# Synthesis and pharmacological characterization of conformationally restricted 2-amino-Adipic acid analogs and carboxycyclopropyl glycines as selective metabotropic glutamate 2 receptor agonists

Markus Staudt, Na Liu, Fanny Malhaire, Yasaman Doroudian, Laurent Prézeau, Emma Renard, Zahra Hasanpour, Jean-Philippe Pin, Lennart Bunch

► To cite this version:

Markus Staudt, Na Liu, Fanny Malhaire, Yasaman Doroudian, Laurent Prézeau, et al.. Synthesis and pharmacological characterization of conformationally restricted 2-amino-Adipic acid analogs and carboxycyclopropyl glycines as selective metabotropic glutamate 2 receptor agonists. European Journal of Medicinal Chemistry, 2024, 266, pp.116157. 10.1016/j.ejmech.2024.116157. hal-04415292

HAL Id: hal-04415292

https://hal.science/hal-04415292v1

Submitted on 4 Nov 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Research paper

# Synthesis and pharmacological characterization of conformationally restricted 2-amino-Adipic acid analogs and carboxycyclopropyl glycines as selective metabotropic glutamate 2 receptor agonists

Markus Staudt<sup>a</sup>, Na Liu<sup>a</sup>, Fanny Malhaire<sup>b</sup>, Yasaman Doroudian<sup>a</sup>, Laurent Prézeau<sup>b</sup>, Emma Renard<sup>a</sup>, Zahra Hasanpour<sup>a</sup>, Jean-Philippe Pin<sup>b</sup>, Lennart Bunch<sup>a,∗</sup>

<sup>a</sup> Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

<sup>b</sup> Institute of Functional Genomics, University of Montpellier, CNRS, Inserm, 34094, Montpellier, France

<figure><img src="image_3.png" /></figure>

## ABSTRACT

The metabotropic glutamate (Glu) receptors (mGluRs) are G-protein coupled receptors, which play a central role in modulating excitatory neurotransmission in the central nervous system (CNS). Thus, the development of tool compounds thereto, continues to interest the scientific community. In this study, we report the design and synthesis of new conformationally restricted 2-amino adipic acid (2AA) 2–4, and glutamic acid 5, 6 analogs, which share the cyclopropane ring as the restrictor. The analogs were characterized at rat mGlu1–8 in an IP-One functional assay. While the 2AA analogs 3a, 4a and CCG-I analog 5a were shown to be selective mGlu2 agonists with low micromolar potencies, CCG-II analog 5b was shown to be a potent full agonist at mGlu2 (EC₅₀ = 82 nM) with ~15-fold selectivity over mGlu3, >25-fold selectivity over group III, and >60-fold selectivity over group I subtypes. An in silico study was performed to address this significant change (>3500 fold) in potency upon introduction of this methyl group (L-CCG-II vs 5b).

## 1. Introduction

(S)-Glutamate (Glu) is the major excitatory neurotransmitter in the central nervous system (CNS), and activates both the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (mGluRs) [1–3]. The former are cationic channels and are further divided into N-methyl-D-aspartic acid (NMDA) receptors, the (S)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and kainic acid (KA) receptors according to their selective actions as agonists [4]. The mGluRs belong to the family C G-protein coupled receptors (GPCRs) and are divided into three subgroups (I-III) based on their sequence homology, cell signaling activation and agonist binding pharmacology [5]. Group I (mGlu1,5) are foremost located post-synaptically and excitatory in action by coupling to Gq proteins leading to activation of the phospholipase C (PLC) pathway, whereas group II (mGlu2,3) and group III (mGlu4,6,7,8) are foremost expressed pre-synaptically and are inhibitory in action, by coupling to Gi/o proteins which leads to inhibition of adenylyl cyclase (AC) [6,7]. The group II subtypes, mGlu2 and mGlu3 are differentially distributed in the CNS and exert distinct functions [8]. While mGlu2,3 are localized pre- and post-synaptically in neurons, mGlu3 is also found in glial cells [9–11]. Extensive pharmacological studies suggest that mGlu2 and mGlu3 are associated with

several neurological and psychiatric disorders such as Parkinsons disease, depression, anxiety, and drug addiction [12–16].

In order to study and understand the detailed function and role of the mGlu2 and mGlu3 subtypes in CNS disorders, the discovery of pharmacological tools capable of differentiating them are of particular interest. Owing to the high degree of protein sequence homology (70 %) between the mGlu2 and mGlu3 receptors with a 100 % residue identity amongst the two orthosteric binding pockets, the discovery of subtype selective orthosteric agonists or antagonists have proved challenging [17]. In the past decades, extensive efforts have been made to identify subtype selective orthosteric agonists or antagonists based on the Glu scaffold [18,19]. The conformationally constrained cyclopentane analog (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD, Fig. 1) was first identified as a selective agonist for mGluRs over iGluRs [20,21]. By incorporation of a cyclopropane ring in the Glu scaffold, it was possible to conformationally restrict Glu in its extended and folded form, providing the four diastereomers of L-2-(carboxycyclopropyl) glycine (L-CCG-I-IV) [22]. Of the two (extended) trans-analogs, (2S,3S, 4S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I, Fig. 1) was found to be a selective agonist for the mGluRs with a preference for group II [23,24]. in contrast, L-CCG-II was only a weak mGluR agonist.

L-CCG-I was further investigated as a lead for potent mGluR agonists by introduction of an additional substituted group at the C3'-position.

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: lebu@sund.ku.dk (L. Bunch).</footnote>
## Abbreviations used

<table><tr><td>CNS</td><td>central nervous system</td></tr><tr><td>dr</td><td>diastereomeric ratio</td></tr><tr><td>DCM</td><td>dichloromethane</td></tr><tr><td>DMAP</td><td>N,N-Dimethyl-4-aminopyridine;</td></tr><tr><td>iGluRs</td><td>ionotropic Glu receptors</td></tr><tr><td>mGluRs</td><td>metabotropic Glu receptors</td></tr><tr><td>GPCRs</td><td>G-protein coupled receptors</td></tr><tr><td>CCG</td><td>L-2-(carboxycyclopropyl)glycine; carboxycyclopropylalanines</td></tr><tr><td>(S)-AA</td><td>(2S)-Aminoadipic acid</td></tr></table>

Agonist (2S,3S,4S,4'R)-2-(2-carboxy-3'-methylcyclopropyl) glycine 1a and 1b holding a methyl and hydroxymethyl group in the C3'-position significantly enhanced potency (micromolar to nanomolar) but selectivity between mGlu2 and mGlu3 was not achieved [25].

A research team at Eli Lilly designed and synthesized a series of bicyclic amino acid analogs, in which the Glu skeleton was incorporated into a fused bicyclo[3.1.0]hexane frame. The constrained bicyclic amino acid (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (1c, LY354740, Fig. 1) possessed high potency and selectivity for mGlu2/3 [26]. Further studies disclosed that a methyl group in the C4α-position of 1c led to the highly potent mGlu2 agonist (1S,2S,4R,5R,6S)-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1d, LY541850, Fig. 1) with partial agonist/antagonist activity at mGlu3 [27]. The analog (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl) bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1e, LY2812223, Fig. 1) with a thiotriazole group at this same position was reported as a functionally selective mGlu2 agonist [28]. Furthermore, (1S,2S,4S,5R, 6S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2, 6-dicarboxylic acid (1f, LY2794193, Fig. 1) with (3-methoxybenzoyl) amino substitution at the C4β-position was recently demonstrated to be a highly potent mGlu3-selective agonist [29]. Interestingly, 4-substituted Glu analog 1g was shown to be a selective mGlu2 agonist and likewise its conformationally restricted analog 1h [30]. In the same series, the Z-isomers 1i and 1j displayed selective and potent agonist activity at Group III subtypes mGlu4,6,8 [30].

(2S)-Aminoadipic acid (AA, 1k, Fig. 2) is the one-carbon homolog of non-selective endogenous mGluR agonist (S)-Glu. Decades ago, it was reported to be a selective agonist for mGlu2 however with only a modest potency (EC₅₀ = 35 µM) by Madsen and co-workers [31]. From the same study, the authors investigated the 4-methyl analog of 1k, which maintained its mGlu2 activity, while the 5-methyl analog as well as the conformationally restricted 5,6-alkene both lost mGluR agonist function [32]. The apparent structural similarity of 1k (and their close analogs) with our previously reported analogs 1g, and 1h-j, the latter which also hold a 2-aminoadipic acid backbone, inspired us to design conformationally restricted hybrid structures 2-4 as a strategy to explore and identify new tool compounds with unprecedented mGluR subtype selectivity profiles (Fig. 2). A preliminary modelling study of 2-4 also inspired the design of two new CCG-I analogs which hold a C4-methyl and a C4-ethyl substituent (5a, 6a) and their corresponding diastereomers L-CCG-II analogs 5b and 6b (Fig. 2).

