7, 119.1, 112.0, 111.3, 97.7, 70.2, 56.7, 55.7, 54.0 (2C), 41.1, 35.7, 32.5 (2C), 31.5; MS (m/z) [M + H]⁺ 493.65–495.66; HRMS (m/z) calcd for C₂₉H₃₄ClN₂O₃ [M + H]⁺ 493.2258, found 493.2254.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-hydroxybenzyl)piperidin-4-yl)propan-1-one (5h)

Following procedure B. Yellow-brown oil (12%); ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.15 (dt, *J* = 8.0, 1.3 Hz, 1H), 6.95 (d, *J* = 7.3 Hz, 1H), 6.80 (d, *J* = 8.1 Hz, 1H), 6.76 (dt, *J* = 7.4, 1.1 Hz, 1H), 6.25 (s, 1H), 4.45 (s, 2H), 3.84 (s, 3H), 3.68 (s, 2H), 3.00–2.97 (m, 2H), 2.90 (t, *J* = 7.8 Hz, 2H), 2.10–2.07 (m, 2H), 1.76–1.73 (m, 2H), 1.63–1.58 (m, 2H), 1.33–1.25 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 198.9, 159.6, 158.3, 147.8, 132.4, 128.7, 128.6, 122.0, 119.1, 119.0, 116.2, 109.9, 97.7, 61.8, 55.8, 53.4 (2C), 40.8, 35.3, 32.2 (2C), 31.1; MS (m/z) [M + H]⁺ 403.60–405.58; HRMS (m/z) calcd for C₂₂H₂₈ClN₂O₃ [M + H]⁺ 403.1788, found 403.1788.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-bromobenzyl)piperidin-4-yl)propan-1-one (5i)

Following procedure A. Yellow-pale oil (34%); ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.47 (br *t*, *J* = 1.6 Hz, 1H), 7.36 (br *dt*, *J* = 1.6, 7.6 Hz, 1H), 7.23 (br *dt*, *J* = 1.6, 7.6 Hz, 1H), 7.16 (t, *J* = 7.6 Hz, 1H), 6.25 (s, 1H), 4.44 (s, 2H), 3.84 (s, 3H), 3.44 (s, 2H), 2.89 (br *t*, *J* = 7.8 Hz, 2H), 2.84 (br *d*, *J* = 10.8 Hz, 2H), 1.93 (br *t*, *J* = 11.0 Hz, 2H), 1.74–1.64 (m, 2H), 1.59 (br *q*, *J* = 6.9 Hz, 2H), 1.34–1.19 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.2, 159.6, 147.8, 141.3, 132.4, 132.1, 130.1, 129.8, 127.9, 122.5, 119.2, 111.4, 97.7, 63.0, 55.8, 54.0, 41.0, 35.7, 32.4, 31.4; HRMS (m/z) calcd for C₂₂H₂₇BrClN₂O₂ [M + H]⁺ 465.093894, found 465.093548.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-fluorobenzyl)piperidin-4-yl)propan-1-one (5j)

Following procedure B. White solid (48%); mp 95°C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.25 (m, 1H), 7.06 (m, 2H), 6.92 (m, 1H), 6.25 (s, 1H), 4.44 (s, 2H), 3.84 (s, 3H), 3.46 (s, 2H), 2.87 (m, 4H), 1.94 (br *t*, *J* = 11.1 Hz, 2H), 1.66 (m, 2H), 1.58 (m, 2H), 1.27 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.1, 162.9 (d, *J* = 243.7 Hz), 147.7, 141.5 (d, *J* = 6.7 Hz), 132.2, 129.5 (d, *J* = 8.1 Hz), 124.6 (d, *J* = 2.9 Hz), 118.9, 115.8 (d, *J* = 21.1 Hz), 113.7 (d, *J* = 20.8 Hz), 111.3, 97.6, 62.9, 55.6, 53.9, 40.9, 35.5, 32.3, 31.3; ¹⁹F NMR (376.10 MHz, CDCl₃) δ –113.97 (s); MS (m/z) [M + H]⁺ 405.58–407.57; HRMS (m/z) calcd for C₂₂H₂₇ClFN₂O₂ [M + H]⁺ 405.1745, found 405.1745.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-methylbenzyl)piperidin-4-yl)propan-1-one (5k)

Following procedure A. Yellow-pale oil (41%); ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.19 (t, *J* = 7.4 Hz, 1H), 7.13 (br *s*, 1H), 7.09 (br *d*, *J* = 7.6 Hz, 1H), 7.06 (br *d*, *J* = 7.2 Hz, 1H), 6.25 (s, 1H), 4.44 (s, 2H), 3.83 (s, 3H), 3.45 (s, 2H), 2.96–2.80 (m, 4H), 2.34 (s, 3H), 1.93 (br *t*, *J* = 10.4 Hz, 2H), 1.73–1.52 (m, 4H), 1.36–1.17 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.6, 147.8, 138.3, 137.9, 132.4, 130.2, 128.1, 127.8, 126.6, 119.2, 111.4, 97.7, 63.7, 55.8, 54.0, 41.0, 35.7, 32.3, 31.5, 21.5; HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₂ [M + H]⁺ 401.1994, found 401.1992.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-methoxybenzyl)piperidin-4-yl)propan-1-one (5l)

Following procedure A. Colorless oil (27%); ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.22 (t, *J* = 8.0 Hz, 1H), 6.93–6.85 (m, 2H), 6.79 (ddd, *J* = 0.8, 2.4, 8.0 Hz, 1H), 6.25 (s, 1H), 4.44 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.47 (s, 2H), 2.95–2.81 (m, 4H), 1.94 (br *t*, *J* = 10.2 Hz, 2H), 1.75–1.52 (m, 4H), 1.36–1.17 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.7, 159.6, 147.7, 140.2, 132.4, 129.2, 121.8, 119.2, 114.9, 112.5, 111.4, 97.7, 63.5, 55.8, 55.4, 54.0, 41.0, 35.7, 32.4, 31.5; HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₃ [M + H]⁺ 417.193947, found 417.193907.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-aminobenzyl)piperidin-4-yl)propan-1-one (5m)

Following procedure A. Hygroscopic solid (36%); ¹H NMR (400 MHz, CD₃OD) δ 7.66 (s, 1H), 7.17 (t, *J* = 7.8 Hz, 1H), 6.87–6.70 (m, 3H), 6.69 (s, 2H), 6.44 (s, 1H), 4.11 (s, 2H), 3.85 (s, 3H), 3.43 (br *d*, *J* = 12.4 Hz, 2H), 3.05–2.76 (m, 4H), 1.95 (br *d*, *J* = 14.0 Hz, 2H), 1.73–1.28 (m, 5H).

