Novel routes to either racemic or enantiopure α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives and biological evaluation of a new promising pharmacological scaffold.

Accepted Manuscript Novel routes to either racemic or enantiopure α-amino-(4-hydroxy-pyrrolidin-3yl)acetic acid derivatives and biological evaluation of a new promising pharmacological scaffold Samy Cecioni, Kaïss Aouadi, Julie Guiard, Sandrine Parrot, Nathalie Strazielle, Sandrine Blondel, Jean-François Ghersi Egea, Christian Chapelle, Luc Denoroy, Jean-Pierre Praly PII:

S0223-5234(15)30044-1

DOI:

10.1016/j.ejmech.2015.05.017

Reference:

EJMECH 7896

To appear in:

European Journal of Medicinal Chemistry

Received Date: 23 February 2015 Revised Date:

23 April 2015

Accepted Date: 11 May 2015

Please cite this article as: S. Cecioni, K. Aouadi, J. Guiard, S. Parrot, N. Strazielle, S. Blondel, J.F. Ghersi Egea, C. Chapelle, L. Denoroy, J.-P. Praly, Novel routes to either racemic or enantiopure α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives and biological evaluation of a new promising pharmacological scaffold, European Journal of Medicinal Chemistry (2015), doi: 10.1016/ j.ejmech.2015.05.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Novel routes to either racemic or enantiopure α-amino-(4-hydroxy-pyrrolidin-

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3-yl)acetic acid derivatives and biological evaluation of a new promising pharmacological scaffold

Samy Cecioni,a Kaïss Aouadi,†,a,# Julie Guiard,a,# Sandrine Parrot,b,# Nathalie Strazielle,c

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Sandrine Blondel,c Jean-François Ghersi Egea,c Christian Chapelle,e Luc Denoroy,*,b,d and Jean-

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Pierre Pralya

a CNRS UMR 5246 ; Université Lyon1 ; Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS) ; F-69622 Villeurbanne, France b INSERM, U1028; CNRS, UMR5292; Université Lyon 1; Lyon Neuroscience Research Center, NeuroDialyTics Unit, Lyon, F-69000, France

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c INSERM, U1028; CNRS, UMR5292; Université Lyon 1; Lyon Neuroscience Research Center, Blood Brain Interfaces Exploratory Platform BIP, Lyon, F-69000, France d INSERM, U1028; CNRS, UMR5292; Université Lyon 1; Lyon Neuroscience Research Center, BioRaN, Lyon, F-69000, France

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e Université de Lyon ; Pulsalys, F-69000 Lyon, France *Corresponding Author: Luc DENOROY, PhD

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CNRS, UMR5292 - INSERM, U1028 - Université Lyon 1; Lyon Neuroscience Research Center, BioRaN team; 8 Avenue Rockefeller; F-69373 Lyon Cedex 08; France; E-mail: [email protected]. Phone: (+33)478777555. Fax: (+33)478777173. †Present Address: Université de Monastir, Laboratoire de Synthèse Hétérocyclique, Produits Naturels et Réactivités, Faculté des Sciences de Monastir, Avenue de l'Environnement, 5000 Monastir, Tunisia. #Author Contributions: K.A., J.G., and S.P. contributed equally to the work.

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GRAPHICAL ABSTRACT

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HIGHLIGHTS Novel α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives are reported

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Synthesis of racemic or enantiopure derivatives requires from 3 to 5 steps.

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Biological evaluation reveals clear-cut glutamate lowering properties for two compounds

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Biological evaluation reveals modest cannabinoid properties for two compounds

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KEYWORDS

Cycloaddition; chiral nitrone; isoxazolidine; pyrrolidine; neuroactive analogs; CNS receptors;

ABBREVIATIONS

Glu,

glutamate;

CB,

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blood brain barrier

cannabinoids;

CSF,

cerebrospinal

fluid;

CNQX,

6-cyano-7-

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nitroquinoxaline-2,3-dione or (IUPAC name) 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-

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carbonitrile; HPLC, high performance liquid chromatography

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ABSTRACT

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Cycloaddition between (+) or (–)-menthone-derived nitrones and N-benzyl-3-pyrroline afforded enantiopure spiro-fused heterocycles. The reaction occurred enantio- and diastereo-selectively on the less hindered side of the nitrone, the 3-pyrroline N-benzyl group being oriented outwards,

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thus controlling the configurations of three simultaneously created chiral centers. From either (+) or (–)-menthone, both enantiomeric cycloadducts were synthesized in excellent yield. Removing

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the chiral auxiliary and the N-benzyl group delivered a series of enantiopure 4-hydroxy-3glycinyl-pyrrolidine derivatives in 3 to 5 steps and 36 to 81 overall yields. Using two other achiral nitrones, shorter routes to racemic analogues were developed. Two of the synthesized compounds markedly lowered extracellular glutamate level and modestly interacted with

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cannabinoid type-1 receptors. As these two neuroactive compounds were devoid of in vitro toxicity and did not cross the blood brain interface, they might represent potential

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pharmacological agents to target peripheral organs.

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1- INTRODUCTION

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Naturally occurring and synthetic alkaloids are long known bioactive products which have found numerous medicinal applications. Consequently, their chemistry is well developed, and they are accessible through a number of methods. Among them, cycloaddition reactions between unsaturated moieties acting as dipolarophiles and various dipoles represent direct and popular

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methods, which can be applied with tight selectivity as exemplified in our recent study using

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chiral nitrones [1]. In addition to their synthetic interest, 5-membered heterocycles display interesting properties and bioactivities. For example, isoxazolidinyl nucleosides can be found among heterosubstituted nucleoside analogs [2], while a new class of small molecule transcriptional inhibitors includes a series of substituted isoxazolines [3-5]. Pyrrolidinecontaining iminosugars are receiving continuous attention as they have high therapeutic potential

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in the treatment of diseases such as diabetes, cancer, and lysosomal storage disorders [6, 7]. Pyrrolidine and proline derivatives have proved useful for the synthesis of inhibitors binding to neuronal nitric oxide synthase [8], the development of new drug candidates for hepatitis C

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treatment [9-11] and the design of insulin-like growth factor-I and its derivatives as potential

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neuroprotective agents [12, 13].

Our studies on the [3+2] cycloaddition of menthone-based nitrones [1, 14] as glycine equivalents reacting with different substituted alkenes first aimed at the synthesis of stereoisomers of 4hydroxy-isoleucine [15-17], a naturally-occurring molecule found in fenugreek (Trigonella foenum-graecum), a plant traditionally used as an antidiabetic remedy, due to the insulinotropic properties of (2S,3R,4S)-4-hydroxy-isoleucine. The menthone-derived spiro-bicyclic nitrones react preferentially through their less hindered face, opposite to the nitrone isopropyl group

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(Scheme 1). With a Z symmetrical alkene, the reaction occurs through one preferred transition state (TS) with minimized steric interactions to afford a stereochemically defined cycloadduct [1, 15, 17]. This design is synthetically attractive because of: a) stereocontrolled creation of up to

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three stereogenic centers in one step; b) atom economy due to recovery of the chiral auxiliary; c) access to both enantiomers depending on the choice of the nitrone; d) mildness of the reaction conditions. The cycloaddition of such chiral nitrones with either E-, or Z-1,4-dichloro-but-2-ene

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afforded enantiopure dichlorinated isoxazolidines suitable for further elaboration. Generating amine groups at two different sites allowed for subsequent cyclizations, to afford two pyrrolidine

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scaffolds, of either the 3-substituted-4-hydroxy-proline, or 3-glycinyl-4-hydroxy-pyrrolidine type [1]. The first ones appeared to be precursors toward the synthesis of kainic acid and kainoids [1, 18]. Kainic acid is a naturally occurring amino acid used in vivo for its neuroexcitatory and epileptogenic properties with induction of seizures [19]. The 3-glycinyl-4-hydroxy-pyrrolidine

structural

analogs

of

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scaffold corresponds to surprisingly unexplored amino acid mimics related to iminocyclitols, and gamma-aminobutyric

acid

(GABA),

an

important

inhibitory

neurotransmitter. Therefore, on a structural basis, these novel molecules are close to classes of

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synthetically and biologically important molecules (Figure 1).

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Compared to our approach with Z-1,4-dichloro-but-2-ene and chiral nitrones [1], accessible 3pyrrolines were obviously advantageous dipolarophiles for more direct syntheses towards the 3glycinyl-4-hydroxy-pyrrolidine scaffold, as disclosed in our recent patent [20]. We now describes

synthetic

routes

to

3-glycinyl-4-hydroxy-pyrrolidine

derivatives,

based

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cycloaddition between N-protected 3-pyrrolines and either menthone-based nitrones or achiral nitrones, to deliver in few steps enantiopure or racemic products (Scheme 1), along with their biological evaluation.

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Because of strong analogies of the designed structures with existing bioactive molecules mimicking amino acids neurotransmitters (Figure 1), effects on the same targets were expected for these pyrrolidine derivatives. More precisely, these new molecules were hypothesized to act

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on brain glutamate (Glu) system or on brain GABA system. In order to test this hypothesis, an in vitro screening was performed to determine on which receptors some of the new molecules obtained may act. Unexpectedly, none of the compounds bound to the various glutamatergic or

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GABA-ergic sites, but two of them exhibited weak interaction with cannabinoid receptors, a result which nevertheless allowed hypothesizing that they may indirectly alter brain Glu level. A

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brain microdialysis experiment, performed to test this second hypothesis, revealed that these two compounds induced a marked fall in extracellular Glu concentration. Finally, bidirectional permeability experiments across an in vitro blood-brain interface were carried out to investigate whether these two compounds could act at both peripheral and central levels after a systemic

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administration.

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2- RESULTS AND DISCUSSION 2.1 Chemical synthesis

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Cycloaddition between the (–)-menthone-derived nitrone 1 and commercial N-benzyl-3-pyrroline (2) was completed after overnight reflux in toluene or within 2 h under microwave activation, to afford cycloadduct 3 in 96 % yield (Scheme 2). This route was straightforward compared to our recent synthesis of the debenzylated analog of 3 [1]. In a similar cycloaddition (see Supplementary Data, S4 and S5), the less reactive N-Boc-3-pyrroline (2’) reacted within 10 h (microwave activation), to afford 3’ the N-Boc analog of 3 (21 % yield), and predominantly an isomeric cycloadduct 3’’ (74 % yield), arising from a regio-, and stereo-selective cycloaddition

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of nitrone 1 to N-Boc-2-pyrroline (Figure 2, Scheme 2). Therefore, the synthetic work was further developed with N-benzyl protected products derived from readily available cycloadduct 3. Based on the experience accumulated in previous syntheses, structure elucidation of the

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cycloadducts 3 and 3’ was straightforward. However, diffraction analysis of crystals obtained for N-benzyl 3 and N-Boc 3’’ were carried out so as to confirm fully their structures (Figure 2). That of 3’’ is best explained by a cycloaddition taking place between nitrone 1 and the N-conjugated

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2-pyrroline arising from an unexpected rearrangement of 3-pyrroline 2’ (Scheme 2). Hence, crystal analysis provided easily unambiguous structural proofs for the obtained cycloadducts, in

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particular for isomeric N-Boc 3’’ whose structure would have been difficult to establish by NMR analysis only. There was no regioselectivity issue in the cycloadditions of 1 to symmetrical 3pyrrolines 2 and 2’. Interestingly, the 74% yield recorded for 3’’ denoted a high regioselectivity for the cycloaddition to the 2-pyrroline isomer, probably due to both steric and electronic factors.

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Removal of the chiral auxiliary in 3 was attempted by acidic (3N HCl, AcOH, 80°C) and basic (LiOH.H2O, THF/H2O) treatments, as developed previously for cycloadducts derived from acyclic alkenes [1, 14-17]. However under such acidic treatment at temperature below 60°C and

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under other mild acidic conditions, no reaction occurred, while with 3N HCl partial conversion

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of 3 occurred (31 and 80 % at 60, and 85°C respectively), to afford the desired product 5, in ~40% yield. Probably the presence of the basic nitrogen atom present in 3 affected the cleavage, which was finally achieved by acetolysis, in a mixture of Ac2O/AcOH and with a catalytic amount of H2SO4. After neutralization with 5M NaOH added dropwise, the N-acetylmethylamide-pyrrolidine 4 was obtained in 86 % yield. Gratifyingly, extended reaction times and slower basic quenching (over 2.5 h) afforded directly the methylamide 5 in a 90 % yield from 3. Based on thin-layer chromatography (TLC) monitoring of these reactions, it was assumed that

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this protocol preserved an acidic medium for a prolonged time, so that protonation of the isoxazolidine nitrogen atom prevented its acetylation in the first period of the treatment before hydrolysis of Ac2O had occurred. Otherwise, selective N-deacetylation of 4 to 5 was

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quantitatively achieved upon treatment in anhydrous MeOH acidified with SOCl2 with brief heating. The chiral auxiliary (–)-menthone was recovered in yield as high as 80% during the

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purification process and has been checked to be reusable for the generation of chiral nitrone. The next steps consisted in reductive treatments for achieving N-debenzylation and/or reductive

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cleavage of the N–O bond. Cleavage of the N–O bond occurred chemoselectively upon treatment with molybdenum hexacarbonyl to afford 6 in 66% yield [21]. N-Debenzylation was achieved by standard catalytic hydrogenolysis at atmospheric pressure in the presence of Pd(OH)2/C in EtOH with another cosolvent (CH2Cl2, H2O, AcOH). Interestingly, depending on their structure, the molecules studied displayed various reactivities. For example, the N-acetyl methylamide 4 was

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debenzylated in excellent yield to afford 7 after a 14 h treatment, without reductive opening of the N–O bond, probably more resistant due to N-acetylation. In contrast, the N-benzyl hydroxypyrrolidine 6 was converted within 2 h to 8 (67 % yield). For achieving both N-debenzylation

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and reductive opening of compound 5, long reaction times were needed. Thus, 5 was converted

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to the rather polar methylamide 9 within 12 h, while enantiopure 3-glycinyl-4-hydroxypyrrolidine 10 required a 24 h reductive treatment. Compound 10 was isolated after a final basic hydrolysis and purification over acidic resin. As stereochemistry can be of crucial importance as regard to bioactivities, the syntheses were also carried out similarly with the (+)-menthone derived nitrone ent-1, so as to deliver the corresponding enantiomers ent-3 to ent-8 and ent-10. All the products were studied by NMR spectroscopy (see Supplementary data). No epimerization was observed. Amino acids 10, ent-10, and (±)-10 (vide infra) gave identical well resolved 1H

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NMR spectra in D2O. When the D2O solution was made basic by adding LiOH, the 1H NMR spectrum of ent-10 changed significantly (see Supplementary data, Figure S1), as the

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zwitterionic form was presumably displaced to the corresponding lithium salt. We also considered a racemic approach to the target molecules, using easily accessible nitrones, like methyl nitrone 11 [22-24], benzyl nitrone 12 [25, 26], and cyano nitrone 13 [27], obtained as

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E / Z mixtures representing achiral glycine equivalents (Scheme 3). Cycloaddition between nitrone 11 and N-benzyl-3-pyrroline 2 proceeded within 1 h under microwave activation, to

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deliver cycloadduct (±)-14 (58 % yield) which was subjected to hydrogenolysis to afford (±)-15 (90 % yield) as ester hydrolysis occurred in aqueous THF. Under the similar conditions nitrone 12 led to cycloadduct (±)-16 (68 % yield). Catalytic hydrogenation achieved both bisdebenzylation and reductive opening of the N–O isoxazolidine bond, affording, due to

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concomitant ester hydrolysis, racemic amino acid (±)-10 in quantitative yield. Cycloaddition of nitrone 13 and 2 proceeded also readily within 2 h to afford two cycloadducts (±)-17 and (±)-18 (45% and 6% isolated yield). While structure (±)-17 was expected, that of (±)-

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18, established by 2D NMR spectroscopy, indicated the involvement of an isomeric nitrone (13’) in the cycloaddition with 2. No evidence of the contamination of 13 by 13’ was observed.