## 2. Results and discussion

### 2.1. Chemistry

The synthesis of the diastereomeric mixture of (S)-AA analog 2 (Scheme 1) commenced by formation of the Weinreb-amide of suitably protected (S)-aspartate to facilitate selective reduction to the corresponding aldehyde 10. The following Horner-Wadsworth-Emmons

reaction gave the desired unsaturated ester 11 as the E-isomer. Unfortunately, Corey-Chaykovsky cyclopropanation [33] of this intermediate failed, necessitating double Boc-protection of the amine. After converting 11 to 12, cyclopropanation was performed with *in situ* formed dimethylsulfoxonium methylide to give 13 in modest yield (1:1 mixture of the two *trans*-diastereomers). Global deprotection in 6 M HCl gave the desired (S)-AA analog 2 as a 1:1 mixture of *trans*-diastereomers.

aReagents and conditions: (a) i. ClCO₂i-Pr, N-methylmorpholine; ii. HN (Me)(OMe), NEt₃, THF, 0 °C to rt, 87%; (b) DIBALH, THF, -78 °C, 92%; (c) NaH, Triethylphosphonoacetate, THF, 0 °C, 75%; (d) Boc₂O, DMAP (cat.), rt, quant.; (e) trimethylsulfoxonium iodide, NaH, DMSO, 80 °C, 18% (1:1 dr); (f) 6 M HCl, reflux, quant. (1:1 dr).

The 5-methyl analog 3 (Scheme 2) was prepared starting from commercially available aldehyde 14. The olefination step provided 15 as a single stereoisomer and the following Corey-Chaykovsky-cyclopropanation gave 5-methyl-cyclopropano analog 16 as a 4:1 diastereomeric mixture. Global deprotection in 6 M HCl under reflux followed by Jones-oxidation gave the desired target analog 3 as a 4:1 mixture of diastereomers.

aReagents and conditions: (a) NaH, triethyl 2-phosphonopropionate, THF, 0 °C, 73%; (b) trimethylsulfoxonium iodide, NaH, DMSO, 80 °C, 51% (4:1 dr); (c) 6 M HCl, reflux, quant. (4:1 dr); (d) H₂SO₄, Na₂Cr₂O₇, NaIO₄, H₂O, 16% (4:1 dr).

The 5-methyl analogs 3a,b (Scheme 3) were prepared from (R)-and (S)-glycidol 18a,b by first trityl-protection of the free alcohol. Following Horner-Wadsworth-Emmons cyclopropanation gave enantiopure intermediates 20a,b [34]. Removal of the trityl group with TFA gave free alcohol 21a,b, which were converted to the desired iodides 22a,b. Unfortunately, enantioselective alkylation of *tert*-butyl 2-((diphenylmethylene)amino)acetate using chiral phase-transfer catalysts failed, presumably due to increased steric hindrance by the *cis*-methyl group. No conversion was observed at temperatures below 0 °C, while elevated temperatures lead to formation of diastereomeric mixtures. Therefore, the achiral phase transfer catalyst benzyltriethylammonium chloride was used, resulting in the α-epimers 23a,b. Mild deprotection, followed by preparative HPLC, gave the TFA-salts 24a,b, which were deprotected in 6 M HCl at 50 °C to the desired AA analogs 3a,b.

aReagents and conditions: (a) DMAP, TrCl, NEt₃, DCM; (b) triethyl 2-phosphonopropionate, NaH, xylene, reflux; (c) TFA, DCM, rt; (d) I₂, PPh₃, imidazole, MeCN: Et₂O (2:3), 0 °C; (e) *tert*-butyl 2-((diphenylmethylene)amino)acetate, benzyltriethylammonium chloride, 50% aq. NaOH, toluene, rt; (f) THF: 1 M HCl (10:1), rt; (g) 6 M HCl, 50 °C.

The synthesis of 4-methyl analogs 4a-d (Scheme 4) commenced with decarboxylative alkenylation of commercially available N-(*tert*-butoxycarbonyl)aspartic acid benzyl ester 27 to give olefin intermediate 28 in 38% yield [35]. Cyclopropanation with ethyl 2-diazoacetate and Rh₂(OAc)₄ as the catalyst, gave a mixture of the four diastereomers 29a-d, which after careful purification on flash column chromatography allowed isolation of pure isomer 29a and pure isomer 29b together with a mixture with two isomers 29c,d in overall 40% yield. After hydrolysis in 6 M HCl and purification by preparative HPLC, the two *cis*-diastereomers 4a,b and the two *trans*-diastereomers 4c,d were obtained all isolated as enantiopure forms (the *cis/-trans* stereochemical configuration was assigned by 2D-NMR (¹H-¹H ROESY, Fig. S1).

aReagents and conditions: (a) i. LiCl, Mg, 1,2-dibromoethane, THF, reflux 1 h; ii. ZnCl₂, THF; (b) i. N-hydroxytetrachlorophthalimide, N,N'-Diisopropylcarbodiimide, DMAP, DCM, rt, 3 h; ii. Ni(acac)₂·xH₂O, 2,2'-bipyridine, alkenylzinc reagent in THF (26), DMF, rt, overnight, 38%; (c) ethyl 2-diazoacetate, Rh₂(OAc)₄, DCM, rt, overnight, 40%; (d) 6 M HCl, 110 °C, 4 h, then preparative HPLC 33-44%.

The synthesis of the L-CCG-I/II analogs 5a,b and 6a,b (Scheme 5) started from Garner's aldehyde, which was converted to olefin 31 by a Wittig reaction. Epoxidation of the alkene by *m*-CPBA gave a 2:1 mixture of the diastereomers 32a and 32b, which were separated by flash column chromatography. The following Horner-Wadsworth-Emmons cyclopropanation reactions provided the methyl- and ethyl-substituted
<figure><img src="image_4.png" /><figcaption>(S)-Glu
non-selectivity
mGlu2:EC₅₀= 4 µM
mGlu3:EC₅₀= 9 µM</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>L-CCG-I
mGlu2,3 agonist
mGlu2:EC₅₀= 0.3 µM
mGlu3:EC₅₀= 0.6 µM</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>L-CCG-II
weak mGlu agonist
mGlu2:EC₅₀= 300 µM</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(1S,3R)-ACPD
mGluR agonist
mGlu2:EC₅₀= 12 µM
mGlu3:EC₅₀= 860 µM</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>1a
mGlu2,3 agonist
mGlu2:EC₅₀= 8 nM
mGlu3:EC₅₀= 38 nM</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>1b
mGlu2,3 agonist
mGlu2:EC₅₀= 8 nM
mGlu3:EC₅₀= 38 nM</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>1c, LY354740
mGlu2,3 agonist
mGlu2:EC₅₀= 5.6 nM
mGlu3:EC₅₀= 27 nM</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>1d, LY541850
mGlu2 agonist
mGlu3 antagonist
mGlu2:EC₅₀= 64 nM
mGlu3:EC₅₀= 3400 nM</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>1e, LY2812223
mGlu2 agonist
mGlu3 partial agonist/antagonist
mGlu2:EC₅₀= 5.6 nM
mGlu3:EC₅₀= 129 nM(61%)
mGlu3:EC₅₀= 2730 nM(40%)</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>1f, LY2794193
mGlu3 agonist
mGlu2:EC₅₀= 47.5 nM
mGlu3:EC₅₀= 0.47 nM</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>1g (1d in pub)
mGlu2 agonist
mGlu2:EC₅₀= 34 µM</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>1h (2a in pub)
mGlu2 agonist
mGlu2:EC₅₀= 34 µM</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>1i (2c in pub)
mGlu4,6,8 agonist
mGlu4:EC₅₀= 2 µM
mGlu6:EC₅₀= 18 µM
mGlu7:EC₅₀= 21 µM</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>1j (2d in pub)
mGlu4,6,8 agonist
mGlu4:EC₅₀= 0.13 µM
mGlu6:EC₅₀= 0.49 µM
mGlu7:EC₅₀= 0.68 µM</figcaption></figure>