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-nitrobenzyl)piperidin-4-yl)propan-1-one (5n)

Following procedure A. Yellow oil (80%); ¹H NMR (400 MHz, CDCl₃) δ 8.18 (br *t*, *J* = 1.6 Hz, 1H), 8.09 (br *d*, *J* = 8.0 Hz, 1H), 7.78 (s, 1H), 7.66 (br *d*, *J* = 7.6 Hz, 1H), 7.47 (t, *J* = 8.0 Hz, 1H), 6.26 (s, 1H), 4.45 (s, 2H), 3.84 (s, 3H), 3.55 (s, 2H), 2.90 (br *t*, *J* = 7.8 Hz, 2H), 2.83 (br *d*, *J* = 11.6 Hz, 2H), 1.99 (br *t*, *J* = 10.2 Hz, 2H), 1.78–1.64 (m, 2H), 1.60 (br *q*, *J* = 6.8 Hz, 2H), 1.35–1.19 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.2, 159.6, 148.4, 147.8, 141.4, 135.2, 132.4, 129.2, 123.9, 122.2, 119.1, 114.4, 97.7, 62.6, 55.8, 54.1, 41.0, 35.6, 32.4, 31.4; HRMS (m/z) calcd for C₂₂H₂₇ClN₃O₄ [M + H]⁺ 432.168460, found 432.168262.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(4-fluorobenzyl)piperidin-4-yl)propan-1-one (5o)

Following procedure B. Yellow pale solid (32%); mp 128°C; ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.26 (m, 2H), 7.06 (m, 2H), 6.98 (m, 2H), 6.25 (s, 1H), 4.48 (s, 2H), 3.83 (s, 3H), 3.43 (s, 2H), 2.86 (m, 4H), 1.91 (br *t*, *J* = 10.6 Hz, 2H), 1.66 (br *d*, *J* = 8.8 Hz, 2H), 1.58 (m, 2H), 1.25 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.1, 161.9 (d, *J* = 243.0 Hz), 159.5, 147.7, 134.3 (d, *J* = 3.0 Hz), 132.2, 130.7 (d, *J* = 7.9 Hz, 2C), 118.9, 114.9 (d, *J* = 21.1 Hz, 2C), 111.2, 97.5, 62.7, 55.6, 53.8, 40.9, 35.6, 32.3, 31.3; ¹⁹F NMR (376.1 MHz, CDCl₃) δ –113.97 (s); MS (m/z) [M + H]⁺ 405.62–407.62; HRMS (m/z) calcd for C₂₂H₂₇ClFN₂O₂ [M + H]⁺ 405.1745, found 405.1740.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(4-hydroxybenzyl)piperidin-4-yl)propan-1-one (5p)

Following procedure B. Yellow oil (13%); ¹H NMR (400 MHz, CD₃OD) δ 7.64 (s, 1H), 7.14–7.12 (m, 2H), 6.74 (m, 2H), 6.43 (m, 4H), 2.34 (s, 3H), 1.93 (br *t*, *J* = 10.4 Hz, 2H), 1.73–1.52 (m, 4H), 1.36–1.17 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.3, 159.6, 147.8, 138.3, 137.9, 132.4, 130.2, 128.1, 127.8, 126.6, 119.2, 111.4, 97.7, 63.7, 55.8, 54.0, 41.0, 35.7, 32.3, 31.5, 21.5; HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₃ [M + H]⁺ 401.1994, found 401.1992.
(s, 1H), 3.84 (s, 3H), 3.44 (s, 2H), 2.93-2.87 (m, 4H), 2.04-1.99 (m, 2H), 1.72-1.70 (m, 2H), 1.57-1.51 (m, 2H), 1.32-1.22 (m, 4H); ¹³C NMR (100 MHz, CD₃OD) δ 201.0, 161.6, 158.1, 151.7, 132.9, 132.4 (2C), 128.2, 117.9, 116.0 (2C), 111.6, 98.1, 63.7, 56.1, 54.4 (2C), 41.7, 36.6, 32.6 (3C); MS (m/z) [M + H]⁺ 403.65-405.66; HRMS (m/z) calcd for C₂₂H₂₈ClN₂O₃ [M + H]⁺ 403.1788, found 403.1786.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(4-methylbenzyl)piperidin-4-yl)propan-1-one (5q)

Following procedure B. White solid (20%); mp 97.0°C; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H), 7.20 (d, *J* = 7.9 Hz, 2H), 7.11 (d, *J* = 7.9 Hz, 2H), 6.24 (s, 1H), 4.49 (*br* s, 2H), 3.81 (s, 3H), 3.47 (s, 2H), 2.90-2.86 (m, 4H), 2.33 (s, 3H), 1.96-1.91 (m, 2H), 1.68-1.65 (m, 2H), 1.60-1.55 (m, 2H), 1.33-1.23 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.2, 159.6, 147.8, 136.7, 135.0, 132.3, 129.5 (2C), 128.9 (2C), 118.9, 111.3, 97.6, 63.2, 55.7, 53.8, 53.5, 41.0, 35.6, 32.2 (2C), 31.4, 21.2; MS (m/z) [M + H]⁺ 401.66-403.65; HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₂ [M + H]⁺ 401.1996, found 401.1996.

### N-(3-((4-(3-(4-Amino-5-chloro-2-methoxyphenyl)-3-oxopropyl)piperidin-1-yl)methyl)-4-bromophenyl) acetamide (5r)

Following procedure A. Yellow-pale oil (70%); ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H), 7.96 (*br* s, 1H), 7.52 (dd, *J* = 2.4, 8.8 Hz, 1H), 7.47-7.39 (m, 2H), 6.25 (s, 1H), 4.50 (s, 2H), 3.82 (s, 3H), 3.50 (s, 2H), 3.01-2.77 (m, 4H), 2.16 (s, 3H), 2.05 (*br* *t*, *J* = 10.8 Hz, 2H), 1.72-1.61 (m, 2H), 1.57 (*br* *q*, *J* = 6.8 Hz, 2H), 1.35-1.18 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 199.4, 168.7, 159.7, 147.9, 138.8, 137.5, 133.0, 132.3, 121.6, 120.0, 118.9, 118.8, 111.3, 97.7, 62.0, 55.8, 54.1, 41.1, 35.7, 32.5, 31.4, 24.7; HRMS (m/z) calcd for C₂₄H₃₀BrClN₃O₃ [M + H]⁺ 522.115358, found 522.114977.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(2-fluoro-5-nitrobenzyl)piperidin-4-yl)propan-1-one (5s)

Following procedure B. Yellow solid (35%); mp 133.4°C; ¹H NMR (400 MHz, CDCl₃) δ 8.36 (dd, *J* = 6.1 Hz, *J* = 2.8 Hz, 1H), 8.13 (ddd, *J* = 8.9, 4.3 Hz, *J* = 3.0 Hz, 1H), 7.78 (s, 1H), 7.15 (*t*ₐpp, *J* = 8.8 Hz, 1H), 6.25 (s, 1H), 4.45 (s, 2H), 3.84 (s, 3H), 3.59 (s, 2H), 2.91-2.84 (m, 4H), 2.08-2.02 (m, 2H), 1.70-1.68 (m, 2H), 1.62-1.57 (m, 2H), 1.29-1.25 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 199.1, 164.8 (d, *J* = 256.4 Hz), 159.6, 147.8, 144.4 (d, *J* = 1.9 Hz), 132.4, 127.8 (d, *J* = 16.2 Hz), 127.2 (d, *J* = 6.9 Hz), 124.6 (d, *J* = 10.2 Hz), 119.1, 116.3 (d, *J* = 25.1 Hz), 111.4, 97.7, 55.8, 55.1, 53.9 (2C), 40.9, 35.4, 32.3 (2C), 31.3; MS (m/z) [M + H]⁺ 450.51-452.50; HRMS (m/z) calcd for C₂₂H₂₆ClFN₃O₄ [M + H]⁺ 450.1596, found 450.1594.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(5-amino-2-fluorobenzyl)piperidin-4-yl)propan-1-one (5t)

