Marion Donnier-Marechal
email: marion.donnier-marechal@univ-lyon1.fr

Pascal Carato
email: pascal.carato@univ-poitiers.fr

Paul-Emmanuel Larchanché
email: emmanuel.larchanche@univ-lille2.fr

Séverine Ravez

Rajâa Boulahjar

Amelie Barczyk
email: amelie.barczyk@univ-lille2.fr

Bénédicte Oxombre

Patrick Vermersch
email: patrick.vermersch@univ-lille2.fr

Patricia Melnyk
email: patricia.melnyk@univ-lille2.fr

Rajaa Boulahjar

Amélie Barczyk

Synthesis and pharmacological evaluation of benzamide derivatives as

Keywords: benzamide, sigma protein, CNS

Introduction

More than 40 years after their discovery and due to their involvement in various CNS pathologies, the sigma proteins continue to be widely studied [1][START_REF] Weissman | Sigma receptors in post-mortem human brains[END_REF][3]. These proteins have been first described as opioid receptors [START_REF] Martin | The effects of morphine-and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog[END_REF] and then classified in the literature as distinct sigma-1 and sigma-2 receptors (S1R and S2R) differing from their pharmacologically and ligand binding profile, anatomical distribution, function and molecular size [START_REF] Maurice | Improving Alzheimer's disease-related cognitive deficits with sigma1 receptor agonists[END_REF][START_REF] Guitart | Sigma receptors: biology and therapeutic potential[END_REF][START_REF] Banister | The therapeutic potential of sigma (σ) receptors for the treatment of central nervous system diseases: evaluation of the evidence[END_REF][START_REF] Walker | Sigma receptors: biology and function[END_REF]. The protein S2R has not been cloned from any species, while the S1R protein was cloned from various mouse [9], rat [START_REF] Seth | Cloning and functional characterization of a sigma receptor from rat brain[END_REF] and human [START_REF] Kekuda | Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1)[END_REF] tissues.

The S1R have been identified as a small-ligand regulated chaperone protein. They are located at the interface between endoplasmic reticulum (ER) and mitochondria called mitochondria associated membrane (MAM) [START_REF] Tsai | Sigma-1 receptor chaperones and diseases[END_REF]13]. They modulate the Ca 2+ homeostasis under conditions of cellular stress [13,[START_REF] Monnet | Sigma-1 receptor as regulator of neuronal intracellular Ca2+: clinical and therapeutic relevance[END_REF], but also the K + and Cl -channels [START_REF] Aydar | The sigma receptor as a ligand-regulated auxiliary potassium channel subunit[END_REF][START_REF] Renaudo | Cancer cell cycle modulated by a functional coupling between sigma-1 receptors and Cl-channels[END_REF]. Additionally, S1R have been shown to regulate the cholinergic [START_REF] Kobayashi | Enhancement of acetylcholine release by SA4503, a novel sigma 1 receptor agonist, in the rat brain[END_REF][START_REF] Matsuno | Increase in extracellular acetylcholine level by sigma ligands in rat frontal cortex[END_REF], adrenergic [START_REF] Gonzalez-Alvear | Sigma receptor regulation of norepinephrine release from rat hippocampal slices[END_REF][START_REF] Lucas | Further evidence for an antidepressant potential of the selective sigma1 agonist SA 4503: electrophysiological, morphological and behavioural studies[END_REF], dopaminergic [START_REF] Debonnel | Modulation of NMDA and dopaminergic neurotransmissions by sigma ligands: possible implications for the treatment of psychiatric disorders[END_REF][START_REF] Gonzalez-Alvear | Regulation of [3H]dopamine release from rat striatal slices by sigma receptor ligands[END_REF], glutamatergic [START_REF] Debonnel | Modulation of NMDA and dopaminergic neurotransmissions by sigma ligands: possible implications for the treatment of psychiatric disorders[END_REF] and serotoninergic [START_REF] Bermack | Modulation of serotonergic neurotransmission by short-and long-term treatments with sigma ligands[END_REF] neurotransmissions.

S1R are involved in the regulation of numerous neurotransmitter systems, the signal transduction pathway is not completely understood but S1R pharmacological modulators have been shown to modulate several CNS diseases such as pain [START_REF] Butera | Current and Emerging Targets To Treat Neuropathic Pain[END_REF]25], depression [START_REF] Bermack | The role of sigma receptors in depression[END_REF] or schizophrenia [START_REF] Itzhak | Clorgyline displays high affinity for sigma binding sites in C57BL/6 mouse brain[END_REF][START_REF] Narita | Interactions of selective serotonin reuptake inhibitors with subtypes of sigma receptors in rat brain[END_REF], but also neurodegenerative disorders like Alzheimer's and Parkinson's diseases [START_REF] Maurice | Neuroprotective and anti-amnesic potentials of sigma () receptor ligands[END_REF][START_REF] Uchida | A variant of the sigma receptor type-1 gene is a protective factor for Alzheimer disease[END_REF][START_REF] Mori | Compromising σ-1 Receptors at the Endoplasmic Reticulum Render Cytotoxicity to Physiologically Relevant Concentrations of Dopamine in a Nuclear Factor-κB/Bcl-2-Dependent Mechanism: Potential Relevance to Parkinson's Disease[END_REF]. Thus, the synthesis of such S1R ligands represents a promising strategy to develop novel treaments for various CNS diseases or even cancers [START_REF] Rui | Synthesis and biological evaluation of new aryl-alkyl(alkenyl)-4-benzylpiperidines, novel Sigma Receptor (SR) modulators, as potential anticancer-agents[END_REF][START_REF] Zampieri | Computer-assisted design, synthesis, binding and cytotoxicity assessments of new 1-(4-(aryl(methyl)amino)butyl)-heterocyclic sigma 1 ligands[END_REF]. Many drugs described in the literature show affinity for S1R, and some of them have undergone clinical trials, but no selective S1R ligands have so far been marketed [START_REF] Zamanillo | Sigma 1 Receptor Chaperone: Pharmacology and Therapeutic Perspectives[END_REF]. A phase 2 study is ongoing to evaluate Anavex 2-73a mixed muscarinic and S1R agonist -in patients with Alzheimer's disease [START_REF] Villard | Anti-amnesic and neuroprotective potentials of the mixed muscarinic receptor/sigma 1 ligand ANAVEX2-73, a novel aminotetrahydrofuran derivative[END_REF][START_REF] Lahmy | Blockade of Tau hyperphosphorylation and Aβ₁₋₄₂ generation by the aminotetrahydrofuran derivative ANAVEX2-73, a mixed muscarinic and σ₁ receptor agonist, in a nontransgenic mouse model of Alzheimer's disease[END_REF].