<caption>**Fig. 1.** Chemical structures of endogenous agonist (S)-Glu, synthetic mGluR selective agonists **1a**, group II selectivity agonists L-CCG-I and **1c**, functionally selective mGlu2 agonists **1d** and **1e**, mGlu3 selective agonist **1f**, mGlu2 selective agonists **1g** and **1h**, mGlu4,6,8 selective agonists **1i** and **1j**.</caption>
## 2-Amino Adipic Acid analogs

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. 2.** Chemical structures of (S)-AA (1k), L-CCG-I and L-CCG-II, and newly designed (S)-AA analogs 2–4 and L-CCG-I analogs 5a,6a, and L-CCG-II analogs 5b,6b.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>**Scheme 1.** Synthesis of conformationally restricted (S)-AA analog 2ª.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Scheme 2.** Synthesis of (S)-AA analog 3ª.</figcaption></figure>

cyclopropanes 33a,b and 35a,b in moderate to good yields [36]. For the four intermediates, Jones oxidation conditions led to both cleavage of the hemiaminal protecting group and oxidation of the primary alcohol to the corresponding carboxylic acid. Finally, global deprotection in 6 M HCl at 50 °C gave the desired L-CCG-I analogs 5a,6a and L-CCG-II analogs 5b,6b in their enantiopure form.

ªReagents and conditions: (a) Ph₃PCH₃Br, KHMDS, THF, -78 °C to rt, 83%; (b) m-CPBA, DCE, reflux; (c) Phosphonate, LiHMDS or BuLi (35b), DME, 130 °C, 18 h or 48 h (35b); (d) 1 M Jones reagent, acetone, rt, 18-48 h; (e) 6 M HCl, 50 °C, 18 h, quant.

The synthesis of 5a was also scaled up using a slightly different route (Scheme 6). Starting from commercially available (S)-2-[(benzyloxy]

methyl]oxirane (37) in gram scale (4.0 g, 24.3 mmol), cyclopropanation was achieved in high yield to give *trans*-cyclopropane 38. Deprotection of benzyl ether was done by Pd/C under a hydrogen atmosphere to yield alcohol 21a. Oxidation gave aldehyde 39 in high yield (90 %), which was following converted to the corresponding diastereomeric mixture of amino-cyanide 40. Finally, global hydrolysis in 6 N HCl gave 5a together with its C1 diastereomer 5a' in 90 %. Enantiopure 5a could following be isolated by preparative HPLC.

ªReagents and conditions: a. Triethylphosphonopropionate, n-BuLi, DME, -78 °C, then microwave oven 130 °C, 90 min (86 %); b. Pd/C 10 % (15 % w/w), H₂, EtOH (degassed), rt, 3 h (67 %); c. PCC, DCM, under N₂, 0 °C to rt, 2 h, 90 %; d. KCN, NH₄OH, NH₄Cl, H₂O: MeOH: DCM (0.5:
<figure><img src="image_22.png" /><figcaption>**Scheme 3.** Synthesis of 5-methyl analogs 3a,b (stereochemistry shown for the series denoted a)ª.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>**Scheme 4.** Synthesis of (S)-AA analogs 4a–4dª.</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>**Scheme 5.** a Synthesis of enantiopure L-CCG-I analogs 5a,6a and enantiopure L-CCG-II analogs 5b,6b.</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>**Scheme 6.** Synthesis of C1 diastereomeric mixture of 5a and 5a', starting from gram scale epoxide 37.</figcaption></figure>
1.3: 0.8), 7 h, rt, 72%; e. HCl (6 N), 6 h, 60 °C, 90%.

## 2.2. Pharmacological characterization

The seven new conformationally restricted 2-aminoadipic acid analogs **2,3,3a,3b,4a-d**, the two new CCG-I analogs **5a,6a** and the two CCG-II analogs **5b,6b** were first evaluated for agonist- and antagonist activity characterized at rat mGlu1–8 in an IP-One functional assay by single-dose stimulation of 30 µM (or the indicated concentrations). For the selected analogs, full dose-response curves (EC₅₀ values) were determined, and all results summarized in Table 1.

## 3. SAR

Regrettably, *trans*-cyclopropane 2-aminoadipic acid analog **2** was completely inactive at all eight mGluR subtypes. Its 5-methyl analog **3** showed low micromolar agonist activity at mGlu2, and this activity to reside in the (4S,5R)-diastereomer **3a**. Repositioning of the methyl group to the 4-position, *trans*-analogs **4c,d**, again lead to a complete loss of activity. However, for the *cis*-analogs **4a,b** low micromolar agonist activity at mGlu2 was observed and to reside in the diastereomer **4a**. The exact stereochemistry of **4a,b** was not determined, as we decided not to pursue this series further due to the low potencies.

Introduction of a methyl group in the 4S-position of L-CCG-I, compound **5a**, led to a significantly loss in agonist potency at mGlu2 and the corresponding ethyl analog, compound **6a**, was completely inactive. But most interestingly, introduction of a methyl group in the 4R-position of L-CCG-II, compound **5b**, led to a nanomolar potent mGlu2 agonist ~15-fold selectivity over mGlu3, >60 fold selectivity over group I subtypes and >25 fold selectivity over group III.

## 4. In silico study

We decided to investigate the unexpected nanomolar potency of **5b** at mGlu2 by an in silico study (Fig. 3A–D). The four compounds L-CCG-I, **5a**, L-CCG-II and **5b** were docked into the mGlu2-LBD crystallized with Glu (PDB code: 5CNI) using an induced fit algorithm with a continuum solvation model (GB/SA). The four compounds docked at comparable docking scores, thus no clear trend could be concluded there upon (dG free energy of binding in kcal/mol: L-CCG-I = -7.5, **5a** = -7.6, L-CCG-II = -7.3 and **5b** = -7.2). However, some interesting differences in binding modes were observed. While the binding mode of docked L-CCG-I and **5a** essentially superimposed onto Glu (Fig. 3A and B), with the 4S-methyl of **5a** clearly in a sterically challenging position near Asp295, the binding mode of L-CCG-II was very different from Glu (Fig. 3C). Most interestingly, introduction of the methyl group in the 4R-position, compound **5b**, docking suggests a binding mode wherein the cyclopropane ring is reoriented and the distal carboxylate group now forms a salt bridge with both Arg61 (as for Glu) and also Arg57 (weak). The latter residue is characteristic for group II subtypes mGlu2/3 (Fig. 3D). Furthermore, the introduced methyl group forms a hydrophobic interaction with Tyr144, which is also characteristic for group II receptors.

To further address the scientific relevance of the binding mode of **5b** in mGlu2 shown in Fig. 3D, we conducted three molecular dynamic simulations (100 ns) with different initial velocities (Fig. S2, supporting information). All three simulations show a stable protein-ligand complex, wherein the distal carboxylate group of **5b** forms salt bridge interactions with Arg61 and Arg57, and in a time-dependent fashion also with Lys377. The latter interaction in correlation with rotation of the C2–C3 bond. In terms of mGlu2/3 subtype selectivity, we have just reported that reorientation of Arg271 to engage in a salt-bridge interaction with the ligand plays an essential role in the observed mGlu2 over mGlu3 selectivity for the potent mGlu2 agonist LBG30300 [37]. However, the Arg271-Asp146 interaction is stable during the simulation of **5b** in mGlu2. Thus the observed 15 fold mGlu2 subtype selectivity must

originate elsewhere from.

Ultimately, we suggest that the significant enhancement of potency of **5b** compared to L-CCG-II, is a result of **5b** binds in low energy conformation, with additional hydrophobic contributions from the introduced methyl group. Also an entropic gain could be significant from additional exclusion of one or more water molecules from the LBD upon ligand binding.

## 5. Conclusion

In conclusion, we have synthesized new conformationally restricted analogs of 2-AA, and methyl analogs of well-known Glu analogs L-CCG-I and L-CCG-II. While the synthesized 2-AA analogs led to identification of micromolar agonists at mGlu2, investigation of the four L-CCG analogs (5a,b and 6a,b) revealed an unexpected 3500 fold increase in potency for 4-methyl L-CCG-II analog **5b** (LBG30120). A binding mode of **5b** in mGlu2 was obtained by induced fit docking and validated by a molecular dynamic simulation. The disclosure of **5b** adds to the pool of empirical data of significant change in potencies and/or target selectivity on the introduction of a methyl group – often referred to as “The Magic Methyl Group” [38].

