New 4-Functionalized Glutamate Analogues Are ... - ACS Publications

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New 4‑Functionalized Glutamate Analogues Are Selective Agonists at Metabotropic Glutamate Receptor Subtype 2 or Selective Agonists at Metabotropic Glutamate Receptor Group III Tri H. V. Huynh,† Mette N. Erichsen,† Amélie S. Tora,‡,§ Cyril Goudet,‡,§ Emmanuelle Sagot,⊥,# Zeinab Assaf,⊥,# Christian Thomsen,∥ Robb Brodbeck,∥ Tine B. Stensbøl,∥ Walden E. Bjørn-Yoshimoto,† Birgitte Nielsen,† Jean-Philippe Pin,‡,§ Thierry Gefflaut,⊥,# and Lennart Bunch*,† †

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark ‡ Institute of Functional Genomics, CNRS, UMR5203, University of Montpellier, 34094 Montpellier, France § INSERM, U1191, 34094 Montpellier, France ∥ H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark ⊥ Institut de Chimie de Clermont-Ferrand, Clermont Université, Université Blaise Pascal, BP 10448, 63000 Clermont-Ferrand, France # CNRS, UMR6296, ICCF, BP 80026, 63177 Aubière, France S Supporting Information *

ABSTRACT: The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6−8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a−d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low−mid nanomolar range.

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INTRODUCTION (S)-Glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system (CNS) where it activates both the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (mGluRs). The iGluRs are divided into three groups, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, kainic acid (KA) receptors, and N-methylD-aspartate (NMDA) receptors. The mGluRs belong to class C of the G-protein coupled receptors and are of particular interest as drug targets. In all, eight mGluR subtypes have been identified (mGlu1−8), and these have been classified into three groups based on their downstream signaling pathway and ligand selectivity. Group I comprises subtypes mGlu1,5, group II comprises subtypes mGlu2,3, and group III comprises subtypes mGlu4,6,7,8. Group II subtypes mGlu2 and mGlu3 are mainly located on the presynaptic neuron and inhibit synaptic release of Glu when activated. In contrast to the mGlu2 subtype, the mGlu3 subtype is also expressed on astrocytes1,2 and exerts neuroprotective effects for cultured cortical neurons upon activation.3 Interestingly, a number of conformationally restricted Glu analogues display selective agonist activity at group II over © 2016 American Chemical Society

groups I and III (Figure 1). Key analogues are (2S,1′S,2′S)-2-(2′carboxycyclo-propyl)glycine (L-CCG-I), which displays low micromolar agonist activity at mGlu2 and mGlu3 as well as at group III subtypes.4 Addition of a second carboxylate group on the cyclopropane ring, (2S,2′R,3′R)-2-(2′,3′dicarboxycyclopropyl)glycine (DCG-IV), advances the agonist potency to the midnanomolar range at mGlu2 and mGlu3.5 A methyl or a hydroxymethyl group also enhances the potency, as (2S,1′S,2′S,3′R)-2-(2′-carboxy-3′-methylcyclopropyl) glycine (I) and (2S,1′S,2′R,3′R)-2-(2′-carboxy-3′-hydroxymethylcyclopropyl) glycine (II) display low nanomolar agonist activity at both mGlu2 and mGlu3.6,7 The rigid Glu analogue (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) displays low nanomolar agonist activity at mGlu2; however, it has only a 3-fold preference over mGlu3.8 The synthesis of analogues thereof has unexpectedly resulted in mixed mGlu2 agonists/mGlu3 antagonists (structures not shown). 9−11 Finally, (±)-1-amino-2-(carboxymethyl)cyclopropane-1-carboxylic acid ((±)-III) is a simple cyclopropyl Received: August 28, 2015 Published: January 27, 2016 914

DOI: 10.1021/acs.jmedchem.5b01333 J. Med. Chem. 2016, 59, 914−924

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of reported mGlu2/3 (group II) selective agonists.

Table 1. Chemical Structures of 4-Substituted Glu Analogues 1a−1k and Pharmacological Characterization at Rat mGlu1−5 and Human mGlu7a

a

All values are in micromolar. SEM calculated from n = 3−4 experiments. --, not tested bIntracellular Ca2+ mobilization assay. c[35S]GTPγS binding assay. dInhibition of forskolin-stimulated cAMP production.26