Numerous studies described the synthesis, as well as the biological and pharmacological activities of S1R ligands that exhibit Ki values in the nanomolar range, Among them, previous work of our laboratory underlines a first series of tetrahydroisoquinoline-hydantoin (tic-hydantoin) compounds (Figure 1) as efficient S1R ligands (Ki = 4.5 to 7.1 nM), with low affinity for S2R (Ki = 500 to 1000 nM) and therefore high S2R/S1R selectivity (100 to 140). These S1R ligands were evaluated in different pharmacological models. One derivative increased cocaine-induced locomotor stimulation and sensitisation in anti-cocaine evaluation [START_REF] Toussaint | Tic hydantoin sigma-1 agonist: Pharmacological characterization on cocaine-induced stimulant and appetitive effects[END_REF], an other one brought about a 57% decrease of infarct volume in an ischaemia model [START_REF] Venna | Neuroprotective and antidepressant-like effects of LC 03/35, a novel sigma-1 receptor ligand[END_REF].

These compounds were described as neuroprotective agents by their strong anti-inflammatory and neuroprotective effects in an experimental model of multiple sclerosis [39]. Despite chemical stability of tic-hydantoin derivatives in neutral and acidic media, they demonstrated a low metabolic stability, preventing from the perspective of drug development. Continuing our efforts in the synthesis of S1R ligands to solve this problem of metabolic stability, other works (Figure 1) were dedicated to replacement of the tic-hydantoin moieties with different tricyclic heterocycles [START_REF] Donnier-Maréchal | Carboline and phenothiazine-derivated heterocycles as potent sigma-1 protein ligands[END_REF] or benzannulated scaffold [START_REF] Donnier-Maréchal | Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective Sigma-1 receptor ligands[END_REF]. Though the S1R affinity was maintained for most of the compounds (Ki = 0.6 to 30 nM), but the S2R/S1R selectivity remained low (< 30).

Thus we report here our efforts to simplify the structure and replace the different heterocycle moieties by benzamide scaffold with a length chain of 2, 3 or 4 methylene functions separated from the amine part with benzylmethylamine to afford compounds 7a-z (Figure 2) [START_REF] Melnyk | Compounds, pharmaceutical composition and their use in treating neurodegenerative diseases[END_REF]. The design of these compounds could be compared to 4-IBP [START_REF] Megalizzi | 4-IBP, a sigma1 receptor agonist, decreases the migration of human cancer cells, including glioblastoma cells, in vitro and sensitizes them in vitro and in vivo to cytotoxic insults of proapoptotic and proautophagig drugs[END_REF], which was recently cocrystallized with S1R [START_REF] Schmidt | Crystal structure of the human σ1 receptor[END_REF]. In a first series, numerous substitutions on the benzamide scaffold were adopted by varying the nature and the position of halogeno atoms (Cl, Br) and groups (alkyl, CF3, OCH3, NO2, N(CH3)2, CN). Considering the good results with compounds 7a-z, we developed a second series of derivatives 8a-h where we kept the 4-chlorobenzamide moieties and modulated the nature of the amine part with N-phenylethyl-N-methylamine, isoindoline, pyrrolidine, morpholine and methylpiperazine (Figure 2). In this second series, compounds 8a-h were synthesized with length chain of 2 or 3 methylene groups between the benzamide and the functionalized amine. In this series, we also evaluated the importance of the hydrophobic part with the phenyl ring and the hydrophilic part with morpholine and piperazine moieties. In a third series, analogues of the promising compound 7i were prepared while modifying the linker. On the benzamide scaffold (7i), the amide function was changed by sulfamide function to give compound 9, the benzamide was reduced in benzylamine to furnish derivative 10 and the retroamide was also synthesized with compound 13 (Figure 2).

N O N N N H O Cl N R2 R1 N H O N m R 3 X Cl N N N N R 1 R 2 N
8a-h 7a-z 9, 10, 13 R 3 : alkyl, halogen, CF In a first time, diamino compounds 5a-f were prepared in two or three steps and final compounds 7a-z were synthesized (Scheme 2). From the first route, diamines 5a-c were synthesized in three steps. 3-Bromopropylamine was protected with Boc group in CH2Cl2 with triethylamine and di-tertbutylcarbonate to afford derivative 3, which reacted with the corresponding substituted amine (HNR1R2) in CH3CN or DMF with potassium carbonate to give compounds 4a-c. These derivatives 4a-c were deprotected in 3M HCl in dry dioxane to give compounds 5a-c. From the second route, the ethyl and butyl analogues of benzylmethylpropane-1,3-diamine (5a) were synthesized in two steps (Scheme 2).

Starting from bromoalkylphtalimides, substitution reaction in DMF with potassium carbonate and benzylmethylamine afforded compounds 6a-b, which were deprotected in ethanol with hydrazine to give intermediates 5d-e. In the series with m= 2 we also synthetize the superior analogue of compound 5d with derivative 5f which contained methylethylbenzylamine group as NR1R2. These diamine intermediates 5a,d,e afforded final compounds 7a-z (Scheme 3). Compounds 7a-z were synthesized according two routes, starting from commercially substituted benzoyl acids, in dichloromethane with hydroxybenzotriazole and O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate at room temperature or with commercially substituted benzoyl chlorides, in dichloromethane with triethylamine The SAR study of compounds 7a-z showed that the substitution of compounds 7h-j with a chlorine atom in para position induced good affinities for S1R, high S2R/S1R selectivities and low cytotoxicities (Table 1). We thus decided to synthesize a second series with 4chlorobenzamide compounds 8a-h using the same experimental conditions. We synthesized compounds 9

N Br O O m N N O O m R 1 R 2 Br N H 2 Br BocHN N BocHN R 1 R 2 N N H 2 R 1 R 2 N R 1 R 2 N N N N H 2 N m R 1 R 2 N N N R 1 R 2 a b c 3
and 10 respectively as the sulfonyl and reduced analogues of the amide compound 7i (Scheme 3).

Derivative 9 was synthesized starting from the 4-chlorophenyl sulfonyl chloride in dichloromethane with trimethylamine and the diamine intermediate 5a. was obtained from compound 11 in benzylmethylamine which reacted both as base and solvent.

Y O Cl H O Cl S Cl O O Cl N H O Cl N R2 R1 N H S O O Cl N N H Cl N N N H 2 N N H 2 N H 2 N m Y O N H O N m R 3 R 3 NH 2 Cl N O Cl N H O N Cl N H O Cl Cl N H 2 N m R 1

Results and Discussion

Biological evaluations and SAR

S1R and S2R affinities were investigated in competition experiments with radioligands according to the methods described by Ganapathy [START_REF] Ganapathy | Molecular and ligandbinding characterization the sigma-receptor in the Jurkat human T lymphocyte cell line[END_REF]. In the S1R assay, the selective ligand [ 3 H] (+)-pentazocine was employed as the radioligand. Since a selective S2R radioligand was not commercially available, the nonselective ligand [ 3 H]-DTG was employed in the S2R assay, in the presence of an excess of non-tritiated (+)-pentazocine, which selectively occupies S1R. In both assays, Jurkat cell membranes were used as a source of receptors.

The Ki values for S1R and S2R were determined from the corresponding IC50 values for each compounds 7a-z, 8a-h, 9, 10, and 13. The S2R/S1R selectivity ratios were also calculated (Table 1).

