# A Dual Approach To Combat Alzheimer's Disease through Concomitant hBChE Inhibition and S1R Activation

Kora Reichau, Lucie Crouzier, Eleonora Poeta, Tina Gehrig, Eva Schaller, Liga Zvejniece, Johann Meunier, Christoph Sotriffer, Barbara Monti, Tangui Maurice, et al.

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## To cite this version:

Kora Reichau, Lucie Crouzier, Eleonora Poeta, Tina Gehrig, Eva Schaller, et al. A Dual Approach To Combat Alzheimer's Disease through Concomitant hBChE Inhibition and S1R Activation. ACS Chemical Neuroscience, In press, 17 (4), pp.805-822. 10.1021/acschemneuro.5c00921. [hal-05460066](https://hal.science/hal-05460066v1)

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1 A Dual Approach To Combat Alzheimer's Disease through
2 Concomitant *hBChE* Inhibition and S1R Activation

3 Kora Reichau, Lucie Crouzier, Eleonora Poeta, Tina Gehrig, Eva Schaller, Liga Zvejniece,

4 Johann Meunier, Christoph Sotriffer, Barbara Monti, Tangui Maurice,* and Michael Decker*

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Cite This: https://doi.org/10.1021/acschemneuro.5c00921

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Supporting Information

5 ABSTRACT: Alzheimer's disease (AD) remains an incurable neurodegenerative
6 disorder, requiring novel therapeutic strategies. We developed multitarget ligands
7 designed to inhibit human butyrylcholinesterase (hBChE) and activate the sigma-1
8 receptor (S1R), addressing both cholinergic dysfunction and neuroinflammation, the
9 latter being reduced through action on both targets. The (pseudo)-irreversible

10 carbamate 18c emerged as the most promising compound, exhibiting potent and
11 selective hBChE inhibition (IC50 = 3.3 nM, 45-fold selectivity over human
12 acetylcholinesterase) and strong S1R agonistic activity (IC50 = 25 nM, EC50 = 99
13 nM) determined in a radioligand binding assay and by S1R-BiP dissociation. Its
14 cleavage product 14c (after carbamate hydrolysis by hBChE) retained dual activity
15 (IC50(hBChE) = 269 nM, IC50(S1R) = 20 nM, and EC50(S1R) = 279 nM). Both
16 compounds reduced lipopolysaccharide (LPS)-induced pro-inflammatory activation
17 profile in the microglial N9 cells while preserving anti-inflammatory marker expression,

18 thereby indicating an overall immunomodulatory effect. In vivo, inhibitor 18c improved cognitive deficits in a mouse model with
19 Aβ<sub>25-35</sub>-induced neurotoxicity, enhancing short- and long-term memory in Y-maze and passive avoidance tests at dosages as low as
20 0.1–1 mg/kg. These findings highlight the potential of dual-targeting hBChE/S1R strategies for AD therapy.
21

21 KEYWORDS: multitarget-directed ligands, neuroinflammation, cholinergic dysfunction, microglial immunomodulation, neuroprotection,

22 4-IBP

<figure><img src="image_8.png" /><figcaption>Neuroprotection in Alzheimer's Disease</figcaption></figure>

1. INTRODUCTION

23 Alzheimer's disease (AD) is a neurodegenerative disorder and
24 the most common cause of dementia, mainly occurring later in
25 life, typically after the age of 65.¹ The disease is progressive in
26 nature and starts with pathophysiological changes in the
27 affected brains, years before any clinical manifestations are
28 observed.¹ Among the characteristic pathological markers are
29 depositions of amyloid β protein (Aβ) and aggregated
30 neurofibrillary tangles (NFTs).²,³ To date, there is no
31 established way to prevent or cure AD.⁴ However, recent
32 advances in antiamyloid immunotherapy, particularly in
33 combination with early diagnosis through biomarker detection,
34 might well present a disease-modifying therapy strategy.⁵,⁶ Still,
35 the clinical effectiveness of these treatments remains
36 controversial.⁷,⁸ While lecanemab represents the first Aβ-
37 targeting monoclonal antibody approved in Europe, other Aβ-
38 antibodies, such as aducanumab, which was approved by the
39 FDA based on its ability to reduce cerebral Aβ levels, did not
40 receive European approval due to insufficient evidence of
41 clinical benefit.⁷,⁹ Due to the large number of dementia
42 patients worldwide (currently more than 55 million), its
43 devastating effect on individuals and healthcare systems, and
44 the lack of treatment strategies to prevent, decelerate, or even

cure AD, the World Health Organization categorized AD as a 45
global public health priority.⁴,¹⁰

46

Besides Aβ aggregation and NFT formation, chronic 47
neuroinflammation is an important pathological feature in 48
AD, which may contribute to the spreading and progression of 49
the pathology. An inflammatory response is commonly 50
triggered by the stimulation of glial cells, particularly microglia, 51
which become activated to defend neurons, clear debris, and 52
maintain brain homeostasis. However, when the activation of 53
microglia is prolonged, cells undergo transcriptional and 54
functional changes, releasing pro-inflammatory cytokines, 55
chemokines, and reactive oxygen species, which contribute to 56
neural damage and synaptic dysfunction. 11–13 In AD, microglia 57
surround Aβ plaques and internalize Aβ aggregates through 58
phagocytosis, but their efficiency declines with disease 59
progression. Dysfunctional and chronically activated microglia 60
release inflammatory mediators and extracellular vesicles that 61

Received: November 25, 2025

Revised: January 7, 2026

Accepted: January 8, 2026

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 1.** (a) Glennon's model for S1R ligands and 4-IBP with accordingly color-coded pharmacophores.⁵⁰ Adapted from ref 54. Available under a CC-BY 4.0 license. Copyright © 2025 Reichau et al. (b) *hBChE-inhibiting carbamates, mechanism, and kinetic parameters of pseudoirreversible BChE inhibition: First, a reversible enzyme-inhibitor complex (EPC) between the enzyme (E) and the inhibitor (PC) is formed and is described by equilibrium constant Kᵢ. In the second step, the carbamate unit is transferred to the enzyme's active site (EC), releasing the corresponding phenol (P) with k₃ as the carbamylation rate constant. Finally, hydrolysis of the carbamate bond leads to reconstitution of the enzyme (E) activity under carbamate release (C') characterized by velocity constant k₄. The decarbamylation step is usually slower than the carbamylation.⁵⁵ Adapted from ref 54. Available under a CC-BY 4.0 license. Copyright © 2025 Reichau et al. (c) Schematic design of S1R/*hBChE* hybrid benzyl piperidines with different chain length (n = 0–1 and m = 0–2) and color-coded pharmacophores.</figcaption></figure>

62 can propagate misfolded Aβ and tau proteins to neighboring neurons, thereby facilitating the spreading of neurodegeneration throughout different brain regions.<sup>14-16</sup> These mediators 64 induce neurotoxicity, synaptic dysfunction, and imbalanced 65 neurogenesis, making neuroinflammation not a "byproduct" of 66 pathophysiological changes like Aβ and NFT formation, but a 67 key driver of AD.<sup>17</sup> Another pathological mechanism is the Aβ-68 induced deficiency of neurotransmitter acetylcholine (ACh). 69 Low levels of ACh impair the function of the cholinergic 70 neurons, which are fundamental for the regulation of cognitive 71 functions, attention, and memory.<sup>6</sup>

73 To preserve cholinergic functions, inhibition of the ACh-74 metabolizing enzyme acetylcholinesterase (*hAChE*) is clin-75 ically used for the symptomatic treatment of AD. Donepezil 76 and rivastigmine are FDA-approved examples of *hAChE* 77 inhibitors.<sup>18-20</sup> However, the *hAChE* level is known to 78 decrease during AD, whereas butyrylcholinesterase (*hBChE*) 79 levels increase up to 120%. *hBChE* is present in plasma and 80 various cells and is a less specific enzyme with a largely 81 unknown physiological function. Studies in *hAChE* knockout 82 mice demonstrated that BChE can take over *hAChEs* function 83 to cleave ACh.<sup>21-24</sup> Additionally, several studies showed that 84 *hBChE* is associated with NFTs and Aβ plaques, making 85 *hBChE* a more reasonable target for AD drug development 86 compared to *hAChE*.<sup>25-28</sup> In our previous work, we already 87 demonstrated pronounced neuroprotective effects of *hBChE* 88 inhibitors in both cellular and in vivo models.<sup>29-31</sup>

89 However, the classical one-target approach is limited in 90 efficacy to combat multifactorial diseases like AD. Instead, 91 addressing several targets using a multitarget-directed ligand 92 seems a more promising strategy.<sup>23,32-34</sup> For example, recently 93 developed *hBChE* inhibitors additionally activating cannabi-94 noid receptor 2 reduced microglia regulated inflammation and 95 could attenuate learning impairments in an Aβ<sub>25-35</sub> mouse 96 model in dosages from 0.3–3 mg/kg. These results regarding 97 neuroprotection proved that such *hBChE* inhibitors exhibit a

disease-modifying mode of action, exceeding purely symptomatic treatment.⁵⁵

Combining *hBChE* inhibition with another neuroprotective target like the sigma-1 receptor (S1R), which modulates several pathways including cholinergic neurotransmitter systems,³⁶ seems to be an auspicious combination to potentially achieve additive or even synergistic pharmacological effects.³⁷

The sigma-1 receptor is a transmembrane chaperone protein, so not actually a receptor, that is particularly enriched in the mitochondria-associated endoplasmic reticulum membranes and interacts with different proteins.³⁸ For instance, S1R ensures bioenergetics and cellular survival by modulation of endoplasmic reticulum-mitochondria calcium transfer,³⁹,⁴⁰ which is disturbed in AD through pores formed by Aβ.⁴¹ Moreover, S1R regulates proper tau phosphorylation and has a number of anti-inflammatory functions in the brain.⁴²,⁴³ For example, S1R regulates microglia response, and activation of S1R was found to suppress lipopolysaccharide (LPS)-induced pro-inflammatory response.⁴⁴

S1R agonists like (+)-pentazocine and PRE-084 could demonstrate improved learning and memory deficits in the Aβ<sub>25-35</sub> mouse model.⁴⁵ The high potential of activating the S1R along with other targets is underlined by the antiamnesic neuroprotectant blarcamesine (ANAVEX2-73) targeting S1R and muscarinic acetylcholine receptors, leading to synergic effects in vivo in a submg/kg range.⁴⁶

In this work, we aimed to develop novel hybrid molecules that inhibit *hBChE* and lead to S1R activation in the same concentration range to obtain a balanced profile for investigating their potential as AD drugs. Therefore, we designed and synthesized new ligands and evaluated the dual-target engagement in vitro, followed by in vivo studies in the Aβ<sub>25-35</sub>-induced learning impairment mouse model.

As a starting point for the development, we used S1R agonist 4-IBP, a benzyl piperidine derivative with high S1R affinity and an available cocrystallized structure.<sup>47-49</sup> The crystal structure
<figure><img src="image_11.png" /><figcaption>Scheme 1. Synthesis of Compound 4<sup>a</sup></figcaption></figure>

<sup>a</sup>Reactions and conditions: (a) 1-benzylpiperidin-4-amine, PPh<sub>3</sub>, I<sub>2</sub>, Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, rt, 48 h, 30%; (b) BBr<sub>3</sub> in CH<sub>2</sub>Cl<sub>2</sub>, CH<sub>2</sub>Cl<sub>2</sub>, 0 °C-rt, 1 h, quant.; (c) heptyl isocyanate, Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, rt, 16 h, 27%.

<figure><img src="image_12.png" /><figcaption>Scheme 2. Synthesis of Compounds 15a–18a–c<sup>a</sup></figcaption></figure>

<sup>a</sup>Reactions and conditions: (a) HBTU, Et<sub>3</sub>N, DMF, rt, 16 h, 91%-quant.; (b) BBr<sub>3</sub> in CH<sub>2</sub>Cl<sub>2</sub>, CH<sub>2</sub>Cl<sub>2</sub>, 0 °C-rt, 1 h, quant.; (c) heptyl isocyanate, Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, rt, 16 h, 34% (d) 4-nitrophenyl azetidine-1-carboxylate, Cs<sub>2</sub>CO<sub>3</sub>, DMF, 55 °C, 2 h, 37%.

135 reveals that the basic amine in the benzyl piperidine moiety
136 forms charge-charge interactions with the glutamic acid
137 residue in the binding pocket.⁴⁸ This interaction is essential
138 for S1R affinity, consistent with Glennon's model,⁵⁰ which
139 defines key structural determinants for S1R binding (Figure
140 1a).