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Catalytic hydrogenation achieved both bis-debenzylation, reductive opening of the N–O isoxazolidine bond, and reduction of the cyano group in (±)-17 to afford diamino alcohol (±)-19 as the main product (3:1 ratio with a monobenzylated impurity, among minor byproducts). Deprotection of (±)-17 was slow even under a 4 bar hydrogen pressure, and the minor product was shown by MS to display a benzyl group. All of the four cycloadducts so obtained showed weak NOE effects between protons H-3 and H-6, thus indicating a trans disposition. As dipoles of the nitrile oxide type were expected to afford related isoxazoline-based cycloadducts, other

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assays (carried out with ethyl chlorooximidoacetate [28]/NEt3 for generating the corresponding nitrile oxide) afforded the desired product (±)-20 which was found stable: when exposed to catalytic hydrogenation under various conditions, it reacted slowly and partially, affording a

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mixture of products, with evidence of the reduction of the benzyl ring to a cyclohexyl ring in one product, as shown by mass spectroscopy [29]. These experiments showed that access to racemic substituted pyrrolidines with achiral dipoles suffered from drawbacks, notably as the nitrones

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exist as E and Z forms, and sometimes as other isomers, thus leading to challenging purifications. Such drawbacks did not occur with menthone-based nitrones which are structurally well defined,

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stable and reactive enough in the presence of a suitable dipolarophile as N-benzyl-3-pyrroline. They afforded in high yield stereochemically defined products, with recovery of the chiral auxiliary in the next steps.

Compounds ent-8, 9, 10 and ent-10 were considered for in vitro assays on structural bases and

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availability. Further tests were carried out with compounds selected when they showed results

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deserving closer investigation.

2.2 Biological activities.

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In order to investigate the type of receptors which could be targeted by the compounds ent-8, 9, 10 and ent-10, including Glu or GABA receptors, a search of specific binding sites was then done using Standard Operative Procedures (See Supplementary data, Table S3). Sixty binding sites were examined in duplicate and data revealed that none of these four compounds significantly bound to any site of ionotropic glutamate receptors (AMPA, kainate, NMDA, PCP, glycine-strychnine-insensitive), metabotropic mGluR5 receptors, nor to GABAA/B receptors and benzodiazepine sites of GABAA receptors. Surprisingly, the compounds 9 and ent-10 altered the

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specific binding (29% of inhibition) of a control agonist of cannabinoid type 1 (CB-1) receptors, whereas ent-8 altered the binding of an agonist of cholecystokinin type 1 (CCK1) receptors (Table I). In contrast to its enantiomer ent-10, compound 10 did not bind to any site of all the

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receptors tested. Although the percentage of agonist binding inhibition was modest, additional tests were performed to determine the agonist/antagonist property of 9 and ent-10 versus cannabinoid receptor subtypes. Experiments showed that the two compounds were not

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antagonists for CB-1 and CB-2 receptors (data not shown), nor agonists for CB-2 receptors (Supplementary data Figure S2 bottom for ent-10). However, ent-10 led to a moderate inhibition

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of the hepatic cell response vs CB-1 control agonist (CP55940), suggesting that it could be a partial agonist of CB-1 receptors (Supplementary data Figure S2 top). In contrast, this approach failed to specify the type of agonistic property of compound 9 towards CB-1 receptors (data not shown).

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The lack of any binding of our novel compounds on Glu or GABA receptors was unexpected because their structures exhibited strong analogies with existing molecules mimicking neurotransmitter amino acids. Nevertheless, the two compounds 9 and ent-10 which act on CB-1

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receptors may indirectly alter brain Glu system owing to the well-known interactions between

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Glu system and CB systems via CB-1 receptors [30]. If this hypothesis is valid, these two compounds are expected to exhibit a sought pharmacological property, i.e. indirectly modulating brain glutamatergic transmission while being devoid of the side-effects encountered with antagonists of ionotropic Glu receptors [31, 32]. In order to determine whether 9 and ent-10 can indirectly affect brain Glu level, an in vivo approach, in which all connections between brain areas and neuronal networks are functionally preserved, is needed. For that purpose, the brain microdialysis technique was used, since it

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allows both the local administration of a drug and the monitoring of drug-induced changes in neurotransmitters concentration in the extracellular medium within the central nervous system of live animals [33-35]. Thus, Glu level was monitored in the striatum, a well-defined brain

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structure containing glutamatergic nerve terminals, when locally infusing the compound 9 or ent10 by reverse dialysis of a 1 mM solution. By using the dialysis yield of amino acids previously determined with our microdialysis probe, i.e. around 7-9 % [36], one may assume that the

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concentration of the perfused 9 and ent-10 compounds in the brain extracellular space is lower than 100 µM. Under such conditions, compounds 9 and ent-10 induced marked 50-60%-

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decreases in Glu levels (Figure 3). In order to test whether Glu could retro-modulate this effect by acting on ionotropic Glu receptors, we co-administered 0.2 mM CNQX (an antagonist of kainate-subtype of ionotropic receptors) 10 min after the beginning of the application of each compound (Figure 3). The data obtained revealed partial reversal of the effects due to ent-10,

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suggesting that kainate receptors may have a modulatory role in the mechanism of the ent-10induced fall in extracellular Glu. In contrast, no significant reversal of compound 9-induced change in Glu levels was observed with CNQX (Figure 3). Such a difference was unlikely to be

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due a difference in sensitivity of kainate receptors between animals receiving either 9 or ent-10,

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since local administration of kainate induced similar effect on Glu levels for those animals. Despite the absence of any binding to Glu or GABA receptors, the two novel pyrrolidine compounds induced a clear-cut lowering of brain extracellular Glu when infused intra-cerebrally. These two compounds were not available in a sufficient quantity to determine whether their biological activity extended to the central nervous system after a systemic administration. To address this issue, we examined the ability of the two compounds 9 and ent-10 to cross the blood-brain interface using a differentiated cellular model of the choroid plexus epithelium that

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reproduces in vivo properties of the blood-cerebrospinal fluid (CSF) barrier [37]. These include tight junctions that restrict paracellular permeability, and efflux mechanisms mediated by multispecific transport proteins. Exposure of the basolateral side of choroidal cells to the

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compounds at a concentration of 150 µM for 60 min did not induce any alteration of the integrity of the monolayer, as assessed by the measurement of [14C]-sucrose permeability across the monolayers (Figure 4A). The permeability coefficients of the two compounds were low, being

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close or similar to sucrose coefficient values (Figure 4B). For comparison, the influx permeability coefficients measured using this model for the highly to mildly lipid soluble

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compounds caffeine and urea are respectively 12 x 10-3 and 1 x 10-3 cm.min-1. The influx Pe value for the antiretroviral drug AZT, whose penetration through the choroidal epithelium is restricted by an active efflux mechanism is around 0.6 x 10-3 cm.min-1 [38]. In order to investigate whether the blood to CSF permeability of our compounds was similarly

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limited by an efflux process, we compared influx and efflux rates for 9 and ent-10. For ent-10, the Pe values measured in the two directions were similar. The efflux permeability for 9 was lower than the influx permeability, and also lower than that of sucrose (not shown). This

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indicates that the poor permeation of the molecules across the blood-CSF barrier results from their hydrophilic properties rather than from a specific transporter-mediated efflux mechanism.

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Given the limited transfer (Pe ≈ 0.3-0.4 × 10-3 cm/min) from the blood peripheral compartment into the cerebral compartment for these compounds, and their absence of toxicity toward the barrier cells, major central side-effects are not to be expected. The permeability study did not reveal alteration of the cellular blood-CSF barrier integrity due to compounds 9 and ent-10. An additional Standard Operative Procedure was carried out in order to verify the absence of toxicity in another cell line. This was done on hepatic HepG2 cells and data

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obtained did not reveal any cytotoxicity of the two compounds tested, namely 9 and ent-10 (data not shown).

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3- CONCLUSION

The cycloaddition between (+) or (–)-menthone-derived nitrones as chiral glycine equivalents and N-benzyl-3-pyrroline opened a direct access to enantiopure fused heterocycles. In agreement

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with previous studies, the reaction occurred on the less hindered face of the nitrone, with the 3pyrroline N-benzyl group oriented outwards, thus minimizing steric interactions in the TS. This

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achieves a tight control of the configurations of the three simultaneously created chiral centers. N-Benzyl-3-pyrroline was more reactive than its N-Boc analogue, which isomerized to 2pyrroline and led preferentially to the corresponding cycloadduct. By choosing either (+) or (–)menthone, both enantiomeric cycloadducts were synthesized in almost quantitative yield. Mild

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hydrolysis, applied for removing the chiral auxiliary, combined with debenzylation by hydrogenolysis, and sometimes saponification, delivered efficiently a series of enantiopure 4hydroxy-3-glycinyl-pyrrolidine derivatives (3 to 5 steps, 36 to 81 overall yields). In the search

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for shorter routes to racemic analogues, achiral N-methyl and N-benzyl nitrones derived from ethyl glyoxalate, and a cyano nitrone were reacted also with N-benzyl-3-pyrroline. Although the

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first nitrones allowed for the synthesis of the desired racemic 4-hydroxy-3-glycinyl-pyrrolidine in 2 steps and a 52-68 % overall yield, cycloadditions with the achiral nitrones employed appeared to be less selective. By contrast, the well-defined menthone-based nitrones allowed for a perfect control of the stereochemistry of the cycloadducts and efficient syntheses of enantiopure 4-hydroxy-3-glycinyl-pyrrolidine derivatives.

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The biological data obtained with derivatives based on this simple yet unexplored scaffold show some promising pharmacological properties. The two compounds 9 and ent-10 lowered brain Glu levels in vivo while not exhibiting any brain or hepatic toxicity. Such a property must be

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emphasized since it may lead to a novel treatment of some psychiatric disorders which are characterized by central glutamatergic hyperactivity, such as depression or addictive states [35, 39-41], while Glu lowering pharmacological agents are presently scarce and actively sought.

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Although these two compounds did not cross the blood-brain interface, one can assume that the modification of active chemical moieties may lead to ability to cross this barrier while keeping

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the brain Glu lowering property. On the other hand, 9 and ent-10 could act on Glu signaling systems which are present at the peripheral level, such as adipose tissues, pancreatic beta cells and heart [42-44]. In conclusion, these new accessible scaffolds have a high pharmacological

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potential that deserves further investigations.

4- Experimental Section

4.1.1 General

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4.1 Chemical synthesis.

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All reagents for synthesis commercially available (highest purity available for reagent grade compounds) were used without further purification. Solvents were distilled over CaH2 (CH2Cl2), Mg/I2 (MeOH), Na/benzophenone (THF) or purchased dry (DMF, DMSO, Toluene). All reactions were performed under an Argon atmosphere. Reactions under microwave activation were performed on a Biotage Initiator system. NMR solvents were purchased from Euriso-Top (Saint Aubin, France). TLC was carried out on aluminum sheets coated with silica gel 60 F254

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(Merck). TLC plates were inspected by UV light (λ = 254 nm). Three staining reagents were used in order to develop TLC plates [10% H2SO4 in EtOH/H2O (95:5 v/v), Ninhydrin in nBuOH and AcOH or p-anisaldehyde in EtOH and H2SO4] followed by heating. Silica gel column

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chromatography was performed with silica gel Si 60 (40–63 µm). Optical rotation was measured using a Perkin Elmer polarimeter. NMR spectra (see Supplementary data) were recorded at 293 K, unless otherwise stated, using a 300 MHz or a 400 MHz spectrometer. Shifts are referenced

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relative to deuterated solvent residual peaks. The NMR signals were identified following the numbering shown (see Table S2 in Supplementary Data). The following abbreviations were used

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to explain the observed multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet (br denotes a broad signal). Coupling constants (J) were expressed in Hertz. Sometimes, geminal (2J) and vicinal (3J) coupling constants were identified. When assigned, the vicinal coupled protons were indicated as subscripts. Complete signal assignments from 1D and 2D NMR were

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based on COSY, HSQC and HMBC correlations. ESI mass spectra were recorded in the positive mode using a Thermo Finnigan LCQ spectrometer. High resolution (HR-ESI-QToF) mass spectra were recorded using a Bruker MicroToF-Q II XL spectrometer. (1S,2S,5R,3'aR,3'bS,7'aS)-2’-Benzyl-2-isopropyl-5,5'-dimethyl-1H-decahydro-

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4.1.2

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spiro[cyclohexane-1,6'-7'-oxa-2',5',6'a-triaza-cyclopenta[a]pentalen-4'-one] (3). In a Biotage Initiator 2 – 5 mL vial, nitrone 1 (392 mg, 1.64 mmol) and N-benzyl-3-pyrroline 2 (314mg, 1.97 mmol, 1.2 eq) were introduced. The vial was flushed with argon and 2.5 mL of anhydrous toluene was added (c = 0.66 mM). The vial was sealed with a septum cap and was sonicated for 20 s. The resulting mixture was irradiated by microwaves (temperature: 140°C). TLC monitoring (EtOAc) showed full conversion after 2 h. After the crude mixture was concentrated, the crude product was purified by flash silica gel column chromatography (EtOAc) to afford cycloadduct 3