In the first series, as all compounds 7b-x exhibited good affinities for S1R (Ki = 1.1 to 47 nM) except for derivative 7a (Ki > 100 nM), we could conclude that substitution on the benzamide scaffold was necessary. The substitution with alkyl group, as propyl, butyl and tert-butyl for compounds 7b-d induced excellent affinities for S1R with Ki = 1.6 to 7.3 nM and good selectivity toward S2R with a S2R/S1R ratio of 126 expecially for derivative 7d. The modulation of the tert-butyl group of compound 7d by trifluoromethyl group with 7e, allowed to keep excellent S1R affinity but led to selectivity decrease with a S2R/S1R ratio of 70.

The substitution with one halogen atom, such as Cl or Br, gave high affinities for S1R (Ki = 1.7 to 47 nM). On the contrary, the substitution with a fluorine decreased S1R affinity dramatically (compound 7n). In this series, the best affinities were obtained when benzamide moiety was substituted at the 4 position with a chlorine or bromine atom (7h, 7m). Substitution in meta position (for 7g Ki = 10 nM, 7l Ki = 3.9 nM) or ortho position (for 7f Ki = 47 nM, 7k Ki = 42 nM) induced a decrease in S1R affinity, but also a meaningful decrease for S2R affinity. Therefore, S2R/S1R selectivities remained modest in the case of substitutions in meta or ortho position compared to the para-substituted compound 7h with excellent affinity for S1R (Ki = 3.6 nM) and selectivity S2R/S1R of 236. According to these results, we decided to introduce two halogens, with two chlorines (compounds 7o-r) or one fluorine and one bromine (compound 7s). In this set of compounds 7n-s, S1R and S2R affinities, and thus selectivities were in the same range. For the last compounds described in table 1 (compounds 7t-x), the nature and the position of the substitutents were modulated with methoxy, dimethylamine, cyano and nitro groups. Substitution with methoxy group (compounds 7t-u) gave moderate affinities for S1R (Ki = 37-40 nM) with a S2R/S1R ratio higher than 27. Compounds 7v-z with dimethylamine, cyano and nitro groups allowed good affinities for S1R (Ki = 1.2-24 nM) and especially derivative 7y with the best S2R/S1R selectivity ratio of 389. Finally, we also noticed the influence of the length chain between the benzamide scaffold and the benzylmethylamine. Indeed the decrease in the chain length (from butyl to ethyl) induced a higher selectivity (compare compounds 7h-j, S2R/S1R = 12 to 236). Moreover, as a comparison, in the case of cyano substitution, a slight modification in the length's chain drastically modified the affinities and selectivity (see compounds 7w and 7x). We then modulated the nature of the amine (HNR1R2) and the length's chain with m = 2, 3 between this amine group and the 4-chlorobenzamide. We preserved the chlorine atom at the 4 position, considering the good and maintained affinities and selectivities obtained with compounds 7h-i. Physicochemical properties of compound 7i were evaluated and shown the potency of this compound with high LLe, solubility and metabolic stability (Figure 3). The modulation of the nature of the amine group, benzylmethylamine (7i) to 2-(phenylethyl)methylamine (8a), led to good affinity S1R (Ki = 4.3 nM), but low selectivity toward S2R (Ki = 200 nM) with a S2R/S1R ratio of 47. The restricted conformation of the benzylmethylamine group with isoindoline moiety for compound 8b afforded acceptable affinity for S1R (Ki = 19 nM) but low selectivity toward S2R (Ki = 350 nM) with a S2R/S1R ratio of 16. For compounds 8c-h, various substituted amine, such as pyrrolidine, morpholine and piperazine, were introduced and we modulated the length's chain with two or three methylene. As expected, the elimination of the phenyl ring with these derivatives 8c-d, identified as an essential hydrophobic part, and the replacement by morpholine or piperazine for derivatives 8e-h, as hydrophilic moieties, induced a dramatic lost of affinity for S1R (Ki ≥100 nM). 1 Starting from 7i, we decided to replace the amide linker with a sulfonylamide function (9), to reduce the amide to amine function [START_REF] Seth | Cloning and functional characterization of a sigma receptor from rat brain[END_REF] and to synthesize the retroamide derivative ( 13) (Table 3). These three compounds 9, 10 and 13 displayed good affinities for S1R (Ki = 2.9-6.5 nM) but low selectivities toward S2R with a S2R/S1R ratio of 26-35.

8c 2 N > 100 > 200 - > 100 - 8d 3 N > 100 > 200 - > 100 - 8e 2 N O > 100 > 200 - > 100 - 8f 3 N O > 100 > 200 - > 100 - 8g 2 N N > 100 > 200 - > 100 - 8h 3 N N > 100 > 200 - > 100 - a-d see footnotes of Table

Docking study of benzamide 7i

To investigate the potential binding mode of benzamide derivatives, compound 7i was docked into the binding site of S1R [START_REF] Schmidt | Crystal structure of the human σ1 receptor[END_REF]. As illustrated in Figure 3, the representative docking solutions highlighted common interactions of S1R ligands. The benzyl moiety interact with hydrophobic regions of the receptor and the p-chlorobenzamide scaffold is stabilized by an aromatic stacking interaction with the Tyr103. In addition, the basic amine of 7i is engaged in a persistent salt bridge with the carboxylate group of the highly conserved Glu172.

Evaluation of cytotoxic effects

To determine the potential cytotoxic effects of our compounds on CNS tissues, a human neuroblastoma cell line was treated with the whole compounds at different concentrations up to 100 μM.

Cell viability was calculated using a colorimetric MTT assay.

All target compounds exhibited very low cytotoxicity (Table 1,2,3) with selectivity index between 2 128 and 76 923 (IC50(SY5Y)/Ki (S1R) ratio). In particular, derivatives 7i and 7p with high affinities for S1R (Ki = 1.3-3.6 nM) and S2R/S1R selectivities (236-238) showed excellent selectivity index of 31 250 and 15 385 respectively. Compound 7y with the best selectivity for S2R (S2R/S1R = 389) also showed excellent selectivity index of 27 778.

Selectivity profile over a panel of 40 recombinant human receptors

Among these compounds, eleven were selected regarding their affinity, selectivity, ligand efficiency and chemical diversity. Compounds (7b, 7e, 7h, 7i, 7l, 7m, 7t, 7u, 7w, 7y, 7z, 8a) were tested on their agonist and antagonist activities on 40 receptors relevant for CNS diseases like serotonine, adenosine, adrenergic, cannabinoide, dopamine, histamine, muscarinic and others receptors. They were tested at 10µM, in duplicate, on the Aequorin [START_REF] Le Poul | Adaptation of Aequorin Functional Assay to High Throughput Screening[END_REF], cAMP HTRF TM [START_REF] Degorce | HTRF: A Technology Tailored for Drug Discovery -A Review of Theoretical Aspects and Recent Applications[END_REF] and GTP 35 S [52] binding assays as described in Table 4.

None of the studied compounds provided significant agonist activity at 10 M, except compound 8a which showed an agonist activity on serotonine receptors (5-HT2B, 5-HT2C and 5-HT7).