141 To achieve activity on *hBChE*, we chose a carbamate motif
142 as a pharmacophore, which has been extensively studied and
143 inhibits ChEs in a pseudoirreversible manner.<sup>30,51-53</sup> In this
144 process, the carbamate unit is covalently transferred to the
145 active-site serine of the enzyme. Concomitantly, the corre-
146 sponding alcohol is released, which may have pharmacological
147 activity, too. Hydrolysis of the carbamate subsequently restores
148 the enzyme's activity over time (Figure 1b).<sup>23</sup> To enhance
149 selectivity for *hBChE* over *hAChE*, a heptyl carbamate was
150 introduced, based on previous reports demonstrating selective
151 inhibition.<sup>30,51-53</sup> As an attachment point for the carbamate,
152 both phenyl rings are, in principle, synthetically favorable.
153 However, substitution at the benzyl piperidine side may cause
154 steric clashes within the narrower secondary hydrophobic
155 pocket, displacing the pharmacophoric amine from glutamic
156 acid and potentially disrupting the essential salt-bridge
157 formation. Consequently, our design focused on the opposite
158 aromatic ring, replacing iodine with a carbamate (Figure 1c).
159 This modification was followed by a stepwise extension of the
160 scaffold through methylene group insertions around the rigid
161 amide side to promote flexibility and potentially facilitate entry
162 of the ligand into the active pocket of *hBChE* for carbamate
163 transfer (Figure 1c).

## 2. RESULTS AND DISCUSSION

### 2.1. Chemistry

To introduce the carbamate unit into the 4-IBP scaffold, 4-methoxybenzoic acid (1) was coupled with 1-benzylpiperidin-4-amine (Scheme 1). The resulting amide 2 was treated with boron tribromide to yield phenol 3. Finally, phenol 3 reacted with heptyl isocyanate to compound 4, fusing the different pharmacophores (Scheme 1).

To study the effects of scaffold flexibility on both targets, 2-(4-methoxyphenyl)acetic acid (5a) was coupled with different benzylamines 6–8 using HBTU (Scheme 2). Amides 9a, 10a, and 11a were obtained in high yields. Deprotection of the phenol group with boron tribromide and reaction with heptyl isocyanate gave compounds 15a, 16a, and 17a with yields of 16–34% (Scheme 2).

For an investigation into the effects of the substitution pattern of compound **17a**, we additionally synthesized *meta* and *ortho*-substituted heptyl carbamates **17b** and **17c**. Applying the same synthetic route as before, using the *meta* and *ortho* substituted phenylacetic acids **5b**, *c*, yielded the respective amides **11b**, *c* and phenols **14b**, *c*, respectively, in high yields. Reaction of phenols **14b**, *c* with heptyl isocyanate yielded heptyl carbamates **17b**, *c* in 32–34% yield. Additionally, we exchanged the heptyl residue of the carbamate with an azetidine. The azetidines were the most effective carbamate class in our previous *hBChE* inhibitor studies,³⁰ resulting in decreased molecular weight and lipophilicity. Azetidine carbamates **18a-c** were formed by reacting the corresponding phenol **14a-c** with 4-nitrophenyl azetidine-1-carboxylate (Scheme 2).
<figure><img src="image_13.png" /><figcaption>Table 1. hChE Inhibitory Activity and S1R Affinity of 4-IBP Derived Benzyl Piperidines 4, 15, 16<sup>a</sup></figcaption></figure>

<table><thead><tr><th></th><th>R<sup>1</sup></th><th>n</th><th>m</th><th>IC<sub>50</sub><br>(hBChE)<br>(pIC<sub>50</sub>±SEM)</th><th>IC<sub>50</sub><br>(hAChE)<br>(pIC<sub>50</sub>±SEM)</th><th>S1R<br>%inhibition<br>@10µM</th><th>S1R<br>%inhibition<br>@100nM</th></tr></thead><tbody><tr><td>4-IBP</td><td>1</td><td>0</td><td>0</td><td>n.d.</td><td>n.d.</td><td>100%<br>IC<sub>50</sub> = 4.2 nM<br>(8.4±0.05)</td><td>94%<br>IC<sub>50</sub> = 4 nM</td></tr><tr><td>4</td><td></td><td>0</td><td>0</td><td>>2500 nM</td><td>>2500 nM</td><td>95%</td><td>2%</td></tr><tr><td>15a</td><td>o<sup>-</sup><sub>NH</sub><br>l<sup>+</sup><sub>l</sub></td><td>1</td><td>0</td><td>>2500 nM</td><td>>2500 nM</td><td>67%</td><td>n.d.</td></tr><tr><td>16a</td><td></td><td>1</td><td>1</td><td>701 nM<br>(6.2±0.07)</td><td>>2500 nM</td><td>95%</td><td>1%</td></tr></tbody></table>

<sup>a</sup>n.d. not determined. Inhibition on *hChE* was determined as triplicates after 20 min of incubation using the Ellman's assay. *IC*<sub>50</sub> values were calculated through a sigmoidal regression using GraphPad Prism 10.<sup>30,35,55</sup> Tacrine was used as a positive control.<sup>59,60</sup> S1R affinity was measured by displacement of radioligand [<sup>3</sup>H](+)-pentazocine using membranes prepared from Wistar rat liver.<sup>56-58</sup> **PRE-084** was used as a positive control.<sup>61</sup>

<figure><img src="image_14.png" /><figcaption>Table 2. *hChE* Inhibitory Activity and S1R Affinity and Efficacy of 4-IBP-Derived Benzyl Piperidines 17a-c, 18a-c, and 14a-c<sup>a</sup></figcaption></figure>

<table><thead><tr><th></th><th>R</th><th>subst.</th><th>IC<sub>50</sub><br>(hBChE)<br>(pIC<sub>50</sub>±SEM)</th><th>IC<sub>50</sub><br>(hAChE)<br>(pIC<sub>50</sub>±SEM)</th><th>SI<br>(IC<sub>50</sub>(hAChE)/IC<sub>50</sub>(hBChE))</th><th>S1R<br>%inhibition<br>@100 nM<br>IC<sub>50</sub><br>(pIC<sub>50</sub>±SEM)</th><th>EC<sub>50</sub> (S1R-BiP)<br>±SEM</th></tr></thead><tbody><tr><td>17a</td><td></td><td>para</td><td>2.1 nM<br>(8.7±0.04)</td><td>>2500 nM</td><td>>1190</td><td>39%</td><td>2.04±0.61 µM</td></tr><tr><td>17b</td><td>o<sup>-</sup><sub>NH</sub><br>l<sup>+</sup><sub>l</sub></td><td>meta</td><td>12 nM<br>(7.9±0.05)</td><td>>2500 nM</td><td>>208</td><td>34%</td><td>2.61±0.97 µM</td></tr><tr><td>17c</td><td></td><td>ortho</td><td>41 nM<br>(7.4±0.04)</td><td>490 nM<br>(6.3±0.20)</td><td>12</td><td>34%</td><td>1.08±0.52 µM</td></tr><tr><td>18a</td><td></td><td>para</td><td>3.4 nM<br>(8.5±0.01)</td><td>550 nM<br>(6.6±0.06)</td><td>162</td><td>17%</td><td>5.15±0.46 µM</td></tr><tr><td>18b</td><td>o<sup>-</sup><sub>NH</sub><br>l<sup>+</sup><sub>l</sub></td><td>meta</td><td>5.5 nM<br>(8.3±0.01)</td><td>498 nM<br>(6.3±0.06)</td><td>91</td><td>33%</td><td>antagonist</td></tr><tr><td>18c</td><td></td><td>ortho</td><td>3.3 nM<br>(8.5±0.01)</td><td>148 nM<br>(6.8±0.05)</td><td>45</td><td>64%<br>IC<sub>50</sub> = 25 nM<br>(7.6±0.05)</td><td>99±21 nM</td></tr><tr><td>14a</td><td></td><td>para</td><td>454 nM<br>(6.3±0.06)</td><td>>2500 nM</td><td>>5.5</td><td>4%</td><td>>10 µM</td></tr><tr><td>14b</td><td>HO<sup>-</sup></td><td>meta</td><td>413 nM<br>(6.4±0.03)</td><td>>2500 nM</td><td>>6.0</td><td>26%</td><td>1.35±0.29 µM</td></tr><tr><td>14c</td><td></td><td>ortho</td><td>269 nM<br>(6.6±0.03)</td><td>>2500 nM</td><td>>9.3</td><td>79%<br>IC<sub>50</sub> = 20 nM<br>(7.7±0.06)</td><td>279±65 nM</td></tr><tr><td>Tacrine</td><td></td><td></td><td>52 mM<br>(7.3±0.02)</td><td>123 nM<br>(6.91±0.04)</td><td>2.4</td><td>n.d.</td><td>n.d.</td></tr><tr><td>PRE-084</td><td></td><td></td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>44%<br>253 nM</td><td>426 nM<br>(6.4 ± 0.02)</td></tr></tbody></table>

<sup>a</sup>n.d. not determined. Inhibition on *hChE* was determined as triplicates after 20 min of incubation using the Ellman's assay. *IC*<sub>50</sub> values were calculated through a sigmoidal regression using GraphPad Prism 10.<sup>30,35,55</sup> Tacrine was used as a positive control.<sup>59,60</sup> S1R affinity was measured by displacement of radioligand [<sup>3</sup>H](+)-pentazocine using membranes prepared from Wistar rat liver.<sup>56-58</sup> **PRE-084** was used as a positive control.<sup>61</sup> S1R-BiP dissociation was measured in S1R-overexpressing CHO cells by immunoprecipitation using a S1R-GFP tag and BiP dosage by Elisa.<sup>62</sup> **PRE-084** was used as a positive control.<sup>63</sup>
<figure><img src="image_15.png" /><figcaption>**Figure 2.** Kinetic studies of carbamate **18c**. Left: Time-dependent *hBChE* inhibition of azetidine carbamate **18c**. Right: Time-dependent reconstitution of the *hBChE* activity after inhibition with azetidine carbamate **18c**. Results are shown as means ± SD of three independent experiments.<sup>30</sup></figcaption></figure>

## 2.2. Enzyme Inhibition

The synthesized compounds were evaluated for their ChE activity using the colorimetric Ellman's assay, applying human isoforms, *hBChE* and *hAChE* (Tables 1 and 2). The results revealed a correlation between *hBChE* inhibition and the length of the aliphatic linker connecting carbamate-substituted phenol to the piperidine moiety. Fused compound 4 and derivative **15a**, both featuring a short aliphatic chain, exhibited no activity on both *hChEs* at 2.5 µM, whereas the more flexible compound **16a** demonstrated selective *hBChE* inhibition with an *IC*<sub>50</sub> value of 701 nM (Table 1). Derivative **17a**, bearing the largest aliphatic chain, possessed the lowest *IC*<sub>50</sub> of 2.1 nM on *hBChE* and lacked activity on *hAChE* at 2.5 µM (Table 2). The substitution pattern among the heptyl carbamates **17a-c** showed a clear tendency in *hBChE* activity (Table 2). While *ortho*-substituted compound **17a** had the lowest *IC*<sub>50</sub> value, the *IC*<sub>50</sub> increased from 12 nM for *meta*-substituted compound **17b** to 41 nM for *ortho* substituted compound **17c**. Inhibition of *hAChE* at concentrations below 2.5 µM was observed only for compound **17c**, with an *IC*<sub>50</sub> of 490 nM, resulting in a selectivity index (SI) of 12. All the azetidine carbamates **18a-c** possessed high activity on *hBChE* with *IC*<sub>50</sub> values in a narrow range of 3.3 to 5.5 nM (Table 2). On *hAChE*, the inhibitory activity of the azetidines **18a-c** increased from *para* to *ortho* substitution, with *IC*<sub>50</sub> values of 550, 498, and 148 nM, respectively. Overall, azetidine carbamates **18a-c** tended to show higher activities toward both *hChEs* compared to heptyl analogues **17a-c**, consistent with the previously proposed higher selectivity of heptyl carbamates. Nevertheless, the selectivity of compounds **18a-c** is acceptable with SI values of 45–162. Given the pronounced inhibition of *hChEs* by carbamates **17a-c** and **18a-c**, the potential activity of their corresponding phenolic cleavage product **14a-c** after carbamate transfer was also examined (Table 2). For phenols **14a-c**, decreasing *IC*<sub>50</sub> values on *hBChE* from *para* to *ortho* substitution were obtained with 454 nM, 148 nM, and 269 nM for **14a**, **14b**, and **14c**, respectively. On *hAChE*, no inhibition was observed at 2.5 µM. Conclusively, the pseudoirreversible carbamates **17a-c** and **18a-c** not only act as selective *hBChE* inhibitors but also release the reversible *hBChE* inhibitors **14a-c** after successful carbamate transfer.