at EAAT1−3, negligible affinity for AMPA and KA receptors, and midrange micromolar affinity for the NMDA receptors (Ki = 52 μM).21 From a comparison of 1d with analogues 1a−k, it is evident that both the side chain length (1c vs 1d) and the carboxylate functionality (1e−k vs 1d) are essentials for the observed mGlu2 selectivity profile. Residue differences within the binding pocket of the three mGluR groups suggest that Arg64 (Figure 2; mGlu3 numbering, PDB code: 2E4U) may play a key role. This amino acid is unique for group II and could participate in a salt bridge to the side chain carboxylate group of 1d. The hypothesis was strengthened by docking of 1d in the Glu-mGlu3 crystal wherein water molecule no. 872 (W872) was first deleted to make space for the 4-side chain. A clear interaction between Arg64 and the side chain carboxylate group was identified (Figure 3), which may explain the observed group II selectivity, but it does not explain the mGlu2 over mGlu3 selectivity. We propose that this intriguing subtype selectivity originates from ligand−host induced differences in the organization of the water matrix within the mGlu2/3 binding pockets. Alternatively, it may arise from disfavored ligand−protein energy states as 1d travels to the mGlu3 binding pocket.25 Most interestingly, the binding mode of 1d suggests a significantly different conformation of the Glu backbone compared to that of Glu crystallized in mGlu3 (Figure 4). The C4-substituent of 1d is rotated about 180° compared with the spatial orientation of the corresponding C4hydrogen on Glu crystallized in mGlu3. Design of Conformationally Restricted Analogues of 1d. A stochastic conformational search of flexible Glu analogue 1d concludes that its global low-energy conformation is approximately 2 kcal/mol lower in energy than its proposed binding conformation from the docking study (Figure 4). We

Glu analogue that displays agonist activity at mGlu2 in the midmicromolar range (EC50 = 10 μM) with greater than 100-fold selectivity over mGlu3 as well as mGluR group I/III subtypes.12 mGlu2/3 agonists have been investigated in a number of CNS diseases and neurological conditions, but it remains to be seen if this field will be fruitful in terms of providing new medicines for unmet needs.13−17 In summary, the search for potent subtype selective mGlu2 (or mGlu3) ligands remains an important objective because such tool compounds can be used to elucidate their individual roles and functions in health and disease.

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RESULTS AND DISCUSSION Chemistry-driven discovery of novel Glu ligands has given access to a large number of L-2,4-syn-substituted Glu analogues as well as cyclic Glu analogues by use of transaminase enzymatic approaches. This work has led to highly potent and selective ligands for the AMPA and KA receptors18−22 as well as a selective excitatory amino acid transporter subtype 2 (EAAT2) inhibitor.23 Introduction of a substituent in the L-2,4-syn position of Glu endorses a folded Glu conformation as the global low-energy conformation. This conformation is identical to the binding conformation of Glu at the iGluRs, and accordingly, L-2,4-synsubstituted Glu analogues are selective iGluR ligands. Nevertheless, we routinely screen our compounds at the mGluRs, and from the group of analogues 1a−k, we identified 1d as a selective mGlu2 agonist with 200-fold selectivity over mGlu3 and no functional activity at mGluR group I or III (Table 1). In support of this interesting finding, the selectivity profile was confirmed in an mGlu2/3 binding assay (mGlu2, Ki = 3.7 ± 0.1 μM; mGlu3, IC50 > 500 μM).24 As previously reported, 1d displays no activity 915



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Figure 4. Superimposition of 1d when docked into Glu-mGlu3 (PDB code: 2E4U; W872 is deleted) with Glu when crystallized in mGlu3 (PDB code: 2E4U).

1d (Figure 4) but in a low-energy conformation would potentially lead to a more potent agonist. On the other hand, since we do not fully understand the origin of the intriguing mGlu2 over mGlu3 subtype selectivity of 1d, we do indeed risk disrupting this attractive pharmacological profile. Introduction of a cyclopropane ring is an attractive strategy to constrain small molecules due to its rigidity, small size, and relative ease of synthesis. On the basis of the suggested binding mode of 1d presented in Figure 4, we designed cyclopropane analogues 2a−d, as shown in Figure 5. To study the legitimacy of this design, we docked 2a−d into the Glu-mGlu3 crystal structure (PDB code: 2E4U; W872 is deleted). The study suggested a number of poses, of which the highest scoring binding modes are presented in Figure 6. Estereoisomer 2a engages in favorable salt bridge interactions with residues Arg68 and Arg64 as well as favorable hydrogen-bond interactions with W810 and W843. On the other hand, Estereoisomer 2b only engages in salt bridge formation with Arg68. Furthermore, a developing steric clash between the cyclopropane methylene group with Arg68 forces 2b into a collapsed state (defined as the formation of a salt bridge between the ammonium group and the Glu backbone distal carboxylate group). The two Z-isomers, 2c and 2d, both engage in salt bridge interactions with Arg68 and Arg64 and hydrogen bonds with W810 and W843. In summary, the in silico study motivated us to pursue the synthesis of the four conformationally restricted glutamate stereoisomers 2a−d as potential mGlu2- and/or mGlu3-selective agonists. Synthesis of 2a/2b. The synthesis of 2a and 2b commenced from readily accessible N-Boc-protected pyroglutamate ester 3,27 which was first converted to the corresponding tertiary amine by treatment with LHMDS followed by Eschenmoser’s salt.28 Subsequent in situ quaternization with methyl iodide in MeOH followed by elimination of the ammonium salt under basic conditions provided 4-methylenepyroglutamate 4 in 66% yield.29 Cyclopropanation with methyl bromoacetate afforded cyclopropane 5 in 49% yield as a (3:4) mixture of diastereomers. At this stage, separation of the two diastereomers 5a/5b was attractive as the absolute configuration could likely be determined by NOE NMR experiments due to the rigid nature of these intermediates. However, separation proved to be unsuccessful on a wide range of HPLC columns. Instead, full hydrolysis under acidic conditions to give 2a/2b was achieved and following separation by prep HPLC gave 2a and 2b in a 1:1 ratio in overall 49% yield (Scheme 1). However, due to the flexible nature of 2a and 2b, it was not possible to assign their respective absolute configuration by NMR.