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(625 mg, 1.57 mmol, 96 %) with no traces of other isomer. Monocrystals of compounds 3 were obtained from a saturated Et2O solution cooled in a freezer. Rf = 0.48 (EtOAc). [α]D + 40.4 (c 1.1, CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.20 (m, 5H, CH-ar), 4.59 (td, 1H, J = 7.0

RI PT

Hz, J = 3.0 Hz, H-4), 3.71 – 3.49 (m, 3H, NCH2Ph, H-6), 3.42 (dd, 1H, J = 10.3 Hz, J = 6.6 Hz, H-3), 2.78 (dd, 1H, J = 10.3 Hz, J = 3.0 Hz, H-5), 2.75 – 2.69 (m, 4H, NCH3, H-2), 2.65 (dd, 1H, J = 9.4 Hz, J = 3.7 Hz, H-2’), 2.58 (dd, 1H, J = 9.9 Hz, J = 6.6 Hz, H-5’), 2.14 – 2.08 (m, 1H, H-

SC

9), 2.00 (dtt, 1H, J = 12.9 Hz, J = 6.5 Hz, J = 3.3 Hz, H-10), 1.90 – 1.78 (m, 2H, H-11, H-12), 1.68 – 1.59 (m, 1H, H-12’), 1.48 (dt, 1H, J = 13.5 Hz, J = 6.7 Hz, H-15), 1.38 (dd, 1H, J = 12.1

M AN U

Hz, J = 3.2 Hz, H-13), 1.18 (t, 1H, J = 12.3 Hz, H-9’), 0.95 – 0.85 (m, 10H, H-11’, H-14, H-16). 13

C NMR (100 MHz, CDCl3) δ 172.8 (C=O), 138.9 (C-ar), 128.6 (CH-ar), 128.3 (CH-ar), 127.1

(CH-ar), 88.0 (C-8), 79.6 (C-4), 71.9 (C-6), 59.6 (NCH2Ph), 59.4 (C-5), 59.3 (C-2), 49.1 (C-3), 48.2 (C-13), 41.0 (C-9), 35.0 (C-11), 29.0 (C-10), 25.9 (NCH3), 24.5 (C-15), 24.2 (CH3), 22.6

TE D

(C-12), 22.4 (CH3), 18.7 (CH3). HR-ESI-QToF MS (positive mode): m/z calcd for C24H36N3O2 [M + H]+ 398.2802, found 398.2806. 4.1.3

(3S,3aR,6aS)-2-Acetyl-5-benzyl-3-(N-methyl-carboxamide)-hexahydro-pyrrolo[3,4-

EP

d]isoxazole (4). In the acetolysis approach for the cleavage of the chiral auxiliary, compound 3

AC C

(226 mg, 0.57 mmol, 1 eq.) was placed in a 50 mL round-bottom flask. The flask was flushed with argon and 3 mL of AcOH were added. Under stirring, 3 mL of Ac2O and 6 drops (~0.3 mL) of H2SO4 (95 %) were added to the suspension. The mixture was then heated to 60°C for 6h30. The flask was cooled down to 0°C and a 5M solution of NaOH was added dropwise until pH 8. The resulting crude mixture was then transferred slowly into 300 mL of saturated NaHCO3. The aqueous medium was extracted with CH2Cl2 (5 x 50 mL) and the combined organic layers were dried with Na2SO4. After concentration, the crude product was purified by silica gel flash

18

ACCEPTED MANUSCRIPT

chromatography (product added on dry silica then direct elution with EtOAc) to afford compound 4 (148 mg, 86 %). Rf = 0.28 (EtOAc). [α]D − 14.2 (c 1, CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.20 (m, 5H, CH-ar), 6.44 (bs, 1H, NHCH3), 4.97 (bs, 1H, H-6), 4.71 (dd, 1H, J

RI PT

= 7.1 Hz, J = 4.8 Hz, H-4), 3.62 (t, 1H, J = 7.1 Hz, H-3), 3.57 (d, 1H, J = 12.7 Hz, NCH2Ph), 3.33 (d, 1H, J = 12.7 Hz, NCH2Ph), 3.10 – 2.99 (m, 2H, H-2, H-5), 2.79 (d, 3H, J = 4.9 Hz, NCH3), 2.34 (dd, 1H, J = 10.0 Hz, J = 7.1 Hz, H-2’), 2.19 (dd, 1H, J = 11.6 Hz, J = 4.8 Hz, H13

C NMR (100 MHz, CDCl3) δ 175.7 (MeNC=O), 169.8

SC

5’), 2.09 (s, 3H, NC(O)CH3).

(C(O)NHCH3), 138.1 (C-ar), 128.9 (CH-ar), 128.5 (CH-ar), 127.5 (CH-ar), 85.0 (C-4), 66.2 (C-

M AN U

6), 60.7 (C-5), 60.4 (C-2), 59.9 (NCH2Ph), 48.4 (C-3), 26.4 (NCH3), 21.2 (C(O)CH3). HR-ESIQToF MS (positive mode): m/z calcd for C16H22N3O3 [M + H]+ 304.1656, found 304.1667. 4.1.4 (3S,3aR,6aS)-5-Benzyl-3-(N-methyl-carboxamide)-hexahydro-pyrrolo[3,4-d]isoxazole (5). Synthesis from 4 by N-deacetylation: In a dry and argon-flushed 25 mL round-bottom flask,

TE D

3 mL of freshly distilled methanol were added. The flask was cooled down to 0°C and 151 µL (5.2 eq.) of SOCl2 were added dropwise. Stirring was maintained for 10 min and compound 4 (122 mg, 0.40 mmol, 1 eq.) in CH2Cl2 (4×1 mL) was added dropwise over 10 min. After 15

EP

additional min at 0°C, the mixture was allowed to warm to rt and was heated (refluxing

AC C

methanol) for 1 h. The crude mixture was cooled to rt and of a 5 % Na2CO3 solution (4 mL) was added. The aqueous layer was extracted with 60 mL of EtOAc and the combined organic layers were washed twice with Na2CO3 (5 %, 40 mL). The obtained aqueous phase was extracted with CH2Cl2 (2 x 20 mL). Rotary evaporation, vacuum – drying, followed by silica gel flash chromatography (elution gradient from EtOAc to EtOAc/IPA, 1/1) afforded pure 5 (104 mg, 0.4 mmol, 99 %). Shorter synthesis from 3: In a shorter acetolysis approach for the cleavage of the chiral auxiliary, compound 3 (542 mg, 1.36 mmol) was placed in a 50 mL round-bottom flask.

19

ACCEPTED MANUSCRIPT

The flask was flushed with argon and 7.2 mL AcOH were added. Under stirring, 7.2 mL Ac2O and 0.4 mL H2SO4 (95-97 %; dropwise addition) were added to the suspension. The mixture was heated to 46°C for 4 h and another 0.4 mL portion of H2SO4 (95-97 %) was added. The resulting

RI PT

mixture was maintained for 2h30 at 46°C and then overnight at rt. The flask was cooled down to 0°C and a 5M solution of NaOH was added dropwise over a period of 2h30 until pH 8. The resulting crude mixture was transferred slowly into 700 mL saturated aqueous NaHCO3. The

SC

aqueous medium was extracted with CH2Cl2 (5 x 100 mL) and the combined organic phase was dried with Na2SO4. After concentration, the crude product (containing trace amount of the N-

M AN U

acetyl analog) was purified by silica gel flash chromatography as before to afford 5 (320 mg, 90 %). Rf = 0.74 (CH2Cl2/IPA, 1/1). [α]D + 10.9 (c 1.3, CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.23 (m, 5H, CH-ar), 7.13 (bs, 1H, NHCH3), 6.22 (d, 1H, J = 3.9 Hz, ONHR), 4.62 (dd, 1H, J = 6.6 Hz, J = 4.8 Hz, H-4), 3.66 (bs, 1H, H-6), 3.62 (t, 1H, J = 6.7 Hz, H-3), 3.48 (d, 2H, J = 6.1

TE D

Hz, NCH2Ph), 3.07 (d, 1H, J = 10.9 Hz, H-5), 3.00 (d, 1H, J = 9.9 Hz, H-2), 2.79 (d, 3H, J = 5.0 Hz, NCH3), 2.37 (dd, 1H, J = 9.9 Hz, J = 6.7 Hz, H-2’), 2.21 (dd, 1H, J = 10.9 Hz, J = 4.9 Hz, H-5’). 13C NMR (100 MHz, CDCl3) δ 171.2 (C=O), 138.4 (C-ar), 128.6 (CH-ar), 128.5 (CH-ar),

EP

127.4 (CH-ar), 83.7 (C-4), 71.0 (C-6), 61.8 (C-5), 60.8 (C-2), 59.9 (NCH2Ph), 50.2 (C-3), 26.0 (NCH3). HR-ESI-QToF MS (positive mode): m/z calcd for C14H20N3O2 [M + H]+ 262.1550,

4.1.5

AC C

found 262.1553.

(α αS,3R,4S)-α α-Acetamido-(1-benzyl-4-hydroxy-pyrrolidin-3-yl)-N-methylacetamide

(6). In a 50 mL round-bottom flask containing 600 mg of compound 4 (1.98 mmol), Mo(CO)6 (417 mg, 1.58 mmol, 0.8 eq.) was added and the flask was flushed with argon. Then, 20 mL of freshly distilled MeCN were added. Upon stirring, further 1.3 mL of water (MilliQ quality) was added and the resulting mixture was heated for 3 h with gentle reflux. Meanwhile, the solution

20

ACCEPTED MANUSCRIPT

turned brown to black. The crude mixture was concentrated after TLC monitoring (CH2Cl2/IPA, 8/2) showed completion of the reaction. The crude slurry was diluted in CH2Cl2 and filtered through a plug of Celite (CH2Cl2/MeOH, 9/1) and concentrated again. The crude product was

RI PT

purified on basic Al2O3 (elution gradient CH2Cl2 then CH2Cl2/MeOH, 8/2) to yield pure 6 (399 mg, 66 %). Rf = 0.18 (Ace/IPA, 1/1). [α]D + 33.1 (c 0.6, DMSO). 1H NMR (400 MHz, CDCl3) δ 7.35 – 7.21 (m, 5H, CH-ar), 7.12 (d, 1H, J = 8.0 Hz, NHAc), 6.92 (d, 1H, J = 4.5 Hz, NHCH3),

SC

4.55 (dd, 1H, J = 8.0 Hz, J = 5.4 Hz, H-6), 4.47 (td, 1H, J = 5.4 Hz, J = 2.8 Hz, H-4), 3.68 (d, 1H, J = 12.9 Hz, NCH2Ph), 3.57 (d, 1H, J = 12.9 Hz, NCH2Ph), 2.93 (dd, 1H, J = 10.3 Hz, J =

3H, NC(O)CH3).

13

M AN U

5.6 Hz, H-5), 2.77 (d, 3H, J = 4.8 Hz, NCH3), 2.66 – 2.50 (m, 4H, H-2, H-2’, H-3, H-5’), 2.02 (s, C NMR (100 MHz, CDCl3) δ 172.4 (NC(O)CH3), 171.3 (C(O)NHCH3),

138.5 (C-ar), 128.9 (CH-ar), 128.5 (CH-ar), 127.3 (CH-ar), 71.5 (C-4), 62.0 (C-5), 60.5 (NCH2Ph), 55.1 (C-2), 53.4 (C-6), 45.4 (C-3), 26.4 (C(O)CH3), 23.3 (NCH3). HR-ESI-QToF MS

TE D

(positive mode): m/z calcd for C16H24N3O3 [M + H]+ 306.1812, found 306.1823. 4.1.6 (3S,3aR,6aS)-2-Acetyl-3-(N-methyl-carboxamide)-hexahydro-pyrrolo[3,4-d]isoxazole (7). A 100 mL round-bottom flask containing compound 4 (157 mg, 0.52 mmol) was subjected

EP

to three vacuum/argon cycles. Under argon, 6 mL of EtOH and 6 mL of CH2Cl2 were added and

AC C

the resulting mixture was subjected to another five vacuum/argon cycles. Then, 110 mg of Pd(OH)2/C 10 % (0.1 mmol, 0.2 eq.) were added. The flask was finally subjected to five vacuum/argon cycles and subsequent five vacuum/hydrogen cycles. Stirring under 1 atm H2 was maintained overnight. The medium was filtered over a plug of Celite (CH2Cl2 and EtOH), concentrated, and the crude product was purified on a C-18 prepacked silica column with a combiflash system (elution gradient from water to water/methanol, 1/1) to yield 7 (110 mg, 99%). Rf = 0.24 (nBuOH/H2O/AcOH, 3/1/1). [α]D + 32.3 (c 0.6, DMSO). 1H NMR (400 MHz,

21

ACCEPTED MANUSCRIPT

D2O) δ 5.23 (br s, 1H, H-4), 4.79* (br s, 1H, H-6), 3.75 – 3.45 (m, 5H, H-2, H-2’, H-3, H-5, H5’), 2.79 (br s, 3H, NCH3), 2.21 (br s, 3H, NC(O)CH3). (*signal partially hidden by solvent residual peak: attribution from 2D experiments).

13

C NMR (100 MHz, D2O) δ 170.9

RI PT

(NC(O)CH3), 170.0 (C(O)NHCH3), 83.5 (C-4), 63.9 (C-6), 49.8 (C-5), 49.6 (C-3), 49.4 (C-2), 26.4 (NCH3), 20.2 (NC(O)CH3). HR-ESI-QToF MS (positive mode): m/z calcd for C9H15N3NaO3 [M + Na]+ 236.1006, found 236.1005.