## 2.3. S1R Binding and Activation

The evaluation of S1R affinity was determined by a [³H](+)-pentazocine binding assay using membrane preparations from male Wistar rats. First, displacement of the radioligand was

measured at drug concentrations of 10 µM and 100 nM (Tables 1 and 2).<sup>56–58</sup> The results indicated that the 4-IBP scaffold loses S1R affinity upon attachment of a large heptyl carbamate moiety to the terminal phenyl ring, as evidenced by the decrease in radioligand displacement from 94% for 4-IBP to only 2% for fused compound 4 at 100 nM each (Table 1). For derivatives bearing one or two additional methylene units within the aliphatic chain, reduced displacement values of 67% at 10 µM for **15a** and 1% at 100 nM for **16a** were observed (Table 1). Further elongation of the aliphatic chain restored affinity, as compound **17a** demonstrated 39% radioligand displacement at 100 nM (Table 2). For the heptyl carbamates, variations in the substitution pattern on the phenyl ring exerted only minor effects on S1R affinity, with compounds **17b**, **17c** exhibiting 34% radioligand displacement at 100 nM (Table 2). In contrast, the corresponding azetidine carbamates **18a-c** displayed a clear trend of increasing the S1R affinity from *para* to *ortho* substitution, as reflected in displacement values of 17, 33, and 64% at 100 nM, respectively. The same trend was observed for the corresponding phenols **14a-c**, which exhibited S1R affinity with 4, 26, and 79%, respectively, displacement at 100 nM (Table 2). Overall, azetidine carbamate **18c** and its corresponding phenol **14c** displayed the highest S1R affinities at 100 nM concentration. Subsequent determination of the full dose-response curve gave *IC*<sub>50</sub> values of 25 nM for **18c** and 20 nM for **14c**, respectively.

For the most promising carbamates **17a-c**, **18a-c**, and their phenols **14a-c**, S1R activation was determined by immunoprecipitation assay using S1R-expressing Chinese hamster ovary (CHO) cells and measuring the binding immunoglobulin protein (BiP)-dissociation through S1R activation (Table 2). All heptyl carbamates acted as S1R agonists, with similar *EC*<sub>50</sub> values of 2.04 and 2.61 µM for *para* and *meta*-substituted derivatives **17a** and **17b**, respectively, and 1.08 µM for the *ortho*-substituted compound **17c**. For azetidines, an *EC*<sub>50</sub> value of 5.15 µM in BiP-dissociation was obtained for **18a**, whereas the *meta*-substituted compound **18b** acted as an antagonist. Azetidine **18c** showed the overall lowest *EC*<sub>50</sub> value with 99 nM. Phenols **14a-c** demonstrated different *EC*<sub>50</sub> values ranging from 10 µM to 279 nM for *ortho*-substituted phenol **14c**.

Comparing all compounds' in vitro data, azetidine carbamate **18c** emerged as the most promising compound showing the best in vitro profile as a multitarget-directed ligand with low nanomolar values on pseudoirreversible *hBChE* inhibition and S1R activation (Table 2). Moreover, the
<figure><img src="image_16.png" /><figcaption>**Figure 3.** (a) Docking poses of agonists **14c** (light green) and **18c** (dark cyan) on human S1R. (b) Docking pose of agonist 4-IBP on S1R (green) in comparison to the crystal structure (orange; PDB code: 5HK2).<sup>48</sup></figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 4.** Docking poses of **14c** and **18c** on *h*ChE. The catalytic triad is marked in pink, the oxyanion hole in yellow, and the acyl binding pocket in orange. (a) Covalent docking pose of carbamate **18c** (dark cyan) and noncovalent docking pose of phenol **14c** (light green) in *h*BChE. (b) Covalent docking pose of carbamate **18c** (dark cyan) in *h*AChE.</figcaption></figure>

281 corresponding phenol **14c** released after carbamate transfer

282 retained significant pharmacological activity on both targets.

## 2.4. Kinetic Investigations of *h*BChE Inhibition Mode

283 To confirm the assumed pseudoirreversible inhibition (Figure

284 1b) of carbamate **18c**, the covalent binding and cleavage of the

285 carbamate on the enzyme's active site serine was investigated

286 (Figure 2). Carbamate bond formation was determined after

287 various incubation times for different inhibitor concentrations.

288 A time-dependent change of enzyme inhibition was obtained,

289 and Michaelis–Menten kinetics applied as described by Hosie

290 et al.<sup>64</sup> and Feaster et al.<sup>55,65</sup> to give a low nanomolar

291 equilibrium constant *K*<sub>i</sub> of 9.0 nM and a carbamylation rate

292 constant *k*<sub>3</sub> of 1.51 min<sup>-1</sup>, representing a comparatively fast

293 carbamylation (Figure 2). Reconstitution of enzyme activity

294 through hydrolysis was performed through dilution experi-

295 ments using high inhibitor concentrations during the *h*BChE

296 incubation. Subsequent dilution then leads to covalent bond

297 hydrolysis, gradually reconstituting the enzyme activity with a

298 half-life time of 80 min and a cleavage constant *k*<sub>4</sub> of 0.009

299 min<sup>-1</sup> for compound **18c** (Figure 2).

## 2.5. Computational Studies

300 To explore the binding modes of compound **18c** and its

301 hydrolysis product **14c**, molecular docking studies were

conducted on S1R and *h*BChE/*h*AChE (Figures 3 and 4).

The PDB structures 5HK1 of S1R (resolution 2.51 Å), 6QAA

of *h*BChE* (resolution 1.90 Å), and 4M0E of *h*AChE

(resolution 2.00 Å) were used as in our previous work.<sup>29,66</sup>

The docking was performed using Schrödinger's Glide

(v10.1).<sup>67,68</sup>

Studies on the S1R show similar binding modes for S1R

308 agonists **14c** and **18c**, which align with the docking pose and

309 crystal structure of the reference S1R agonist 4-IBP (Figure

310 3).<sup>48</sup> The central interaction of all compounds is the formation

311 of a hydrogen bond between the basic amine of the benzyl

312 piperidine and Glu172. Referring to Glennon's model, this

313 interaction with Glu172 is crucial for binding.<sup>50</sup> The phenyl

314 substituent of the benzyl piperidine is located in the smaller,

315 secondary hydrophobic pocket and exhibits a T-shaped “edge-

316 to-face” stacking with His154. The aliphatic amide chain and

317 the substituted phenyl ring of all structures occupy the larger

318 primary hydrophobic pocket. Furthermore, pi-stacking inter-

319 actions between the phenyl ring and Tyr206 are observed for

320 **14c** and **18c**, leading to an improved docking score compared

321 to 4-IBP. For phenol **14c**, additional hydrogen-bond formation

322 to Thr181 occurs, which matches the slightly higher S1R

323 affinity of phenol **14c** observed in vitro.

302

f3f4

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<figure><img src="image_18.png" /><figcaption>**Figure 5.** Effects of phenol 14c on N9 microglia cell activation after LPS (100 ng/mL) treatment. Cells were treated with 1, 2.5, and 5 µM of 14c. Expression of the phenotypic markers NOS2 (b), TREM2 (c), TGFβ2 (d), and IL1β (f) was evaluated by Western blot (a, e); GAPDH and Ponceau were used for cell lysates and media endogenous normalization, respectively. Nitrite release was quantified using the Griess test (g). All quantitative data are presented as means ± SEM from at least three independent experiments. Statistical significance between different treatments</figcaption></figure>
Figure 5. continued

was calculated by using one-way analysis of variance (ANOVA) followed by post hoc comparison through Bonferroni's test. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to LPS-activated microglia; *p < 0.05, **p < 0.01, and ***p < 0.001, compared to control.

For the docking on ChEs, two distinct docking protocols were applied due to the compound's pseudoirreversible and reversible modes of inhibition, respectively (Figure 4). For pseudoirreversible inhibitor **18c**, the covalent tetrahedral intermediate, formed by reaction of the catalytic serine with the carbamate carbonyl in accordance with the reaction mechanism, was modeled. In contrast, noncovalent docking was conducted for reversible inhibitor **14c** on ChEs.

On *hBChE*, a folded position of molecule **18c** is observed (Figure 4a). The basic amine of the benzyl piperidine forms a hydrogen bond with Asp70, which is known to play a key role in the association of positively charged ligands,⁶⁹ whereas the attached benzyl group shows pi-stacking interactions with Trp82. The substituted phenyl moiety on the other side of the molecule occupies the hydrophobic acyl binding pocket. For phenol **14c**, a pose similar to that of **18c** is observed (Figure 4a). As for inhibitor **18c**, a hydrogen bond between the piperidine and Asp70 is formed, pi-stacking interactions of the benzyl group and Trp82 occur, and the substituted phenyl moiety occupies the acyl binding pocket. Since the same interaction motifs are observed in both covalent and noncovalent docking, the binding mode can be attributed to good noncovalent interactions in the binding pocket, which are associated with high affinity, and not only to the covalent reaction with the catalytic serine.

On *hAChE*, the binding of compound **18c** is characterized by hydrogen-bond formation between the amide nitrogen and Tyr124 (Figure 4b). Moreover, the protonated nitrogen of the piperidine moiety stabilizes the pose via cation-pi-stacking to Trp286, while the azetidine on the other side of the molecule occupies the acyl binding pocket. These strong interaction patterns contribute to compound **18c** observed activity on *hAChE*.

## 2.6. Microglia Activation

To evaluate the immunomodulatory potential of lead compound **18c** and its corresponding phenol **14c**, their effects on LPS-induced microglial activation were investigated. Activated microglia can be classified into two phenotypic states: the classically activated or pro-inflammatory M1 phenotype, in response to infection or tissue injury and is characterized by the release of pro-inflammatory mediators such as Interleukin-1 beta (IL1β), NO and nitric oxide synthase (NOS2) expression; and the alternatively activated or anti-inflammatory M2-phenotype, typically associated with tissue repair and neuroprotection, marked by the release of transforming growth factor-β2 (TGF-β2) and the expression of triggering receptor expressed on myeloid cells 2 (TREM2).⁷⁰,⁷¹ For this purpose, murine N9 microglial cells were stimulated with LPS (100 ng/mL) for 24h in the presence of increasing concentrations (1–5 µM) of compounds **14c** and **18c** (Figures 5 and 6). Phenotypic markers were analyzed by Western blot, and nitrite accumulation was measured using the Griess assay.

Treatment with phenol **14c** markedly reduced NOS2 expression at 2.5 and 5 µM, reaching levels comparable to those of untreated cells (Figure 5b). Similarly, **14c** significantly decreased the level of release of IL1β and nitrite at 5 µM

(Figure 5f,g), while showing no significant effect on the anti-inflammatory markers TREM2 and TGF-β2 up to 5 µM (Figure 5c,d). Carbamate **18c** exhibited a stronger and dose-dependent reduction in the release of pro-inflammatory mediators, including IL-1β and NO (Figure 6b,f,g), together with a significant downregulation of NOS2 expression at 2.5 µM and 5 µM (Figure 6b). In contrast, **18c** did not significantly affect the expression of anti-inflammatory markers TREM2 or TGF-β2 (Figure 6c,d).

Overall, both compounds displayed a similar immunomodulatory profile, attenuating the LPS-induced pro-inflammatory activation of microglia while preserving anti-inflammatory and homeostatic functions.

## 2.7. Lack of Neurotoxicity

To exclude the potential neurotoxicity of hybrid molecule **18c** and its phenol **14c**, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using murine hippocampal neuronal (HT22) cells was used. For compounds **18c** and **14c**, no neurotoxicity up to a concentration of 10 µM was observed (Figure 7).

399 f7

## 2.8. Solubility

Furthermore, the hydrochloric salt of compound **18c**, showing the best in vitro results, was prepared for in vivo application using HCl in Et₂O. The solubility of the salts in phosphate-buffered saline (PBS) was assessed using the continuous shake protocol.⁷² Analysis was conducted with high-performance liquid chromatography (HPLC) followed by UV quantification. The carbamate **18c·HCl** possessed a solubility of 62 µg/mL.

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406

## 2.9. In Vivo Studies

The neuroprotective effects of the compound **18c** were evaluated in the established Aβ mouse model, which was previously utilized for studies on both S1R agonists and BChE inhibitors.³¹,³⁵,⁶³ Male mice were used to minimize variability associated with hormonal fluctuations across the estrous cycle, which can affect behavioral readouts.⁷³ Oligomeric Aβ₂₅⁻₃₅ peptide was administered intracerebroventricularly (ICV) in Swiss-OF1 mice on day 1. Mice were treated intraperitoneally once a day with the compounds dissolved in 10% dimethyl sulfoxide (DMSO) in saline between days 1 and day 7. Effects on short-term memory of the mice were assessed on day 8 using spontaneous alternation in the Y-maze. Long-term memory was tested on day 9 (training) and day 10 (test) using the step-through type passive avoidance test (Figure 8).

421 f8

A significant attenuation of Aβ₂₅⁻₃₅-induced deficits in spontaneous alternation was observed in a dose range of 0.3–1 mg/kg for hybrid compound **18c** (Figure 9a). Additionally, the compound exhibited beneficial effects on Aβ₂₅⁻₃₅-induced impairments in the passive avoidance test. Here, hybrid compound **18c** showed disease-modifying effects at doses of 0.3 mg/kg (Figure 9b). In summary, compound **18c** ameliorated Aβ₂₅⁻₃₅-induced learning impairments in both short- and long-term memory.