Following procedure B. Yellow-brown oil (11%); ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 1H), 6.83 (dd, *J* = 9.5 Hz, *J* = 8.8 Hz, 1H), 6.72 (dd, *J* = 6.2, 2.8 Hz, 1H), 6.65 (ddd, *J* = 8.7, 4.1, 2.8 Hz, 1H), 6.44 (s, 1H), 3.85 (s, 3H), 3.55 (d, *J* = 1.2 Hz, 2H), 2.98-2.95 (m, 2H), 2.91-2.87 (m, 2H), 2.16-2.11 (m, 2H), 1.73-1.71 (m, 2H), 1.59-1.52 (m, 2H),

1.33-1.26 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 200.1, 161.7, 156.3 (d, *J* = 234.0 Hz), 151.7, 144.8, 132.9, 124.1 (d, *J* = 16.4 Hz), 119.7 (d, *J* = 3.3 Hz), 117.9, 117.3 (d, *J* = 7.7 Hz), 116.4 (d, *J* = 23.8 Hz), 111.6, 98.1, 56.3 (d, *J* = 1.3 Hz), 56.1, 54.4 (2C), 41.6, 36.4, 32.6 (3C); MS (m/z) [M + H]⁺ 420.60-422.60; HRMS (m/z) calcd for C₂₂H₂₈ClFN₃O₂ [M + H]⁺ 420.1854, found 420.1857.

### 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-(3-methylbenzyl)piperidin-4-yl)propan-1-one fumaric acid salt (10)

To a solution of 85 mg of compound **5k** (0.212 mmol) in 3 mL of iPrOH was added 25 mg of fumaric acid (0.212 mmol, 1 equivalent). The solution was refluxed for 2 h. The mixture was then concentrated *in vacuo*. The residue is triturated in Et₂O and then filtrated. Sixty milligram of desired compound were obtained as a beige solid (55%). Yellow solid; mp 185.4°C; ¹H NMR (400 MHz, CD₃OD) δ 7.56 (s, 1H), 7.28-7.16 (m, 4H), 6.60 (s, 2H), 6.35 (s, 1H), 4.11 (s, 2H), 3.75 (s, 3H), 3.32 (*br* *d*, *J* = 12.4 Hz, 2H), 2.88-2.81 (m, 4H), 2.28 (s, 3H), 1.86 (*br* *d*, *J* = 13.9 Hz, 2H), 1.55-1.52 (m, 3H), 1.38-1.32 (m, 2H); ¹³C NMR (100 MHz, CD₃OD) δ 198.7, 169.8 (2C), 160.3, 150.4, 139.0, 134.8 (2C), 131.5, 130.4, 129.3, 128.8, 127.9, 116.3, 110.3, 96.6, 60.2, 54.7 (2C), 52.1, 48.2, 39.8, 33.1, 30.1, 28.9, 20.0 (2C). HRMS (m/z) calcd for C₂₃H₃₀ClN₂O₂ [M + H]⁺ 401.1994, found 401.1996.

## *In vitro* Biological Studies

### Pharmacological Characterization of Drugs on Human 5-HT₄R

For competition studies, 2.5 µg of proteins (5-HT₄(b) membrane preparations, HTS110M, Eurofins. Eurofins' 5-HT₄(b) membrane preparations are crude membrane preparations made from 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 drug (9 was used as a reference standard) and 0.2 nM [³H]-GR113808 (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.

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.

### Pharmacological Characterization of Drugs on Human 5-HT₆R

Drugs were evaluated through their possibility to compete for the binding of [³H]-LSD on membranes of HEK-293 cells transiently expressing the human 5-HT₆ receptors (ref. RBHS6M, Perkin Elmer). In brief, 4 µg of proteins were incubated at 37°C for 60 min in duplicate in the absence or the presence of 10⁻⁶ or 10⁻⁸M of each drug (2 was used as a reference
standard) and 2.5 nM [³H]-LSD (ref. NET638250UC, Perkin Elmer), in 25 mM Tris-HCl buffer (pH 7.4) supplemented with 0.5 mM EDTA. At the end of the incubation, the homogenates were then filtered through Whatman GF/C filters and washed five times with ice-cold 25 mM Tris-HCl buffer. Non-specific binding was evaluated in the presence of 100 µM serotonin. Radioactivity associated to proteins was then quantified and expressed as the percentage of inhibition of the drugs under study.

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.

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 plasmids encoding HA-tagged 5-HT₄R (100 ng/10⁶ cells) or HA-tagged 5-HT₆R (70 ng/10⁶ cells or 300 ng/10⁶ cells), then seeded in 96-well plates (16,000 cells/well).

5-HT₄R-induced cAMP production: 24 h after transfection, cells were exposed to the indicated concentrations of 5-HT₄R ligands in the presence of 0.1 mM of the phosphodiesterase inhibitor RO-20-1724, at 37°C in 100 µL of HBS (20 mM HEPES; 150 mM NaCl; 4.2 mM KCl; 0.9 mM CaCl₂; 0.5 mM MgCl₂; 0.1% glucose; 0.1% BSA). After 10 min, cells were then lysed by addition of the same volume of Triton-X100 (0.1%).

Blockade of the 5-HT<sub>6</sub>R-induced cAMP production: 24 h after transfection, cells were exposed to the indicated concentrations of 5-HT<sub>6</sub>R ligands at 37°C in 50 µL of HBS. After 7 min, 5-HT (5.10<sup>-7</sup>M final concentration) in the presence of RO-20-1724 (0.1 mM final concentration), in 50 µL HBS, was added to the wells. After 10 min at 37°C, cells were then lysed by addition of 100 µL of Triton-X100 (0.1%).

Reversion of the basal 5-HT<sub>6</sub>R-induced cAMP production: the 5-HT<sub>6</sub>R expression was augmented by transfection of higher quantities of cDNA (300 ng/10<sup>6</sup> cells) to increase the basal cAMP production linked to the constitutive activity of the receptor. Twenty-four hours after transfection, cells were exposed to 10<sup>-4</sup>M of 5-HT<sub>6</sub>R ligands in the presence of RO-20-1724 (0.1 mM final concentration), at 37°C in 100 µL of HBS. This experiment is performed in the absence of 5-HT. After 10 min at 37°C, cells were then lysed by addition of 100 µL 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.

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 et al. (1961). Acetylthiocholine iodide and 5,5-dithiobis-(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 was added into the 96-well plate followed by 50 µL of test compound solution and 50 µL of enzyme (0.05 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 (BioTek, Synergy 2). Test compounds were dissolved in analytical grade DMSO. Donepezil (DPZ) 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/v₀ × 100) 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 10⁻⁶M concentration of compounds under study. For the compounds with significant inhibition (≥50%), IC₅₀ values were determined graphically by plotting the % inhibition vs. the logarithm of six inhibitor concentrations in the assay solution using the GraphPad Prism 6 software.

In vivo Biological Studies

Animals

Adult male NMRI mice (3 months old, weighing 35–40 g) from
Janvier labs (Le Genest-Saint-Isle, France) were used to perform
experiments. Mice were housed by ten in standard polycarbonate
cages in standard controlled conditions (22 ± 2°C, 55 ± 10%
humidity) with a reversed 12 h light/dark cycle (light on at
7 pm). Food and water were available *ad libitum* in the home
cage. All experiments were conducted (between 9 am and 3 pm)
during the active-dark-phase of the cycle, were approved by the
ethics committee of Normandy (CENOMEXA, n°5161) and were
in agreement with the European Directives and French law on
animal experimentation (personal authorization n° 14–17 for
MB and 14–60 for TF).