## 6. Experimental section

**Chemistry:** All reactions involving dry solvents or sensitive agents were performed under an argon atmosphere and glassware was flame dried under vacuum prior to use. Commercially available chemicals were used without further purification. DCM, THF and DMF were dried using a SG WATER solvent purification system (commercialized by Pure Process Technology). MeOH was dried by standing over 4 Å molecular sieves for a minimum of 48h. Reactions were monitored by analytical thin-layer chromatography (TLC, Merck silica gel 60 F₂₅₄ aluminum sheets) or by HPLC. Flash chromatography was carried out using Merck silica gel 60A (40–63 µm). For dry column vacuum chromatography (DCVC), Merck silica gel 60 (15–40 µm) and a standard setup was used. ¹H NMR spectra were recorded at 400 or 600 MHz and ¹³C NMR spectra at 100 or 150 MHz on a Bruker Avance III or Bruker Avance III HD, respectively. Chemical shifts (δ) are reported in ppm relative to TMS. For ¹³C NMR in D₂O was added 1 % CD₃OD as internal reference. HPLC was performed using a Dionex UltiMate 3000 pump and photodiode array detector (210 and 254 nm, respectively) installed with an XTerra MS C 18 3.5 µm, 4.6 mm × 150 mm column, using a 5 → 95 % MeCN gradient in H₂O containing 0.1 % TFA. For HPLC control, data collection and data handling, Chromeleon software v. 6.80 was used. Preparative HPLC was carried out on an Agilent Prep HPLC systems with Agilent 1100 series pump, Agilent 1200 series diode array, multiple wavelength detector (G1365B), and Agilent PrepHT High Performance Preparative Cartridge Column (Zorba × 300 SB-C18 Prep HT, 21.2 × 250 mm, 7 µm). LC-MS spectra were recorded using either an Agilent 1200 series solvent delivery system equipped with an autoinjector coupled to an Agilent 6400 series triple quadrupole mass spectrometer equipped with an electrospray ionization source or Waters Aquity UPLC-MS with dual wavelength detection with electrospray ionization. Gradients of 5 % aqueous MeCN +0.1 % HCO₂H (solvent A), and 95 % aqueous MeCN + 0.05 % HCO₂H (solvent B) were employed. IR spectra were recorded on a PerkinElmer 801 spectrophotometer. Optical rotation was measured using a PerkinElmer 241 Spectrometer or an Anton Paar Modular Circular Polarimeter (MCP) 100/150 with Na lamp (D line, 589 nm). Compounds were dried under high vacuum or freeze dried using a Holm & Halby, Heto LyoPro 6000 freeze drier. The purity of compounds submitted for pharmacological characterization was determined by HPLC to be >95 % until further notification.

2-((S)-2-Amino-2-carboxyethyl)cyclopropane-1-carboxylic acid hydrochloride (2). Compound **13** (70.0 mg, 0.14 mmol) was suspended in 6 M HCl (25 mL) and the suspension was heated to reflux for 3d. The solvent was removed at the rotary evaporator, to give **2** as a pale white
<figure><img src="image_26.png" /><figcaption>**Table 1**

Chemical structures of cyclopropyl adipic acid analogs 2,3,3a,3b,4a-d and L-CCG-I analogs 5a,6a and L-CCG-II analogs 5b,6b, and pharmacological characterization at rat mGlu1-8 by IP-One functional assay.ª</figcaption></figure>

<table><thead><tr><th rowspan="2">Compound</th><th rowspan="2">Cα</th><th colspan="2">Group I</th><th colspan="2">Group II</th><th colspan="4">Group III</th></tr><tr><th>mGlu1 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu5 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu2 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu3 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu4 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu6 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu7 EC<sub>50</sub> (pEC<sub>50</sub>)</th><th>mGlu8 EC<sub>50</sub> (pEC<sub>50</sub>)</th></tr></thead><tbody><tr><td>LY379268</td><td></td><td>-</td><td>-</td><td>0.0006 (-9.21 ± 0.09)</td><td>0.0095 (-8.03 ± 0.19)<br/>~100 %@<br/>3 µM</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>LAP4</td><td></td><td>-</td><td>-</td><td>-</td><td>-</td><td>~30</td><td>2.5</td><td>~30</td><td>~30</td></tr><tr><td>2</td><td></td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td></tr><tr><td>3</td><td>S</td><td>NE@100</td><td>NE@100</td><td>10 (4.8 ± 0.11)</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td><td>NE@100</td></tr><tr><td>3a</td><td>SR</td><td>NE@30</td><td>NE@30</td><td>8.9 (5.05 ± 0.08)</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>3b</td><td>SR</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>4a</td><td>S</td><td>NE@30</td><td>NE@30</td><td>7.5 (5.12 ± 0.07)</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>4b</td><td>S</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>4c</td><td>S</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>4d</td><td>S</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>5a</td><td>S</td><td>NE@30</td><td>NE@30</td><td>4.1 (5.39 ± 0.06)</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>6a</td><td>S</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td><td>NE@30</td></tr><tr><td>5b (LBG30120)</td><td>S</td><td>5.21 (5.29 ± 0.09)</td><td>10.60 (4.95 ± 0.10)</td><td>0.082 (7.09 ± 0.04)</td><td>1.22 (5.91 ± 0.06)</td><td>NE@30</td><td>2.18 (5.67 ± 0.02)</td><td>NE@30</td><td>NE@30</td></tr><tr><td>6b</td><td>S</td><td>>30</td><td>>30</td><td>1.798 (5.74 ± 0.26)</td><td>>30</td><td>>30</td><td>26.3 (4.58 ± 0.03)</td><td>>30</td><td>>30</td></tr></tbody></table>

NE@100, NE@30: no effect at respectively 100 and 30 µM.

ª All values as EC₅₀ in µM with pEC₅₀ ± SEM in parentheses. Estimated values from a 30 µM single-dose stimulation compared to full agonists reference compounds; Quis for mGlu1,5; LY-37 for mGlu2,3; LAP4 for mGlu4,6–8). Mean values calculated from 2 or 3 experiments. –, not tested. No antagonistic effect was observed for any of the compounds.
<figure><img src="image_27.png" /><figcaption>**Fig. 3A.** Induced-fit docking of L-CCG-I (green) into mGlu2-LBD (5cni, Glu in magenta). The distal carboxylate groups both form dual salt-bridge interaction with Arg61 and Lys377.</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Fig. 3B.** Induced-fit docking of 4-methyl L-CCG-I analog 5a (green) docked into mGlu2-LBD (5cni, Glu in magenta). The distal carboxylate group forms a salt-bridge with Arg61.</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>**Fig. 3C.** Induced-fit docking of L-CCG-II (green) into mGlu2-LBD (5cni, Glu in magenta). The distal carboxylate group of forms a salt-bridge with Arg61.</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>**Fig. 3D.** Induced-fit docking of L-CCG-II analog 5b (green) into mGlu2-LBD (5cni, Glu in magenta). The distal carboxylate group of 5b forms a strong salt-bridge with Arg61 and a weaker one with Arg57. A salt bridge to Lys377 is not observed. The methyl group forms a hydrophobic interaction with Tyr144.</figcaption></figure>

solid (28 mg, 0.13 mmol, 94 %, 1:1 mixture of *trans*-diastereomers). ¹H NMR (600 MHz, D₂O) δ 4.22–4.18 (m, 1H), 2.15–2.04 (m, 1H), 2.03–1.90 (m, 1H), 1.64–1.57 (m, 1H), 1.56–1.47 (m, 1H), 1.30–1.23 (m, 1H), 1.03–0.97 (m, 1H). ¹³C NMR (151 MHz, D₂O) (1:1 diastereomers) δ (178.3, 171.6), (171.6, 171.5), (52.7, 52.6), (32.7, 32.5), (19.6, 19.5), (18.2, 17.8), (15.0, 14.8). LC-MS (*m/z*) calcd. for C₇H₁₂NO₄ [M + H]⁺ 174.1, found: 174.1.