Significant antagonist activity was observed at 10 M for serotonine , adrenergic (all subtypes), histamine H1, dopamine D4 and muscarinic M1 receptors for compounds 7b, 7e, 7i, 7l, 7m, 8a with propyl, trifluoromethyl groups or chlorine, bromine atoms. In these cases, the position of the substitution (meta or para) or the length's chain played a significant part in the antagonist activities of the compounds. For exemple, 4-bromo benzamide 7m was more selective than the 3-bromo derivative 7l, which showed significant antagonist activity on 5-HT2B, 5-HT2C, α2A and H1 receptors. For 4chlorobenzamide derivatives, propyl chain and benzylmethylamine seemed optimal (compound 7i) as the decrease from propyl to ethyl (compound 7h) induced activity on 5-HT2A, 5-HT2C and D2 receptors. In a similar manner, the stretching from benzylamine (compound 7i) to phenethylamine (compound 8a) led to an increased activity on 5-HT1B, 5-HT2B, 5-HT2C, 5-HT3, α2A, α2B, H1 and M2 receptors. Compounds 7t, 7u, 7w, 7y could be considered as selective compounds because they show excellent profil toward all receptors and hight selectivities for S1R. In particular, methoxy derivatives 7t and 7u showed no activity upon near all tested receptors at a concentration of 10 M. Compounds 7w, 7y with cyano and nitro group showed low affinities for H1, 5-HT3 and µ opioid receptors. Once again, the length's chain between the benzylmethylamine and the benzamide scaffold was important. When we compared the nitro compounds 7y and 7z the elongation of the ethyl (7y) to propyl chain (7z) induced higher affinities for antagonist activities for 1a and M1. It is important to emphasize that compound 7y showed an excellent profil S1R toward S2R (S1R : Ki = 3.6 nM, S2R : Ki > 1400 nM, S2R/S1R= 389) and an excellent profil for agonist and antagonist activities against all the tested receptors.

a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [
[a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [

Conclusion

Starting from previous work in our lab and in the literature, we used benzamide moiety as tic-hydantoin surrogate and various modulations were realized. In the first series, we modulated the nature and the position of atoms or groups on the benzamide moiety and kept the benzylmethylamine separated with alkyl chain with 2 to 4 methylene group from the heterocycle, to give compounds 7a-z. In a second series, 4-chlorobenzamide moiety was kept and the nature of the amine group was modulated (HNR1R2) to afford derivatives 8a-h. Then, three compounds were prepared as the sulfonyl (9), reduced [START_REF] Seth | Cloning and functional characterization of a sigma receptor from rat brain[END_REF] and retroamide (13) analogues of derivative 7i. The Ki values for S1R and S2R were determined from the corresponding IC50 values for each compound. The modulation with chlorine or bromine atoms gave good affinities for S1R and selectivities toward S2R. The length's chain also played an important part from these 2 parameters. As expected, the introduction of hydrophilic moieties in compounds 8c-h induced a dramatic lost of affinities for S1R, underlining the major importance of both hydrophobic moieties highlighted in the Glennon's pharmacophore. All together this study underlined the interest of compounds 7i, w, y with Cl, CN or NO2 groups at the 4-position of the benzamide scaffold showed excellent affinity for S1R (Ki = 1.2 to 3.6 nM), selectivity for S2R (Ki up to 1400 nM) and high selectivity index (from 28 000 to 83 000). Physicochemical properties of compound 7i are very compatible with drug development. Futhermore, these compounds showed a highly selective profile over 40 other receptors. These derivatives will be considered for further biological investigations.

Experimental

Chemistry

Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise noted. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil

Preparation of N-(3-bromopropyl)-4-chlorobenzamide (1)

To a solution of 3-bromopropylamine hydrobromide (2.86

2-(4-Chlorophenyl)-5,6-dihydro-4H-1,3-oxazine (2)

To a solution of isoindoline (20.4 µL, 0.18 mmol)

Preparation of diamine intermediates (5a-e)

tert-Butyl-3-bromopropylcarbamate (3)

3-Bromopropylamine (13.70 mmol, 3.0 g) was dissolved in 80 mL of DCM. After addition of triethylamine (16.4 mmol, 2.32 mL) and di-tert-butylcarbonate (13.70 mmol, 3.0 g), the resulting mixture was stirred at room temperature overnight. Then, it was washed with 2 x 60 mL of a citric acid solution (5%) and 50 mL of saturated brine solution. The organic fraction was dried over magnesium sulphate and concentrated to give the desired product as yellow oil of sufficient purity for use without purification in the next step. Yield: 97%. 1 H NMR (300 MHz, CDCl3), δ: 4.67 (br s, 1H, NH); 3.45 (t, J = 7.4 Hz, 2H, CH2); 3.28 (q, J = 7.1 Hz, 2H, CH2); 2.06 (p, 3 J = 7 Hz, 2H, CH2); 1.46 (s, 9H, C(CH3)3).

General procedure for the synthesis of substituted compounds 4a-c

To a mixture of tert-butyl-3-bromopropylcarbamate 1.2 (5.6 mmol, 1.3 eq, 1.3 g) and N,N-disubstituted amine (4.3 mmol, 1 eq) in 30 mL of DMF was added potassium carbonate (13.0 mmol, 3 eq, 1.8 g). The resulting mixture was heated at 70°C for 24 hours. Then, the solvent was removed under reduced pressure and water (80 mL) added to the residue. The crude product was extracted with 3 x 60 mL of DCM. The combined organic fractions were washed with 60 mL of water and dried over magnesium sulphate. The solvent was evaporated and the crude product collected as yellow oil. Purification by column chromatography (DCM: MeOH(NH3), 9.7:0.3 (v/v)) was performed.

tert-Butyl-3-[benzyl(methyl)amino]propyl carbamate (4a)

Yield: 74%.

tert-Butyl-3-[N-methyl-N-(2-phenylethyl)amino] propyl carbamate (4b)

Yield: 81%.

General procedure for the synthesis of substituted compounds 6a-c

To a mixture of N-(bromoethyl or bromobutyl)phtalimide (5.9 mmol) and benzylmethylamine (7.1 mmol, 0.8 mL) or 2-phenylethylamine (7.1 mmol, 0.9 mL) in 30 mL of DMF was added potassium carbonate (11.80 mmol, 1.6 g). The resulting mixture was heated at 90°C for 16 hours. Then, inorganics were eliminated by filtration and the solvent was removed under reduced pressure. Purification by column chromatography (DCM: MeOH(NH3), 9.9:0.1 (v/v)) was performed.

2-[2-(Benzylmethylamino)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (6a)

Yield 52%.

2-[2-(N-methyl-N-(2-phenylethyl)amino)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (6c)

Yield: 64%.

General procedure for the synthesis of substitutedcompounds 5a-c

The compound 1.3 (1 eq) was dissolved in 72 mL of 3M HCl in dry 1,4-dioxane. The resulting mixture was stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give the desired product of sufficient purity for use without purification in the next step.

N 1 -Benzyl-N 1 -methylpropane-1,3-diamine (5a)
Yield 98%. Mp: 77°C.