CNS-Activity and Acute Toxicity Test

Behavioral and neurological changes induced by graded doses (1, 10, 100 mg/kg) of the tested derivatives were evaluated in mice, in groups of four, by a standardized observation technique at different times (30 min, 3 and 24 h) after intraperitoneal administration (Morpugo, 1971). Major changes of behavioral data (for example, hypo- or hyperactivity, ataxia, tremors, convulsion, etc...) were noted in comparison to the control group. The approximate DL₅₀ of the compounds were also calculated through the quantification of mortality after 24 h. Amphetamine (2 mg/kg), chlorpromazine (10 mg/kg) were used as a stimulant and depressive references, respectively.
## Locomotor Activity

Locomotion of mice was measured using an actimeter (Imetronic®) through infrared detection. Eight individual removable polycarbonate cages (21 cm length, 7 cm wide and 12 cm high), where each mouse was placed, are disposed in the actimeter. Locomotor activity was measured by recording the number of interruption of beams of the red light over a period of 30 min through an attached recording system to the actimeter. Compound 10 was tested at 1, 3 and 10 mg/kg. Amphetamine (2 mg/kg), chlorpromazine (3 mg/kg) were used as a stimulant and depressive references, respectively (Freret et al., 2012).

## Spatial Working Memory

Anti-amnesic activity of tested compounds was evaluated by reversal of scopolamine (0.5 mg/kg)-induced deficit on spontaneous alternation behavior in the Y maze test (Hooper et al., 1996). The Y maze made of gray plastic consisted of three equally spaced arms (21 cm long, 7 cm wide with walls 15-cm high). The mouse was placed at the end of one of the arms and allowed to move freely through the maze during a 5 min session while the sequence of arm entries was recorded by an observer. An arm entry was scored when all four feet crossed into the arm. An alternation was defined as entries into all three arms on a consecutive occasion. The number of possible alternation is thus the total number of arm entries minus two; the percentage of alternation was calculated as (actual alternation/ possible alternation) × 100. Compound 10 was tested at 0.3, 1 and 3 mg/kg. DPZ (1 mg/kg) was tested as a clinical reference.

## Pharmacological Treatments

Amphetamine, (+)-α-Methylphenethylamine hemisulfate, chlorpromazine hydrochloride, imipramine hydrochloride and scopolamine hydrobromide were purchased from Sigma (France). All those pharmacological compounds were

dissolved in NaCl 0.9% as the vehicle were administered IP 30 min before tests, except scopolamine which was subcutaneously administered 20 min before spontaneous alternation test.

## Statistical Analysis

Results were expressed as mean ± SD and were analyzed by one-way analysis of variance (ANOVA), with Statview® software. In case of significance, a SNK (Student-Newman-Keuls) post hoc test was realized. Additionally, for the spontaneous alternation test, the percentage of alternation was compared to a theoretical 50% value (random alternation) by an univariate *t*-test. Differences were considered as statistically significant if the *p*-value was strictly under 0.05.

# RESULTS

## Chemistry

The targeted compounds **5a-t** were obtained starting from 4-amino-5-chloro-2-methoxybenzoic acid (6) as reported for the synthesis of **5a** (*Scheme* 1; Rochais et al., 2015). The synthesis of a β-ketoester (7) was achieved in 62% yield using the carbonyldiimidazole (CDI) activation of the carboxylic acid group of 6. A nucleophilic substitution, using N-Boc 4-(iodomethyl) piperidine at room temperature to avoid the risk of a double substitution, allowed the installation of the methylpiperidine moiety. The latter was immediately followed by a saponification-decarboxylation sequence using hydroalcoholic potassium hydroxide. The reaction yielded 8 in a 86% yield. Finally, the TFA-N-deprotection of 8, immediately followed by another nucleophilic substitution with various benzyl bromides yielded compounds **5a-t**. The fumaric acid salt 10 was synthesized starting from **5k** and using fumaric acid in iPrOH.

<figure><img src="image_6.png" /><figcaption>**SCHEME 1** | Synthetic pathways for access to compounds **5a-t**, 10. Conditions and reagents: (a) CDI, THF; (b) $KO_2CCH_2CO_2Et$, $MgCl_2$, THF; (c) N-Boc 4-iodomethylpiperidine, $K_2CO_3$, DMF; (d) KOH, $EtOH/H_2O$; (e) TFA, 1,4-dioxan or DCM; (f) R-benzyl bromide, $K_2CO_3$, 1,4-dioxane or DMF; (g) fumaric acid, iPrOH.</figcaption></figure>
<table><caption>**TABLE 1** | (h)AChE inhibitory activity, (h)5-HT<sub>6</sub>R and (h)5-HT<sub>4</sub>R affinity for DPZ, **2**, **9** and compounds **5a-t** (% inhibition at 10<sup>-6</sup>M, 10<sup>-8</sup>M and 10<sup>-8</sup>M, respectively).</caption><thead><tr><th rowspan="2">Cmpd</th><th rowspan="2">R</th><th>(h)AChE</th><th colspan="2">(h)5-HT<sub>6</sub>R</th><th colspan="2">(h)5-HT<sub>4</sub>R</th></tr><tr><th>%10<sup>-6</sup>M</th><th>%10<sup>-6</sup>M</th><th>%10<sup>-8</sup>M</th><th>%10<sup>-6</sup>M</th><th>%10<sup>-8</sup>M</th></tr></thead><tbody><tr><td>DPZ</td><td>-</td><td>97</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>2</td><td>-</td><td>-</td><td>106</td><td>88</td><td>-</td><td>-</td></tr><tr><td>9</td><td>-</td><td>-</td><td>-</td><td>-</td><td>100</td><td>42</td></tr><tr><td>5a</td><td>H</td><td>98</td><td>29</td><td>0</td><td>102</td><td>41</td></tr><tr><td>5b</td><td>2-Cl</td><td>91</td><td>92</td><td>27</td><td>94</td><td>16</td></tr><tr><td>5c</td><td>2-Br</td><td>80</td><td>65</td><td>34</td><td>96</td><td>17</td></tr><tr><td>5d</td><td>2-F</td><td>96</td><td>68</td><td>21</td><td>94</td><td>10</td></tr><tr><td>5e</td><td>2-I</td><td>37</td><td>81</td><td>25</td><td>96</td><td>1</td></tr><tr><td>5f</td><td>2-Me</td><td>87</td><td>85</td><td>24</td><td>98</td><td>9</td></tr><tr><td>5g</td><td>2-OBn</td><td>33</td><td>18</td><td>0</td><td>70</td><td>7</td></tr><tr><td>5h</td><td>2-OH</td><td>94</td><td>24</td><td>0</td><td>98</td><td>10</td></tr><tr><td>5i</td><td>3-Br</td><td>44</td><td>77</td><td>7</td><td>94</td><td>5</td></tr><tr><td>5j</td><td>3-F</td><td>97</td><td>59</td><td>9</td><td>98</td><td>12</td></tr><tr><td>5k</td><td>3-Me</td><td>89</td><td>80</td><td>9</td><td>98</td><td>25</td></tr><tr><td>5l</td><td>3-OMe</td><td>35</td><td>78</td><td>6</td><td>94</td><td>9</td></tr><tr><td>5m</td><td>3-NH<sub>2</sub></td><td>93</td><td>74</td><td>31</td><td>100</td><td>35</td></tr><tr><td>5n</td><td>3-NO<sub>2</sub></td><td>64</td><td>51</td><td>0</td><td>90</td><td>3</td></tr><tr><td>5o</td><td>4-F</td><td>92</td><td>58</td><td>13</td><td>102</td><td>19</td></tr><tr><td>5p</td><td>4-OH</td><td>98</td><td>24</td><td>0</td><td>100</td><td>23</td></tr><tr><td>5q</td><td>4-Me</td><td>87</td><td>21</td><td>21</td><td>100</td><td>24</td></tr><tr><td>5r</td><td>2-Br;5-NHCOMe</td><td>13</td><td>35</td><td>7</td><td>96</td><td>6</td></tr><tr><td>5s</td><td>2-F;5-NO<sub>2</sub></td><td>63</td><td>18</td><td>5</td><td>62</td><td>4</td></tr><tr><td>5t</td><td>2-F;5-NH<sub>2</sub></td><td>79</td><td>65</td><td>3</td><td>98</td><td>18</td></tr></tbody></table>