SC

4.1.7 (α αS,3R,4S)-α α-Acetamido-(4-hydroxy-pyrrolidin-3-yl)-N-methylacetamide (8). A 25 mL

M AN U

round-bottom flask containing compound 6 (19 mg, 0.06 mmol) was subjected to three vacuum/argon cycles. Under argon, 5 mL of EtOH were added and the resulting mixture was subjected to another five vacuum/argon cycles. Then, 7 mg of Pd(OH)2/C 20 % (0.012 mmol, 0.2 eq.) were added. The flask was finally subjected to five vacuum/argon cycles and subsequent five vacuum/hydrogen cycles. After stirring for 2 h under 1 atm H2, the medium was filtered over

TE D

a plug of Celite (CH2Cl2 and EtOH), and concentrated. The crude product was purified on a C-18 prepacked silica column with a combiflash system (elution water) to yield 8 (9 mg, 67%). Rf =

EP

0.31 (nBuOH/H2O/AcOH, 3/1/1). [α]D + 34.0 (c 0.2, DMSO). 1H NMR (400 MHz, D2O) δ 4.44 – 4.41 (m, 1H, H-4), 4.39 – 4.23 (m, 1H, H-6), 3.43 – 3.16 (m, 2H, H-2, H-5), 3.11 – 2.89 (m,

AC C

2H, H-2’, H-5’), 2.77 (d, 3H, J = 4.1 Hz, NCH3), 2.54 – 2.46 (m, 1H, H-3), 2.07 (bs, 3H, NC(O)CH3). 13C NMR (100 MHz, D2O) δ 174.6 (NC(O)CH3), 174.0 (C(O)NHCH3), 70.8 (C-4), 54.0 (C-5), 52.8 (C-6), 46.2 (C-3), 45.9 (C-2), 26.1 (NCH3), 22.0 (NC(O)CH3). HRMS ESIQToF, positive mode: m/z calcd for C9H15N3NaO3 [M + Na]+ 236.1006, found 236.1005. 4.1.8 (αS,3R,4S)-α-Amino-(4-hydroxy-pyrrolidin-3-yl)-N-methylacetamide (9). A 50 mL round-bottom flask containing compound 5 (100 mg, 0.38 mmol) was subjected to three vacuum/argon cycles. Under argon, 10 mL EtOH and 6 mL CH2Cl2 were added and the resulting

22

ACCEPTED MANUSCRIPT

mixture was subjected to another five vacuum/argon cycles. Then, 100 µL AcOH and 40 mg of Pd(OH)2/C 20 % (0.076 mmol, 0.2 eq) were added. The flask was finally subjected to five vacuum/argon cycles and subsequent five vacuum/hydrogen cycles. Stirring was maintained

RI PT

overnight under 1 atm H2. TLC monitoring showed a complete conversion. The catalyst was removed by filtration over a plug of Celite (EtOH/MeOH). After concentration, addition of MeOH (2 mL) and toluene (5 mL) precipitated the crude 9 as a foam (36.7 mg, 55 % yield)

SC

containing 10 % AcOH. White foam; Rf = 0.19 (n-BuOH, CH3CO2H, H2O, 3:1:1); [α]22D +3.0 (c 0.5, H2O). 1H NMR (D2O, 400 MHz) δ 4.61 (m, 1H, J3,4 = 3.7 Hz, H-4), 3.50 (d, 1H, J3,6 = 9.9

2

M AN U

Hz, H-6), 3.40 (m, 2H, H-5, H-5’), 3.23 (dd, 1H, J2’,3 = 8.5 Hz, 2J = 11.6 Hz, H-2’), 3.20 (t, 1H, J = J2,3 = 11.8 Hz, H-2), 2.74 (s, 3H, NCH3), 2.40 (m, 1H, H-3). 13C NMR (D2O, 100 MHz) δ

175.9 (CO), 69.31 (CH), 53.70 (CH2), 53.07 (CH), 47.29 (CH), 45.37 (CH2), 26.07 (CH). MS: ESI positive current: 174.0 [M+H]+, 347.0 [2M+H]+; HRMS calcd for C7H15N3O2 Na1, m/z =

TE D

196.1056; found: 196.1051 (also visible : 174.1228 [M+H]+ ; 212.0788 [2M+H]+). 4.1.9 (α αS,3R,4S)-α α-Amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid (10). A 100 mL round-

EP

bottom flask containing compound 5 (112 mg, 0.43 mmol) was subjected to three vacuum/argon cycles. Under argon, 10 mL of EtOH and 6 mL of CH2Cl2 were added and the resulting mixture

AC C

was subjected to another five vacuum/argon cycles. Then, 45 mg of Pd(OH)2/C 20 % (0.086 mmol, 0.2 eq.) were added. The flask was finally subjected to five vacuum/argon cycles and subsequent five vacuum/hydrogen cycles. Stirring was maintained under 1 atm H2 for 3 h. As TLC monitoring showed an incomplete conversion, 100 µL of AcOH were added (ca. 4 eq., additional 5 vacuum/hydrogen cycles) and stirring was maintained for 24 h. The catalyst was removed by filtration over a plug of Celite (EtOH/MeOH). After concentration, the crude product (156 mg, Rf = 0.17 in nBuOH/H2O/AcOH, 3/1/1) was subjected to basic hydrolysis using

23

ACCEPTED MANUSCRIPT

180 mg of hydrated LiOH (4.3 mmol, 10 eq.) in 4 mL of THF and 4 mL of ultrapure water in a 50 mL round-bottom flask. Stirring was maintained at rt for 3 h and the solvents were evaporated off. The crude carboxylate salt was purified on a C-18 prepacked silica column with a

RI PT

combiflash system (elution water) and a subsequent purification on a Ion Exchange Resin (Dowex 50W X8). After incorporation of the product into a resin column, the column was extensively washed with water. Addition of 2.5 % and then 5 % of NH4OH into the eluent

SC

allowed the elution of the carboxylate ammonium. Evaporation and high-vacuum drying provided the zwitterionic form of compound 10 (47 mg, 0.29 mmol, 57% over two steps). Rf =

M AN U

0.18 (nBuOH/H2O/AcOH, 3/1/1). [α]D + 4.2 (c 0.5, DMSO/H20, 4/1). 1H NMR (400 MHz, D2O) δ 4.58 (t, 1H, J = 3.0 Hz, H-4), 3.54 (d, 1H, J = 9.5 Hz, H-6), 3.44 (dd, 1H, J = 8.6 Hz, J = 11.8 Hz, H-2), 3.39 – 3.36 (m, 2H, H-5, H-5’), 3.29 (d, 1H, J = 11.8 Hz, H-2’), 2.46 (ddd, 1H, J = 3.8 Hz, J = 9.1 Hz, J = 12.4 Hz, H-3). 13C NMR (100 MHz, D2O) δ 178.6 (C=O), 69.8 (C-4), 54.0

TE D

(C-6), 53.7 (C-5), 46.4 (C-3), 46.1 (C-2). HR-ESI-QToF MS (positive mode): m/z calcd for C6H13N2O3 [M + H]+ 161.0921, found 161.0924. 4.1.10

(±)-cis

2H-5-Benzyl-2-methyl-3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-d]isoxazole-3-

EP

carboxylic acid, ethyl ester ((±)-14).

AC C

A solution of nitrone 11 (300 mg, 2.29 mmol) and N-benzyl-3-pyrroline 2 (0.8 mL, 4.20 mmol) in toluene (3 mL) was stirred for 1 h at 80°C under micro-wave irradiation. TLC (1:3 EtOAc/petroleum ether) showed complete conversion of the nitrone into a major product, and unidentified polar byproducts. The solvent was evaporated and the residue was subjected to silica gel column chromatography (mobile phase: 1:4 EtOAc/toluene) to afford cycloadduct (±)-14 (390 mg, 58%). Colourless oil. Rf = 0.4 (1:4 EtOAc/toluene). 1H NMR (500 MHz, CDCl3) δ 7.35-7.20 (m, 5H, H-Ar), 4.60 (dd, 1H, J = 4.9 Hz, J = 7.4 Hz, H-4), 4.20 (q, 2H, J = 7.1 Hz, H-

24

ACCEPTED MANUSCRIPT

8), 3.70 (d, 1H, J = 13.2 Hz, NCH2Ph), 3.58 (d, 1H, J = 13.2 Hz, NCH2Ph), 3.25 (q, 1H, J = 7.1 Hz, H-3), 3.17 (br s, 1H, H-6), 3.00 (d, 1H, J = 11 Hz, H-5’), 2.94 (d, 1H, J = 9.8 Hz, H-2’), 2.78 (s, 3H, NCH3), 2.28 (br t, 1H, J = 7.6 Hz, H-2), 2.19 (br d, 1H, J = 6.6 Hz, H-5), 1.27 (t, 3H, J =

RI PT

7.1 Hz, H-9). 1D NOE experiments with selective irradiations (H-3, H-4, or H-6), showed signals enhancements as follows : H-3 irradiation : enhancements of H-2 : 4 %, H-2’ : 1.8 %, H4 : 4 % ; irradiation of H-4 : enhancements of H-3 : 3.5 %, H-5 : 3.7 %, H-5’ : 1.9 % ; irradiation 13

C NMR (126 MHz, CDCl3) δ 170.03

SC

of H-6 : enhancements of H-2 : 1.3 %, H-2’ : 2.7 %.

(CO), 138.58 (C-ar), 128.73, 128.41, 127.17 (5CH-ar), 81.03 (C-4), 75.48 (C-6), 61.26 (C-8),

M AN U

59.08 (C-11), 58.77 (C-5), 56.79 (C-2), 52.54 (C-3), 43.85 (NCH3), 14.24 (C-9). HRMS-ESI, positive mode: m/z calcd for C16H23N2O3 [M+H]+ : 291.1703; found: 291.1702. 4.1.11

(±)-3,4-cis

α-N-Methylamino-(4-hydroxy-pyrrolidin-3-yl)acetic

acid

((±)-15).

Cycloadduct (±)-14 (150 mg, 517 µmol) was dissolved in a 1:1 THF/water mixture (15 mL). The

TE D

medium was stirred overnight under H2 atmosphere at rt in the presence of Pd(OH)2/C 20% (20 mg) as catalyst. Under these conditions, fast N-debenzylation, slower N–O bond reductive cleavage and hydrolysis of the ethyl ester occurred cleanly, within ~15 h. Solids were removed

EP

by filtration over Celite and the solvent was evaporated under reduced pressure. Purification was

AC C

achieved by reversed phase column chromatography (elution: H2O, then MeOH/H2O 1/10) and the material (85 mg, 95 % yield) was recrystallized from MeOH to afford (±)-15 (80 mg, 89%) as white crystals, mp 192 °C (MeOH). 1H NMR (400 MHz, D2O) δ 4.54 (t, 1H, 3J = 3.2 Hz, H4), 3.36 (dd, 1H, 3J = 3.3 Hz, 2J = 12.8 Hz, H-5), 3.31 (dd, 1H, 3J = 0.9 Hz, 2J = 12.7 Hz, H-5’), 3.26 (dd, 1H, 3J = 8.5 Hz, 2J = 11.6 Hz, H-2), 3.18 (t, 1H, 2J = 3J = 11.7 Hz, H-2’), 3.17 (d, 1H, J = 10 Hz, H-6), 2.34 (s, 3H, NCH3), 2.32 (m, 1H, H-3).

13

C NMR (101 MHz, D2O) δ 178.99

25

ACCEPTED MANUSCRIPT

(CO), 69.41 (C-4), 62.78 (C-6), 53.87 (C-5), 46.04 (C-3), 45.79 (C-2), 33.47 (NCH3). HRMSESI, positive mode: m/z calcd for C7H15N2O3 [M+H]+: 175.1078; found : 175.1077.

RI PT

4.1.12 (±)-cis 2H-2,5-Dibenzyl-3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-d]isoxazole-3-carboxylic acid, ethyl ester ((±)-16). Nitrone 12 (149 mg, 0.72 mmol) and N-benzyl-3-pyrroline 2 (170 µL, 0.89 mmol) were dissolved in toluene (2 mL) containing a few drops of CH2Cl2 for

SC

solubilization. Cycloaddition was completed after stirring for 1 h at 80°C under micro-wave irradiation. The solvent was evaporated under reduced pressure and purification of the residue by

M AN U

silica gel column chromatography (mobile phase: 1/9 EtOAc/toluene) afforded cycloadduct (±)16 (180 mg, 68% yield). Colourless oil; Rf = 0.6 (EtOAc/toluene 1/4). 1H NMR (500 MHz, 50°C, CDCl3) δ 7.25-7.45 (m, 10H, H-Ar), 4.62 (dd, 1H, J = 4.9 Hz, J = 7.6 Hz, H-4), 4.22 (d, 1H, 2J = 13.7 Hz, ONCH2Ph), 4.12 (dq, 2H, 3J8,9 = 7.1 Hz, J = 1.6 Hz, H-8), 4.05 (d, 1H, 2J = 13.7 Hz, ONCH2Ph), 3.68 (d, 1H, 2J = 13.2 Hz, NCH2Ph), 3.61 (d, 1H, 2J = 13.2 Hz, NCH2Ph),

TE D

3.39 (d, 1H, 3J = 7.0 Hz, H-6), 3.28 (q, 1H, 3J = 7.0 Hz, H-3), 3.00 (d, 1H, 3J = 11 Hz, H-5’), 2.95 (d, 1H, 3J = 9.6 Hz, H-2’), 2.32 (br s, 1H, H-2), 2.21 (br s, 1H, H-5), 1.24 (t, 3H, 3J = 7.1 Hz, H-9). 13C NMR (126 MHz, 50°C, CDCl3) δ 170.28 (C=O), 138.64, 136.93, 129.50, 128.76,

EP

128.44, 128.30, 127.47, 127.23 (12C-ar), 81.05 (C-4), 73.09 (C-6), 61.24 (C-8), 60.92 (C-10),

AC C

59.06 (C-11), 58.88 (C-5), 56.94 (C-2), 52.23 (C-3), 14.23 (C-9). 2D NOE correlations were confirmed by 1D NOE experiments with selective irradiations (H-3, H-4, or H-6), showing signals enhancements as follows : H-3 irradiation : enhancements of H-2 : 3.7 %, H-2’ : 1.7 %, H-4 : 5.2 % ; irradiation of H-4 : enhancements of H-3 : 3.9 %, H-5 : 2.9 %, H-5’ : 2.0, H-6 : 1.0 % ; irradiation of H-6 : enhancements of H-2 : 0.9 %, H-2’ : 3.9 %, H-4 : 1.4 %. HRMS-ESI, positive mode: m/z calcd for C22H27N2O3 [M+H]+ 367.2016; found 367.2009.