3-(Isoindolin-2-yl)propan-1-amine (5c)

Yield: 83%.

General procedure for the synthesis of substituted compounds 5d-f

In 50 mL of ethanol 96°, was added a mixture of 2-[N,N-disubstitutedaminoalkyl]-2,3-dihydro-1Hisoindole-1,3-dione (6.2 mmol) and hydrazine hydrate (62.0 mmol, 3.1 mL). The resulting mixture was heated at reflux for 16 hours. The solution was removed under reduced pressure. The mineral was eliminated by filtration and washed with ethyl acetate. The filtrate was evaporated under reduced pressure to give an oily residue. Purification by column chromatography (DCM: MeOH(NH3), 9:1 (v/v)) was performed.

N 1 -Benzyl-N 1 -methylethane-1,2-diamine (5d)
Yield 55%.

General procedure E1

A solution of N,N-disubstitutedalkyldiamine 5a-f or commercially available amine (1 eq), and triethylamine (3 eq) in 10 mL of DCM was cooled at 0°C. A solution of substituted benzoylchloride (1 eq) in 5 mL of DCM was added slowly. The resulting mixture was stirred at room temperature overnight.

The solution was evaporated under reduced pressure. An aqueous solution of 3% sodium hydroxide (20 mL) was added and the mixture was stirred for 1 hour. The solution was extracted with DCM. The organic layer was dried over magnesium sulphate and concentrated to give an oily product. The residue was purified by thick layer chromatography (DCM:MeOH(NH3), 9:1 (v/v)) or column chromatography (DCM:MeOH(NH3), 9:1 (v/v)).

General procedure E2

Reaction was carried out under nitrogen atmosphere. In 10 mL of DCM, benzoic acid (1 eq), HOBt (1.2 eq) and HBtu (1.2 eq) were added and stirred at room temperature for 10 minutes. A solution of N,N-disubstitutedalkyldiamine 5a-f or commercially available amine (1 eq) and DIEA (15 eq) in DCM was added to the reacting mixture. After stirring at room temperature for 24 hours, the solvent was removed under reduced pressure and DCM was added to the residue. The solution was washed with a solution of NaHCO3 (5%) then saturated NaCl solution. The organic layer was dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by thick layer chromatography (DCM:MeOH(NH3), 9:1 (v/v)) or column chromatography (DCM:MeOH(NH3), 9:1 (v/v)).

N-[3-(N-benzyl-N-methylamino)propyl]benzamide (7a)

Procedure E2. Purification by thick layer chromatography was performed. Yield: 75%.

N-[3-(benzylmethylamino)propyl]-4-propylbenzamide (7b)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH3), 9:1 (v/v)) was performed.

Yield: 61%.

N-[3-(benzylmethylamino)propyl]-4-butylbenzamide (7c)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH3), 9:1 (v/v)) was performed.

Yield: 30%.

N-(4-chlorobenzyl)-3-(benzylmethylamino)propanamine (10)

A solution of 4-chlorobenzaldehyde (0.55 mmol, 77 mg) and N 1 -benzyl-N 1 -methylpropane-1,3-diamine 5a (0.55 mmol, 100 mg) in MeOH (10 mL) was stirred at room temperature for 16 hours. Then, sodium borohydride (0.82 mmol, 32 mg) was added and the reaction media was stirred for 3 hours. The mixture was quenched with water (5 mL) and evaporated under reduce pressure. The crude product was purified by thick layer chromatography (DCM:MeOH(NH3), 9:1 (v/v)). Yield: 35%. IR (cm -1 ): 2950 (NH). 1 H NMR (300 MHz, CDCl3), δ: 7.80 (br s, 1H, NH); 7.48 (d,J = 8.4 Hz,2H,H2,H6);7.33 (d,J = 8.5 Hz,2H,H3,H5);3H,Haro);7.13 (m,2H,Haro)

4-(benzylmethylamino)-N-(4-chlorophenyl)butanamide (13)

A solution of compound 11 (1.29 mmol) in N-benzylmethylamine (7.75

In vitro testing 5.2.1 Assay for binding to sigma receptors

The binding assays were performed by CEREP (Poitiers, France), according to Ganapathy [START_REF] Megalizzi | 4-IBP, a sigma1 receptor agonist, decreases the migration of human cancer cells, including glioblastoma cells, in vitro and sensitizes them in vitro and in vivo to cytotoxic insults of proapoptotic and proautophagig drugs[END_REF]. Rapidely, the S1R binding assay was carried out by incubating Jurkat cell membranes (10-20 mg protein per tube)

with Nonspecific binding was determined, in both assays, under similar conditions, but in the presence of 10 µM unlabelled haloperidol. Inhibition constants (Ki) were calculated from the IC50 values according to the method of Cheng and Prusoff [START_REF] Cheng | Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction[END_REF].

Cell culture and cytotoxicity assay

The human neuroblastoma cell line (SY5Y) was cultured in DMEM (Dulbecco's Modified Eagle Medium) (Gibco) supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 IU/mL penicillin, 1 mM non-essential amino acids and 10% (v/v) heat-inactivated foetal bovine serum (Sigma Aldrich), and grown at 37°C in a humidified incubator with 5% CO2.

Cells were starved for 24 hours to obtain synchronous cultures, and were then incubated in culture medium that contained various concentrations of test compounds (100, 50, 10, 5, 1, 0.5, 0.1, 0.05 µM), each dissolved in less than 0.1% DMSO. After 72 hours of incubation, cell growth was estimated by the colorimetric MTT (thiazolyl blue tetrazolium bromide) assay.

Physicochemical properties

Standardised ADME experiments were performed by CEREP (Paris, France). Bioavailabily-related profile was measured according to Lipinski et al. for aqueous solubility in PBS (pH 7.4, [48]); Sangster et al. [START_REF] Ganapathy | Molecular and ligandbinding characterization the sigma-receptor in the Jurkat human T lymphocyte cell line[END_REF] for partition coefficient (logD, n-octanol-PBS, pH 7.4) and Kuhnz and Gieschen [49] for metabolic stability (human liver microsomes). The LC/MS system for microsomal stability and metabolites identification consisted of an Orbitrap Exactive instrument (Thermo) equipped with an electrospray ionization source used in positive mode (M+H + ). The apparatus was managed with Xcalibur software.

Selectivity profile (agonist and antagonist activities) over a panel of 40 recombinant human receptors

Rapidely, the tests were performed by Euroscreen (www.ogeda.com) with recombinant human G coupled receptor expressing cell lines using aequorin functional assays for serotonin 5-HT2A, 2B, 2C, 3 and 5A, adrenergic alpha1a and 1b, angiotensin AT1, cholesistokinin CCFK1, endothelin ETA, histamine H1, muscarinic M1 and 3, neurokinin NK1 and 2, vasopressin V1a and vasoactive intestinal peptide VPAC1

receptor analysis [START_REF] Le Poul | Adaptation of Aequorin Functional Assay to High Throughput Screening[END_REF]. cAMP HTRF TM assays were used to analyse serotonin 5-HT1A, 4E, 6 and 7, adenosine A1, 2A and 3, adrenergic beta1 and 2, cannabinoid CB1, dopamine D1, histamine H2 and neuropeptide Y NPY receptors [START_REF] Degorce | HTRF: A Technology Tailored for Drug Discovery -A Review of Theoretical Aspects and Recent Applications[END_REF]. GTPγS 35 assays were used to analyse serotonin 5-HT1B, adrenergic alpha2a and b, dopamine D2L, muscarinic M2 and 4, opïod delta, kappa and mu, and somatostatine STT4

receptor [START_REF] Ferrer | A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors[END_REF].