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430
<figure><img src="image_19.png" /><figcaption>**Figure 6.** Effects of azetidine carbamate **18c** on N9 microglia cell activation after LPS (100 ng/mL) treatment. Cells were treated with 1, 2.5, and 5 µM of compound **18c**. Expression of the phenotypic markers NOS2 (b), TREM2 (c), TGFβ2 (d), and IL1β (f) was evaluated by Western blot (a, e); GAPDH and Ponceau were used for cell lysates and media endogenous normalization, respectively. Nitrite release was quantified using the Griess test (g). All quantitative data are presented as means ± SEM from at least three independent experiments. Statistical significance between different treatments was calculated by using one-way analysis of variance (ANOVA) followed by post hoc comparison through Bonferroni's test. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to LPS-activated microglia; *p < 0.05, **p < 0.01, and ***p < 0.001, compared to control.</figcaption></figure>

### 3. CONCLUSION

431 In this study, we developed and characterized a series of novel

432 benzylpiperidine-based multitarget ligands using the S1R

433 agonist 4-IBP as a template to introduce hBChE inhibitory

activity for potential therapeutic treatment of AD. Among the 434 synthesized compounds, the pseudoirreversibly acting inhibitor 435 **18c** emerged as the most promising compound with 436 nanomolar dual-target activity in vitro at both targets, releasing 437
<figure><img src="image_20.png" /><figcaption>**Figure 7.** Lack of neurotoxicity of compounds **14c** and **18c** in the MTT assay using neuronal HT22 cells. Results are shown as means ± SEM of three independent experiments. Viability of treated cells was compared with untreated control cells (100%). ANOVA: *F*<sub>(5,15)</sub> = 0.901; *p* < 0.0001 for **14c**; *F*<sub>(6,13)</sub> = 0.993; *p* < 0.0001 for **18c**. Levels of significance: **** *p* < 0.0001.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Figure 8.** Schematic application protocol for the Aβ mouse model. Reproduced from ref. 54. Available under a CC-BY 4.0 license. Copyright © 2025 Reichau et al.</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 9.** Effects of compound **18c** (a, b) on Aβ<sub>25-35</sub> induced learning impairments in mice. (a) Spontaneous alternation performance in the Y-maze and (b) retention latency in the passive avoidance test. Mice were treated with Aβ<sub>25-35</sub> (9 nmol, intracerebroventricularly (ICV)) or vehicle (3 µL of ddH<sub>2</sub>O, V) on day 1, then received either vehicle (DMSO 10% in saline) or the compound (0.1–1 mg/kg) intra-peritoneally (IP) between day 1 and 7. Mice were then tested for spontaneous alternation on day 8 and passive avoidance on days 9 (training) and 10 (test). Group sizes are shown beneath each bar graph. Data show ± SEM (a) or median and interquartile range (b). ANOVA: *F*<sub>(4,90)</sub> = 10.45; *p* < 0.0001 in (a); Kruskal–Wallis ANOVA: *H* = 18.66, *p* = 0.0009 in (b). ** *p* < 0.01, *** *p* < 0.001 vs V-treated group; # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs Aβ<sub>25-35</sub>-treated group; Dunnett's test in (a); Dunn's test in (b).</figcaption></figure>

the less active phenol **14c** after carbamate transfer to the
enzyme. Similar binding poses of lead compound **18c** and its
**438** corresponding phenol **14c** were found on both targets in
**440** molecular docking studies. Further in vitro studies demon-
**441** strated that both compounds **18c** and **14c** reduce pro-
**442** inflammatory microglial activation without affecting anti-
**443** inflammatory markers, suggesting beneficial immunomodula-
**444** tory effects in AD. In vivo, lead compound **18c** significantly

ameliorated Aβ<sub>25-35</sub>-induced cognitive deficits in mice at low dosages of 0.1–1 mg/kg, supporting its neuroprotective efficacy. Since the present study is limited to investigations into murine cells and in vivo models, species-specific differences between murine and human systems cannot be excluded. Therefore, further studies aim at validating the neuroprotective and immunomodulatory effects of hBChE/S1R dual ligands in human-derived cellular models. Compared to therapies using combinations of separate hBChE inhibitors and S1R agonists, multitarget-directed ligands such as compound **18c** offer several potential advantages, including a reduced risk of drug-drug interactions, simplified dose regimes, more predictable pharmacokinetics, and possible improved patient compliance.<sup>34,74,75</sup> Overall, compound **18c** emerged as a promising multitarget ligand, combining selective hBChE inhibition with potent S1R agonism and immunomodulatory activity, illustrating dual hBChE/S1R modulation as a rational approach to mitigate cholinergic and inflammatory dysfunction in Alzheimer's disease.<sup>464</sup>

4. METHODS

**4.1. General Information**

General experimental information was compiled as previously 465
described.⁵⁴ All reagents were purchased from Sigma-Aldrich (St. 466
Louis, Missouri), ABCR (Karlsruhe, Germany), BLDpharm (Shang- 467
hai, China), and enamine (Kyiv, Ukraine) and used without further 468
purification. N,N-dimethylformamide (DMF) and CH₂Cl₂ used as 469
reaction solvents were purchased as dry solvents with a safe seal 470
(Sigma-Aldrich, St. Louis, Missouri) and used under an argon 471
atmosphere. Thin-layer chromatography was performed on silica gel 472
60 alumina foils with a fluorescent indicator (254 nm) (Macherey 473
Nagel GmbH & Co. KG, Düren, Germany). Spots were detected by 474
UV light (254 and 366 nm) or through staining with ninhydrin or 475
KMnO₄. Normal phase purification was performed using a silica gel 476
with a particle size of 40–63 µm (Macherey Nagel GmbH & Co. KG, 477
Düren, Germany) as the stationary phase and PE/EtOAc or CH₂Cl₂/ 478
MeOH as the liquid phase with optional addition of 1% Et₃N. Reverse 479
phase purification was performed using flash column chromatography 480
with C18 silica gel with 30 µm particle size (Büchi, Flawil, 481
Switzerland) as the stationary phase and water/MeOH with 0.1% 482
formic acid (FA) or water/MeCN with 0.1% FA as the liquid phase. 483
Nuclear magnetic resonance spectra were measured with a Bruker 484
AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in 485
deuterated solvents (DMSO; CDCl₃, CD₃OD, and D₂O). Chemical 486
shifts are expressed in parts per million (ppm) relative to the 487
respective solvent residues. Purity was determined by HPLC 488
(Shimadzu Products), containing aDGU-20A3R degassing unit, an 489
LC20AB solvent delivery unit, and aSPD-20A UV/vis detector. UV 490
detection was measured at 254 nm. Mass spectra were obtained by a 491
492 liquid chromatography-mass spectrometry 2020 (Shimadzu Products). As a stationary phase, a Synergi 4U fusion-RP (150 × 4.6 mm) 493 column (Synergi, Aschaffenburg, Germany) was used, and as a mobile 494 phase, a gradient of H₂O/MeOH with 0.1% FA was used. Parameters: 495 A = water, B = MeOH, V(B)/[V(A) + V(B)] = from 5 to 90% over 496 10 min, V(B)/[V(A) + V(B)] = 90% for 5 min, and V(B)/[V(A) + 497 V(B)] = from 90 to 5% over 3 min. The method was performed with a 498 flow rate of 1.0 mL/min. Compounds were only used for biological 499 evaluation if the purity was ≥95%. 500

## 4.2. Chemistry. General Procedure for Amide Formation (GP-A)

501 Acid (1.0 equiv) was dissolved in DMF (0.1 M), and HBTU (1.2 502 equiv), Et₃N (3.0 equiv), and the corresponding amine (2.0 equiv) 503 were added successively. The reaction mixture was stirred for 16 h at 504 rt. The reaction was terminated by the addition of water. The aquatic 505 layer was basified with aq. KOH (1 M, pH 11) and extracted with 506 EtOAc. The combined organic layers were washed with brine, dried 507 over Na₂SO₄, and filtered, and the solvent was removed under 508 reduced pressure. If necessary, the product was purified by column 509 chromatography. 510

## 4.3. General Procedure for Methyl Ether Cleavage (GP-B)

511 The procedures described below were performed as previously 512 reported.⁵⁴ Methyl ether (1.0 equiv) was dissolved in CH₂Cl₂ (0.1 513 M), and BBr₃ (4.0 eq., 1 M in CH₂Cl₂) was added dropwise. The 514 reaction mixture was warmed to rt and stirred for 1 h. The reaction 515 was terminated by the addition of sat. NaHCO₃. The aquatic layer 516 was extracted with CH₂Cl₂. The combined organic layers were dried 517 over Na₂SO₄, filtered, and the solvent was removed under reduced 518 pressure. The crude product was obtained and used without further 519 purification. 520

## 4.4. General Procedure for Heptyl Carbamate Formation (GP-C)

521 The procedures described below were performed as previously 522 reported.⁵⁴ Phenol (1.0 equiv) was dissolved in CH₂Cl₂ (0.04 M), 523 heptyl isocyanate (1.5 equiv) and Et₃N (1.5 equiv) were added 524 successively. The reaction mixture was stirred for 16 h at rt. The 525 solvent was removed under reduced pressure, and the crude product 526 was loaded onto Celite. The product was obtained after purification 527 by reversed-phase flash column chromatography. 528

## 4.5. General Procedure for Azeditine Carbamate Formation (GP-D)

529 The procedures described below were performed as previously 530 reported.⁵⁴ Phenol (1.0 equiv) was dissolved in DMF (0.02 M), and 531 Cs₂CO₃ (2.0 equiv) and azetidine (2.0 equiv) were added 532 successively. The reaction mixture was stirred at 55 °C for 16 h. 533 The reaction was terminated by the addition of water. The aquatic 534 layer was extracted with EtOAc. The combined organic layers were 535 washed with brine, dried over Na₂SO₄, filtered, and the solvent was 536 removed under reduced pressure. The product was obtained after 537 purification by reverse-phase flash column chromatography. 538

539 **4.5.1. N-(1-Benzylpiperidin-4-yl)-4-methoxybenzamide (2).** 540 I₂ (542 mg, 2.13 mmol, 1.3 equiv) was dissolved in CH₂Cl₂ (7 mL). 541 PPh₃ (560 mg, 2.13 mmol, 1.3 equiv) and 1-benzylpiperidin-4-amine 542 (750 mg, 2.46 mmol, 1.5 equiv) were added successively at 0 °C. The 543 reaction mixture was stirred for 5 min, then 4-methoxybenzoic acid 544 (250 mg, 1.64 mmol, 1.0 equiv) was added and stirred for another 5 545 min before Et₃N (684 µL, 4.92 mmol, 2.0 equiv) was added. The 546 reaction mixture was warmed to rt and stirred for 48 h. The reaction 547 was terminated by the addition of CH₂Cl₂ (50 mL) and aq. NaOH 548 (50 mL, 1 M). The organic layer was washed with aq. NaOH (3 × 40 549 mL, 1 M), and then with aq. HCl (3 × 50 mL, 1 M). The acidic layer 550 was collected and basified with aq. NaOH (30%) to pH 14 and 551 extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers 552 were dried over Na₂SO₄, filtered, and the solvent was removed under 553 reduced pressure. The product 2 (159 mg, 0.49 mmol, 30%) was 554 obtained as a colorless solid after purification by column 555 chromatography (CH₂Cl₂/MeOH 3% + 0.5% Et₃N).⁷⁶ ¹H NMR

(400 MHz, CDCl₃) δ = 7.64 (d, J = 8.8 Hz, 2H), 7.25-7.23 (m, 2H), 555 7.24-7.17 (m, 3H), 6.83 (d, J = 8.8 Hz, 2H), 5.88 (d, J = 7.8 Hz, 556 1H), 3.99-3.86 (m, 1H), 3.76 (s, 3H), 3.47 (s, 2H), 2.84-2.75 (m, 557 2H), 2.19-2.05 (m, 2H), 2.01-1.89 (m, 2H), 1.59-1.44 (m, 2H) 558 ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 166.4, 162.2, 138.2, 129.3, 559 128.8, 128.4, 127.3, 127.2, 113.8, 63.1, 55.5, 52.4, 47.0, 32.4 ppm. 560 ESI-MS: *m/z* calculated for [M + H]⁺: 325.18, found: 325.15. m.p. = 561 175 °C. 562

563 **4.5.2. N-(1-Benzylpiperidin-4-yl)-4-hydroxybenzamide (3).** 564 Compound 3 was synthesized according to GP-C from 2. The crude 565 product 3 (88 mg, 0.28 mmol, 84%) was obtained as a colorless oil 566 and was used without further purification. ¹H NMR (400 MHz, 567 DMSO) δ = 7.70 (d, J = 8.7 Hz, 2H), 7.36-7.23 (m, 5H), 6.77 (d, J = 568 8.7 Hz, 2H), 3.78-3.67 (m, 1H), 3.45 (s, 2H), 2.84-2.78 (m, 2H), 569 2.05-1.97 (m, 2H), 1.77-1.71 (m, 2H), 1.61-1.48 (m, 2H) ppm. 570 ¹³C NMR (101 MHz, DMSO) δ = 165.5, 160.0, 138.7, 129.3, 128.9, 571 128.3, 127.0, 125.5, 114.8, 62.3, 52.4, 46.9, 31.7 ppm. ESI-MS: *m/z* 572 calculated for [M + H]⁺: 311.17, found: 311.20. 573