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4.1.13 (±)-3,4-cis α-Amino-(4-hydroxy-pyrrolidin-3-yl)-acetic acid ((±)-10). Cycloadduct (±)16) (30 mg, 82 µmol) was dissolved in a 1:1 THF/water mixture (3 mL). After addition of Pd(OH)2/C 20% (10 mg) as catalyst, the medium was stirred overnight under H2 atmosphere at

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rt. Solids were removed by filtration over Celite and the solvent was evaporated under reduced pressure to afford a residue (20 mg). HRMS ESI, positive mode: m/z Calcd for C6H13N2O3 [M+H]+ 161.0921; found 161.0927. NMR data was found in agreement with 10 and ent-10 (see

(±)-cis

((±)-17)

and

2H-2,5-Dibenzyl-3-cyano-3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-d]isoxazole

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4.1.14

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Supplementary data, NMR data).

(±)-cis

2H-5-Benzyl-2-cyanomethyl-3-phenyl-3,3a,4,5,6,6a-hexahydro-

pyrrolo[3,4-d]isoxazole ((±)-18). Cyanonitrone 13 (550 mg, 3.44 mmol) and N-benzyl-3pyrroline 2 (1.3 mL, 6.83 mmol) were dissolved in toluene (4 mL) and the medium was stirred for 2 h at 80°C under micro-waves irradiation. The solvent was removed under reduced pressure

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before purification by silica gel column chromatography (eluent: EtOAc/toluene 1/10). This allowed the separation of a minor more mobile regioisomer (60 mg, 6%) from the main racemic cycloadduct expected (±)-17 (500 mg, 45%). Compound (±)-17: Yellowish oil. Rf = 0.4 (1:4

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EtOAc/toluene). 1H NMR (500 MHz, 80°C, toluene-d8) δ 7.4-7.0 (m, 10H, H-Ar), 4.28 (ddd, 1H,

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J = 3.4 Hz, J = 6.0 Hz, J = 7.5 Hz, H-4), 4.15 (d, 1H, J = 13.4 Hz, H-8), 3.80 (d, 1H, J = 13.4 Hz, H-8), 3.29 (d, 1H, J = 13.1 Hz, H-9), 3.23 (d, 1H, J = 13.1 Hz, H-9), 3.07 (d, 1H, J = 3.6 Hz, H-6), 2.79 (ddd, 1H, J = 4.0 Hz, J = 7.8 Hz, J = 11.6 Hz, H-3), 2.48 (dd, 1H, J = 3.1 Hz, J = 10.1 Hz, H-5’), 2.22 (br s, 1H, H-5), 2.17 (br m, 1H, H-2’), 2.09 (br m, 1H, H-2). 1D NOE experiments with selective irradiations (H-3, H-4, or H-6), showed signals enhancements as follows : H-3 irradiation : enhancements of H-2 : 2.5 %, H-2’ : 1.9 %, H-4 : 2 %, H-6 : 1.1 % ; irradiation of H-4 : enhancements of H-3 : 2.1 %, H-5 : 1.9 %, H-5’ : 1.4 % ; irradiation of H-6 :

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enhancements of H-2 : 2 %, H-2’ : 1.8 %. 13C NMR (126 MHz, 80°C, toluene-d8) : δ 139.8 (Caar), 137.2 (Ca’-ar), 130.2, 129.5, 129.4, 129.3, 128.6, 128.1 (10C-ar), 116.9 (C-7), 81.9 (C-4),

m/z calcd for C20H22N3O [M+H]+ : 320.1757; found: 320.1767.

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60.0 (C-9), 59.9 (C-6), 59.8, 59.7 (C-5, C-8), 57.5 (C-2), 53.6 (C-3). HRMS-ESI, positive mode:

Compound (±)-18: yellow clear oil. Rf = 0.6 (1/4 EtOAc/toluene). 1H NMR (500 MHz, toluene-

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d8) δ 7.5-7.0 (m, 10H, H-Ar), 4.40 (dd, 1H, J = 4.7 Hz, J = 7.7 Hz, H-4), 3.71 (d, 1H, J = 8.1 Hz, H-6), 3.39 (s, 2H, H-9), 3.20 (d, 1H, J = 17.2 Hz, H-7), 3.01 (d, 1H, J = 10.8 Hz, H-5’), 2.77 (d,

(m, 2H, H-2, H-5).

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1H, J = 17.2 Hz, H-7), 2.71 (d, 1H, J = 9.7 Hz, H-2’), 2.64 (app. q, 1H, J = 7.3 Hz, H-3), 1.73 C NMR (126 MHz, toluene-d8) δ 140.02 (Ca-ar), 138.41 (Ca’-ar), 129.84,

129.35, 129.35, 129.31, 128.93, 128.02 (10C-ar), 115.00 (C-8), 82.19 (C-4), 76.02 (C-6), 59.54 (C-9), 59.28 (C-5), 58.04 (C-3), 56.60 (C-2), 42.16 (C-7). 2D NMR (HMBC) showed correlations between H-6 and aromatic carbons, as well as correlations between methylene H-7

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and C-8 in the nitrile group. 1D NOE experiments with selective irradiations (H-3, H-4, or H-6), showed signals enhancements as follows : H-3 irradiation : enhancements of H-2 : 4.6 %, H-4 : 4.2 %, H-6 : 1.1 %, H-Ar : 3.5% ; irradiation of H-4 : enhancements of H-3 : 3.8 %, H-5 : 4.1 %,

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H-5’ : 1.2 % ; irradiation of H-6 : enhancements of H-2’ : 2.9 %, H-5’ : 0.3 %, H-Ar : 5.4 %.

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HRMS-ESI, positive mode: m/z calcd for C20H22N3O [M+H]+ 320.1757 ; found 320.1750. 4.1.15 (±)-3,4-cis 3-(1,2-Diaminoethyl)-4-hydroxy-pyrrolidine ((±)-19). The cycloadduct (±)17 (40 mg, 125 µmol) was dissolved in a 10:10:1 THF/water/AcOH mixture (3 mL). After adding Pd(OH)2/C 20% (10 mg)as catalyst, the medium was stirred at rt for 40 h under H2 (4 bars). TLC monitoring (3:1:1 nBuOH/water/AcOH) showed the complete transformation of (±)17 into four more polar products. The medium was filtered through a pad of Celite, and the solvent was evaporated under reduced pressure. The crude residue (23 mg, 90 % crude yield)

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showed by mass spectrometry a predominant ion (m/z 146[M+H]+) corresponding to the expected amino alcohol (±)-19 and another one, for a minor benzylated product (m/z 221 [M+H]+).

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C NMR confirmed the existence of these 2 products in 1:3 to 1:4 ratio. (±)-19 : 1H

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NMR (400 MHz, D2O) δ 4.59 (br s, 1H, H-4), 3.47 (dd, 1H, 2J = 11.6 Hz, 3J = 8.6 Hz, H-2), 3.34 (m, 2H, H-5), 3.21 (dt, 1H, 3J = 3.2 Hz, 3J = 9.8 Hz, 3J = 9.8 Hz, H-6), 3.15 (t, 1H, 2J = 11.6 Hz, 3

J = 11.6 Hz, H-2), 3.04 (dd, 1H, 3J = 3.2 Hz, 2J = 13.3 Hz, H-7), 2.87 (dd, 1H, 3J = 10.0 Hz, 2J

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= 13.3 Hz, H-7), 2.30 (m, 1H, H-3). 13C NMR (101 MHz, D2O) δ 69.41 (C-4), 53.66 (C-5), 48.04 (C-6), 47.01 (C-3), 45.15 (C-2), 43.38 (C-7). HRMS-ESI, positive mode: m/z calculated for

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C6H16N3O [M+H]+ 146.1288 ; found : 146.1281. The other product showed the following signals: 13C NMR (101 MHz, D2O) δ 132.9 (1C-ar), 129.5 (2CH-ar), 129.48 (1CH-ar), 129.1 (2CH-ar), 68.81 (CH, C-4), 60.62 (CH2, C-7), 53.84 (CH2, C-5), 51.15 (CH, C-6), 45.29 (CH2, C-2), 43.29 (CH2-benzyl), 42.24 (CH, C-3). MS m/z = 221 [M+H]+ corresponded to C13H20N2O,

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indicating loss of one amino residue (probably the α-amino group) and the presence of a benzyl group (seen by NMR) probably attached to the pyrrolidine nitrogen. 4.1.16

(±)-cis

3aH-5-N-Benzyl-4,5,6,6a-tetrahydro-pyrrolo[3,4-d]-isoxazole-3-carboxylic

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acid, ethyl ester ((±)-20). N-Benzyl-3-pyrroline 2 (504 µL, 2.64 mmol) was added to a solution

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of ethyl chlorooximidoacetate (1 g, 6.62 mmol, 2.5 eq) in toluene (20 mL). The flask was heated to 110°C (oil bath) while a solution of Et3N (276.8 µL, 19.8 mmol, 7.5 eq) in toluene (15 mL) was added over 16h using a syringe pump. After concentration under reduced pressure, the crude product was purified by silica gel column chromatography (4:1 petroleum ether/EtOAc) to afford compound (±)-20 (370 mg, 1.35 mmol, 51%). Yellow oil Rf = 0.52 (7:3 petroleum ether/EtOAc). 1

H NMR (400 MHz, CDCl3) : δ 7.26 (m, 5H, phenyl), 5.20 (dd, 1H, J5,4 = 4.5 Hz, J3,4 = 9.6 Hz,

H-4), 4.31 (2 complex q, 2H, J = 7.2 Hz, diastereotopic OCH2), 3.91 (dt, 1H, J2’,3 = 1.0 Hz, J2,3 =

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7.1 Hz, J3,4 = 9.5 Hz, H-3), 3.68 (d, 1H, 2J = 13.3 Hz, NCH2Ph), 3.55 (d, 1H, 2J = 13.3 Hz, NCH2Ph), 3.24 (d, 1H, 2J = 11.2 Hz, H-5’), 3.19 (d, 1H, 2J = 9.9 Hz, H-2’), 2.44 (dd, 1H, J2,3 = 7.2 Hz, 2J = 9.8 Hz, H-2), 2.37 (dd, 1H, J5,4 = 4.6 Hz, 2J = 11.2 Hz, H-5), 1.31 (t, 3H, J = 7.2 Hz, 13

C NMR (100 MHz, CDCl3): δ 160.7, 152.2 (C=N, C=O), 137.8 (C-ar), 128.5 (2CH-ar),

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Me).

128.2 (2CH-ar), 127.1 (1CH-ar), 87.4 (C-4), 61.9 (OCH2), 61.1 (C-5), 58.3 (CH2 benzyl), 56.9 (C-2), 51.0 (C-3), 14.1 (CH3). MS m/z = 274.13 for C15H18N2O3; MS-ESI positive mode: 275.1

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[M+H]+, 297.1 [M+Na]+.

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4.1.17 Attempted synthesis of (±)-3,4-cis α-Amino-(1-benzyl-4-hydroxy-pyrrolidin-3yl)acetic acid, ethyl ester ((±)-21). A 25 mL round-bottom flask containing compound (±)-20 (60 mg, 0.21 mmol) was subjected to three vacuum/argon cycles. Under argon, 6 mL MeOH was added and the resulting mixture was subjected to another five vacuum/argon cycles. Pd(OH)2/C 20 % (20 mg, 0.038 mmol) was added. The flask was subjected to five vacuum/argon cycles and

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five vacuum/hydrogen cycles. Stirring was maintained overnight under H2 (10 bar) and the medium was filtered over a plug of Celite (H2O/MeOH). Compound (±)-21 was not detected by MS, but instead an ion m/z 285 [M+H]+ indicated a product formed by reduction of the

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isoxazoline and displaying a cyclohexyl group (reduction of the benzyl group), while the crude

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reaction mixture led to complex NMR spectra. When the reduction was attempted without AcOH and under a 4 bar H2 pressure, the starting material was still present after 4 days, and TLC showed a multicomponent mixture (6 spots). 4.1.18 Synthesis of enantiomers from the (+)-menthone-based nitrone : Appling the protocols reported above for the cycloaddition with the (+)-menthone-based nitrone, and for further reactions delivered in similar yields the corresponding enantiomers, and with identical NMR data

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with those obtained initially from the (–)-menthone-based nitrone, except for their optical rotations (see Supplementary data Table S1).

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4.1.19 X-Ray diffraction analysis of compounds 3 and 3’’ : Suitable crystals of 3 and 3’’ were selected and mounted on a Gemini kappa-geometry diffractometer (Agilent Technologies UK Ltd) equipped with an Atlas CCD detector and using Mo radiation ( λ = 0.71073 Å).

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Intensities were collected at 110 K for 3 and 100 K for 3’’ by means of the CrysalisPro software (Agilent Technologies, Version 1.171.34.49, release 20-01-2011 CrysAlis171.NET, compiled

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Jan 20 2011,15:58:25). Reflection indexing, unit-cell parameters refinement, Lorentzpolarization correction, peak integration and background determination were carried out with the CrysalisPro software. An analytical absorption correction was applied using the modeled faces of the crystal [45]. The resulting set of hkl was used for structure solution and refinement. The

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structures were solved by direct methods with SIR97 [46] and the least-square refinement on F2 was achieved with the CRYSTALS software [47]. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were all located in a difference map, but those attached to

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carbon atoms were repositioned geometrically. The H atoms were initially refined with soft restraints on the bond lengths and angles to regularize their geometry (C---H in the range 0.93--

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0.98 Å) and Uiso(H) (in the range 1.2-1.5 times Ueq of the parent atom), after which the positions were refined with riding constraints. Crystal data for compound 3: C24H35N3O2, Mr = 397.56 g mol-1, Monoclinic, P21,a = 9.718 (1) Å, b = 8.2101 (8) Å, c = 13.979 (2) Å, α = γ = 90° and β = 101.79(1)°, Dcalc = 1.209 g cm-3, Z = 2, independent reflections = 5111 (Rint = 0.038), Rvalues [I>2(I), 4575 reflections]: R1 = 0.056, wR2 = 0.129; S = 0.94. Crystal data for compound 3’’: C22H37N3O4, Mr = 407.55 g mol-1, Monoclinic, C2,a = 15.752 (2) Å, b = 11.156 (1) Å, c = 13.719 (2) Å, α = γ = 90° and β = 108.15(1)°, Dcalc = 1.182 g cm-3, Z = 2, independent reflections

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= 4822 (Rint = 0.043), Rvalues [I >2σ(I), 4001 reflections]: R1 = 0.054, wR2 = 0.110; S = 0.92. Crystallographic data (excluding structure factors) for the structures 3 and 3’’ in this report have been deposited with the Cambridge Crystallographic Data Centre as supplementary publications

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nos. CCDC 981868 and CCDC 981867. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44 (0)1223 336033 or e-

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mail: [email protected]). 4.2 Biological activities.