Agonist and antagonist activities of the test compound are expressed as the percentage of the activity of the reference agonist at its EC100 concentration or the percentage of inhibition of the reference agonist activity at its EC80 concentration, respectively.

For the aequorin assays, different cell lines coexpressing the receptors were grown in specific culture media and incubated for 4 hours with coelenterazine h. For agonist testing, cell suspensions were mixed with test or reference agonist in a 96-well plate. For antagonist testing, reference agonist at its EC80 (final concentration) was injected to the mix containing cells and tested compound. The resulting emission of light was recorded using Hamamatsu Functional Drug Screening System 6000 [START_REF] Le Poul | Adaptation of Aequorin Functional Assay to High Throughput Screening[END_REF].

For the cAMP HTRF TM assays, different cell lines coexpressing the receptors were grown in specific culture media and resuspended in assay buffer. For agonist testing on Gs coupled receptor, the tested compound was added at increasing concentrations. For antagonist testing, the tested compound and the reference agonist at its EC80 (final concentration) were added. For agonist and antagonist testing on Gi coupled receptor, forskolin was added to the reagents listed above. After incubation, cells were lysed and cAMP concentrations were estimated with the HTRF kit, according to the manufacturer specifications [START_REF] Degorce | HTRF: A Technology Tailored for Drug Discovery -A Review of Theoretical Aspects and Recent Applications[END_REF].

For the GTPS scintillation proximity assay, optimized specific conditions were determinated for each receptor. Membrane extracts were prepared from different cell lines coexpressing the receptors, mixed with GDP and incubated on ice. For agonist testing, the tested compound or the reference agonist, GDP mix and GTP[ 35 S] were added. For antagonist testing, the tested compound or the reference antagonist, the reference agonist at historical EC80, GDP mix and GTP[ 35 S] were added. GTP concentrations were estimated with PerkinElmer TopCount reader [START_REF] Ferrer | A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors[END_REF].

Molecular docking

The compound was built using the Chembiodraw 3D module and minimized.

Figure 1 :

 1 Figure 1: Tic-hydantoin, benzannulated and tricyclic series with propyl-N-methyl-N-benzylamine or isoindoline chains

Figure 2 :Scheme 1 :

 21 Figure 2: Structure of final compounds 7a-z, 8a-h, 9, 10, 13

Scheme 2 :

 2 Scheme 2: Preparation of diamine compounds 5a-f Reagents and conditions: (a): di-tert-butylcarbonate, triethylamine, CH2Cl2, 18h, rt, 97%; (b): HNR1R2, potassium carbonate, DMF or CH3CN, 24h, 70°C, 65-81%; (c): 3M HCl in dry dioxane, 18h, rt, 83-98%; (d): benzylmethylamine, potassium carbonate, DMF, 16h, 90°C, 52-64%; (e): H2NNH2, EtOH 96°, 16h, reflux, 55-73%.

Scheme 3 :

 3 Scheme 3: Synthesis of final compounds 7a-x, 8a-h, 9, 10 and 13

  = 6.0[START_REF] Lle | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF] Solubility (pH7.4) = 173µM [48] Metabolic stability (hLM) = 77%[START_REF] Kuhnz | Predicting the oral bioavailability of 19-nortestosterone progestins in vivo from their metabolic stability in human liver microsomal preparations in vitro[END_REF]

Figure 3 :

 3 Figure 3: Physicochemical properties of compound 7i

Figure 3 :

 3 Figure 3: Docking of compound 7i in the binding site of S1R (PDB Entry: 5HK2)

[

  60/UV254 sheets (thickness 0.2mm). Purification of products was carried out by either column chromatography or thick layer chromatography. Column chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). Thick layer chromatography was performed on glass plates coated with Macherey-Nagel Sil/UV254 (thickness 2 mm), from which the pure compounds were extracted with the following solvent system: DCM/MeOH(NH3), 90:10. IR spectra were performed on a Bruker Alpha FT-IR Spectrometer. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1 H and 75 MHz for 13 C). Chemical shifts are expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position ( in ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = double doublet, br = broad and m = multiplet), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of 2D experiments (COSY, HSQC and HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionisation and a UV detector (diode array). HRMS experiments were performed on Q Exactive Benchtop LC-MS/MS (Thermo Scientific). The purity of final compounds was verified by two types of high pressure liquid chromatography (HPLC) columns: C18 Interchrom UPTISPHERE and C4 Interchrom UPTISPHERE. Analytical HPLC was performed on a Shimadzu LC-2010AHT system equipped with a UV detector set at 254 nm and 215 nm. Compounds were dissolved in 50 µL methanol and 950 µL buffer A, and injected into the system. The following eluent systems were used: buffer A (H2O/TFA, 100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention times (HPLC tR) were obtained at a flow rate of 0.2 mL/min for 35 min using the following conditions: a gradient run from 100% of buffer A over1 min, then to 100% of buffer B over the next 30 min.

  1 H NMR (300 MHz, CDCl3), δ: 7.90 (br s, 1H, NH) ; 7.60(d, J = 8.2 Hz, 2H, H2, H6); 5H, Haro); 7.13 (d, J = 8.1 Hz, 2H, H3, H5); 3.55 (t, J = 6.0 Hz, 2H, CH2); 3.50 (s, 2H, CH2); 2.60 (m, 4H, 2 CH2); 2.23 (s, 3H, CH3); 1.80 (p, J = 6.0 Hz, 2H, CH2); 1.62 (s, J = 7.5 Hz, 2H, CH2); 0.95 (t, J = 7.3 Hz, 2H, CH3). 13 C NMR (75 MHz, CDCl3) δ: 167.2 (CO); 146.1 (Caro); 138.4 (Caro); 132.2 (Caro); 129.3 (Caro); 128.5 (Caro); 128.4 (Caro); 127.2 (Caro); 126.9 (Caro); 63.3 (CH2); 57.1 (CH2); 42.1 (CH3); 40.2 (CH2); 37.9 (CH2); 25.5 (CH2); 24.3 (CH2); 14.2 (CH3). LCMS m/z calc for [M+H + ]: 325.2; found: 325.0. HRMS: calcd for (C21H28ON2)H + : 325.22744; found: 325.22617. HPLC (C4, 35 min): tR 16.5 min, PHPLC 96%; HPLC (C18, 35 min): tR 22.5 min, PHPLC 97%.