## In vitro Results

The biological evaluation of the synthesized compounds as potential inhibitors of human AChE was performed using the Ellman assay (Ellman et al., 1961), as well as their affinity for human 5-HT<sub>6</sub>R and 5-HT<sub>4</sub>R using a radioligand displacement assay (Table 1). In these tests, DPZ was used as a reference AChE inhibitor (AChEI), 2 as a 5-HT<sub>6</sub>R ligand and 9 (RS67333; Eglen et al., 1995) as a 5-HT<sub>4</sub>R ligand.

The results of these *in vitro* evaluations are reported in a 3-fold-entry figure (Figure 3).

Among the 20 tested compounds, 12 novel derivatives appear as moderate to potent AChEIs with %inhibition at 10<sup>-6</sup>M ≥ 80%. Their IC<sub>50</sub> values ranged from 450 (5q) to 6 nM (5p; Table 2). Some of these AChEI showed an additional affinity towards 5-HT<sub>6</sub>R with %inhibition at 10<sup>-6</sup>M > 70% (5b, f, k, m) and Ki ranging from 1,150 to 110 nM. Three derivatives (5e, i, l) displayed affinity for 5-HT<sub>6</sub>R, while being devoid of inhibitory effect towards AChE. Finally, among the four dual compounds, 5k and 5m further exhibited an affinity towards 5-HT<sub>4</sub>R with %inhibition at 10<sup>-8</sup>M ≥ 25% and Ki of 168 and 43 nM respectively. Three other compounds (5o-q) displayed good affinity towards 5-HT<sub>4</sub>R but are devoid of such an affinity towards 5-HT<sub>6</sub>R.

By virtue of its well-balanced *in vitro* activities towards the three targets, 5k was selected for further investigation. According to the concept of MTDL and the synergy theoretically displayed by the association of activities into a sole compound, 5k has been preferred to more potent compounds (e.g., 5m) but with unbalanced levels of activity towards the three targets. This derivative has then been used as its fumaric

acid salt 10. The *in vitro* activities of 10 are depicted in Table 3.

The pharmacological profile of 10 was established towards (h)5-HT<sub>4</sub>R and (h)5-HT<sub>6</sub>R, respectively. It acts as a partial agonist towards (h)5-HT<sub>4</sub>R, in a similar manner as RS67333 (9), and as an inverse agonist towards (h)5-HT<sub>6</sub>R, in a similar manner as SB271046, used as a control 5-HT<sub>6</sub>R antagonist, and idalopirdine (2; Figure 4 and Table 3). Indeed, in our hands, these three compounds decreased cAMP production below the basal, in the absence of agonist (Figure 4C).

## In vivo Results

Whatever the dose tested (1, 10 and 100 mg/kg), preliminary toxicological and pharmacological screening of 10 (Table 4) did not show any deleterious signs, suggesting thus a LD<sub>50</sub> quite higher than 100 mg/kg.

None of the tested doses of 10 (1, 3 and 10 mg/kg) did neither modify spontaneous locomotor activity (Figure 5).

Moreover, an anti-amnesic effect was observed in the scopolamine-induced spontaneous alternation deficit test, after IP administration of 10 at doses starting from 0.3 mg/kg (Figure 6). The degree of effect was similar to that of the dual-targeted compound donecopride in the same dose range (Rochais et al., 2015).

## DISCUSSION

Trying to establish SAR for MTDL is always quite challenging taking into consideration that the structural requirements can be divergent between the expected interactions with the different targets. Considering the three present targets, the SAR appeared similar between AChE and 5-HT<sub>4</sub>R, but partially different from those observed for 5-HT<sub>6</sub>R.

Indeed, with regard to AChE and 5-HT<sub>4</sub>R activities, the best substituent of the piperidine moiety of compounds 5 is the unsubstituted benzyl group (5a). According to our previous work (Yahiaoui et al., 2016), the nature of the substitution of a phenyl group distant from the piperidine moiety could influence in a great manner the affinity of the ligands towards both 5-HT receptors. For the two targets, poly or bulky substituents (Br, I, OBn, OMe, NO<sub>2</sub>), appear detrimental to the activities (5c, g, i, l, n) while a smaller one (OH, NH<sub>2</sub>, F, Me) seems likely to relatively maintain the activity (5h, p, m, d, j, o), especially when present in para position (5p).

Conversely, the 5-HT<sub>6</sub>R affinity appears linked to the substitution of the benzyl group within the studied scaffold, since the unsubstituted derivative 5a was devoid of such an activity. Further, small substituents (OH, F) do not favor, this time, the affinity (5d, h, j, o, p), whatever their position on the phenyl ring, while bulkier ones (Cl, Br, I, Me, OMe) promote it, especially in *ortho* or *meta* positions (5b, c, e, f, i, k, l).

These contradictory requirements explain that 5k with a methyl group in *meta* position displayed balanced activities towards the three targets and was chosen for further investigation under its fumarate form (10). Indeed, according to the MTDL concept (Cavalli et al., 2008),
<figure><img src="image_7.png" /><figcaption>FIGURE 3 | 3D representation of (h)5-HT<sub>6</sub>R and (h)5-HT<sub>4</sub>R affinity and (h)AChE inhibitory activity for compounds 5a–t.</figcaption></figure>