**2-((S)-2-Amino-2-carboxyethyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (3).** A solution of sodium dichromate (2.2 mg, 7.5 µmol, 0.05 eq.), sulfuric acid (0.043 mL) and sodium periodate (144 mg, 0.68 mmol, 4.5 eq.) in 1.158 mL of deionized water was added to the crude 2-((S)-2-Amino-3-hydroxypropyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (31.5 mg, 0.15 mmol, 1.0 eq.). The solution was stirred overnight at rt. DOWEX 50WX2 (815 mg), suspended in 3.5 mL of deionized water was added and the reaction mixture was stirred for an additional hour. The resin was filtered, washed with water and the product was eluted with 1 M ammonium hydroxide solution. Removal of solvent furnished a pale-yellow oil, which was dissolved in 1 M HCl and the solvent was removed under reduced pressure. The crude product was purified using preparative HPLC (isocratic 100 % A) to furnish the desired product as a 4:1 mixture of diastereomers (white solid, 5.3 mg, 23.7 µmol, 16 %). Major diastereomer: ¹H NMR (600 MHz, D₂O) δ 4.08 (t, *J* = 6.1 Hz, 1H), 2.21 (dt, *J* = 14.7, 6.2 Hz, 1H), 1.93 (ddd, *J* = 14.9, 8.2, 6.6 Hz, 1H), 1.49–1.56 (m, 1H), 1.45 (br dd, *J* = 9.0, 4.2 Hz, 1H), 1.27 (s, 3H), 0.63–0.73 (m, 1H). ¹³C NMR (151 MHz, D₂O) δ 180.4, 172.3, 53.3, 29.1, 22.3, 22.2, 21.9, 13.2. Minor diastereomer: ¹H NMR (600 MHz, D₂O) δ 4.08 (t, *J* = 6.1 Hz, 1H), 2.21 (dt, *J* = 14.7, 6.2 Hz, 1H), 1.93 (ddd, *J* = 14.9, 8.2, 6.6 Hz, 1H), 1.56–1.62 (m, 1H), 1.45 (br dd, *J* = 9.0, 4.2 Hz, 1H), 1.25 (s, 3H), 0.63–0.73 (m, 1H). ¹³C NMR (151 MHz, D₂O) δ 180.4, 172.3, 53.4, 29.1, 22.6, 22.2, 21.8, 13.1. LC-MS (*m/z*) calcd. for C₈H₁₄NO₄ [M + H]⁺ 188.1, found: 188.1.

**(1S,2R)-2-(2RS)-2-amino-2-carboxyethyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (3a).** 2-Amino-3-((1R,2S)-2-(2RS)-((ethoxycarbonyl)-2-methylcyclopropyl)propanoic acid hydrotrifluoro-methylsulfonate (24a) (27.5 mg, 83.5 µmol) was dissolved in 6 M HCl (1 mL) and stirred at 50 °C for 18 h. The solvent was removed under vacuum and the white residue was re-dissolved in 1 M HCl (1 mL). This step was repeated once more to yield **3a** a white solid (19 mg, quant.). Diastereomer 1: ¹H NMR (600 MHz, D₂O) δ 3.98–4.07 (m, 1H), 1.99–2.12 (m, 1H), 1.74–1.87 (m, 1H), 1.39–1.46 (m, 1H), 1.27 (dt, *J* = 9.2, 4.6 Hz, 1H), 1.09 or 1.06 (s, 3H), 0.50 (ddd, *J* = 10.5, 6.5,
4.6 Hz, 1H). Diastereomer 2: ¹H NMR (600 MHz, D₂O) δ 3.98–4.07 (m, 1H), 1.99–2.12 (m, 1H), 1.74–1.87 (m, 1H), 1.32–1.39 (m, 1H), 1.27 (dt, *J* = 9.2, 4.6 Hz, 1H), 1.09 or 1.06 (s, 3H), 0.50 (ddd, *J* = 10.5, 6.5, 4.6 Hz, 1H). ¹³C NMR (151 MHz, D₂O) (1:1 diastereomers) δ (180.3, 180.2), (171.5, 171.4), (52.8, 52.6), (28.9, 28.8), (22.4, 22.0) (22.3, 22.2), (21.8, 21.7), (13.1, 13.0). LC-MS (*m/z*) calcd. for C₈H₁₄NO₄ [M + H]⁺ 188.1, found 188.2.

(1R,2S)-2-(2RS)-(2-Amino-2-carboxyethyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (3b). 2-Amino-3-(1S,2R)-2-(2RS)-(ethoxycarbonyl)-2-methylcyclopropyl)propanoic acid hydrotrifluoro-methylsulfonate (24b) (36.1 mg, 83.5 µmol) was dissolved in 1 mL of 6 M HCl and stirred at 50 °C for 18 h. The solvent was removed under vacuum and the white residue was redissolved in 1 M HCl (1 mL). This step was repeated once more to yield a white solid (24.5 mg, quant.). Diastereomer 1: ¹H NMR (600 MHz, D₂O) δ 4.12 (m, 1H), 2.19–2.25 (m, 1H), 1.90–1.97 (m, 1H), 1.55–1.61 (m, 1H), 1.45 (dt, *J* = 9.0, 4.3 Hz, 1H), 1.26 or 1.24 (s, 3H), 0.66 (td, *J* = 7.1, 4.6 Hz, 1H). Diastereomer 2: ¹H NMR (600 MHz, D₂O) δ 4.12 (m, 1H), 2.19–2.25 (m, 1H), 1.90–1.97 (m, 1H), 1.49–1.55 (m, 1H), 1.45 (dt, *J* = 9.0, 4.3 Hz, 1H), 1.26 or 1.24 (s, 3H), 0.66 (td, *J* = 7.1, 4.6 Hz, 1H). ¹³C NMR (151 MHz, D₂O) (1:1 diastereomers) δ (180.4, 180.4), (172.0, 172.0), (53.1, 53.0), (29.1, 29.0), (22.6, 22.1), (22.4, 22.2), (21.8, 21.8), (13.2, 13.1). LC-MS (*m/z*) calcd. for C₈H₁₄NO₄ [M + H]⁺ 188.1, found 188.2.

(1S,2S)-2-((S)-2-Amino-2-carboxyethyl)-2-methylcyclopropane-1-carboxylic acid or (1R,2R)-2-((S)-2-amino-2-carboxyethyl)-2-methylcyclopropane-1-carboxylic acid (4a,b). Isomer 29a (110 mg, 0.27 mmol) was dissolved in 6 M HCl (5.4 mL) The resulting solution was heated to reflux for 4 h and then evaporated under reduced pressure at 40 °C. The residue was purified by preparative HPLC to give the cis-compound 4a (27 mg, 44 %). Purity: ≥95 %, Rt: 5.05 min. LC-MS (*m/z*) calcd for C₈H₁₃NO₄ [M + H]⁺, 188.2; found,188.5. ¹H NMR (400 MHz, Deuterium Oxide) δ 4.17 (t, *J* = 7.52 Hz, 1H), 2.27–2.14 (m, 2H), 1.72 (dd, *J* = 5.90, 7.85 Hz, 1H), 1.28 (s, 3H), 1.19–1.12 (m, 2H). ¹³C NMR (101 MHz, D₂O) δ 177.1, 173.4, 52.4, 33.6, 26.9, 24.1, 23.2, 21.6. [α]<sup>25</sup><sub>D</sub> +52.8 (c 0.108, H₂O).

The cis-product **4b** was obtained as a HCl salt. Yield from 29b (15 mg, 33 %). Purity: ≥90 %, Rt: 4.98 min ¹H NMR (400 MHz, Deuterium Oxide) δ 4.25 (t, *J* = 7.89 Hz, 1H), 2.38 (dd, *J* = 7.28, 14.67 Hz, 1H), 2.15 (dd, *J* = 8.17, 14.80 Hz, 1H), 1.73 (t, *J* = 6.86 Hz, 1H), 1.31 (s, 3H), 1.17 (d, *J* = 7.34 Hz, 2H). ¹³C NMR (101 MHz, D₂O) δ 177.2, 173.0, 51.8, 33.5, 26.5, 24.1, 22.7, 21.8. [α]<sup>25</sup><sub>D</sub> -59.9 (c 0.104, H₂O).

(1R,2S)-2-((S)-2-Amino-2-carboxyethyl)-2-methylcyclopropane-1-carboxylic acid or (1S,2R)-2-((S)-2-amino-2-carboxyethyl)-2-methylcyclopropane-1-carboxylic acid (4c,d).