573 **4.5.3. 4-((1-Benzylpiperidin-4-yl)carbamoyl)phenyl heptylcarbamate (4).** Compound 4 was synthesized according to GP-C 574 from 3. The product 4 (16 mg, 0.04 mmol, 27%) was obtained as a 575 colorless solid after purification by flash column chromatography as 576 (H₂O/MeOH 5-60-95% + 0.1% FA). ¹H NMR (400 MHz, 577 CD₂Cl₂) δ = 7.76 (d, J = 8.6 Hz, 2H), 7.41-7.31 (m, 5H), 7.16 (d, J 578 = 8.6 Hz, 2H), 6.47 (d, J = 8.0 Hz, 1H), 5.24 (t, J = 6.0 Hz, 1H), 579 4.08-3.97 (m, 1H), 3.77 (s, 2H), 3.28-3.19 (m, 2H), 3.15-3.05 (m, 580 2H), 2.48-2.38 (m, 2H), 2.06-1.98 (m, 2H), 1.87-1.74 (m, 2H), 581 1.61-1.53 (m, 2H), 1.38-1.27 (m, 8H), 0.94-0.86 (m, 3H) ppm. 582 ¹³C NMR (101 MHz, CD₂Cl₂) δ = 166.2, 154.3, 154.1, 135.1, 131.9, 583 130.4, 128.9, 128.6, 128.6, 121.9, 62.1, 52.1, 46.6, 41.7, 32.2, 31.1, 584 30.2, 29.4, 27.1, 23.0, 14.2 ppm. ESI-MS: *m/z* calculated for [M + 585 H]⁺: 452.18, found: 452.30. m.p. = 122 °C. 586

587 **4.5.4. N-(1-Benzylpiperidin-4-yl)-2-(4-methoxyphenyl)-acetamide (9a).** Compound 9a was synthesized according to GP-588 A from 2-(4-methoxyphenyl)acetic acid and 1-benzylpiperidin-4-589 amine. The product 9a (530 mg, 1.56 mmol, quant.) was obtained as 590 an orange solid and converted without further purification.⁷⁷ ¹H NMR (400 MHz, CDCl₃) δ = 7.34-7.19 (m, 5H), 7.13 (d, J = 8.7 591 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.42 (d, J = 8.1 Hz, 1H), 3.78 (s, 592 3H), 3.77-3.73 (m, 1H), 3.46 (s, 2H), 3.43 (s, 2H), 2.76-2.64 (m, 593 2H), 2.16-1.99 (m, 2H), 1.87-1.75 (m, 2H), 1.37-1.26 (m, 2H) 594 ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 170.8, 158.8, 138.4, 130.5, 595 129.1, 128.2, 127.0, 127.0, 114.4, 63.0, 55.3, 52.1, 46.5, 43.0, 32.1 596 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 339.20, found: 339.20. 597 m.p. = 152 °C. 598

599 **4.5.5. N-(1-Benzylpiperidin-4-yl)-2-(4-hydroxyphenyl)-acetamide (12a).** Compound 12a was synthesized according to 600 GP-B from 9a. The product 12a (355 mg, 1.09 mmol, 81%) was 601 obtained as a colorless oil and converted without further purification. 602 For the Ellman's assay, a small amount of the crude was purified by 603 flash chromatography (H₂O/MeCN 5-20-95% + 0.1% FA).⁷⁷ ¹H NMR (400 MHz, MeOD) δ = 6.91 (s, 1H), 5.94-5.84 (m, 5H), 5.53 604 (d, J = 8.5 Hz, 2H), 5.17 (d, J = 8.5 Hz, 2H), 2.55 (s, 2H), 2.35-2.22 605 (m, 1H), 1.82 (s, 2H), 1.79-1.70 (m, 2H), 1.36-1.26 (m, 2H), 606 0.51-0.41 (m, 2H), 0.26-0.10 (m, 2H) ppm. ¹³C NMR (101 MHz, 607 MeOD) δ = 174.3, 157.5, 132.4, 132.0, 131.0, 130.6, 130.1, 127.6, 610 116.3, 61.9, 52.3, 46.1, 42.9, 30.2 ppm. ESI-MS: *m/z* calculated for 611 [M + H]⁺: 325.18, found: 325.15. 612

613 **4.5.6. 4-[(1-Benzylpiperidin-4-yl)amino]-2-oxoethylphenyl heptylcarbamate (15a).** Compound 15a was synthesized 614 according to the GP-C from 12a. The product 15a (28 mg, 0.06 615 mmol, 20%) was obtained as a colorless solid after purification by 616 flash chromatography (H₂O/MeCN 5-40-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.31-7.27 (m, 4H), 7.25 (d, J = 2.0 617 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 5.27 (d, J = 618 8.0 Hz, 2H), 5.04 (t, J = 6.1 Hz, 1H), 3.86-3.70 (m, 1H), 3.51 (s, 620 2H), 3.45 (s, 2H), 3.33-3.20 (m, 3H), 2.78-2.66 (m, 2H), 2.18-621 2.00 (m, 2H), 1.88-1.79 (m, 2H), 1.64-1.52 (m, 2H), 1.40-1.26 622 (m, 10H), 0.94-0.83 (m, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ 623 = 170.5, 154.6, 150.5, 135.2, 131.8, 130.3, 129.9, 128.7, 128.1, 122.2, 624
625 62.0, 51.7, 46.0, 43.3, 41.5, 31.9, 30.9, 30.0, 29.1, 26.9, 22.7, 14.2 ppm.
626 ESI-MS: m/z calculated for [M + H]⁺: 466.31, found: 466.30.
627 m.p. = 189 °C.

628 **4.5.7.** N-((1-Benzylpiperidin-4-yl)methyl)-2-(4-630 methoxyphenyl)acetamide (10a). Compound 10a was synthesized according to GP-A from 2-(4-methoxyphenyl)acetic acid and 631 (1-benzylpiperidin-4-yl)methanamine. The product 10a (620 mg, 632 1.75 mmol, quant.) was obtained as an orange solid and converted 633 without further purification.⁷⁸ ¹H NMR (400 MHz, CDCl₃) δ = 7.33–7.24 (m, 5H), 7.15 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.6 Hz, 634 2H), 5.49 (t, J = 6.2 Hz, 1H), 3.80 (s, 3H), 3.50 (s, 2H), 3.48 (s, 2H), 635 3.08 (t, J = 6.4 Hz, 2H), 2.89–2.81 (m, 2H), 1.93 (td, J = 2.5, 11.7 636 Hz, 2H), 1.62–1.47 (m, 2H), 1.48–1.36 (m, 1H), 1.26–1.13 (m, 637 2H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 171.6, 159.0, 138.2, 638 130.6, 129.3, 128.3, 127.1, 127.1, 114.6, 63.3, 55.4, 53.3, 45.2, 43.1, 639 35.9, 29.9 ppm. ESI-MS: m/z calculated for [M + H]⁺: 353.22, found: 640 353.26. m.p. = 117 °C.

642 **4.5.8.** N-((1-Benzylpiperidin-4-yl)methyl)-2-(4-643 hydroxyphenyl)acetamide (13a). Compound 13a was synthesized 644 according to GP-B from 10a. The product 13a (386 mg, 1.14 mmol, 645 87%) was obtained as a colorless oil and was converted without 646 further purification. For the Ellman's assay, a small amount of the 647 crude was purified by flash chromatography (water/MeCN 5–20– 648 95% + 0.1% FA).⁷⁸ ¹H NMR (400 MHz, MeOD) δ = 7.46 (s, 5H), 649 7.09 (d, J = 8.1 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 4.19 (s, 2H), 3.39 650 (s, 4H), 3.11 (s, 2H), 2.95–2.82 (m, 2H), 1.91–1.79 (m, 2H), 1.79– 651 1.69 (m, 1H), 1.50–1.34 (m, 2H) ppm. ¹³C NMR (101 MHz, 652 MeOD) δ = 175.0, 169.4, 157.5, 132.2, 131.1, 130.9, 130.2, 127.6, 653 116.4, 61.5, 53.0, 44.7, 43.1, 35.2, 28.0 ppm. ESI-MS: m/z calculated 654 for [M + H]⁺: 339.20, found: 339.26.

655 **4.5.9.** 4-(2-(((1-Benzylpiperidin-4-yl)methyl)amino)-2-656 oxoethyl)phenyl heptylcarbamate (16a). Compound 16a was 657 synthesized according to GP-C from 13a. The product 16a (22 mg, 658 0.05 mmol, 16%) was obtained as a colorless oil after purification by 659 flash chromatography (water/MeCN 5–40–95% + 0.1% FA). ¹H 660 NMR (400 MHz, CDCl₃) δ = 7.37–7.28 (m, 5H), 7.22 (d, J = 8.2 661 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H), 5.74 (s, 1H), 5.07 (t, J = 6.0 Hz, 662 1H), 3.70 (s, 2H), 3.53 (s, 2H), 3.25 (q, J = 6.7 Hz, 2H), 3.12–2.99 663 (m, 4H), 2.15 (t, J = 11.6 Hz, 2H), 1.66–1.50 (m, 5H), 1.47–1.23 664 (m, 10H), 0.89 (t, J = 6.5 Hz, 3H) ppm. ¹³C NMR (101 MHz, 665 CDCl₃) δ = 171.5, 167.3, 154.6, 150.5, 132.0, 130.9, 130.4, 129.3, 666 129.0, 122.3, 60.8, 51.6, 44.3, 43.1, 41.5, 34.1, 31.9, 29.9, 29.1, 27.0, 667 26.8, 22.7, 14.2 ppm. ESI-MS: m/z calculated for [M + H]⁺: 480.31, 668 found: 480.30.

669 **4.5.10.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(4-670 methoxyphenyl)acetamide (11a). Compound 11a was synthesized according to GP-A from 2-(4-methoxyphenyl)acetic acid and 2-671 (1-benzylpiperidin-4-yl)ethan-1-amine. The product 11a (305 mg, 672 0.83 mmol, 91%) was obtained as an orange solid and was used 673 without further purification. ¹H NMR (400 MHz, CDCl₃) δ = 7.32– 674 7.27 (m, 4H), 7.25–7.21 (m, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.88 (d, J 675 8.7 Hz, 2H), 5.27 (s, 1H), 3.80 (s, 3H), 3.49 (s, 2H), 3.47 (s, 2H), 676 3.25–3.17 (m, 2H), 2.87–2.82 (m, 2H), 1.96–1.84 (m, 2H), 1.61– 677 1.54 (m, 2H), 1.38–1.29 (m, 3H), 1.27–1.22 (m, 1H), 1.21–1.16 678 (m, 2H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 171.4, 158.9, 138.4, 679 130.6, 129.3, 128.2, 127.1, 127.0, 114.5, 63.5, 55.3, 53.7, 43.0, 38.7, 680 38.7, 36.2, 33.6, 32.2 ppm. ESI-MS: m/z calculated for [M + H]⁺: 681 367.23, found: 367.25. m.p. = 89 °C.

683 **4.5.11.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(3-684 methoxyphenyl)acetamide (11b). Compound 11b was synthesized 685 sized according to GP-A from 2-(3-methoxyphenyl)acetic acid and 2-686 (1-benzylpiperidin-4-yl)ethan-1-amine. The product 11b (309 mg, 687 0.84 mmol, 92%) was obtained as an orange solid and was used 688 without further purification. ¹H NMR (400 MHz, CDCl₃) δ = 7.34– 689 7.28 (m, 5H), 7.25–7.23 (m, 1H), 6.86–6.75 (m, 3H), 5.30 (s, 1H), 690 3.80 (s, 3H), 3.53 (s, 2H), 3.47 (s, 2H), 3.28–3.15 (m, 2H), 2.87– 691 2.81 (m, 2H), 1.92–1.83 (m, 2H), 1.70–1.62 (m, 2H), 1.62–1.55 692 (m, 2H), 1.39–1.31 (m, 2H), 1.23–1.16 (m, 2H) ppm. ¹³C NMR 693 (101 MHz, CDCl₃) δ = 170.8, 160.0, 138.3, 136.6, 130.0, 129.2, 694 128.1, 127.0, 121.6, 115.0, 112.7, 63.4, 55.2, 53.7, 43.9, 37.5, 36.1,

33.5, 32.1 ppm. ESI-MS: m/z calculated for [M + H]⁺: 367.23, found: 695 367.25. m.p. = 91 °C.