<table><caption>TABLE 2 | (h)AChE inhibitory activity (IC<sub>50</sub>) and (h)5-HT<sub>6</sub>R and (h)5-HT<sub>4</sub>R affinity (K<sub>i</sub>) for DPZ, 2, 9, 5b–f, h–m, o–q, t and the fumarate 10.</caption><thead><tr><th rowspan="2">Cmpd</th><th rowspan="2">R</th><th>(h)AChE</th><th>(h)5-HT<sub>6</sub>R</th><th>(h)5-HT<sub>4</sub>R</th></tr><tr><th>IC<sub>50</sub> (nM) n = 2</th><th>K<sub>i</sub> (nM) n = 3</th><th>K<sub>i</sub> (nM) n = 3</th></tr></thead><tbody><tr><td>DPZ</td><td>-</td><td>7 ± 1.5</td><td>ND</td><td>ND</td></tr><tr><td>2</td><td>-</td><td>ND</td><td>6.9 ± 1.2</td><td>ND</td></tr><tr><td>9</td><td>-</td><td>ND</td><td>ND</td><td>5.1 ± 0.5 (Irving et al., 2010)</td></tr><tr><td>5b</td><td>2-Cl</td><td>156 ± 71</td><td>110 ± 19</td><td>ND</td></tr><tr><td>5c</td><td>2-Br</td><td>317 ± 115</td><td>ND</td><td>ND</td></tr><tr><td>5d</td><td>2-F</td><td>101.6 ± 65.4</td><td>ND</td><td>ND</td></tr><tr><td>5e</td><td>2-I</td><td>ND</td><td>186 ± 71</td><td>ND</td></tr><tr><td>5f</td><td>2-Me</td><td>225.2 ± 57.3</td><td>270 ± 14</td><td>ND</td></tr><tr><td>5h</td><td>2-OH</td><td>57.4 ± 9.4</td><td>ND</td><td>ND</td></tr><tr><td>5i</td><td>3-Br</td><td>ND</td><td>169 ± 44</td><td>ND</td></tr><tr><td>5j</td><td>3-F</td><td>91 ± 53.8</td><td>ND</td><td>ND</td></tr><tr><td>5k</td><td>3-Me</td><td>161 ± 43</td><td>230 ± 49</td><td>168 ± 15</td></tr><tr><td>5l</td><td>3-OMe</td><td>ND</td><td>265 ± 64</td><td>ND</td></tr><tr><td>5m</td><td>3-NH<sub>2</sub></td><td>41 ± 5</td><td>1150 ± 559</td><td>42.5 ± 2.6</td></tr><tr><td>5o</td><td>4-F</td><td>78.4 ± 12.1</td><td>ND</td><td>ND</td></tr><tr><td>5p</td><td>4-OH</td><td>6.0 ± 0.4%</td><td>ND</td><td>ND</td></tr><tr><td>5q</td><td>4-Me</td><td>450.5 ± 89.5</td><td>ND</td><td>ND</td></tr><tr><td>5t</td><td>2-F;5-NH<sub>2</sub></td><td>152.7 ± 19.9</td><td>ND</td><td>ND</td></tr><tr><td>10</td><td>3-Me, fumarate salt</td><td>33.7 ± 1.7</td><td>219 ± 42</td><td>210 ± 43</td></tr></tbody></table>

ND, not determined.
<table><caption>TABLE 3 | (h)5-HT<sub>6</sub>R and (h)5-HT<sub>4(a)</sub>R pharmacological profile of compounds 2, 9 and 10.</caption><thead><tr><th rowspan="2"></th><th colspan="3">(h)5-HT<sub>6</sub>R</th><th colspan="3">(h)5-HT<sub>4(a)</sub>R</th></tr><tr><th>Log(IC<sub>50</sub>)</th><th>% control antagonist response</th><th>Profile</th><th>Log(EC<sub>50</sub>)</th><th>% control agonist response</th><th>Profile</th></tr></thead><tbody><tr><td>2</td><td>-6.2 ± 0.2 n = 2</td><td>101.2 ± 3.2 n = 2</td><td>Inverse agonist</td><td>-</td><td>-</td><td>-</td></tr><tr><td>9</td><td>-</td><td>-</td><td>-</td><td>-8.8 ± 0.2 n = 2</td><td>48.7 ± 5.2 n = 3*</td><td>Partial agonist</td></tr><tr><td>10</td><td>-5.0 ± 0.1 n = 2</td><td>93.4 ± 1.8 n = 2</td><td>Inverse agonist</td><td>-7.7 ± 0.0 n = 2</td><td>59.8 ± 2.5 n = 2</td><td>Partial agonist</td></tr></tbody></table>

SB271046 was used as a control 5-HT<sub>6</sub>R antagonist and 5-HT as control 5-HT<sub>4(a)</sub>R agonist. *Result reported from Lecoutey et al. (2014).

<figure><img src="image_8.png" /><figcaption>TABLE 4 | Pharmacological and toxicological properties of 10.</figcaption></figure>

<table><thead><tr><th>Compound</th><th>Doses (mg/kg)</th><th>LD<sub>50</sub> (mg/kg)</th><th>Symptoms (subtoxic doses)</th></tr></thead><tbody><tr><td>10</td><td>1–10–100</td><td rowspan="5">&gt;100</td><td>No symptoms</td></tr><tr><td rowspan="4">Amphetamine</td><td rowspan="4">2</td><td>Hyperactivity</td></tr><tr><td>Exophthalmia</td></tr><tr><td>Irritability</td></tr><tr><td>Hypoactivity</td></tr><tr><td rowspan="2">Chlorpromazine</td><td rowspan="2">10</td><td>Ataxia</td></tr><tr><td>Sleep</td></tr></tbody></table>

pleiotropic ligand could present moderate *in vitro* activities toward their targets that could, however, lead to potent and promising *in vivo* activities explained by their synergistic effect (Prati et al., 2015).

Of interest, compound **10**, idalopirdine and SB271046 were able to reverse the constitutive activity of the 5-HT<sub>6</sub> receptor, thus demonstrating an inverse agonist profile. Such a property is of high interest regarding this receptor as it is known to possess a high constitutive activity in the brain, exerting a tonic activity (Duhr et al., 2014; Deraredj Nadim et al., 2016). Regarding the 5-HT<sub>4R</sub>, **10** demonstrated a partial agonist activity similar to the one of **9**, which had proven its efficiency to prevent amyloid production in a mouse

model of AD (Hashimoto et al., 2012; Giannoni et al., 2013; Baranger et al., 2017).

The main result of the behavioral study concerns the anti-amnesic effect of **10** in a model of scopolamine-induced deficit of working memory from the dose of 0.3 mg/kg.

Although no pharmacokinetic data are available for compound **10** yet, this result indicates that it reaches the general circulation and crosses the blood-brain barrier upon IP administration. The anti-amnesic effect of compound **10** suggests that the co-modulation of the three targets could be a therapeutic strategy of interest in the field of memory impairments. The anti-amnesic properties of the compound **10**, together with the absence of any deleterious signs during the pharmacological screening (even when tested at high dose: 100 mg/kg), are arguing in favor of **10** as a new promising drug, with excellent general tolerance.

## CONCLUSION

We have synthesized a variety of compounds possessing both *in vitro* and *in vivo* activities towards three therapeutic targets (5-HT<sub>4R</sub>/5-HT<sub>6R</sub>, AChE) in the potential treatment
<figure><img src="image_9.png" /></figure>

**FIGURE 5** | Effect of compound 10 on spontaneous locomotor activity. Data are expressed as the mean ± standard deviation (n = 8). Drugs were administered intraperitoneally (IP) 30 min before the behavioral test. Compound 10: 1–3–10 mg/kg; AMP: amphetamine 2 mg/kg; CPZ: chlorpromazine 3 mg/kg (*p* < 0.05 NaCl, Student-Newman-Keuls (SNK) test).