Figure 2. Glu crystallized in mGlu3 (PDB code: 2E4U) and subunit residue difference for Arg64 (mGlu3).

Figure 3. Docking of 1d in the Glu-mGlu3 X-ray structure (PDB code: 2E4U; W872 is deleted).

therefore envisaged that a redesigned rigid scaffold that would orient the side chain carboxylate group in a similar way as that in 916



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Figure 5. Chemical structures of 1d and conformationally restricted analogues 2a−d.

Figure 6. Superimposition of the highest scoring binding modes of 1d (green) and 2a−d (magenta) when docked into the Glu-mGlu3 X-ray structure (type code) (PDB code: 2E4U; W872 is deleted).

Scheme 1. Synthesis of Diastereomeric Pair 2a and 2ba

Scheme 2. Synthesis of Spirolactams 6a/6b from 5a/5b Followed by HPLC Separation and Hydrolysis to Target Compounds 2a and 2ba

a

Reagents and conditions: (a) LHMDS, dimethylmethy-lidene ammonium iodide, THF, −30 °C, 2 h; (b) MeI, MeOH, sat. aq. NaHCO3, rt, 3 days, 66%; (c) methyl bromoacetate, DABCO, Cs2CO3, MeCN, 80 °C, 23 h, 57%; (d) 4 M HCl, 100 °C, 17 h, 49%, then preparative HPLC.

a

Reagents and conditions: (a) 4 M HCl, rt, 4 h, 48%, then prep HPLC; (b) 4 M HCl, 100 °C, 17 h, quantitative yields.

We therefore sought to assign the stereochemical configurations of 2a/2b by means of conformationally restricted lactams 6a and 6b (Scheme 2) in the hope that these analogues could be separated by HPLC. Protected spirolactams 5a/5b were treated with 4 M HCl at room temperature for 4 h, which hydrolyzed the ester and BOC functionalities without cleaving

the lactam ring. Subsequently, it was a pleasant finding that the diastereomeric mixture of 6a/6b was easily separated by HPLC to afford spirolactams 6a and 6b in 48% total yield. The stereochemical configurations of spirolactams 6a and 6b were assigned by use of NMR (NOE) experiments (Figure 7) in 917



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Scheme 3. Synthesis of Analogues 2c and 2da

Figure 7. Assignment of the stereochemical configuration of spirolactams 6a and 6b by NMR (NOE) experiments.