696 **4.5.12.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(2-697 methoxyphenyl)acetamide (11c). Compound 11c was synthesized according to GP-A from 2-(2-methoxyphenyl)acetic acid and 2-698 (1-benzylpiperidin-4-yl)ethan-1-amine. The product 11c (316 mg, 700 0.86 mmol, 94%) was obtained as an orange solid and was used 701 without further purification. ¹H NMR (400 MHz, CDCl₃) δ = 7.28– 702 7.26 (m, 3H), 7.25–7.22 (m, 2H), 7.22–7.20 (m, 1H), 7.18 (dd, J = 703 1.7, 7.5 Hz, 1H), 6.90 (td, J = 1.1, 7.5 Hz, 1H), 6.85 (dd, J = 1.1, 8.2 704 Hz, 1H), 5.67 (t, J = 5.8 Hz, 1H), 3.79 (s, 3H), 3.51 (s, 2H), 3.44 (s, 705 2H), 3.22–3.12 (m, 2H), 2.84–2.77 (m, 2H), 1.87–1.80 (m, 2H), 706 1.58–1.48 (m, 2H), 1.36–1.26 (m, 2H), 1.23–1.11 (m, 3H) ppm. 707 ¹³C NMR (101 MHz, CDCl₃) δ = 171.1, 157.1, 138.3, 131.3, 129.2, 708 128.8, 128.1, 126.9, 123.9, 121.1, 110.7, 63.4, 55.5, 53.7, 38.8, 37.2, 709 36.1, 33.4, 32.1 ppm. ESI-MS: m/z calculated for [M + H]⁺: 367.23, 710 found: 367.25. m.p. = 94 °C.

711 **4.5.13.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(4-712 hydroxyphenyl)acetamide (14a). Compound 14a was synthesized 713 according to GP-B from 11a. The product 14a (200 mg, 0.56 mmol, 714 92%) was obtained as an orange oil and was converted without further 715 purification. For the Ellman's assay, a small amount of the crude was 716 purified by flash chromatography (water/MeCN 5–95% + 0.1% FA). 717 ¹H NMR (400 MHz, CDCl₃) δ = 8.66 (s, 1H), 7.41–7.33 (m, 5H), 718 7.04 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 5.55 (t, J = 5.9 Hz, 719 1H), 3.84 (s, 2H), 3.44 (s, 2H), 3.19 (q, J = 6.7 Hz, 2H), 3.15–3.08 720 (m, 2H), 2.30–2.21 (m, 2H), 1.65–1.55 (m, 2H), 1.47–1.38 (m, 721 2H), 1.37–1.30 (m, 2H), 1.27–1.18 (m, 1H) ppm. ¹³C NMR (101 722 MHz, CDCl₃) δ = 172.1, 169.1, 156.5, 132.5, 130.6, 128.9, 723 126.2, 116.6, 52.2, 43.2, 37.3, 35.6, 32.7, 31.1, 29.8 ppm. ESI-MS: m/z 724 z calculated for [M + H]⁺: 367.23, found: 367.25.

725 **4.5.14.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(3-726 hydroxyphenyl)acetamide (14b). Compound 14b was synthesized 727 according to GP-B from 11b. The product 14b (160 mg, 0.45 728 mmol, 74%) was obtained as an orange oil and converted without 729 further purification. For the Ellman's assay, a small amount of the 730 crude was purified by flash chromatography (water/MeCN 5–95% + 731 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ= 8.65 (s, 1H), 7.39–7.32 732 (m, 5H), 7.10 (t, J = 7.8 Hz, 1H), 6.81–6.72 (m, 2H), 6.69–6.65 (m, 733 1H), 6.13 (t, J = 5.8 Hz, 1H), 3.89 (s, 2H), 3.42 (s, 2H), 3.23–3.13 734 (m, 4H), 2.35 (t, J = 12.0 Hz, 2H), 1.66–1.59 (m, 2H), 1.54–1.39 735 (m, 2H), 1.37–1.24 (m, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 736 171.6, 169.1, 158.1, 136.6, 130.9, 130.2, 129.3, 129.1, 120.6, 116.5, 737 115.1, 61.0, 52.2, 43.9, 37.1, 35.3, 32.4, 29.4 ppm. ESI-MS: m/z 738 calculated for [M + H]⁺: 367.23, found: 367.25.

739 **4.5.15.** N-(2-(1-Benzylpiperidin-4-yl)ethyl)-2-(2-740 hydroxyphenyl)acetamide (14c). Compound 14c was synthesized 741 according to GP-B from 11c. The product 14c (216 mg, 0.61 mmol, 742 quant) was obtained as an orange oil and converted without further 743 purification. For the Ellman's assay, a small amount of the crude was 744 purified by flash chromatography (water/MeCN 5–95% + 0.1% FA). 745 ¹H NMR (400 MHz, CDCl₃) δ = 8.65 (s, 1H), 7.39–7.32 (m, 5H), 746 7.10 (t, J = 7.8 Hz, 1H), 6.81–6.72 (m, 2H), 6.69–6.65 (m, 1H), 747 6.13 (t, J = 5.8 Hz, 1H), 3.89 (s, 2H), 3.42 (s, 2H), 3.23–3.13 (m, 748 4H), 2.35 (t, J = 12.0 Hz, 2H), 1.66–1.59 (m, 2H), 1.54–1.39 (m, 749 2H), 1.37–1.24 (m, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 750 173.0, 168.6, 156.1, 130.9, 130.7, 129.1, 129.0, 128.9, 122.0, 120.2, 751 117.4, 61.5, 52.6, 40.7, 37.3, 34.9, 32.8, 29.6 ppm. ESI-MS: m/z 752 calculated for [M + H]⁺: 353.22, found: 353.20.

753 **4.5.16.** 4-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-754 oxoethyl)phenyl ethylpentamethyl- λ²-azanecarboxylate (17a). Compound 17a was synthesized according to GP-C from 755 14a. The product 17a (30 mg, 0.06 mmol, 34%) was obtained as a 756 colorless oil after purification by flash column chromatography (water/MeCN 5–40–95% + 0.1% FA). ¹H NMR (400 MHz, 757 CDCl₃) δ = 7.36–7.28 (m, 4H), 7.25–7.23 (m, 1H), 7.20 (d, J = 8.3 758 5.9 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 5.65 (t, J = 5.9 Hz, 1H), 5.24 (t, J = 759 7.1 Hz, 1H), 3.55 (s, 2H), 3.49 (s, 2H), 3.28–3.14 (m, 4H), 2.93– 760 2.85 (m, 2H), 2.03–1.93 (m, 2H), 1.64–1.53 (m, 4H), 1.39–1.20 761 (m, 13H), 0.88 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (101 MHz, 762
765 CDCl₃) δ= 170.9, 154.6, 150.4, 132.0, 130.3, 129.6, 128.3, 127.4, 122.1, 63.1, 53.5, 46.1, 43.2, 41.4, 37.4, 36.0, 33.3, 31.8, 31.7, 29.9, 29.0, 26.8, 22.7, 14.1 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 494.30, found: 194.33.

769 **4.5.17.** 3-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-oxoethyl)phenyl ethylpentamethyl-λ⁸-azanecarboxylate (17b). Compound 17b was synthesized according to GP-C from 14b. The product 17b (28 mg, 0.06 mmol, 32%) was obtained as a colorless oil after purification by flash column chromatography (water/MeCN 5-40-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.30 (dd, *J* = 3.4, 6.6 Hz, 6H), 7.24 (dd, *J* = 3.1, 5.4 Hz, 1H), 7.09-7.03 (m, 1H), 7.02 (s, 1H), 5.61 (t, *J* = 5.9 Hz, 1H), 5.23 (t, *J* = 5.9 Hz, 1H), 3.53 (s, 2H), 3.50 (s, 2H), 3.29-3.14 (m, 4H), 2.92-2.85 (m, 2H), 2.02-1.92 (m, 2H), 1.65-1.52 (m, 4H), 1.39-1.17 (m, 13H), 1.01-0.83 (m, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 170.5, 154.6, 151.5, 136.5, 129.8, 129.6, 128.3, 127.4, 126.2, 122.8, 120.6, 63.1, 53.6, 43.6, 41.4, 37.5, 36.0, 33.3, 31.8, 31.7, 29.9, 29.0, 26.8, 22.7, 14.2 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 494.30, found: 194.33.

781 **4.5.18.** 2-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-oxoethyl)phenyl heptylcarbamate (17c). Compound 17c was synthesized according to GP-C from 14c. The product 17c (38 mg, 0.08 mmol, 34%) was obtained as a colorless oil after purification by flash column chromatography (water/MeCN 5-40-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.33-7.27 (m, 6H), 7.26-7.22 (m, 1H), 7.21-7.13 (m, 1H), 7.11 (d, *J* = 7.9 Hz, 1H), 5.94 (t, *J* = 5.7 Hz, 1H), 5.39 (t, *J* = 6.0 Hz, 1H), 3.53 (s, 2H), 3.50 (s, 2H), 3.25-3.19 (m, 2H), 3.19-3.13 (m, 2H), 2.91-2.82 (m, 2H), 2.01-1.89 (m, 2H), 1.63-1.51 (m, 4H), 1.36-1.22 (m, 12H), 1.19-1.14 (m, 1H), 0.88 (t, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 170.5, 155.1, 149.3, 131.1, 129.6, 128.7, 128.4, 128.4, 127.4, 126.4, 123.2, 63.2, 55.6, 53.6, 41.6, 38.2, 37.4, 35.8, 33.3, 31.8, 31.7, 29.9, 29.0, 26.8, 22.7, 14.2 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 494.30, found: 194.33.

### 4.6. 4-Nitrophenyl azetidine-1-carboxylate

799 Azetidine (354 µL, 5.25 mmol, 1.0 equiv) was dissolved in CH₂Cl₂ (51 mL), Et₃N (2.1 mL, 15.7 mmol, 3.3 equiv) and 4-nitrophenyl chloroformate (1.16 g, 5.77 mmol, 1.1 equiv) were added successively. The reaction mixture was stirred at rt for 2. The reaction mixture was diluted with CH₂Cl₂ (30 mL) and washed with aq. HCl (2 × 20 mL, 1 M) and brine (30 mL). The organic layer was dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The product (921 mg, 4.14 mmol, 79%) was obtained as a yellow solid after purification by column chromatography (PE:EtOAc 3:1-2:1).³⁵ ¹H NMR (400 MHz, CDCl₃) δ = 8.22 (d, *J* = 9.2 Hz, 2H), 7.30 (d, *J* = 9.2 Hz, 2H), 4.19 (d, *J* = 41.9 Hz, 4H), 2.36 (d, *J* = 7.4 Hz, 2H) ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 223.06, found: 223.10.

801 **4.6.1.** 4-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-oxoethyl)phenyl azetidine-1-carboxylate (18a). Compound 18a was synthesized according to the GP-D from 14a. The product 18a (18 mg, 0.04 mmol, 7%) was obtained as a yellow oil after purification by flash column chromatography (water/MeCN 0-20-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.41-7.32 (m, 5H), 7.22 (d, *J* = 8.5 Hz, 2H), 7.07 (d, *J* = 8.5 Hz, 2H), 5.66-5.60 (m, 1H), 4.26-4.17 (m, 2H), 4.15-4.07 (m, 4H), 3.86 (s, 2H), 3.50 (s, 2H), 3.20 (q, *J* = 6.8 Hz, 2H), 3.17-3.12 (m, 2H), 2.33 (d, *J* = 7.6 Hz, 2H), 2.29-2.21 (m, 2H), 1.70-1.61 (m, 2H), 1.54-1.42 (m, 2H), 1.37 (q, *J* = 7.0 Hz, 2H), 1.27-1.15 (m, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 171.0, 167.9, 154.2, 150.5, 132.7, 132.1, 130.7, 130.3, 128.8, 128.8, 122.3, 61.2, 52.2, 43.4, 37.0, 35.5, 32.3, 29.9, 15.9 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 436.25, found: 436.25.
812 **4.6.2.** 3-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-oxoethyl)phenyl azetidine-1-carboxylate (18b). Compound 18b was synthesized according to GP-D from 14b. The product 18b (40 mg, 0.09 mmol, 16%) was obtained as a beige oil after purification by flash column chromatography (water/MeCN 0-20-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.40-7.33 (m, 5H), 7.31-7.27 (m, 1H), 7.05 (dt, *J* = 1.3, 7.6 Hz, 1H), 7.03-7.00 (m, 2H), 4.20 (s, 2H), 4.10 (s, 2H), 3.86 (s, 2H), 3.51 (s, 2H), 3.20 (q, *J* = 6.8 Hz, 2H), 3.15 (d, *J* = 11.8 Hz, 2H), 2.39-2.23 (m, 4H), 1.71-1.63 (m, 2H), 1.54-1.41 (m, 2H), 1.37 (q, *J* = 6.9 Hz, 2H), 1.30-1.18 (m, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 170.7, 167.9, 154.2, 151.6, 136.5, 132.6, 130.7, 129.9, 128.8, 128.8, 126.3, 122.8, 120.7, 61.2, 52.2, 43.7, 37.1, 35.5, 32.2, 29.8, 15.9 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 436.25, found: 436.25.