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4.2.1 Determination of target receptors. Experiments based on inhibition of specific binding have been conducted in vitro on standardized assays for sixty potential pharmacological targets including G-protein coupled receptors, ion channels and transporters (please see Supplementary data, Table S3) using specific radioligands. This study has been carried out by Eurofins Cerep-

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Panlabs (Celle L’Evescault, France). The percentage of inhibition of specific binding was determined for the 4 compounds at a concentration of 10 µM (n=2). Results are expressed as percent of control specific binding [(measured specific binding / control specific binding)×100]

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and as percent of inhibition of the control specific binding [100-(measured specific binding / control specific binding)×100], obtained in presence of four synthesis compounds (ent-8, 9, ent-

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10 and 10). Data analysis was performed on the sixty receptor targets using a « box and whisker » representation being performed for each compound. The properties agonist /antagonist were thereafter determined only for the compounds exhibiting at least 25 % of inhibition of specific binding, i.e. for 9 and ent-10 vs both CB1- and CB2-receptors using specific agonists or antagonist ligands and measuring the release of cAMP by human recombinant CHO cells [48]. Data analysis was performed using one-way ANOVA using the software developed by Institut Pierre Louis INSERM UMR S1193 (http://marne.u707.jussieu.fr/biostatgv/?module=tests).

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Cytotoxicity was evaluated by Cerep Inc. (Redmond, WA, USA) using a hepatic HepG2 cell line exposed 72h to each compound at 37°C and five criteria were followed: cell number, intracellular free calcium, nuclear size, membrane permeability, mitochondrial membrane

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potential [49].

4.2.2 In vivo extracellular glutamate concentration using microdialysis and analysis by

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capillary electrophoresis. Concentric microdialysis probes were constructed from regenerated cellulose dialysis tubing (Spectra/Por hollow fiber, MWCO 6000 Da, 225 mm o.d., 3 mm active

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dialysis length; Spectrum Medical Industries, Los Angeles, CA, USA) and fused-silica capillary tubing (40 µm i.d×105 µm o.d.; Polymicro Technology, Phoenix, AZ, USA), the body of the probe being made of a 3-cm 26G stainless steel tube, as previously designed [50]. Before implantation, the probes were perfused at a rate of 1 µL/min with artificial cerebrospinal fluid (aCSF) (145.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.2 mM CaCl2, 0.45 mM NaH2PO4, 1.55

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mM Na2HPO4, pH 7.4) using a pump from Harvard (Model 22, South Natick, MA, USA). In vivo microdialysis experiments were carried out on male Wistar rats (weighting ≈ 300 g;

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Charles River Laboratories, L’Arbresle, France). The animals were kept and used in experiments according to the European Communities Council Directives. Our protocol was approved by the

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local ethical committee of Université Lyon 1 (CEEA-55). Rats were anaesthetized with 1.4 g/kg i.p. urethane (Sigma Chemicals, St. Louis, MO, USA) and probes were implanted in the dorsolateral striatum according to the atlas of Paxinos and Watson at the following coordinates (relative to bregma): anterior 0 mm, lateral +3.5 mm, ventral +6.5 mm below the brain surface. The rate of infusion was 1 µL/min. The collection of basal samples was initiated 2 h after microdialysis probe implantation. At the end of the experiment, the rats were killed with a lethal dose of anesthetic.

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Samples were collected every 5-min in 0.2 mL PCR tubes (Axygene, Union City, CA, USA) and immediately stored at -30°C until analysis. New compounds were administered in situ at 1 mM through reverse microdialysis by switching the inlet of the probe from normal aCSF to aCSF

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containing the tested chemicals [50, 51].

Glu levels were determined by capillary electrophoresis with laser-induced fluorescence

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detection [52]. On the day of analysis, 5 µL of sample and 5 µL of standard solutions were derivatized at room temperature by adding 2 µL of a mixture (1:2:1 v/v/v) of (i) internal standard

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(10-4 M cysteic acid in 0.117 M perchloric acid), (ii) a borate/NaCN solution (100:20 v/v mixture of 500 mM borate buffer, pH 8.7, and 87 mM NaCN in water), and (iii) a 2.925 mM solution of naphthalene-2,3-dicarboxaldehyde in acetonitrile/water (50:50 v/v). The samples were diluted in 5 µL of water and then analyzed for amino acid content using an automatic capillary electrophoresis P/ACE™ MDQ system (Beckman, USA) equipped with a ZETALIF laser-

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induced fluorescence detector (Picometrics, France). Excitation was performed using a laser diode (Oxxius, France) at a wavelength of 410 nm, the emission wavelength being 490 nm. Separations were carried out on a 63 cm × 50 µm i.d. fused-silica capillary (Composite Metal

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Services, Worcester, England) with an effective length of 52 cm. Each day, before the analyses,

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the capillary was sequentially flushed with 0.25 M NaOH (15 min), ultra-pure water (15 min), and running buffer (75 mM sodium borate, pH 9.20 ± 0.02) (5 min). The separation conditions were an applied voltage of 25 kV, hydrodynamic sample injection (10 s at 0.2 psi), and a temperature, between 41 and 43°C. The capillary was sequentially flushed for 30 s each with running buffer between analyses. Electropherograms were acquired at 15 Hz using P/ACE™ MDQ software. The Glu levels in striatal microdialysates were far above the limits of detection and were expressed as percent of the four baseline values preceding compound administration.

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All the reagents of the microdialysis experiments (except our synthesis compounds) were purchased from Sigma Chemicals (St Louis, MO, USA). Standard solutions of 1 or 0.1 mM of our synthesized compounds were aliquoted in aCSF and stored at -30°C. Before administration,

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they were diluted in aCSF. 4.2.3 Blood-CSF barrier permeability.

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Primary cell cultures of choroid plexus epithelial cells were prepared from neonate rat choroid plexuses as previously described [38, 53] The cells were seeded on microporous Transwell

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polycarbonate filter inserts (6.5 mm diameter, 0.33 cm2 surface, 0.4 µm pore size) and grown in culture medium consisting of Ham’s F-12 and DMEM (1:1) supplemented with 10% FCS, 2 mM glutamine, 50 µg/mL gentamycine, and several growth factors as previously reported [53]. The medium was renewed every other day until the day of the permeability studies.

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Permeability studies were performed on a rotating platform (200 rpm) at 37°C, in Ringer-Hepes buffer (RH) as previously described [37]. The volumes used in the two compartments of the inserts (the lower one mimicking blood and the upper one mimicking brain CSF) were chosen as

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to be in equilibrium and to avoid any hydrostatic pressure. Inserts were rinsed with RH buffer before initiating permeability studies. For each synthesized compound, transfer was carried out

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in the same conditions on three laminin-coated inserts (without cells) and three inserts covered by a confluent monolayer of epithelial cells. Blood-to-CSF flux and CSF-to-blood flux were measured for 9 and ent-10 as previously described [37, 38]. Samples were assayed by HPLC. [14C]-sucrose permeability was measured as an index of the barrier integrity. At the end of the transfer of the compound of interest, [14C]-sucrose was added in the upper compartment of inserts. The radioactivity of aliquots sampled at regular intervals from each well as well as an

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aliquot of the upper chamber was determined by liquid scintillation counting using a β-counter (Canberra Packard TRI-CARB 1600TR).

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Data were expressed as permeability coefficient Pe (cm.min-1) calculated from the clearance curves obtained from the transfer experiments performed over one hour, as previously described in details [37, 38].

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4.2.4 HPLC analysis of two new synthetized compounds. The primary amine of compound ent-10 was derivatized at room temperature by adding one volume of OPA (o-phthaldialdehyde;

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5 mg/mL in 100/5/895 (v/v/v) methanol/2β-mercaptoethanol/200 mM borate buffer, pH 9.5). Reaction time was set to 2 min. Derivatized samples were stable for 30 to 40 min. The analytical HPLC conditions were as follows: RP-C18 column (150 mm, 5 µm), mobile phases with elution gradient in min (A:B): 0-5 (83 :17) ; 6-11 (35 :65) ; 12-17 (83 :17), with A being a 90:10 (v:v)

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mixture of 50 mM potassium phosphate buffer pH 6 and methanol and B being 100% methanol; spectrophotometric detection at 340 nm; flow rate at 1 mL/min; injection volume of 20 µL. The retention time for ent-10 was of 6.1 min.

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The secondary amine of compound 9 was derivatized at room temperature with a 0.1 volume of FMOC (fluorenylmethyloxycarbonyl chloride; 5 mg/mL in acetonitrile) and 20 µL of 200 mM

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borate buffer, pH 9.5. Reaction time was set to 30 min and derivatized samples were stable for two hours. The analytical conditions were as follows: RP-C18 column (15 mm × 5 µm), mobile phase with elution gradient in min (A:B): 0 (65:35) ; 18 (40:60) ; 19-29 (20:80) ; 30-35 (35:35), with A being a 90:10 (v:v) mixture of 50 mM sodium acetate buffer pH 4,2 and acetonitrile and B being 100% acetonitrile; spectrophotometric detection at 230 nm; flow rate at 1 mL/min; injection volume of 20 µL. Retention time for derivative 9 was of 14.8 min.

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ACKNOWLEDGEMENTS Financial support (in particular as post-doctoral stipends for SC and KA) from Lyon Science Transfert and University of Lyon is thankfully acknowledged. The authors are also grateful for

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supports from the University Lyon 1, CNRS, CPE, and INSERM. The authors kindly thank Dr. F. Albrieux, C. Duchamp and N. Henriques (Centre Commun de Spectrométrie de Masse of ICBMS UMR-5246) for technical assistance, and Gabriel Debilly (Centre de Recherche en

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Neurosciences de Lyon, INSERM U1028) for the biostatistical analysis of binding data.

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FIGURE LEGENDS

Figure 1. Target molecules and structural analogs mimicking neurotransmitters as excitatory

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glutamate and inhibitory GABA. Figure 2. View of the atomic structures of 3 and 3’’ determined by single-crystal X-ray diffraction analysis (displacement ellipsoids are plotted at the 50 % probability level).

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Figure 3. Effects of compounds 9 and ent-10 on glutamate level (black circles) in the striatum of

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anesthetized rats. The new synthetic compounds (hatched bars) were administered at 1 mM by reverse dialysis. CNQX, a kainate receptor antagonist, was administered at 0.2 mM (black bars) by reverse dialysis. Data are expressed as percentage of the mean of the four baseline values preceding the beginning of the application of the synthetic compound.

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Figure 4. Blood-to-CSF influx of compounds ent-10 and 9 across the choroidal epithelial cell monolayer. A. The integrity of the physical barrier during exposure of the cells to the compounds at a concentration of 150 µM, assessed by the measurement of the paracellular permeability to

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sucrose, is preserved. The dotted line represents the mean Pe value for sucrose determined in the absence of any compound. B. Blood-to-CSF permeability coefficients are presented for

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compounds. They are close or similar to the sucrose permeability coefficients. Data are expressed as mean ± SD in both graphs.

38

Figure 1

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39

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Figure 2

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Figure 3

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B

ent-10

9

Figure 4

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Scheme 1. Routes to 3-glycinyl-4-hydroxy-pyrrolidines: racemic and chiral design from 3-

CHIRAL APPROACH

N

O N

H N

H

O

O

Hydrolysis Reduction

N

O

R = Me, Bn R' = CO2Et

Z N

-O

H

HO H2N

[3+2]

R'

N [3+2]

R

OH

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(–)-menthone based nitrone

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N

RACEMIC APPROACH

Z N

Z N

-O

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pyrrolines [1].

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3-glycinyl-4-hydroxy-pyrrolidine scaffold

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Scheme 2. Synthesis of enantiopure α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives by cycloaddition of (–)-menthone-derived nitrone 1 and N-benzyl-3-pyrroline 2.a H

BocN O N

O

3''

N

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74 % BocN

2'

O N

Bn N

i

O N

+

1

2

BnN

O N

H

ii

(–)-menthone

4

iv 90 %

(+)-menthone

1

+

(–)-menthone

v 66 % H

vi 99 %

O

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O NH HN

O

EP H N

H

HO

a

AC C

H3N (αS,3R,4S)

O-

10

H

O

vii 67 %

7

HN

HN H HO

O

O N HN

viii 55 %

O

6

HN

5

ix 57 % (2 steps)

O

HO HN HN

BnN

3 - 10

O

O

BnN

iii 99 %

H

ENANTIOMERS OF

O

O N HN

86 %

96 %

3

ENANTIOMER OF

21 %

BnN H

N

3'

N

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+

-O

O N

Boc N

Boc N

O

SC

H

i

O

HO HN HN

H2N HN

(αS,3R,4S)

H

O

9

O

8

Reagents and conditions: (i) toluene, µW, 140°C, 2h and 10.5h with, respectively, 2 and 2’; (ii)

1/ 1:1 Ac2O/AcOH, ε H2SO4, 60°C; 2/ 5M aq NaOH added dropwise; (iii) SOCl2, MeOH, reflux, 30 min; (iv) 1/ 1:1 Ac2O/AcOH, ε H2SO4, 46°C, 6h30; 2/ 5M aq NaOH added over 2h30, 0°C; (v) Mo(CO)6, 15:1 CH3CN/H2O; (vi) H2, Pd(OH)2/C, EtOH, CH2Cl2, 14 h ; (vii) H2, Pd(OH)2/C, EtOH, 2 h ; (viii) H2, Pd(OH)2/C, EtOH/CH2Cl2 and AcOH, ~12 h; (ix) 1/ 1atm H2, Pd(OH)2/C, EtOH/CH2Cl2 and AcOH, 24 h; 2/ LiOH, THF/H2O, H+ Dowex resin.

44

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Scheme 3. Synthesis of racemic α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives.a

O

i

ii

58%

90%

11

O

N

Bn

O

CO2Et

(±)-14

3:1 Z/E

O N

H N HO + MeH2N

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O

Bn N

O

i O

68%

12

O

CN O N

O

13 3:1 Z/E

iv

CN

N Bn

Bn N

O

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N

13'

O

(±)-15

H N

iii quant. CO2Et

(±)-16

O

HO

H CN

+

H3N

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5:3 Z/E

N Bn Bn N

O-

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O N

Bn N

O-

(±)-10

H N

v

HO H2N

(±)-17 45 %

NH2

(±)-19

crude yield: 90 % ~ 70 % purity

H

(±)-18

N

6%

EP

NC

a

C

N

AC C

EtO2C

Bn N

vi

H N

vii

O

51% O

N

(±)-20

CO2Et

HO H2N

CO2R

(±)-21 R = Et, H

Reagents and conditions: (i) 2, µW, 80°C, 1 h; (ii) H2, Pd(OH)2/C, 1:1 THF/H2O, 24 h; (iii) H2, Pd(OH)2/C, 1:1 THF/H2O, 2 h (iv) 2, µW, 80°C, 2 h; (v) H2, Pd(OH)2/C, 10:10:1 THF/H2O/AcOH, 40 h ; (vi) 2, EtO2CCCl=NOH, Et3N, toluene, reflux, overnight; (vii) H2, pressure from 1 to 10 atm, Pd(OH)2/C, various solvents.