  [ 3 H](+)-pentazocine (15 nM) and a range of concentrations of test compounds, at 37°C for 2 hours, in 5 mM Tris/HCl buffer (pH = 7.4). The S2R binding assay was performed by incubating Jurkat cell membranes (10-20 mg protein per tube) with [ 3 H]-DTG (25 nM) in the presence of (+)-pentazocine (1 µM) to saturate S1R, and a range of concentrations of test compounds, at room temperature for 1 hour in 5 mM TrisHCl buffer (pH = 7.4). Bound radioactivity was measured using liquid scintillation counting.

  Docking simulation was then performed into S1R (RCSB Protein Data Bank 5HK2) with the automated GOLD program. The active site was defined around 4-IBP. Marion Donnier-Marechal : Contributed to the compounds synthesis Pascal Carato : Contributed to the design of the study and wrote the manuscript Paul-Emmanuel Larchanché : Contributed to the compounds synthesis Amélie Barczyk : Contributed to the cytotoxicity assays Bénédicte Oxombre : Contributed to the analyses of biological data and wrote the manuscript Patrick Vermersch : Contributed to the design of the study Patricia Melnyk : Contributed to the design of the study and wrote the manuscript Reference NMR and mass spectra of compounds 7a-z, 8a-h, 9, 10 and 13 are available as supplementary data.

  3 , OCH 3 , NO 2 , N(CH 3 ) 2, CN

	X: SO 2 NH, CH 2 NH, NHCO
	m= 2,3,4

  Derivative 10 was obtained by a reductive amination of diamine intermediate 5a in MeOH with 4-chlorobenzaldehyde, followed by the addition of sodium borohydride. We also decided to synthesize the retroamide analogue 13 of compound 7i.

	Starting from 4-
	chloroaniline, reaction in dichloromethane with 4-chlorobutyryl chloride gave compound 11. This
	derivative 11 never afforded derivative 13 in DMF with potassium carbonate or in CH3CN with
	triethylamine and benzylmethylamine, but gave compound 12 after cyclization. The desired derivative 13

Table 1 :

 1 Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 7a-z

	Compounds	R3	m	Ki (nM) a	Ki (σ2)/	IC50	IC50/Ki
				σ1	σ2	Ki (σ1)	(µM)	(σ1)
							SY5Y b
	7a	H	3	> 100	>> 400 a	-	> 100	-
	7b	4-Pr	3	3.7	160	43	> 100	> 27 027
	7c	4-Bu	3	7.3	49	7	90.2	12 356
	7d	4-tBu	3	1.6	200	126	> 100	> 62 500
	7e	4-CF3	3	1.4	98	70	> 100	> 71 429
	7f	2-Cl	3	47	> 1200	> 26	> 100	> 2 128
	7g	3-Cl	3	10	340	34	> 100	> 10 000
	7h	4-Cl	2	3.6	850	236	> 100	> 27 778
	7i	4-Cl	3	3.2	190	60	> 100	> 31 250
	7j	4-Cl	4	1.7	20	12	> 100	> 58 824
	7k	2-Br	3	42	> 1200	> 29	> 100	> 2 381
	7l	3-Br	3	3.9	350	90	> 100	> 25 641
	7m	4-Br	3	2.1	160	76	60.2	28 667
	7n	4-F	3	20	470	24	> 100	> 5 000
	7o	2,3-Cl	3	10	310	31	> 100	> 10 000
	7p	2,3-Cl	2	6.5	110	17	> 100	> 15 385
	7q	3,4-Cl	3	1.1	59	54	56.6	51 455
	7r	3,5-Cl	3	23	48	2	60.0	2 609
	7s	2-F-4-Br	3	2.9	200	69	> 100	> 34 483
	7t	3-OCH3	3	40	> 1200	> 30	> 100	> 2 500
	7u	4-OCH3	3	37	> 1200	> 27	100.0	2 702
	7v	3-N(CH3)2	3	1.9	321	159	> 100	> 52 632
	7w	4-CN	2	1.2	170	142	> 100	> 83 334
	7x	4-CN	3	24	> 1200	> 50	> 100	> 4 167
	7y	4-NO2	2	3.6	1400	389	> 100	> 27 778
	7z	4-NO2	3	4.3	360	84	> 100	> 23 256

a Mean Ki values for 2-3 independent experiments are shown with less than 10% deviation. b Compound concentration causing 50% of cell death after 24 h treatment.

Table 2 :

 2 Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 8a-h

	Compounds m	-NR1R2	Ki (nM) a	Ki (σ2)/	IC50	IC50/Ki (σ1)
			σ1		σ2	Ki (σ1)	(µM)
							SY5Y b
	8a	3	N	4.3	200	47	> 100	> 23 256
	8b	3	N	19	350	16	> 100	> 5 264

Table 3 :

 3 Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 9

, 10, 13

	Compounds	X		Ki (nM) a	Ki (σ2)/	IC50 (µM)	IC50/Ki (σ1)
			σ1	σ2	Ki (σ1)	SY5Y b
	9	SO2NH	5.8	200	35	> 100	> 17 242
	10	CH2NH	2.9	95	33	> 100	> 34 483
	13	NHCO	6.5	170	26	> 100	> 15 385

a-d see footnotes of Table

1

Table 4 :

 4

[a]

: Agonist activity at 10 µM in duplicate;

[b]

: Antagonist activity at 10 µM in duplicate. For the agonist activity, data are expressed as the percentage of the activity of the reference agonist. For the antagonist activity, data are expressed as the percentage of inhibition of the reference agonist activity In green, 0-50%, in white 51-80%, in red 81-100%.

Numerical value are indicated over 51%. ND, not determined.

  in 1 mL of DMF was added a 24 mg(0.18 mmol) amount of potassium carbonate. The resulting mixture was stirred at 70°C. After 30 minutes, the 4chloro-N-(3-bromopropyl)-4-chlorobenzamide (50 mg, 0.18 mmol) was added and the reacting mixture was stirred at 70°C overnight. The solvent was removed under reduced pressure and 15 mL of water added to the residue. The crude product was extracted with 3x15 mL of DCM. The combined organic

	fractions were washed with water and dried over magnesium sulphate. The compound was purified by
	column chromatography (DCM:MeOH(NH3), 99:1 (v/v)) and obtained as a colourless oil. Yield: 70%.
	Mp= 84°C. 1 H NMR (300 MHz, CDCl3) : 7.83 (d, J = 9.2 Hz, 2H, H2, H6); 7.34 (d, J = 9.1 Hz, 2H, H3,
	H5); 4.36 (t, J = 6.3 Hz, 2H, CH2); 3.61 (t, J = 6.1 Hz, 2H, CH2); 1.99 (p, J = 6.3 Hz, 2H, CH2). 13 C NMR
	(75 MHz, CDCl3) : 154.7 (OCN); 136.4 (Caro); 132.6 (Caro); 128.3 (2 Caro); 128.2 (2 Caro); 65.2 (CH2);
	42.6 (CH2); 21.9 (CH2). LCMS (ESI + ): LCMS m/z calc for [M+H + ]: 196.0, 198.0; found: 196.0; 198.0.