834 (q, *J* = 6.8 Hz, 2H), 3.15 (d, *J* = 11.8 Hz, 2H), 2.39-2.23 (m, 4H), 1.71-1.63 (m, 2H), 1.54-1.41 (m, 2H), 1.37 (q, *J* = 6.9 Hz, 2H), 1.30-1.18 (m, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃) δ = 170.7, 167.9, 154.2, 151.6, 136.5, 132.6, 130.7, 129.9, 128.8, 128.8, 126.3, 122.8, 120.7, 61.2, 52.2, 43.7, 37.1, 35.5, 32.2, 29.8, 15.9 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 436.25, found: 436.25.

839 **4.6.3.** 2-(2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-2-oxoethyl)phenyl azetidine-1-carboxylate (18c). Compound 18c was synthesized according to the GP-D from 14c. The product 18c (92 mg, 0.21 mmol, 37%) was obtained as a beige oil after purification by flash column chromatography (water/MeCN 0-20-95% + 0.1% FA). ¹H NMR (400 MHz, CDCl₃) δ = 7.37-7.35 (m, 4S), 7.26-7.23 (m, 1H), 7.21 (dd, *J* = 1.9, 7.8 Hz, 1H), 7.11 (td, *J* = 8.46, 1.4, 7.5 Hz, 1H), 7.05 (dd, *J* = 1.4, 8.0 Hz, 1H), 6.11-6.04 (m, 1H), 4.26-4.16 (m, 2H), 4.08-3.99 (m, 2H), 3.94 (s, 2H), 3.47 (s, 2H), 3.29-3.19 (m, 2H), 3.16-3.09 (m, 2H), 2.39-2.24 (m, 4H), 1.71-1.62 (m, 2H), 1.56-1.41 (m, 2H), 1.33 (q, *J* = 6.9 Hz, 2H), 1.26-1.12 (m, 1H) ppm ¹³C NMR (101 MHz, CDCl₃) δ = 170.6, 167.4, 154.1, 149.2, 131.0, 129.2, 128.9, 128.7, 128.4, 126.3, 123.2, 60.5, 51.7, 50.4, 49.4, 38.1, 36.9, 35.0, 31.7, 29.2, 15.9 ppm. ESI-MS: *m/z* calculated for [M + H]⁺: 436.25, found: 436.25.

### 4.7. ChE Inhibition

The experimental procedures for ChE inhibition, IC₅₀ determination, carbamylation, and decarbamylation were performed as previously described.³⁰,³⁵,⁵⁴,⁵⁵ hBChE (E.C. 3.1.1.8, from humans) was kindly provided by Oksana Lockridge from the University of Nebraska Medical Center. hAChE, acetylthiocholiniodin (ATC), butyrylthiocholiniodin (BTC), bovine serum albumin (BSA), and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich (St. Louis, Missouri). Phosphate buffer (buffer A) was prepared by dissolving NaH₂PO₄ in water (55 mM) and adjusting a pH to 8.0 by the addition of NaOH (0.1 M). A second buffer (buffer B) was prepared by dissolving DNTB in buffer A (0.3 mM). ATC and BTC solutions (75 mM and 45 mM) were prepared with water. Enzyme stock solutions (1.76 µg/mL) were prepared by dilution with buffer A and addition of 1 mg/mL BSA. Compounds' stock solutions were prepared in DMSO (4 mM). All experiments were carried out at 25 °C and in triplicate.

870 **4.7.1.** **IC₅₀ Determination.** Test compound stock solutions were diluted with buffer A to a starting concentration of 10 µM or 100 µM according to the compound's activity. Further dilutions were prepared with buffer B on a 96-well plate (>1% DMSO) to obtain 12 different concentrations with a volume of 130 µL in each well. Negative control was performed in each plate using buffer A instead of the compound solution and diluted with buffer B as for compounds. Ten microliters of the respective enzyme solution (1.76 µg/mL) were added and incubated for 20 min. Then, 3 µL of the respective substrate solution (ATC or BTC, 75 mM) was added rapidly with a dispenser and enzyme activity was measured immediately by UV absorption (λ = 881 412 nm). Read out was carried out every 30 s over 3 min (Spectramax 882 190 microplate reader, Molecular Devices, San José, California). The respective enzyme activity for each concentration was calculated via the slope of all 7 measurement points. Enzyme activity was plotted against the logarithmic compound concentration, and IC₅₀ values were calculated by Graphpad using a sigmoidal fit and presented as means ± SEM of independent triplicates.

881 **4.7.2.** **Carbamylation.** To determine the on-kinetics, the experimental setup for determining the IC₅₀ values was replicated, with the incubation time of the enzyme varying (5, 10, 15, 40, and 60 min).

892 **4.7.3.** **Decarbamylation.** To determine the off-kinetic, compounds were diluted in buffer and 30 µL hBChE stock solution (2.2 µg/mL) to a final volume of 200 µL (1000-fold concentration of the respective IC₅₀ value for enzyme inhibition >85%). For the negative control, buffer A and the respective percentage of DMSO were used instead of the compound stock solution. After 1 h of incubation, 2 µL of each compound solution was rapidly added to 2 mL of buffer B (0.9 mM DTNB) by dispenser. The obtained solutions were transferred to a 96-well plate, and 2 µL BTC (45 mM) was added
902 to one row for each of eight different time points. Readouts were
903 performed as described above for IC₅₀ determination. Enzyme activity
904 was plotted against the time after dilution to determine the half-life of
905 inhibition.

## 4.8. S1R Activation

906 The experimental procedures for membrane preparation, IC₅₀
determination, and Bip dissociation were performed as previously
907 described.⁵⁴ Haloperidol (4-[4-(4-Chlorophenyl)-4-hydroxy-1-piper-
908 idyl]-1-(4-fluorophenyl)-butan-1-one) was purchased from Alfa Aesar
909 (Karlsruhe, Germany). [³H](+)-pentazocine was purchased from
910 Biotrend Chemikalien GmbH (Köln, Germany).

911 **4.8.1. Membrane Preparation.** Male Wistar rats (ENVIGO, 912 UK) were decapitated, and their livers were dissected and removed to 913 ice-cold 10 mM Tris-HCl, pH 7.4, containing 0.32 M sucrose. Livers 914 were homogenized (after mincing with scissors) using a Potter- 915 Elvehjem Teflon-glass homogenizer in 10 mL/g tissue wet weight of 916 ice-cold 10 mM Tris-HCl/0.32 M sucrose, pH 7.4. The crude 917 homogenate was centrifuged for 10 min at 1000 g, and the pellets 918 were discarded. The resultant supernatant was centrifuged at 31,000 g 919 for 15 min. The pellet was resuspended in 3 mL/g of 10 mM Tris- 920 HCl, pH 7.4, by vortexing, and the suspension was allowed to 921 incubate at 25 °C for 15 min. Following centrifugation at 31,000 g for 922 15 min, the pellet was resuspended to 1.53 mL/g in 10 mM Tris-HCl, 923 pH 7.4, by gentle Potter-Elvehjem homogenization, and aliquots were 924 stored at -80 °C until use. Protein concentration was determined by 925 the method of Lowry et al.⁵⁶ using bovine serum albumin as a 926 standard. 927

928 **4.8.2. [³H](+)-pentazocine Binding Assay.** The binding assay 929 buffer consisted of 60 µL of incubation buffer (50 mM TRIS-HCl, pH 930 8.0, 25 °C), 100 µL of membrane aliquots, 20 µL of the tested 931 substances, or incubation buffer for the control, and 20 µL of 932 [³H](+)-pentazocine. The final concentration of [³H](+)-pentazocine 933 was 1.2 nM. All compounds were dissolved in dimethyl sulfoxide at a 934 concentration of 10 mM as stock solutions. These stock solutions 935 were diluted with 100% DMSO at a concentration of 1 mM and 936 following dilutions with 10% DMSO TRIS-HCl at 50 mM to the 937 required concentrations. Nonspecific binding was assessed by adding 938 haloperidol (10 µM). The radioligand and tested compounds were 939 added to the membranes and were then incubated for 150 min at 37 940 °C under dark conditions using a Thermo-Shaker (BioSan, PST- 941 60HL-4). The bound and free radioligands were separated by rapid 942 filtration under vacuum using Millipore GF/C filter paper (Merck 943 Millipore, Billerica, Massachusetts, USA) presoaked with 0.5% 944 polyethylenimine in 10 mM TRIS-HCl, pH 7.4, for at least 1 h 945 before use. The filters were washed four times with 0.25 mL of 10 946 mM TRIS-HCl (pH 8.0, 4 °C). The radioactivity of the samples was 947 measured with a liquid scintillation microplate counter, MicroBeta2 948 (PerkinElmer, Waltham, Massachusetts, USA), with 30% efficiency. 949 Each experiment was repeated at least three times, and each assay was 950 conducted in duplicate.⁵⁷,⁵⁸

951 **4.8.3. S1R-BiP Dissociation.** Green fluorescent protein (GFP)- 952 tagged S1R protein-overexpressing CHO cells (gift from Drs Yuko 953 Yasui and Tsung-Ping Su, NIDA/NIH, Baltimore, MD, USA) were 954 expanded in a minimum essential medium culture medium 955 supplemented with 10% heat-inactivated fetal bovine serum and 2 956 mmol/L Glutamax (Invitrogen, Carlsbad, CA, USA). After plating the 957 cells, different concentrations of each compound were added for 30 958 min (37 °C), and the culture medium was replaced by phosphate 959 buffered saline (3 mL). The cells were harvested, suspended in 50 960 mmol/L Hepes pH 7.4, and then cross-linked with 50 µg/mL of 961 dithiobissuccinimidyl propionate (Thermoscientific, Waltham, MA, 962 USA). The reaction was terminated by adding Tris/HCl 50 mmol/L, 963 pH 8.8. After incubating 15 min on ice, the cells were lysed in 50 964 mmol/L Tris pH 7.4 buffer, containing 150 mmol/L NaCl, 1% Triton 965 X-100, 0.3% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 966 protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). After centrifugation at 16,000 g for 1 min, the supernatant was 967 incubated with Chromotek GFP-trap agarose (Proteintech, United 968 Kingdom) overnight at 4 °C. After centrifugation at 16,000 g for 1

min, the supernatant was discarded and the pellet was suspended in 970 0.5 mL buffer (50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% 971 Triton X/100, 0.3% sodium deoxycholate, 0.1% SDS), rinsed twice, 972 and analyzed by Heat Shock 70 kDa Protein 5 ELISA assay (#CL- 973 SEC343Mu, Euromedex, Souffelweyersheim, France).⁶³ 974

## 4.9. Computational Studies

The experimental procedures for neurotoxicity were performed as 975 previously described.⁵⁴ Ligand structures were built in Molecular 976 Operating Environment (MOE version 2024.0601, Chemical 977 Computing Group, Canada) and energy-minimized with the 978 MMFF94x force field⁷⁹ to a root-mean-square (rms) gradient of 979 0.001 kcal/(mol Å). The substituents of the piperidine substructure 980 were modeled in the equatorial position of the chair conformation. 981

Noncovalent docking studies at the S1R were carried out with 982 Glide v10.1 using standard precision (SP) with GlideScore version 983 SP5.0 of Schrödinger release 2023-4 (Glide, Schrödinger, LLC, New 984 York, NY. 2023). The crystal structure 5HK1⁴⁸ with the best 985 resolution of 2.51 Å in the Protein Database (PDB) was selected, and 986 only chain A of the homotrimer was used. Prior to docking, all water 987 and nonprotein molecules were removed, and hydrogens were added 988 to the protein residues with MOE at physiological pH (7.4). Asp126 989 was included in the protonated state, as it interacts with Glu172 via a 990 hydrogen bond. The grid used for docking was centered at the 991 centroid of Val84 and Glu172, the inner box was set to a size of 15 Å, 992 and the outer box was set to 45 Å. By default, the OPLS_2005 force 993 field⁸⁰ was applied. Docking was performed with enhanced conforma- 994 tional sampling by a factor of 3. A maximum of ten poses per ligand 995 were generated and ranked by their GlideScore. Using GlideScore, 996 which is an empirical scoring function approximating ligand binding 997 free energy, enables a comparison to experimental results. The two 998 top-ranked poses were visually analyzed, and the preferred poses were 999 selected based on interaction patterns. 1000

Covalent docking studies to hBChE and hAChE were performed 1001 using Glide v10.1 with Covalent Docking v1.3 in Pose Prediction 1002 Mode⁶⁸ of Schrödinger Release 2023-4 (CovDock, Schrödinger, 1003 LLC, New York, NY, 2023). The crystal structure 4MOE⁸¹ for AChE 1004 (resolution 2.00 Å) and 6QAA⁸² for BChE (resolution 1.90 Å), which 1005 has been used in our previous work,²⁹,⁶⁶ was employed. All water and 1006 nonprotein molecules were removed, and hydrogens were added to 1007 the protein residues with MOE at pH = 7. The catalytic histidine 1008 (His447 for AChE and His438 for BChE) was maintained in its 1009 protonated state, as the following covalent docking aims to model the 1010 covalent tetrahedral intermediate. The grid was generated on the run 1011 during docking and was centered at the centroid of reactive Ser203 1012 and Phe338 for AChE, which corresponds to Ser198 and Phe329 in BChE. A box size of 15 Å for the inner box and 45 Å for the outer box 1013 was selected. Constraints were applied and had to be satisfied by at 1014 least one hydrogen bond between the carbonyl-oxygen of the ligand 1015 and the backbone-nitrogen of the glycines that form the oxyanion 1016 hole. As a reaction type, a custom reaction ("index 6") was obtained 1017 from Schrödinger's covalent reactions repository.⁸³ A maximum of ten 1018 poses per ligand were generated. Poses were ranked by their Prime 1019 Energy. For geometry optimization, a short energy minimization 1020 of the ligand was performed by using the OPLS_2005 force field. The 1021 two top-ranked poses were visually analyzed, and the preferred one 1022 was selected based on interaction patterns. 1023