The trans-products **4c** and **4d** were separated by preparative HPLC and obtained as HCl salt. Yield from mixture of 29c/d.

**4c** (28 mg, 18 %). Purity: ≥90 %, Rt: 3.78 min. LC-MS (*m/z*) calcd for C₈H₁₃NO₄ [M + H]⁺, 188.2; found,188.5. ¹H NMR (400 MHz, Deuterium Oxide) δ 4.32–4.21 (m, 1H), 2.18–2.08 (m, 1H), 1.97–1.86 (m, 1H), 1.69 (ddd, *J* = 1.14, 5.84, 8.39 Hz, 1H), 1.31 (s, 3H), 1.18 (t, *J* = 5.43 Hz, 1H), 1.11 (dd, *J* = 5.12, 8.31 Hz, 1H). ¹³C NMR (101 MHz, D₂O) δ 177.0, 173.0, 51.9, 41.0, 26.1, 23.5, 20.8, 15.3. [α]<sup>25</sup><sub>D</sub> +62.8 (c 0.103, H₂O).

**4d** (26 mg, 16 %). Purity: ≥90 %, Rt: 4.58 min. LC-MS (*m/z*) calcd. for C₈H₁₃NO₄ [M + H]⁺, 188.2; found, 188.5. ¹H NMR (400 MHz, Deuterium Oxide) δ 4.21 (d, *J* = 7.33 Hz, 1H), 2.12 (dd, *J* = 6.76, 14.57 Hz, 1H), 1.98 (dd, *J* = 7.46, 14.60 Hz, 1H), 1.73 (dd, *J* = 5.79, 8.27 Hz, 1H), 1.30 (s, 3H), 1.16 (t, *J* = 5.42 Hz, 1H), 1.10 (dd, *J* = 5.19, 8.28 Hz, 1H). ¹³C NMR (101 MHz, D₂O) δ 176.8, 173.2, 52.2, 40.9, 26.3, 23.6, 20.7, 15.5. [α]<sup>25</sup><sub>D</sub> -60.0 (c 0.100, H₂O).

(1S,2S)-2-((S)-Amino(carboxy)methyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (5a). (S)-2-((tert-Butoxycarbonyl) amino)-2-((1S,2S)-2-(ethoxycarbonyl)-2-methylcyclopropyl)acetic acid (34a) (12.0 mg, 39.8 µmol) was suspended in 6 M HCl (1 mL) and the reaction mixture was stirred at 50 °C for 18 h. Removal of the solvent furnished the title compound (8.32 mg, quant.) as a white solid. ¹H NMR (600 MHz, D₂O) δ 3.81 (d, *J* = 11.3 Hz, 1H), 1.96 (dddd, *J* = 11.3, 9.1,

6.6, 0.8 Hz, 1H), 1.60 (dd, *J* = 9.1, 5.0 Hz, 1H), 1.45 (s, 3H), 1.00 (dd, *J* = 6.6, 5.1 Hz, 1H). ¹³C NMR (151 MHz, D₂O) δ 178.7, 171.0, 53.0, 25.9, 24.1, 20.0, 14.1. [α]<sup>20</sup><sub>D</sub> +30.2 (c 0.08, H₂O). LC-MS (*m/z*) calcd. for C₇H₁₂NO₄ [M + H]⁺ 174.1, found 174.2.

Compound 5a (together with its C1 diastereomer 5a') was also obtained from hydrolysis of 40. A solution of α-aminocyanide 40 (0.52 mmol, 0.25 g) in aq. HCl (6 M, 10 mL) was heated at 60 °C for 6h. After cooling to r.t., the aq HCl was removed under reduced pressure to give a white solid (if an oil is obtained, adding Et₂O, followed by its removal under reduced pressure led to solidification). ¹H NMR (600 MHz, in D₂O) δ (ppm): 3.69–3.65 (m, 2 diastereomer, 1H), 1.91–1.86 (m, 2 diastereomers, 1H), 1.58–1.52 (m, 2 diastereomers, 1H), 1.36 (s, 2 diastereomers, 3H), 1.11–1.07 (m, 1 diastereomers, 0.55H), 0.94–0.90 (m, 1 diastereomers, 0.45H).

The two diastereomers 5a and 5a' were separated by HPLC equipped with a crown column as stationary phase and H₂O/ACN/TFA as the mobile phase. (1S)-Diastereomer 5a: ¹H NMR (600 MHz, in D₂O) δ (ppm): 3.56 (d, *J* = 11.1 Hz, 1H), 1.91–1.86 (m, 1H), 1.34 (s, 3H), 1.57 (m, 1H), 1.08–1.06 (m, 1H), ¹³C NMR (151 MHz, D₂O) δ 178.7, 171.0, 53.0, 25.9, 24.1, 20.0, 14.1. (1R)-Diastereomer 5a': ¹H NMR (600 MHz, in D₂O) δ (ppm): 3.52 (d, *J* = 11.3 Hz, 1H), 1.86–1.81 (m, 1H), 1.50–1.46 (m, 1H), 1.37 (s, 3H), 0.91–0.80 (m, 1H); ¹³C NMR (151 MHz, D₂O) δ 178.5, 170.8, 54.3, 25.5, 23.6, 20.5, 13.7.

(1R,2R)-2-((S)-Amino(carboxy)methyl)-1-methylcyclopropane-1-carboxylic acid hydrochloride (5b). (S)-2-((tert-Butoxycarbonyl) amino)-2-((1R,2R)-2-(ethoxycarbonyl)-2-methylcyclopropyl)acetic acid (34b) (31.0 mg, 0.10 mmol) was suspended in 6 M HCl (1 mL) and the reaction mixture was stirred at 50 °C for 18 h. Removal of the solvent furnished the title compound (21.6 mg) as a white solid. ¹H NMR (600 MHz, D₂O) δ = 3.72 (d, *J* = 11.0 Hz, 1H), 1.96 (ddd, *J* = 11.0, 9.7, 6.5 Hz, 1H), 1.64 (dd, *J* = 9.7, 5.2 Hz, 1H), 1.42 (s, 3H), 1.16 (dd, *J* = 6.5, 5.2 Hz, 1H). ¹³C NMR (151 MHz, D₂O) δ = 178.7, 171.6, 53.0, 26.6, 23.1, 20.6, 13.1. [α]<sup>20</sup><sub>D</sub> +57.9 (c 0.11, H₂O). LC-MS (*m/z*) calcd. for C₇H₁₂NO₄ [M + H]⁺ 174.1, found 174.2.

(1S,2S)-2-((S)-Amino(carboxy)methyl)-1-ethylcyclopropane-1-carboxylic acid hydrochloride (6a). (S)-2-((tert-Butoxycarbonyl) amino)-2-((1S,2S)-2-(ethoxycarbonyl)-2-ethylcyclopropyl)acetic acid (36a) (11.0 mg, 34.9 µmol) was suspended in 6 M HCl (1 mL) and the reaction mixture was stirred at 50 °C for 18 h. Removal of the solvent furnished the title compound (7.76 mg, quant.) as a white solid. ¹H NMR (600 MHz, D₂O) δ 3.84 (d, *J* = 11.2 Hz, 1H), 2.16 (dqd, *J* = 14.3, 7.2, 1.2 Hz, 1H), 1.92 (ddd, *J* = 11.2, 9.0, 6.7 Hz, 1H), 1.61 (ddd, *J* = 9.1, 5.2, 1.3 Hz, 1H), 1.34 (dq, *J* = 14.7, 7.4 Hz, 1H), 1.08 (t, *J* = 7.3 Hz, 3H), 0.96 (dd, *J* = 6.8, 5.2 Hz, 1H). ¹³C NMR (151 MHz, D₂O) δ 177.9, 171.0, 53.0, 30.3, 27.0, 21.9, 17.2, 11.0. [α]<sup>20</sup><sub>D</sub> +46.2 (c 0.08, H₂O). LC-MS (*m/z*) calcd. for C₈H₁₄NO₄ [M + H]⁺ 188.1, found 188.2.