<figure><img src="image_10.png" /></figure>

**FIGURE 6** | Effect of compound 10 on scopolamine-induced impairment during the spontaneous alternation test. Data are expressed as the mean ± standard deviation (n = 8). Drugs were administered IP 30 min before the test, and scopolamine was administered SC 20 min before the test. Compound 10: 0.3, 1, 3 mg/kg; DPZ: Donepezil; 1 mg/kg; Scopolamine: 0.5 mg/kg (*p* < 0.05 vs. 50%; univariate *t*-test).

of AD. The use of these innovative MTDL which may exhibit a greater benefit than single-targeted drugs towards the neuropathological hallmarks of the disease and the associated behavioral deficits will require further studies in animal

## REFERENCES

Baranger, K., Giannoni, P., Girard, S. D., Girot, S., Gaven, F., Stephan, D., et al. (2017). 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. *Neuropharmacology* 126, 128–141. doi: 10.1016/j.neuropharm.2017.08.031

models and potential future clinical trials to demonstrate their efficacy.

## ASSOCIATED CONTENT

Analytical data of final compounds. This material is available free of charge *via* the Internet at http://pubs.acs.org.

## DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the **Supplementary Files**.

## ETHICS STATEMENT

All experiments were conducted in agreement with the European Directives and French law on animal experimentation (personal authorization n° 14-17 for MB and 14-60 for TF).

## AUTHOR CONTRIBUTIONS

The manuscript was written through contributions of SC, TF, MB, CR and PD. All authors have given approval to the final version of the manuscript.

## FUNDING

This work was supported by funding from the Fondation Vaincre Alzheimer (#FR-15072) and 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.

## ACKNOWLEDGMENTS

We would like to thank Steve Dubel for final proofreading of the manuscript.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2019.00148/full#supplementary-material

Benhamú, B., Martín-Fontecha, M., Vázquez-Villa, H., Pardo, L., and López-Rodríguez, M. L. (2014). Serotonin 5-HT₆ receptor antagonists for the treatment of cognitive deficiency in Alzheimer's disease. *J. Med. Chem.* 57, 7160–7181. doi: 10.1021/jm5003952

Cavalli, A., Bolognesi, M. L., Minarini, A., Rosini, M., Tumiatti, V., Recanatini, M., et al. (2008). Multi-target-directed ligands to combat neurodegenerative diseases. *J. Med. Chem.* 51, 347–372. doi: 10.1021/jm7009364
Cho, S., and Hu, Y. (2007). Activation of 5-HT₄ receptors inhibits secretion of β-amyloid peptides and increases neuronal survival. *Exp. Neurol.* 203, 274–278. doi: 10.1016/j.expneurol.2006.07.021

Claeysen, S., Bockaert, J., and Giannoni, P. (2015). Serotonin: a new hope in Alzheimer's disease? *ACS Chem. Neurosci.* 6, 940–943. doi: 10.1021/acschemneuro.5b00135

Consolo, S., Arnaboldi, S., Giorgi, S., Russi, G., and Ladinsky, H. (1994). 5-HT₄ receptor stimulation facilitates acetylcholine release in rat frontal cortex. *Neuroreport* 5, 1230–1232. doi: 10.1097/00001756-199406020-00018

Dawson, L. A., Nguyen, H. Q., and Li, P. (2000). *In vivo* effects of the 5-HT₆ antagonist SB-271046 on striatal and frontal cortex extracellular concentrations of noradrenaline, dopamine, 5-HT, glutamate and aspartate. *Br. J. Pharmacol.* 130, 23–26. doi: 10.1038/sj.bjp.0703288

Dawson, L., Nguyen, H., and Li, P. (2001). The 5-HT(6) receptor antagonist SB-271046 selectively enhances excitatory neurotransmission in the rat frontal cortex and hippocampus. *Neuropsychopharmacology* 25, 662–668. doi: 10.1016/s0893-133x(01)00265-2

de Bruin, N. M., and Kruse, C. G. (2015). 5-HT<sub>6</sub> receptor antagonists: potential efficacy for the treatment of cognitive impairment in schizophrenia. *Curr. Pharm. Des.* 21, 3739–3759. doi: 10.2174/13816128216661506051 12105

Deraredj Nadim, W., Chaumont-Dubel, S., Madouri, F., Cobret, L., De Tauzia, M. L., Zajdel, P., et al. (2016). Physical interaction between neurofibromin and serotonin 5-HT<sub>6</sub> receptor promotes receptor constitutive activity. *Proc. Natl. Acad. Sci. U S A* 113, 12310–12315. doi: 10.1073/pnas. 1600914113

Dolles, D., and Decker, M. (2017). “Dual-acting compounds acting as receptor ligands and enzyme inhibitors,” in *Design of Hybrid Molecules for Drug Development*, ed. M. Decker (Oxford: Elsevier), 137–165.

Dolles, D., Hoffmann, M., Gunesch, S., Marinelli, O., Möller, J., Santoni, G., et al. (2018). Structure-activity relationships and computational investigations into the development of potent and balanced dual-acting butyrylcholinesterase inhibitors and human cannabinoid receptor 2 ligands with pro-cognitive *in vivo* profiles. *J. Med. Chem.* 61, 1646–1663. doi: 10.1021/acs.jmedchem.7b 01760

Duhr, F., Déléris, P., Raynaud, F., Séveno, M., Morisset-Lopez, S., Mannoury la Cour, C., et al. (2014). Cdk5 induces constitutive activation of 5-HT₆ receptors to promote neurite growth. *Nat. Chem. Biol.* 10, 590–597. doi: 10.1038/nchembio.1547

Eglen, R. M., Bonhaus, D. W., Johnson, L. G., Leung, E., and Clark, R. D. (1995). Pharmacological characterization of two novel and potent 5-HT₄ receptor agonists, RS67333 and RS67506, *in vitro* and *in vivo*. *Br. J. Pharmacol.* 115, 1387–1392. doi: 10.1111/j.1476-5381.1995.tb16628.x

Ellis, J. M., and Fell, M. J. (2017). Current approaches to the treatment of Parkinson's disease. *Bioorg. Med. Chem. Lett.* 27, 4247–4255. doi: 10.1016/j. bmlc.2017.07.075

Ellman, G. L., Courtney, K. D., Andres, V. Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. *Biochem. Pharmacol.* 7, 88–95. doi: 10.1016/0006-2952(61) 90145-9

Freret, T., Bouet, V., Quiedeville, A., Nee, G., Dallemagne, P., Rochais, C., et al. (2012). Synergistic effect of acetylcholinesterase inhibition (donepezil) and 5-HT(4) receptor activation (RS67333) on object recognition in mice. *Behav. Brain Res.* 230, 304–308. doi: 10.1016/j.bbr.2012.02.012

Ge, J., and Barnes, N. M. (1996). 5-HT₄ receptor-mediated modulation of 5-HT release in the rat hippocampus *in vivo*. *Br. J. Pharmacol.* 117, 1475–1480. doi: 10.1111/j.1476-5381.1996.tb15309.x

Giannoni, P., Gaven, F., De Bundel, D., Baranger, K., Marchetti-Gauthier, E., Roman, F., et al. (2013). Early administration of RS 67333, a specific 5-HT₄ receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer's disease. *Front. Aging Neurosci.* 5:96. doi: 10.3389/fnagi.2013.00096

Hashimoto, G., Sakurai, M., Teich, A. F., Saeed, F., Aziz, F., and Arancio, O. (2012). 5-HT₄ receptor stimulation leads to soluble AβPPα production through MMP-9 upregulation. *J. Alzheimers Dis.* 32, 437–445. doi: 10.3233/jad-2012- 111235

Hirst, W. D., Stean, T. O., Rogers, D. C., Sunter, D., Pugh, P., Moss, S. F., et al. (2006). SB-399885 is a potent, selective 5-HT₆ receptor antagonist

with cognitive enhancing properties in aged rat water maze and novel object recognition models. *Eur. J. Pharmacol.* 553, 109–119. doi: 10.1016/j.ejphar. 2006.09.049