45

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ent-10

9

ent-8

CB1 receptors

+29 %

+29%

n.s.

CCK1 receptors

n.s.

n.s.

-46%

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% of inhibition of specific binding

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Table 1: Significant values of the percent inhibition of control specific binding determined at 10 µM (n=2) for compounds ent-10, 9 and ent-8

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n.s. not significant

46

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Supplementary data. Additional figures illustrating NMR data, properties of (+)-menthone based ent-series, and in

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vitro determination methods of target receptors (including CB-1 and CB-2 receptors) are available.

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Authors’ contribution

JPP and SP designed experiments; SC, KA and JG performed chemical synthesis and analyzed

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data; SP performed brain microdialysis study; SP and LD analyzed neurochemical data; JFGE, NS and SB performed blood brain interface study and analyzed data; SC, KA, SP, LD, JFGE and

REFERENCES

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JPP wrote the manuscript, JPP and CC supervised the research.

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[1] K. Aouadi, J. Abdoul-Zabar, M. Msaddek, J.-P. Praly, A cycloaddition–cyclization combined approach to enantiopure 3-glycinyl-4-hydroxypyrrolidines and 3-substituted 4-hydroxyprolines, Eur. J. Org. Chem. (2014) 4107-4114. [2] F. Casu, M.A. Chiacchio, R. Romeo, G. Gumina, Chiral synthesis of heterosubstituted nucleoside analogs from non-carbohydrate precursors, Curr. Org. Chem. 11 (2007) 1017-1032. [3] C.E. Taylor, Q.T. Pan, A.K. Mapp, Synergistic Enhancement of the Potency and Selectivity of Small Molecule Transcriptional Inhibitors, ACS Med. Chem. Lett. 3 (2012) 30-34. [4] C.A. Bates, W.C. Pomerantz, A.K. Mapp, Transcriptional Tools: Small Molecules for Modulating CBP KIX-Dependent Transcriptional Activators, Biopolymers 95 (2011) 17-23. [5] R.J. Casey, J.P. Desaulniers, J.W. Hojfeldt, A.K. Mapp, Expanding the repertoire of small molecule transcriptional activation domains, Bioorg. Med. Chem. 17 (2009) 1034-1043. [6] B.L. Stocker, E.M. Dangerfield, A.L. Win-Mason, G.W. Haslett, M.S.M. Timmer. Recent Developments in the Synthesis of Pyrrolidine-Containing Iminosugars, Eur. J. Org. Chem. (2010) 1615-1637. [7] A. Trapero, A. Llebaria, A Prospect for Pyrrolidine Iminosugars as Antidiabetic alphaGlucosidase Inhibitors, J. Med. Chem. 55 (2012) 10345-10346.

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[8] F.T. Xue, J.M. Kraus, K.J. Labby, H.T. Ji, J. Mataka, G.Y. Xia, H.Y. Li, S.L. Delker, L.J. Roman, P. Martasek, T.L. Pouos, R.B. Silverman, Improved Synthesis of Chiral Pyrrolidine Inhibitors and Their Binding Properties to Neuronal Nitric Oxide Synthase, J. Med. Chem. 54 (2011) 6399-6403. [9] M. Martin-Rodriguez, C. Najera, J.M. Sansano, A. de Cozar, F.P. Cossio, Chiral gold(I) vs chiral silver complexes as catalysts for the enantioselective synthesis of the second generation GSK-hepatitis C virus inhibitor, Beilstein J. Org. Chem. 7 (2011) 988-996. [10] C. Najera, J.M. Sansano, Proline and pyrrolidine derivatives: New drug candidates for hepatitis C treatment, Actual Chimique 370 (2013) 28-30. [11] U. Chiacchio, L. Borrello, L. Crispino, A. Rescifina, P. Merino, B. Macchi, E. Balestrieri, A. Mastino, A. Piperno, G. Romeo, Stereoselective Synthesis and Biological Evaluations of Novel 3 '-Deoxy-4 '-azaribonucleosides as Inhibitors of Hepatitis C Virus RNA Replication, J. Med. Chem. 52 (2009) 4054-4057. [12] J. Guan, S. Mathai, H.P. Liang, A.J. Gunn, Insulin-like growth factor-I and its derivatives: potential pharmaceutical application for treating neurological conditions, in: V. Di Marzo (Ed.) Recent patents on CNS Drug Discovery, Bentham Science Publishers Ltd., 2013, pp. 142-160. [13] J. Guan, Insulin-like growth factor-I and its derivatives: potential pharmaceutical application for ischemic brain injury, in: V. Di Marzo (Ed.) Recent patents on CNS Drug Discovery, Bentham Science Publishers Ltd., 2008, pp. 112-127. [14] B. Westermann, A. Walter, U. Florke, H.J. Altenbach, Chiral auxiliary based approach toward the synthesis of C-glycosylated amino acids, Org. Lett. 3 (2001) 1375-1378. [15] K. Aouadi, E. Jeanneau, M. Msaddek, J.-P. Praly,.New synthetic routes toward enantiopure (2S,3R,4R)-4-hydroxyisoleucine by 1,3-dipolar cycloaddition of a chiral nitrone to C4 alkenes, Synthesis (2007) 3399-3405. [16] K. Aouadi, E. Jeanneau, M. Msaddek, J.-P. Praly, 1,3-Dipolar cycloaddition of a chiral nitrone to (E)-1,4-dichloro-2-butene: a new efficient synthesis of (2S,3S,4R)-4hydroxyisoleucine, Tetrahedron Lett. 53 (2012) 2817-2821. [17] K. Aouadi, E. Jeanneau, M. Msaddek, J.-P. Praly, Analogues of insulin secretagogue (2S,3R,4S)-4-hydroxyisoleucine: synthesis by 1,3-dipolar cycloaddition reactions of chiral nitrones to alkenes, Tetrahedron-Asymmetr. 19 (2008) 1145-1152. [18] J.-F. Poisson, A. Orellana, A.E. Greene, Stereocontrolled synthesis of (-)-kainic acid from trans-4-hydroxy-L-proline, J. Org. Chem. 70 (2005) 10860-10863. [19] R.S. Fisher, Animal models of the epilepsies, Brain Res. Rev. 14 (1989) 245-278. [20] J.-P. Praly, K. Aouadi, S. Cecioni, L. Denoroy, S. Parrot, Nouveaux dérivés de pyrrolidine, in, PCT/FR2013/051524, WO2014006307 A1, France, 2014. [21] S. Cicchi, A. Goti, A. Brandi, A. Guarna, F. Desarlo, 1,3-aminoalcohols by reductive cleavage of isoxazolidines with molybdenum hexacarbonyl, Tetrahedron Lett. 31 (1990) 33513354. [22] P. Deshong, C.M. Dicken, R.R. Staib, A.J. Freyer, S.M. Weinreb, Determination of configuration and conformation of isoxazolidines by nuclear overhauser effect difference spectroscopy, J. Org. Chem. 47 (1982) 4397-4403. [23] P. Deshong, J.M. Leginus, Nitrone cyclo-additions - An efficient method for the homologation of aldehydes to alpha,beta-unsaturated aldehydes, J. Org. Chem. 49 (1984) 34213423. [24] Z.Y. Hong, L. Liu, C.C. Hsu, C.H. Wong, Three-step synthesis of sialic acids and derivatives, Angew. Chem. Int. Edit. 45 (2006) 7417-7421.

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[25] T.B. Nguyen, A. Martel, R. Dhal, G. Dujardin, A Large-Scale Low-Cost Preparation of NBenzylhydroxylamine Hydrochloride, Synthesis (2009) 3174-3176. [26] F. Machetti, F.M. Cordero, F. De Sarlo, A. Brandi, Practical synthesis of both enantiomers of protected 4-oxopipecolic acid, Tetrahedron 57 (2001) 4995-4998. [27] H. Tokuyama, T. Kuboyama, A. Amano, T. Yamashita, T. Fukuyama, A novel transformation of primary amines to N-monoalkylhydroxylamines, Synthesis (2000) 1299-1304. [28] A.P. Kozikowski, M. Adamczyk, Methods for the stereoselective cis cyanohydroxylation and carboxyhydroxylation of olefins, J. Org. Chem. 48 (1983) 366-372. [29] P. Conti, M. De Amici, A. Pinto, L. Tamborini, G. Grazioso, B. Frolund, B. Nielsen, C. Thomsen, B. Ebert, C. De Micheli, Synthesis of 3-hydroxy- and 3-carboxy-Delta(2)-isoxazoline amino acids and evaluation of their interaction with GABA receptors and transporters, Eur. J. Org. Chem. (2006) 5533-5542. [30] B. Lutz, Molecular biology of cannabinoid receptors, Prostag. Leukotr. Ess. 66 (2002) 123142. [31] H. Gunduz-Bruce, The acute effects of NMDA antagonism: from the rodent to the human brain, Brain Res. Rev. 60 (2009) 279-286. [32] C. Pittenger, G. Sanacora, J.H. Krystal, The NMDA receptor as a therapeutic target in major depressive disorder, CNS & neurological disorders drug targets 6 (2007) 101-115. [33] L. Ståhle, Chapter 7 - The use of microdialysis in pharmacokinetics and pharmacodynamics, in: T.E. Robinson, J.B. Justice (Eds.) Techniques in the Behavioral and Neural Sciences, Elsevier, 1991, pp. 155-174. [34] L. Denoroy, L. Bert, S. Parrot, F. Robert, B. Renaud, Assessment of pharmacodynamic and pharmacokinetic characteristics of drugs using microdialysis sampling and capillary electrophoresis, Electrophoresis 19 (1998) 2841-2847. [35] S. Parrot, B. Renaud, L. Zimmer, L. Denoroy, Monitoring Neurotransmitter Amino Acids by Microdialysis: Pharmacodynamic Applications, in: Applications of Microdialysis in Pharmaceutical Science, John Wiley & Sons, Inc., 2011, pp. 151-206. [36] S. Parrot, V. Sauvinet, V. Riban, A. Depaulis, B. Renaud, L. Denoroy, High temporal resolution for in vivo monitoring of neurotransmitters in awake epileptic rats using brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection, J. Neurosci. Meth. 140 (2004) 29-38. [37] N. Strazielle, J.E. Preston, Transport across the choroid plexuses in vivo and in vitro, Methods Mol. Med. 89 (2003) 291-304. [38] N. Strazielle, M.-F. Belin, J.-F. Ghersi-Egea, Choroid plexus controls brain availability of anti-HIV nucleoside analogs via pharmacologically inhibitable organic anion transporters, AIDS 17 (2003) 1473-1485. [39] D.C. Javitt, Glutamate as a therapeutic target in psychiatric disorders, Mol. Psychiatr. 9 (2004) 984-997, 979. [40] K. Hashimoto, Emerging role of glutamate in the pathophysiology of major depressive disorder, Brain Res. Rev. 61 (2009) 105-123. [41] P.W. Kalivas, R.T. Lalumiere, L. Knackstedt, H. Shen, Glutamate transmission in addiction, Neuropharmacology 56 (2009) 169-173. [42] N.L. Brice, A. Varadi, S.J. Ashcroft, E. Molnar, Metabotropic glutamate and GABA(B) receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells, Diabetologia 45 (2002) 242-252.

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[43] A. Nicolaysen, R. Gammelsaeter, J. Storm-Mathisen, V. Gundersen, P.O. Iversen, The components required for amino acid neurotransmitter signaling are present in adipose tissues, J. Lipid Res. 48 (2007) 2123-2132. [44] X. Gao, X. Xu, J. Pang, C. Zhang, J.M. Ding, X. Peng, Y. Liu, J.M. Cao, NMDA receptor activation induces mitochondrial dysfunction, oxidative stress and apoptosis in cultured neonatal rat cardiomyocytes, Physiol. Res. 56 (2007) 559-569. [45] R.C. Clark, J.S. Reid, The analytical calculation of absorption in multifaceted crystals, Acta Crystallogr. A 51 (1995) 887-897. [46] A. Altomare, M.C. Burla, M. Camalli, G.L. Cascarano, C. Giacovazzo, A. Guagliardi, A.G.G. Moliterni, G. Polidori, R. Spagna, The analytical calculation of absorption in multifaceted crystals, J. Appl. Crystallogr. 32 (1999) 115-119. [47] P.W. Betteridge, J.R. Carruthers, R.I. Cooper, K. Prout, D.J. Watkin, CRYSTALS version 12: software for guided crystal structure analysis, J. Appl. Crystallogr. 36 (2003) 1487-1487. [48] C.C. Felder, K.E. Joyce, E.M. Briley, J. Mansouri, K. Mackie, O. Blond, Y. Lai, A.L. Ma, R.L. Mitchell, Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors, Mol. Pharmacol. 48 (1995) 443-450. [49] V.C. Abraham, D.L. Taylor, J.R. Haskins, High content screening applied to large-scale cell biology, Trends Biotechnol. 22 (2004) 15-22. [50] L. Bert, F. Robert, L. Denoroy, L. Stoppini, B. Renaud, Enhanced temporal resolution for the microdialysis monitoring of catecholamines and excitatory amino acids using capillary electrophoresis with laser-induced fluorescence detection. Analytical developments and in vitro validations, J. Chromatogr. A 755 (1996) 99-111. [51] S. Parrot, L. Bert, B. Renaud, L. Denoroy, Glutamate and aspartate do not exhibit the same changes in their extracellular concentrations in the rat striatum after N-methyl-D-aspartate local administration, J. Neurosci.Res. 71 (2003) 445-454. [52] S. Parrot, L. Bert, L. Mouly-Badina, V. Sauvinet, J. Colussi-Mas, L. Lambas-Senas, F. Robert, J.P. Bouilloux, M.F. Suaud-Chagny, L. Denoroy, B. Renaud, Microdialysis monitoring of catecholamines and excitatory amino acids in the rat and mouse brain: recent developments based on capillary electrophoresis with laser-induced fluorescence detection--a mini-review, Cell. Mol. Neurobiol. 23 (2003) 793-804. [53] N. Strazielle, J.-F. Ghersi-Egea, Demonstration of a coupled metabolism-efflux process at the choroid plexus as a mechanism of brain protection toward xenobiotics, J. Neurosci. 19 (1999) 6275-6289.