(1R,2R)-2-((S)-Amino(carboxy)methyl)-1-ethylcyclopropane-1-carboxylic acid hydrochloride (6b). (S)-2-((tert-Butoxycarbonyl) amino)-2-((1R,2R)-2-(ethoxycarbonyl)-2-ethylcyclopropyl)acetic acid (36b) (29.0 mg, 92 µmol) was suspended in 1 mL of 6 M HCl and the reaction mixture was stirred at 50 °C for 18 h. Removal of the solvent furnished the title compound (20.6 mg, quant.) as a white solid. ¹H NMR (600 MHz, D₂O) δ 3.85–3.76 (m, 1H), 2.19 (dqd, *J* = 13.9, 7.0, 1.5 Hz, 1H), 1.91 (ddd, *J* = 11.1, 9.7, 6.6 Hz, 1H), 1.67 (ddd, *J* = 9.7, 5.4, 1.5 Hz, 1H), 1.23–1.17 (m, 1H), 1.15 (dd, *J* = 6.7, 5.4 Hz, 1H), 1.10 (t, *J* = 7.2 Hz, 3H). ¹³C NMR (151 MHz, D₂O) δ 178.0, 171.3, 52.8, 29.4, 27.8, 20.5, 17.8, 11.2. [α]<sup>20</sup><sub>D</sub> +24.9 (c 0.21, H₂O). LC-MS (*m/z*) calcd. for C₈H₁₄NO₄ [M + H]⁺ 188.1, found 188.2.

## 7. Pharmacological characterization

**Cell Culture and Transfection.** HEK293 cells were transiently transfected with rat mGluRs encoding plasmids by electroporation and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Cergy Pontoise, France) supplemented with 10 % of fetal bovine serum. Cells were seeded in polyornithine-coated 96-well plates at a density of
150 000 cells/well. In order to minimize extracellular Glu concentra-
tion, receptors were cotransfected with the high-affinity Glu transporter
EAAC1 and medium was replaced by Glutamax (Life Technologies) at
least 2 h before stimulation.

**IP-One Functional Assay.** To monitor receptor signalling activity through inositol monophosphate (IP1) production, group II and group III mGluRs were cotransfected with a chimeric Gi/Gq protein to allow coupling to the phospholipase C pathway. IP1 production was measured 24 h after transfection using the IP-One HTRF kit (Cisbio Biossays, Codolet, France) according to the manufacturer's recommendations. For mGlu3, cells were first incubated with 1 µM LY341495 for 10 min at 37 °C to prevent the high basal activity. Then, cells were washed two times and stimulated with the test compounds at the indicated concentration. The reported agonist potency on mGlu3 was calculated considering the left-shifting factor of potency (9.5 fold) due to the presence of the antagonist, which was estimated with the same analysis performed on mGlu2. Experiments were performed at least in duplicates, and data were analysed using Prism software (GraphPad, La Jolla, CA, USA).

8. In silico study

Docking was performed using MOE (2022.02). Firstly the PDB file
was prepared by isolation of chain A and then submitted to the built-in
function protonate 3D. Docking was performed by standard setup,
induced fit and with GB/SA continuum model as solvation. Scoring
function, S, returns calculated free energy of binding dG in kcal/mol.

9. Associated content

Supporting Information available: Synthetic procedures of in-
termediates. 2D-NMR(¹H-¹HROESY) of analogs **4a–d**. PDB files of
Fig. 3A–D. NMR spectra of final compounds. HPLC traces of 4a-d.

CRediT authorship contribution statement

**Markus Staudt:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization. **Na Liu:** Writing – review & editing, Investigation, Conceptualization. **Fanny Malhaire:** Writing – review & editing, Investigation. **Yasaman Doroudian:** Investigation, Methodology, Writing – original draft, Writing – review & editing. **Laurent Prézeau:** Writing – review & editing, Investigation. **Emma Renard:** Writing – review & editing, Investigation. **Zahra Hasanpour:** Writing – review & editing, Investigation. **Jean-Philippe Pin:** Writing – review & editing, Investigation. **Lennart Bunch:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

Lennart Bunch reports financial support was provided by University of Copenhagen. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgments

The authors gratefully acknowledge the financial support from the
Danish Research Council (DFF/FSS) and the China Scholarship Council
(CSC) foundation.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejmech.2024.116157.

**References**

[1] V. Parpura, T.A. Basarsky, F. Liu, K. Jeftinija, S. Jeftinija, P.G. Haydon, Glutamate-mediated astrocyte-neuron signalling, Nature 369 (1994) 744–747, https://doi.org.10.1038/369744a0.

[2] S. Ozawa, H. Kamiya, K. Tsuzuki, Glutamate receptors in the mammalian central nervous system, Prog. Neurobiol. 54 (1998) 581–618.

[3] R.W. Gereau, G. Swanson, The Glutamate Receptors, Springer Science & Business Media, 2008.

[4] S. Ozawa, H. Kamiya, K. Tsuzuki, Glutamate receptors in the mammalian central nervous system, Prog. Neurobiol. 54 (1998) 581–618.

[5] S.S. Willard, S. Koochekpour, Glutamate, glutamate receptors, and downstream signaling pathways, Int. J. Biol. Sci. 9 (2013) 948–959, https://doi.org/10.7150/ijbs.6426.

[6] C.M. Niswender, P.J. Conn, Metabotropic glutamate receptors: physiology, pharmacology, and disease, Annu. Rev. Pharmacol. Toxicol. 50 (2010) 295–322, https://doi.org/10.1146/annurev.pharmtox.011008.145533.

[7] S.S. Willard, S. Koochekpour, Glutamate, glutamate receptors, and downstream signaling pathways, Int. J. Biol. Sci. 9 (2013) 948–959, https://doi.org/10.7150/ijbs.6426.

[8] C.M. Niswender, P.J. Conn, Metabotropic glutamate receptors: physiology, pharmacology, and disease, Annu. Rev. Pharmacol. Toxicol. 50 (2010) 295–322, https://doi.org/10.1146/annurev.pharmtox.011008.145533.

[9] R.S. Petralia, Y.-X. Wang, A.S. Niedzielski, R.J. Wenthold, The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations, Neuroscience 71 (1996) 949–976.

[10] Y. Tamaru, S. Nomura, N. Mizuno, R. Shigemoto, Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites, Neuroscience 106 (2001) 481–503.

[11] A. Neki, H. Ohishi, T. Kaneko, R. Shigemoto, S. Nakanishi, N. Mizuno, Pre-and postsynaptic localization of a metabotropic glutamate receptor, mGluR2, in the rat brain: an immunohistochemical study with a monoclonal antibody, Neurosci. Lett. 202 (1996) 197–200.

[12] L. Musazzi, Targeting metabotropic glutamate receptors for rapid-acting antidepressant drug discovery, Expet Opin. Drug Discov. 16 (2021) 147–157, https://doi.org/10.1080/17460441.2020.1822814.

[13] K. Domanegg, W.H. Sommer, M.W. Meinhardt, Psychedelic targeting of metabotropic glutamate receptor 2 and its implications for the treatment of alcoholism, Cells 12 (2023), https://doi.org/10.3390/cells12060963.

[14] C.G. Modrak, C.S. Wilkinson, H.L. Blount, M. Schwendt, L.A. Knackstedt, The role of mGlu receptors in susceptibility to stress-induced anhedonia, fear, and anxiety-like behavior, Int. Rev. Neurobiol. 168 (2023) 221–264, https://doi.org/10.1016/bs.irn.2022.10.006.

[15] C. Kwan, W. Kang, E. Kim, S. Belliveau, I. Frouni, P. Huot, Metabotropic glutamate receptors in Parkinson's disease, Int. Rev. Neurobiol. 168 (2023) 1–31, https://doi.org/10.1016/bs.irn.2022.10.001.

[16] S. Chaki, M. Watanabe, mGlu2/3 receptor antagonists for depression: overview of underlying mechanisms and clinical development, Eur. Arch. Psychiatr. Clin. Neurosci. 273 (2023) 1451–1462, https://doi.org/10.1007/s00406-023-01561-6.

[17] J. Hao, Q. Chen, Insights into the structural aspects of the mGlu receptor orthosteric binding site, Curr. Top. Med. Chem. (Sharjah, United Arab Emirates) 19 (2019) 2421–2446, https://doi.org/10.2174/1568026619666191011094935.

[18] D. Ma, Conformationally constrained analogues of glutamate as subtype-selective modulators of metabotropic glutamate receptors, Bioorg. Chem. 27 (1999) 20–34.

[19] F.C. Acher, Metabotropic glutamate receptors, Tocris Biosci. Rev. Lett. (2019) 1–24.