  1 H NMR (300 MHz, CDCl3), δ: 7.85(m, 2H, Haro); 7.70 (m, 2H, Haro); 5H,

	Haro); 3.80 (t, J = 6.6 Hz, 2H, CH2); 2.62-2.75 (m, 6H, 3CH2); 2.38 (s, 3H, CH3). 13 C NMR (75 MHz,
	CDCl3) δ: 168.3 (CO); 140.3 (Caro); 133.8 (Caro); 132.2 (Caro); 128.7 (Caro); 128.2 (Caro); 125.8 (Caro);
	123.1 (Caro); 59.4 (CH2); 54.7 (CH2); 42.2 (CH3); 35.8 (CH2); 33.7 (CH2). LCMS m/z calc for [M+H + ]:
	309.1; found: 309.0.

  To a solution of 4-chloroaniline (7.8 mmol, 1.0 g) in 15 mL of DCM at 0°C was added slowly a solution of 4-chlorobutyryl chloride (7.8 mmol, 878 µL) in 5 mL of DCM. The resulting mixture was stirred at room temperature for 12 hours. Then the reaction was quenched with 25 mL of water and the product extract with 3 x 25 mL of DCM. The combined organic layers were dried over magnesium sulphate and concentrated to give the desired product 2.2 with sufficient purity to be used without purification in the next step. Yield: 87%. Mp = 98°C.

	; 4.00 (s, 2H, CH2); 3.53 (s, 2H, CH2); 2.94 (t, J =
	6.5 Hz, 2H, CH2); 2.60 (t, J = 6.3 Hz, 2H, CH2); 2.27 (s, 3H, CH3); 2.02 (p, J = 6.2 Hz, 2H, CH2). 13 C
	NMR (75 MHz, CDCl3) δ: 136.5 (Caro); 135.3 (Caro); 131.2 (2 Caro); 130.3 (Caro); 129.4 (2 Caro); 129.3 (2
	Caro); 128.6 (2 Caro); 127.8 (Caro); 62.2 (CH2); 55.4 (CH2); 50.9 (CH2); 47.3 (CH2); 41.5 (CH3); 22.6
	(CH2). LCMS m/z calc for [M+H + ]: 303.1, 305.1; found: 303.0, 305.0. HRMS: calcd for (C18H23N2Cl)H + :
	303.16225; found: 303.16180. HPLC (C4, 35 min): tR 12.4 min, PHPLC 95%; HPLC (C18, 35 min): tR 21.1
	min, PHPLC 95%.
	4-Chloro-N-(4-chlorophenyl)butyramide (11)
	232.0, 234.0, 236.0; found: 232.0, 234.0, 236.0.
	1-(4-Chlorophenyl)pyrrolidin-2-one (12)
	To a solution of N-methyl-N-benzylamine (106 µL, 0.82 mmol) in 10 mL of DMF was added a 340 mg
	(2.46 mmol) amount of potassium carbonate. The resulting mixture was stirred at 70°C. After 30 minutes,
	the 4-chloro-N-(4-chlorophenyl)butyramide 11 (210 mg, 0.91 mmol) was added and the reacting mixture
	was stirred at 70°C overnight. The solvent was removed under reduced pressure and 15 mL of water
	added to the residue. The crude product was extracted with 3x15 mL of DCM. The combined organic
	layers were washed with water and dried over magnesium sulphate. Purification by column
	chromatography (DCM:MeOH(NH3), 99:1 (v/v)) was performed. Yield: 67%. Mp = 98°C. 1 H NMR (300
	MHz, CDCl3), δ: 7.63-7.55 (m, 2H, H2 and H6); 7.36-7.29 (m, 2H, H3, H5); 3.84 (t, J = 7.3 Hz, 2H, CH2);
	2.62 (t, J = 8.5 Hz, 2H, CH2); 2.16 (p, J = 7.3 Hz, 2H, CH2). 13 C NMR (75 MHz, CDCl3) δ: 174.2 (CO);
	138.0 (Caro); 129.4 (Caro); 128.8 (2 Caro);120.9 (2 Caro); 48.6 (CH2); 32.6 (CH2); 17.8 (CH2).

1 H NMR (300 MHz, CDCl3), δ: 7.75 (br s, 1H, NH); 7.46 (d, J = 9.1 Hz, 2H, H2, H6); 7.26 (d, J = 9.0 Hz, 2H, H3, H5); 3.65 (t, J = 6.2 Hz, 2H, CH2); 2.55 (t, J = 7.3 Hz, 2H, CH2); 2.18 (p, J = 6.3 Hz, 2H, CH2). 13 C NMR (75 MHz, CDCl3) δ: 170.0 (CO); 136.2 (Caro); 129.4 (Caro); 129.0 (2 Caro); 121.1 (2 Caro); 44.4 (CH2); 34.1 (CH2); 27.8 (CH2). LCMS m/z calc for [M+H + ]:

  mmol, 1 mL) was stirred at room temperature for 12 hours. A 5 mL amount of hexane was added and the white solid, identified as disubstitutedamine hydrochloride, was filtered. The filtrate was concentrated and purified by thick layer

	chromatography (cyclohexane: ethyl acetate: MeOH(NH3), 4.5:4.5:1 (v/v)) to afford compound 13. Yield:
	70%. IR (cm -1 ): 1686 (C=0). 1 H NMR (300 MHz, CDCl3), 9.63 (br s, 1H, NH); 7.45 (d, J = 9.1 Hz, 2H,
	H2, H6); 7.34-7.27 (m, 5H, Haro); 7.21 (d, J = 9.0 Hz, 2H, H3, H5); 3.69 (s, 2H, CH2); 2.62 (t, J = 6.2 Hz,
	2H, CH2); 2.51 (t, J = 7.0 Hz, 2H, CH2); 2.35 (s, 3H, CH3); 1.96 (p, J = 6.1 Hz, 2H, CH2). 13 C NMR (75
	MHz, CDCl3) δ: 171.4 (CO); 137.2 (Caro); 135.9 (Caro); 129.7 (2 Caro); 128.8 (2 Caro); 128.7 (2 Caro); 128.5
	(2 Caro); 128.0 (Caro); 120.8 (Caro); 62.2 (CH2); 56.5 (CH2); 41.4 (CH3); 36.2 (CH2); 22.2 (CH2). LCMS
	m/z calc for [M+H + ]: 317.1, 319.1; found: 317.2, 319.2. HRMS: calcd for (C18H21ON2Cl)H + : 317.14152;
	found: 317.14026. HPLC (C4, 35 min): tR 8.6 min, PHPLC >99%; HPLC (C18, 35 min): tR 20.3 min, PHPLC
	>99%.

Acknowledgments

The authors acknowledged Nicolas Renault for docking experiments. The 300 MHz NMR facilities were funded by the Région Nord-Pas de Calais (France), the Ministère de la Jeunesse, de l'Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER). This work was supported by Lille 2 University, FRI "J'innove", PRES Univ Lille Nord de France and Nord de France SATT Grants. MDM is the recipient of a fellowship from Lille 2 University.

Conflict of Interest:

The authors declare no competing financial interests. All authors have approved the final article.

Authors contribution