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Supplementary Data for

Novel routes to either racemic or enantiopure α-

SC

amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid

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derivatives and biological evaluation of a new promising pharmacological scaffold Samy Cecioni,a Kaïss Aouadi,†,a Julie Guiard,a Sandrine Parrot,b Nathalie Strazielle,c

Jean-Pierre Pralya

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Sandrine Blondel,c Jean-François Ghersi Egea,c Christian Chapelle,e Luc Denoroy,*,b,d and

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1/ Figure S1 NMR of final products 10, ent-10, (±)-10 and zwitterion

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deprotonation using LiOH…………………………………….…….………..S3 2/ Experimental data for N-Boc-pyrrolidine compounds…….……....S4-S5 3/ Table S1- Properties of (+)-menthone derived ent-series…….……...…..S6 4/ Table S2- Atom numbering applied for assigning the NMR signals……S7 5/ NMR Spectra: 1

H and 13C NMR of compound 3…………………………………………..…...S8 S1

ACCEPTED MANUSCRIPT H and 13C NMR of compound 4…………………………………………..…...S9

1

H and 13C NMR of compound 5………………………………………..….......S10

1

H and 13C NMR of compound 6…………………………………………..…...S11

1


1

H and 13C NMR of compound 8………………………………………..……...S13

1


1

H and 13C NMR of compound 10……………………………………..…..…...S15

1


1


1

H and 13C NMR of compound 16………………………………………....…...S18

1

H and 13C NMR of compound 17…………………..……………………..…...S19

1

H and 13C NMR of compound 18…………………………………………..….S20

1


1


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1

6/ Table S3- In vitro determination methods of target receptors……S23-S27 7/ Figure S2 - Percent inhibition of CB-1 and CB-2 control agonists…….S28

S2

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Figure S1: Similar 1H NMR spectra recorded in D2O for 10, ent-10 and changed 1H NMR spectrum recorded in D2O + LiOH for ent-10 (Top). 1H NMR spectra recorded in D2O for the racemic compound (±)-10 (Bottom).

S3

ACCEPTED MANUSCRIPT

2/ Experimental data for N-Boc-pyrrolidine compounds (1S,2S,5R,3'aR,3'bS,7'aS)-2'-t-Butoxycarbonyl-2-isopropyl-5,5'-dimethyl-1H-decahydro-

SC

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spiro[cyclohexane-1,6'-7'-oxa-2',5',6'a-triaza-cyclopenta[a]pentalen-4'-one] (3’)

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In a Biotage Initiator 0.5 – 2 mL vial, (–)-menthone derived nitrone 1 (146 mg, 0.61 mmol) and N-Boc-3-pyrroline 2’ (124mg, 0.73 mmol, 1.2 eq) were introduced. The vial was flushed with argon and 1.5 mL of anhydrous toluene was added. The vial was sealed with a septum cap and was sonicated for 20 seconds. The resulting crude mixture was submitted to

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sequential irradiation (140°C) runs under microwaves (from 30 min to 3 h). Monitoring the reaction by TLC (PE/EtOAc, 1/1) showed complete conversion after 10 h 30 min. The crude mixture was concentrated and purified by silica gel flash chromatography (elution gradient

EP

from PE/EtOAc, 7/3 to 1/1) to yield three fractions as white foams. After evaporation, the first one provided 177 mg of pure compound 3’’; the second yielded 31 mg of pure cycloadduct 3’

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and the third 29 mg of a mixture of compounds 3’’ and 3’ (composed of 78 % of 3’ as estimated by NMR analysis). These data represent a global yield of 95 % with a 3.4/1 ratio for compounds 3’’ / 3’. Compound 3’ (54 mg, 21 % yield) : Rf = 0.20 (PE/EtOAc, 1/1). [α]D = + 31.6 (c 1.2, CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 4.47 (t, J = 4.4 Hz, 1H, H-4), 3.78 – 3.74 (m, 1H, H-6)*, 3.72 – 3.41 (m, 5H, H-2, H-3, H-5)*, 2.71 (s, 3H, NCH3), 2.00 – 1.94 (m, 2H, ½ H-9, H-10), 1.82 – 1.78 (m, 1H, ½ H-11), 1.71 – 1.65 (m, 1H, ½ H-12), 1.60 – 1.55 (m, 1H, ½ H-12), 1.41 (s, 9H, tBu), 1.38 – 1.31 (m, 2H, H-13, H-15), 1.26 – 1.18 (m, 1H, ½ H-9), 0.95 – 0.85 (3×d, J = 6.5 Hz, 10H, ½ H-11, H-14, H-16). (* weak and broadened signals due S4

ACCEPTED MANUSCRIPT to tBu rotamers). 13C NMR (100 MHz, CDCl3) δ 171.8 (MeNC=O), 154.0 (tBuOC=O), 89.4 (C-8), 80.5, 79.5 (C-4 and CIV-tBu), 70.9 (C-6), 50.5, 50.0, 49.9, 49.6, 49.0 (5×s, C-2, C-3, C5)*, 48.1 (C-13), 40.8 (C-9), 34.7 (C-11), 29.3 (C-10), 28.5 (tBu), 26.1 (NCH3), 24.4 (C-15),

calcd for C22H37N3NaO4 [M + Na]+ 430.2676, found 430.2672.

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24.2 (CH3), 22.4 (CH3), 22.3 (C-12), 18.6 (CH3). HR-ESI-QToF MS (positive mode): m/z

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(1S,2S,5R,3'aR,3'bS,7'aS)-1'-t-Butoxycarbonyl-2-isopropyl-5,5'-dimethyl-1H-decahydro-

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spiro[cyclohexane-1,6'-7'-oxa-1',5',6'a-triaza-cyclopenta[a]pentalen-4'-one] (3’’).

3’’ (183 mg, 74 % yield). Monocrystals of compounds 3’’ were obtained from a saturated Et2O solution stored in a freezer. Rf = 0.34 (PE/EtOAc, 1/1). [α]D = + 58.4 (c 1, CH2Cl2). 1H

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NMR (400 MHz, CDCl3) δ 5.57 (d, J = 6.0 Hz, 1H, H-2), 3.63 – 3.60 (m, 1H, ½ H-5), 3.57 (bs, 1H, H-6), 3.52 (t, J = 6.7 Hz, 1H, H-3), 3.45 – 3.38 (m, 1H, ½ H-5), 2.72 (s, 3H, NCH3),

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2.14 – 2.09 (m, 1H, ½ H-9), 2.05 – 1.92 (m, 3H, H-4, H-10), 1.80 – 1.76 (m, 1H, ½ H-11), 1.75 – 1.58 (m, 2H, H-12), 1.45 (bs, 10H, H-15, tBu), 1.38 (dd, J = 12.4 Hz, J = 3.0 Hz, 1H, H-13), 1.27 – 1.15 (m, 1H, ½ H-9), 0.90 (d, J = 6.6 Hz, 4H, H-14, ½ H-11), 0.85 (d, J = 6.6 Hz, 3H, H-16), 0.79 (d, J = 6.6 Hz, 3H, H-16).

13

C NMR (100 MHz, CDCl3) δ 172.4

(MeNC=O), 153.9 (tBuOC=O), 88.1 (C-8), 87.7 (C-2), 79.9 (CIV-tBu), 72.0 (C-6), 48.7 (C-3), 48.0 (C-13), 45.4 (C-5), 41.3 (C-9), 34.8 (C-11), 30.1 (C-4), 28.9 (C-10), 28.5 (tBu), 25.9 (NCH3), 24.5 (C-15), 24.3 (CH3), 22.5 (CH3), 22.5 (C-12), 18.8 (CH3). HR-ESI-QToF MS (positive mode): m/z calcd for C22H37N3NaO4 [M + Na]+ 430.2676, found 430.2672. S5

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3/ Table S1 Main features of the synthesis of enantiomers from the (+)menthone-derived nitrone compound

yield %

reaction time

first series from

optical rotation

(–)-menthone

2 h, 140°C, µW

85

− 40.9 (c 1.2, CH2Cl2)

ent-4

50°C, 6 h 30

90

+ 14.7 (c 1.7, CH2Cl2)

ent-5

acidic MeOH, ∆, 1 h

98

− 11.8 (c 1.9, CH2Cl2)

ent-6

Mo(CO)6, ∆, 3 h

56

− 31.4 (c 0.6, DMSO)

ent-7

H2, 2 h

99

ent-8

H2, 1 h

82

ent-9

not prepared

ent-10

H2, 28 h

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ent-3

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conditions

− 14.2 (c 1, CH2Cl2) + 10.9 (c 1.3, CH2Cl2) + 33.1 (c 0.6, DMSO) + 32.3 (c 0.6, DMSO).

− 35.1 (c 0.6, DMSO)

+ 34.0 (c 0.2, DMSO)

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− 31.5 (c 0.6, DMSO)

− 4.1 (c 0.9, DMSO/H2O, 4/1)

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+ 40.4 (c 1.1, CH2Cl2)

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+3.0 (c 0.5, H2O) + 4.2 (c 0.5, DMSO/H20, 4/1)

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3/ Table S2 Atom numbering applied for assigning the NMR signals

7

8

(±)-14

(±)-15

(±)-19 (±)-18

(±)-17

(±)-20

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H and 13C NMR of compound 3

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5) Table S3. Supporting Information on the in vitro determination methods of target receptors Experiments based on inhibition of specific binding have been conducted in vitro on standardized

and transporters using specific radioligands as listed below.

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assays1 for sixty potential pharmacological targets including G-protein coupled receptors, ion channels

Biological system involved

Reference compound

5-HT transporter (h) (antagonist radioligand)

serotonine

imipramine

5-HT1A (h) (agonist radioligand)

serotonine

8-OH-DPAT

5-HT1B (antagonist radioligand)

serotonine

serotonin

serotonine

kétansérine

serotonine

mésulergine

serotonine

MDL 72222

serotonine

serotonin

serotonine

serotonin

serotonine

sérotonine

A1 (h) (antagonist radioligand)

adenosine

DPCPX

A2A (h) (agonist radioligand)

adenosine

NECA

A3 (h) (agonist radioligand)

adenosine

IB-MECA

alpha 1 (non-selective) (antagonist radioligand)

adrenaline/noradrenaline

prazosin

alpha 2 (non-selective) (antagonist radioligand)


yohimbine

AMPA (agonist radioligand)

glutamate

AMPA

AT1 (h) (antagonist radioligand)

angiotensine

saralasin

B2 (h) (agonist radioligand)

bradykinine

NPC 567

beta 1 (h) (agonist radioligand)


atenolol

5-HT2A (h) (antagonist radioligand)

5-HT2B (h) (agonist radioligand)

5-HT5a (h) (agonist radioligand)

5-HT6 (h) (agonist radioligand)

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5-HT3 (h) (antagonist radioligand)

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ICI 118551

BZD (central) (agonist radioligand)

GABA

diazepam

Ca2+ channel (L, verapamil site) (phenylalkylamine) (antagonist radioligand)

ion channel

D 600

CB1 (h) (agonist radioligand)

cannabinoids

WIN 55212-2

CCK1 (CCKA) (h) (agonist radioligand)

cholecystokinin

CCR1 (h) (agonist radioligand)

chemokine

Cl- channel (GABA-gated) (antagonist radioligand)

ion channel

CXCR2 (IL-8B) (h) (agonist radioligand)

C-X-C chemokine

IL-8

D1 (h) (antagonist radioligand)

dopamine

SCH 23389

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beta 2 (h) (agonist radioligand)

dévazépide

MIP-1alpha

picrotoxinin

dopamine

(+)butaclamol

opioids

DPDPE

dopamine

nomifensine

prostaglandines

PGE2

endothelin

endothelin-1

GABA (non-selective) (agonist radioligand)

GABA

GABA

GAL2 (h) (agonist radioligand)

galanin

galanin

glycine (strychnine-insensitive) (antagonist radioligand)

glutamate

MDL 105,519

H1 (h) (antagonist radioligand)

histamine

pyrilamine

H2 (h) (antagonist radioligand)

histamine

cimetidine

kainate (agonist radioligand)

glutamate

kainate

kappa (KOP) (agonist radioligand)

opioids

U50,488

KV channel (antagonist radioligand)

ion channel

alpha -dendrotoxin

M1 (h) (antagonist radioligand)

nicotine

pirenzepine


nicotine

methoctramine

delta 2 (DOP) (h) (agonist radioligand)

dopamine transporter (h) (antagonist radioligand)

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EP4 (h) (agonist radioligand)

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4-DAMP

MC4 (h) (agonist radioligand)

melanocortin

NDP-alpha -MSH

MT1 (ML1A) (h) (agonist radioligand)

melatonin

melatonin

mu (MOP) (h) (agonist radioligand)

opioids

DAMGO

Na+ channel (site 2) (antagonist radioligand)

ion channel

NK2 (h) (agonist radioligand)

tachykinin

NK3 (h) (antagonist radioligand)

neurokinin

NMDA (antagonist radioligand)

glutamate

CGP 39653

NOP (ORL1) (h) (agonist radioligand)

opioids

nociceptin

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veratridine

[Nle10]-NKA(4-10)

[Me-Phe]-NKB

norepinephrine transporter (h) (antagonist radioligand)

noradrenaline

protriptylin

NTS1 (NT1) (h) (agonist radioligand)

neurotensin

neurotensin

glutamate

TCP

ion channel

apamin

sst (non-selective) (agonist radioligand)

somatostatin

somatostatin-14

V1a (h) (agonist radioligand)

vasopressin

AVP

vasoactive intestinal polypeptide

VIP

Y1 (h) (agonist radioligand)

neuropeptide Y

NPY

Y2 (h) (agonist radioligand)

neuropeptide Y

NPY

PCP (antagonist radioligand)

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SKCa channel (antagonist radioligand)

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VPAC1 (VIP1) (h) (agonist radioligand)

This study has been carried out by Cerep SA facility (Celle L’Evescault, France).

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Figure S2. Percent inhibition of control agonist response obtained in the presence of compound ent-10 (n=3 for each concentration tested). 30 nM CP

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2 receptor agonist. *p

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