[20] E. Palmer, D.T. Monaghan, C.W. Cotman, Trans-ACPD, a selective agonist of the phosphoinositide-coupled excitatory amino acid receptor, Eur. J. Pharmacol. 166 (1989) 585–587.

[21] D.D. Schoepp, B.G. Johnson, C.R. Salhoff, J.W. McDonald, M. V Johnston, In vitro and in vivo pharmacology of trans- and cis-(±)-1-amino-1,3-cyclopentanedicarboxylic acid: dissociation of metabotropic and ionotropic excitatory amino acid receptor effects, J. Neurochem. 56 (1991) 1789–1796, https://doi.org/10.1111/j.1471-4159.1991.tb02082.x.

[22] K. Yamanoi, Y. Ohfune, K. Watanabe, P.N. Li, H. Takeuchi, Synthesis of trans and cis-α-(carboxycyclopropyl) glycines. Novel neuroinhibitory amino acids as L-glutamate analogue, Tetrahedron Lett. 29 (1988) 1181–1184.

[23] K. Shimamoto, Y. Ohfune, Syntheses and conformational analyses of glutamate analogs: 2-(2-carboxy-3-substituted-cyclopropyl) glycines as useful probes for excitatory amino acid receptors, J. Med. Chem. 39 (1996) 407–423.

[24] K. Yamanoi, Y. Ohfune, K. Watanabe, P.N. Li, H. Takeuchi, Synthesis of trans and cis-α-(carboxycyclopropyl) glycines. Novel neuroinhibitory amino acids as L-glutamate analogue, Tetrahedron Lett. 29 (1988) 1181–1184.

[25] I. Collado, C. Pedregal, A. Mazón, J.F. Espinosa, J. Blanco-Urgóiti, D.D. Schoepp, R. A. Wright, B.G. Johnson, A.E. Kingston, (2 S, 1 S, 2 S, 3 R)-2-(2'-Carboxy-3'-methylcyclopropyl) Glycine is a potent and selective metabotropic group 2 receptor agonist with anxiolytic properties, J. Med. Chem. 45 (2002) 3619–3629.

[26] J.A. Monn, M.J. Valli, S.M. Massey, R.A. Wright, C.R. Salhoff, B.G. Johnson, T. Howe, C.A. Alt, G.A. Rhodes, R.L. Robey, Design, synthesis, and pharmacological characterization of (+)-2-aminobicyclo [3.1.0] hexane-2, 6-
dicarboxylic acid (LY354740): a potent, selective, and orally active group 2
metabotropic glutamate receptor agonist possessing anticonvulsant and anxiolyti,
J. Med. Chem. 40 (1997) 528–537.

[27] J.A. Monn, M.J. Valli, S.M. Massey, J. Hao, M.R. Reinhard, M.G. Bures, B.A. Heinz, X. Wang, J.H. Carter, B.G. Getman, G.A. Stephenson, M. Herin, J.T. Catlow, S. Swanson, B.G. Johnson, D.L. McKinzie, S.S. Henry, Synthesis and pharmacological characterization of 4-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates: identification of new potent and selective metabotropic glutamate receptor 2/3 receptor agonists, J. Med. Chem. 56 (2013) 4442–4455, https://doi.org/10.1021/jm4000165.

[28] J.A. Monn, L. Prieto, L. Taboada, C. Pedregal, J. Hao, M.R. Reinhard, S.S. Henry, P. J. Goldsmith, C.D. Beadle, L. Walton, T. Man, H. Rudyk, B. Clark, D. Tupper, S. R. Baker, C. Lamas, C. Montero, A. Marcos, J. Blanco, M. Bures, D.K. Clawson, S. Atwell, F. Lu, J. Wang, M. Russell, B.A. Heinz, X. Wang, J.H. Carter, C. Xiang, J. T. Catlow, S. Swanson, H. Sanger, L.M. Broad, M.P. Johnson, K.L. Knopp, R.M. A. Simmons, B.G. Johnson, D.B. Shaw, D.L. McKinzie, Synthesis and pharmacological characterization of C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: identification of a potent, selective metabotropic glutamate receptor agonist and determination of agonist-bound human, J. Med. Chem. 58 (2015) 1776–1794, https://doi.org/10.1021/jm501612y.

[29] J.A. Monn, S.S. Henry, S.M. Massey, D.K. Clawson, Q. Chen, B.A. Diseroad, R. M. Bhardwaj, S. Atwell, F. Lu, J. Wang, M. Russell, B.A. Heinz, X.S. Wang, J. H. Carter, B.G. Getman, K. Adragni, L.M. Broad, H.E. Sanger, D. Ursu, J.T. Catlow, S. Swanson, B.G. Johnson, D.B. Shaw, D.L. McKinzie, J. Hao, Synthesis and Pharmacological Characterization of C4beta-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1 S,2 S,4 S,5 R,6 S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a, J Med Chem 61 (2018) 2303–2328, https://doi.org/10.1021/acs.jmedchem.7b01481.

[30] T.H. V Huynh, M.N. Erichsen, A.S. Tora, C. Goudet, E. Sagot, Z. Assaf, C. Thomsen, R. Brodbeck, T.B. Stensbøl, W.E. Bjørn-Yoshimoto, B. Nielsen, J.P. Pin, T. Geflaut, L. Bunch, New 4-functionalized glutamate analogues are selective agonists at metabotropic glutamate receptor subtype 2 or selective agonists at metabotropic

glutamate receptor group III, J. Med. Chem. 59 (2016) 914–924, https://doi.org/
10.1021/acs.jmedchem.5b01333.

[31] M. Guldbrandt, T.N. Johansen, K. Frydenvang, H. Brauner-Osborne, T.B. Stensbol, B. Nielsen, R. Karla, F. Santi, P. Krogsgaard-Larsen, U. Madsen, Glutamate receptor ligands: synthesis, stereochemistry, and enantiopharmacology of methylated 2-aminoadipic acid analogs, Chirality 14 (2002) 351–363, https://doi.org/10.1002/chir.10104.

[32] M. Guldbrandt, T.N. Johansen, K. Frydenvang, H. Brauner-Osborne, T.B. Stensbol, B. Nielsen, R. Karla, F. Santi, P. Krogsgaard-Larsen, U. Madsen, Glutamate receptor ligands: synthesis, stereochemistry, and enantiopharmacology of methylated 2-aminoadipic acid analogs, Chirality 14 (2002) 351–363, https://doi.org/10.1002/chir.10104.

[33] E.J. Corey, M. Chaykovsky, Dimethyloxosulfonium methylide ((CH3)2SOCH2) and dimethylsulfonium methylide ((CH3)2SCH 2). Formation and application to organic synthesis, J. Am. Chem. Soc. 87 (1965) 1353–1364, https://doi.org/10.1021/ja01084a034.

[34] A. Armstrong, J.N. Scutt, Stereocontrolled synthesis of 3-(trans-2-Aminocyclopropyl)alanine, a key component of belactosin A, Org. Lett. 5 (2003) 2331–2334, https://doi.org/10.1021/ol0346887.

[35] J.T. Edwards, R.R. Merchant, K.S. McClymont, K.W. Knouse, T. Qin, L.R. Malins, B. Vokits, S.A. Shaw, D.H. Bao, F.L. Wei, T. Zhou, M.D. Eastgate, P.S. Baran, Decarboxylative alkenylation, Nature 545 (2017) 213–218, https://doi.org/10.1038/nature22307.

[36] C.D. Bray, F. Mincone, Stereocontrolled Synthesis of Quaternary Cyclopropyl Esters W, 2010, pp. 5867–5869, https://doi.org/10.1039/c0cc01333a.

[37] N. Liu, F. Eshak, F. Malhaire, I. Brabet, L. Prézeau, E. Renard, J.-P. Pin, F.C. Acher, M. Staudt, L. Bunch, Design, synthesis, pharmacology, and in silico studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300): a picomolar potency subtype-selective mGlu2 receptor agonist, J. Med. Chem. (2024), https://doi.org/10.1021/ACS.JMEDCHEM.3C01811.

[38] H. Schönherr, T. Cernak, Profound methyl effects in drug discovery and a call for new C-H methylation reactions, Angew. Chem. Int. Ed. 52 (2013) 12256–12267, https://doi.org/10.1002/anie.201303207.TOC.