Hooper, N., Fraser, C., and Stone, T. W. (1996). Effect of purines analogues on spontaneous alternation in mice. *Psychopharmacology* 123, 250–257. doi: 10.1007/bf02246579

Irving, H. R., Tochon-Danguy, N., Chinkwo, K. A., Li, J. G., Grabbe, C., Shapiro, M., et al. (2010). Investigations into the binding affinities of different human 5-HT₄ receptor splice variants. *Pharmacology* 85, 224–233. doi: 10.1159/000280418

Karila, D., Freret, T., Bouet, V., Boulouard, M., Dallemagne, P., and Rochais, C. (2015). Therapeutic potential of 5-HT₆ receptor agonists. *J. Med. Chem.* 58, 7901–7912. doi: 10.1021/acs.jmedchem.5b00179

Kilbinger, H., and Wolf, D. (1992). Effects of 5-HT₄ receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus. *Naunyn Schmiedebergs Arch. Pharmacol.* 345, 270–275. doi: 10.1007/bf00168686

Lalut, J., Karila, D., Dallemagne, P., and Rochais, C. (2017). Modulating 5-HT₄ and 5-HT₆ receptors in Alzheimer's disease treatment. *Future Med. Chem.* 9, 781–795. doi: 10.4155/fmc-2017-0031

Lecoutey, C., Hédou, D., Freret, T., Giannoni, P., Gaven, F., Since, M., et al. (2014). Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment. *Proc. Natl. Acad. Sci. U S A* 111, E3825–E3830. doi: 10.1073/pnas.1410315111

Lelong, V., Lhonneur, L., Dauphin, F., and Boulouard, M. (2003). BIMU 1 and RS 67333, two 5-HT₄ receptor agonists, modulate spontaneous alternation deficits induced by scopolamine in the mouse. *Naunyn Schmiedebergs Arch. Pharmacol.* 367, 621–628. doi: 10.1007/s00210-003-0743-2

Lezoualc'h, F. (2007). 5-HT₄ receptor and Alzheimer's disease: the amyloid connection. *Exp. Neurol.* 205, 325–329. doi: 10.1016/j.expneurol.2007. 02.001

Lucas, G., Di Matteo, V., De Deurwaerdère, P., Porras, G., Martín-Ruiz, R., Artigas, F., et al. (2001). Neurochemical and electrophysiological evidence that 5-HT₄ receptors exert a state-dependent facilitatory control *in vivo* on nigrostriatal, but not mesoaccumbal, dopaminergic function. *Eur. J. Neurosci.* 13, 889–898. doi: 10.1046/j.0953-816x.2000.01453.x

Marcos, B., Gil-Bea, F. J., Hirst, W. D., Garcia-Alloza, M., and Ramirez, M. J. (2006). Lack of localization of 5-HT₆ receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5-HT₆ receptor-mediated acetylcholine release. *Eur. J. Neurosci.* 24, 1299–1306. doi: 10.1111/j.1460-9568. 2006.05003.x

Morpugo, C. (1971). A new design for the screening of CNS-active drugs in mice. *Arzneimittelforschung* 11, 1727–1734.

Nirogi, R., Mohammed, A. R., Shinde, A. K., Gagginapally, S. R., Kancharla, D. M., Middekadi, V. R., et al. (2018). Synthesis, structure-activity relationships, and preclinical evaluation of heteroaromatic amides and 1,3,4-oxadiazole derivatives as 5-HT₄ receptor partial agonists. *J. Med. Chem.* 61, 4993–5008. doi: 10.1021/acs.jmedchem.8b00457

Prati, F., De Simone, A., Bisignano, P., Armirotti, A., Summa, M., Pizzirani, D., et al. (2015). Multitarget drug discovery for Alzheimer's disease: triazinones as BACE-1 and GSK-3β inhibitors. *Angew. Chem. Int. Ed Engl.* 54, 1578–1582. doi: 10.1002/anie.201410456

Quiedeville, A., Boulouard, M., Hamidouche, K., Da Silva Costa-Aze, V., Nee, G., Rochais, C., et al. (2015). Chronic activation of 5-HT₄ receptors or blockade of 5-HT₆ receptors improve memory performances. *Behav. Brain Res.* 293, 10–17. doi: 10.1016/j.bbr.2015.07.020

Riemer, C., Borroni, E., Levet-Trafit, B., Martin, J. R., Poli, S., Porter, R. H. P., et al. (2003). Influence of the 5-HT₆ receptor on acetylcholine release in the cortex: pharmacological characterization of 4-(2-bromo-6-pyrrolidin-1-ylpyridine-4-sulfonyl)phenylamine, a potent and selective 5-HT₆ receptor antagonist. *J. Med. Chem.* 46, 1273–1276. doi: 10.1021/jm021085c

Rochais, C., Lecoutey, C., Gaven, F., Giannoni, P., Hamidouche, K., Hedou, D., et al. (2015). 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. *J. Med. Chem.* 58, 3172–3187. doi: 10.1021/acs.jmedchem. 5b00115
Russo, O., Cachard-chastel, M., Giner, M., Soulier, J., Berthouze, M., Richard, T., et al. (2009). Design, synthesis, and biological evaluation of new 5-HT₄ receptor agonists: application as amyloid cascade modulators and potential therapeutic utility in Alzheimer's disease design. *J. Med. Chem.* 52, 2214–2225. doi: 10.1021/jm801327q

Shirazi-Southall, S., Rodriguez, D. E., and Nomikos, G. G. (2002). Effects of typical and atypical antipsychotics and receptor selective compounds on acetylcholine efflux in the hippocampus of the rat. *Neuropsychopharmacology* 26, 583–594. doi: 10.1016/s0893-133x(01)00400-6

Steward, L. J., Ge, J., Stowe, R. L., Brown, D. C., Bruton, R. K., Stokes, P. R. A., et al. (1996). Ability of 5-HT₄ receptor ligands to modulate rat striatal dopamine release *in vitro* and *in vivo*. *Br. J. Pharmacol.* 117, 55–62. doi: 10.1111/j.1476-5381.1996.tb15154.x

Wilkinson, D., Windfeld, K., and Colding-Jørgensen, E. (2014). Safety and efficacy of idalopirdine, a 5-HT₆ receptor antagonist, in patients with moderate Alzheimer's disease (LADDER): a randomised, double-blind, placebo-controlled phase 2 trial. *Lancet Neurol.* 13, 1092–1099. doi: 10.1016/s1474-4422(14)70198-x

Yahiaoui, S., Hamidouche, K., Ballandonne, C., Davis, A., De Oliveira Santos, J. S., Freret, T., et al. (2016). Design, synthesis, and pharmacological evaluation of multitarget-directed ligands with

both serotonergic subtype 4 receptor (5-HT4) partial agonist and 5-HT6 antagonist activities, as potential treatment of Alzheimer's disease. *Eur. J. Med. Chem.* 121, 283–293. doi: 10.1016/j.ejmech.2016.05.048

Zhang, M. Y., Hughes, Z. A., Kerns, E. H., Lin, Q., and Beyer, C. E. (2007). Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples. *J. Pharm. Biomed. Anal.* 44, 586–593. doi: 10.1016/j.jpba.2007.02.024

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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