For the docking of compound 14c, a noncovalent docking protocol 1024 similar to the one used for the S1R was applied. The catalytic histidine 1025 (His447 for AChE and His438 for BChE) was maintained in its 1026 deprotonated state, in contrast to the covalent docking protocol. The 1027 grid was centered at the centroid of the reactive Ser203 and Phe338 1028 for AChE, which corresponds to Ser198 and Phe329 in BChE. A box 1029 size of 15 Å for the inner and 45 Å for the outer box was selected. By 1030 default, the OPLS_2005 force field was applied. Docking was 1031 performed with enhanced conformational sampling by a factor of 3. 1032 A maximum of ten poses per ligand were generated and ranked by 1033 their GlideScore. In addition, the poses were visually inspected for a 1034 reasonable binding mode. 1035

1036
1037 Validation was carried out by redocking of the crystallized ligands.
1038 The RMSD of the top-ranked pose for the redocking of PD144418 in
1039 5HK1 was 0.860 Å; for dihydrotanshinone I in 4MOE an RMSD of
1040 0.424 Å was obtained, and for (S)-2-(butylamino)-N-(2-cyclo-
1041 heptylethyl)-3-(1H-indol-3-yl)propanamide in 6QAA (including the
1042 crystallized and interacting DMSO molecule in the binding site), an
1043 RMSD of 0.637 Å was obtained.

1044 All figures of docking poses were generated with PyMol (The
1045 PyMOL Molecular Graphics System, version 2.6.1; Schrödinger,
1046 LLC).

## 4.10. Effects on Microglia

1047 All experiments on mouse N9 microglial cells were conducted as
1048 previously described.<sup>84</sup> using concentrations of 1, 2.5, and 5 µM of
1049 compound **14c** or **18c**, respectively.

## 4.11. Neurotoxicity

1050 The experimental procedures for neurotoxicity were performed as
1051 previously described.<sup>54</sup> HT22 cells were grown in Dulbecco's
1052 modified Eagle medium (Sigma-Aldrich, St. Louis, Missouri)
1053 supplemented with 10% (v/v) fetal calf serum and 1% (v/v)
1054 penicillin-streptomycin. Cells were subcultured every 2 days and
1055 incubated at 37 °C with 5% CO₂ in a humidified incubator.
1056 Compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-
1057 Aldrich, Munich, Germany) as stock solutions and further diluted into
1058 the culture medium. For the determination of cell viability, a
1059 colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
1060 bromide (MTT, Sigma-Aldrich, Munich, Germany) assay was used.
1061 MTT solution (4 mg mL⁻¹ in PBS) was diluted 1:10 with medium
1062 and added to the wells after removal of the old medium. Cells were
1063 incubated for 3 h, and then the lysis buffer (10% SDS) was applied.
1064 The next day, absorbance at 560 nm was determined with a multiwell
1065 plate photometer (Tecan, SpectraMax 250).<sup>30,35</sup>

1066 Five × 10³ cells per well were seeded into sterile 96-well plates and
1067 incubated overnight. For the neurotoxicity assay, medium was
1068 removed, and 1.25, 2.5, 5, or 10 µM of the compound diluted with
1069 medium from a 10 mM stock solution was added to the wells. 0.05%
1070 DMSO in DMEM served as a control. Cells were incubated for 24 h,
1071 and neurotoxicity was determined by using a colorimetric MTT assay.

## 4.12. Solubility

1072 The experimental procedures for solubility were performed as
1073 previously described.<sup>54</sup> For solubility measurements, a 10 mg/mL
1074 stock solution in DMSO was prepared for each HCl salt. A dilution
1075 row with concentrations of 100, 80, 40, 20, and 10 µg/mL in MeOH
1076 was prepared for the calibration curve. The dilutions were measured
1077 via HPLC using the method described in the general information. The
1078 UV peak areas were plotted against the concentration to give the
1079 calibration curve. For solubility determination, 8 µL of sample stock
1080 solution was added to 792 µL of PBS and shaken overnight. After
1081 centrifugation (2000, 2 min) and filtration, the samples were analyzed
1082 by HPLC.<sup>72</sup> Data are representative of three independent experi-
1083 ments.

## 4.13. In Vivo Studies

1084 The experimental procedures for all in vivo studies were performed as
1085 previously described.<sup>54</sup> The neuroprotective effects of the compounds
1086 were determined using the pharmacological in vivo mouse model of
1087 AD induced by intracerebroventricular (ICV) injection of oligo-
1088 merized Aβ<sub>25-35</sub> peptide at day 1 into Swiss mice.<sup>30,31,85,86</sup> Mice were
1089 treated intraperitoneally (IP) with the compounds dissolved in 10%
1090 DMSO in saline, once-a-day between days 1 and day 7. On day 8, the
1091 short-term memory of the mice was tested by using spontaneous
1092 alternation in the Y-maze. Effects on long-term memory were assessed
1093 on day 9 (training) and day 10 (test) using the step-through type
1094 passive avoidance test.

1095 **4.13.1. Animals.** Male Swiss mice, 51 days old, weighing 32 ± 5 g,
1096 from JANVIER laboratories (Saint Berthevin, France) were kept in for
1097 housing, and experiments took place in the animal facility building of
1098 the University of Montpellier (CECEMA, Office of Veterinary
1099 Services agreement # B-34-172-23). Upon arrival, animals were

divided into groups and housed with access to food and water ad libitum except during behavioral tests. Mice were kept in a temperature- and humidity-controlled animal facility on a 12/12 h light/dark cycle (lights off at 07:00 PM). Behavioral experiments were conducted between 8:00 AM and 5:00 PM in a sound-attenuated and air-regulated experimental room. All animal procedures were conducted in strict adherence to the European Union directive of September 22, 2010 (2010/63/EU) and the ARRIVE guidelines. The project was authorized by the French National Ethic Committee (APAFIS no. 148515034).<sup>31,87</sup>

1010 **4.13.2. Preparation and Administration of Injection Solutions.** The Aβ<sub>25-35</sub> peptide was solubilized at 3 µM in water (vehicle solution V1) and oligomerized by incubation at 37 °C for 4 days. Mice were anesthetized with 2.5% isoflurane, and ICV was injected with Aβ<sub>25-35</sub> (9 nmol in 3 µL). Compounds were dissolved in 100% DMSO at a concentration of 6 mg/mL, further diluted with physiological saline (0.9% NaCl) to a final percentage of 10% DMSO, and injected IP. The vehicle solution (V2) was prepared with 10% DMSO in physiological saline and injected IP. After all injections, the behavior of the mice in their home cage was checked visually.<sup>31</sup>

1011 **4.13.3. Behavioral Tests.** At day 7 after injection, animals were tested in the Y-maze on their spontaneous alternation behavior. On days 8 and 9, the passive avoidance test was performed to measure the nonspatial contextual long-term memory.<sup>35</sup>

## ■ ASSOCIATED CONTENT

### ■ Supporting Information

The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.5c00921.

NMR spectra, HPLC chromatograms of target compounds, hBChE inhibition, hBChE kinetics, S1R binding, and solubility (PDF)

## ■ AUTHOR INFORMATION

### Corresponding Authors

Tangui Maurice – MMDN, University of Montpellier, EPHE, INSERM, 34095 Montpellier, France; ● orcid.org/0000-0002-4074-6793; Email: tangui.maurice@umontpellier.fr

Michael Decker – *Pharmaceutical and Medicinal Chemistry*, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, 97074 Würzburg, Germany; ● orcid.org/0000-0002-6773-6245;

Email: michael.decker@uni-wuerzburg.de

### Authors

Kora Reichau – *Pharmaceutical and Medicinal Chemistry*, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, 97074 Würzburg, Germany; ● orcid.org/0000-0003-3919-7667

Lucie Crouzier – MMDN, University of Montpellier, EPHE, INSERM, 34095 Montpellier, France

Eleonora Poeta – IRCCS, Istituto delle Scienze Neurologiche di Bologna, 40139 Bologna, Italy

Tina Gehrig – *Pharmaceutical and Medicinal Chemistry*, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, 97074 Würzburg, Germany

Eva Schaller – *Pharmaceutical and Medicinal Chemistry*, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, 97074 Würzburg, Germany

Liga Zvejniece – *Laboratory of Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, LV-1006 Riga, Latvia*
1161 Johann Meunier - MMDN, University of Montpellier, EPHE, INSERM, 34095 Montpellier, France

1162 Christoph Sotriffer - Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, 97074 Würzburg, Germany

1167 Barbara Monti - IRCCS, Istituto delle Scienze Neurologiche di Bologna, 40139 Bologna, Italy; Department of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy; orcid.org/0000-0003-0330-482X

1171 Complete contact information is available at:

1172 https://pubs.acs.org/10.1021/acschemneuro.5c00921

## 1173 Author Contributions

1174 The manuscript was written through the contributions of all

1175 authors. All authors have given approval to the final version of

1176 the manuscript. K.R.: Conceptualization, Data curation,

1177 Investigation, Methodology, Visualization, Writing—original

1178 draft. L.C.: Formal analysis, Investigation, Methodology,

1179 Writing, review, and editing. T.G.: Formal analysis, Invest-

1180 tigation, Methodology, Visualization, Writing, review, and

1181 editing. E.S.: Formal analysis, Investigation, Writing, review,

1182 and editing. E.P.: Formal analysis, Investigation, Writing,

1183 review, and editing. L.Z.: Formal analysis, Investigation,

1184 Resources, Writing, review, and editing. J.M.: Formal analysis,

1185 Investigation, Methodology, Writing—review and editing.

1186 C.S.: Resources, Supervision, Writing, review, and editing.

1187 B.M.: Resources, Writing, review, and editing. T.M.:

1188 Conceptualization, Data curation, Funding acquisition, Project

1189 administration, Resources, Supervision, Writing—review and

1190 editing. M.D.: Conceptualization, Funding acquisition, Project

1191 administration, Resources, Supervision, Writing—review and

1192 editing.

## 1193 Funding

1194 M.D. and T.M. acknowledge a binational funding from the

1195 German Deutsche Forschungsgemeinschaft (DFG) and the

1196 French Agence National de la Recherche (ANR) through a

1197 cooperative DFG/ANR grant (project DFG DE1546/12-1,

1198 ANR-22-CE92-0080). Campus France (PHC Procope),

1199 BayFrance (Franco-Bavarian University cooperation, FK03-

1200 2020), and German Academic Exchange Service (DAAD,

1201 Deutscher Akademischer Austauschdienst, 57702280) are

1202 gratefully acknowledged for funding of travel costs. The work

1203 has been supported through the European Cooperation in

1204 Science and Technology (COST Association), SIGMA1_EU-

1205 ROPE action under No. CA23156 and by RRF grant No. 53/

1206 OSI/PA (RRF project No.5.2.1.1.i.0/2/24/I/CFLA/001).

## 1207 Notes

1208 The authors declare no competing financial interest.

## 1209 ■ ACKNOWLEDGMENTS

1210 We thank Dr. O. Lockridge (University of Nebraska Medical

1211 Center) for kindly donating hBChE and the CECEMA-UM

1212 animal facility for animal housing and experiments. Table of

1213 content graphic was created in BioRender. Decker, M. (2025)

1214 https://BioRender.com/5c3utoq

## 1215 ■ ABBREVIATIONS

1216 ACh, acetylcholine; Aβ, Amyloid-β; AD, Alzheimer's disease;

1217 ANOVA, analysis of variance; APP, amyloid precursor protein;

1218 BiP, binding immunoglobulin protein; ChE, Cholinesterase;

CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide; 1219 DMF, N,N-dimethylformamide; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); FA, formic acid; GP, general procedure; hAChE, 1220 human acetylcholinesterase; hBChE, human butyrylcholinesterase; HPLC, high-performance liquid chromatography; 1221 HT22, murine hippocampal neuronal cell line; ICV, intracerebroventricular; IL1β, Interleukin-1 beta; IP, intraperitoneal; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFT, neurofibrillary tangle; NOS2, nitric oxide synthase; PBS, phosphate-buffered saline; rt, room temperature; SI, selectivity index; S1R, sigma-1 receptor; TGFβ2, transforming growth factor-beta 2; TREM2, 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231

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