accordance with the following analysis: The 1H NMR spectra of compounds 6a and 6b showed the expected double doublet for H5 at 4.45 and 4.48 ppm, respectively. For both isomers, a strong NOE was observed between H5 ↔ H4b with a large coupling constant (Jcis = 10 Hz) and between H5 ↔ H4a with a smaller coupling constant (Jtrans = 3.6 Hz). In addition, no NOE was observed between H6 ↔ H4a/H4b. This clearly shows that H6 is oriented in the opposite direction relative to H4a/H4b, confirming the anti-configuration at the cyclopropane ring. Finally, a NOE effect was observed between H4a ↔ H7 for 6a and between H4b ↔ H7 for 6b. The relative orientation of the cyclopropane ring was thereby unequivocally determined, and exact stereochemistry could be assigned to 6a, (1R,3S,6S)-4-oxo-5-azaspiro[2.4]heptane-1,6-dicarboxylic acid, and 6b, (1S,3R,6S)-4-oxo-5azaspiro[2.4]heptane-1,6-dicarboxylic acid (Figure 7). With this information in hand, spirolactam 6a was hydrolyzed to give a product with HPLC retention identical to that of 2b, allowing for the assignment of (1S,2R) stereochemistry to 2b. Spirolactam 6b was hydrolyzed to give a product with HPLC retention time identical to that of 2a, allowing (1R,2S) stereochemistry to be assigned to 2a (Scheme 2). Synthesis of 2c/2d. To establish a syn relationship between the two distal carboxylate groups in 2c/2d, we had to take a different approach (Scheme 3). Commercially available BocAsp(OtBu)-OH (7) was treated with N-methyl morpholine, isobutyl chloroformate, and then 2-mercaptopyridine N-oxide. Carrying on immediately with the crude product, irradiating with two tungsten lamps (2 × 100 W) in the presence of Nmethylmaleimide afforded intermediate 8 (Scheme 3).30,31 Subsequent oxidative elimination afforded imide 9 in 50% yield. From here, a 2 + 3 cycloaddition reaction of 9 with an ethereal solution of diazomethane installed the desired syn relationship of the two distal carboxylate groups in pyrazoline 10a/10b. Without further purification, crude pyrazoline 10a/ 10b was dissolved in acetonitrile in a round-bottomed quartz flash and irradiated with mercury lamps (6 × 24 W) in the presence of 3 equiv of benzophenone to afford cyclopropanes 11a/11b in 86% yield as a (∼1:1) mixture of diastereomers.32 At this stage, we attempted to separate diastereomers 11a/11b by HPLC for subsequent stereochemical assignment by NMR. Regrettably, this approach was not successful, and cyclopropanes 11a/11b were therefore fully deprotected to give the final products 2c/2d in 35% yield as a (1:1) mixture of diastereomers. Due to the flexible nature of 2c and 2d, the separation of the two diastereomers by HPLC was not possible (Scheme 3). Given the success for the separation of 6a/6b, we decided to cyclize cyclopropane 2c/2d with thionyl chloride in methanol followed by in situ aqueous acidic hydrolysis of methyl esters to afford pyroglutamate derivatives 6c/6d in 48% yield (Scheme

a

Reagents and conditions: (a) N-methyl morpholine, isobutyl chloroformate, then 2-mercaptopyridine N-oxide, TEA, THF, −20 °C in the dark, 4 h; (b) N-methylmaleimide, tungsten lamp, 2 × 110 W, 1 h; (c) mCPBA, DCM, rt, 3 h, then toluene, 120°, 1.5 h, 50%; (d) diazomethane, diethyl ether, rt, 15 min; (e) benzophenone, MeCN, irradiation (mercury lamps), 21−48 °C, 1.5 h, 86%; (f) conc. HCl, acetic acid, 120 °C, 19 days, 35%.

4).33 Again, separation of the diastereomers was possible by preparative HPLC to afford 6c and 6d in 10 and 23% yields, Scheme 4. Synthesis of 2c and 2d via Lactams 6c and 6da

a

Reagents and conditions: (a) thionyl chloride, MeOH, rt, 1 h and at 70 °C, 3 h, then HCl (aq) 3 h, then preparative HPLC, 48%; (b) 6 M HCl, reflux, 19 h, HPLC purification. 2c, 53%; 2d, 80%.

respectively. The stereochemical configuration was then determined by NMR. For both of the two spirolactams 6c and 6d, strong NOE effects were observed between H5 ↔ H4a, H5 ↔ H4b, and H6 ↔ H7ab. For 6c, the H4a and H6 signals are collapsed (same ppm value). Thus, it was not possible to unequivocally confirm a NOE effect between H4b ↔ H6 because a NOE effect will always be observed between H4a ↔ H4b. Fortunately, for 6d, a NOE effect was observed between H4b ↔ H7ab, whereas for 6c, no NOE effect was observed between H4b ↔ H7ab. In all, the analysis allowed for the assignment of the stereochemical configuration of 6c and 6d in accordance with Figure 8. With 918



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this information in hand, the two spirolactams 6c and 6d were hydrolyzed in 6 M HCl to afford 2c and 2d in 53 and 80% yields, respectively.

Figure 9. Activity of compound 1d and new conformational restricted analogues 2a−d on mGlu2 and mGlu3 receptors. Prior to IP1 accumulation measurement, cells were preincubated with 3 μM LY341495. Then, receptor activity upon ligand binding was measured with IP-One HTRF kit after 1 mM compound stimulation. Compounds 1d, 2a, and 2d showed selective agonist activity at mGlu2 and no activity onat mGlu3. Statistical differences: ***, p < 0.001; **, p < 0.01, as determined by one-way ANOVA with Dunnett’s post test in comparison to vehicle.

Figure 8. Assignment of the stereochemical configuration of spirolactams 6c and 6d by NMR (NOE) experiments.

Table 3. Pharmacological Characterization of Glu, DCG-IV, 1d, and New Conformational Restricted Analogues 2a−d at Rat mGlu2,3 in a FRET-Based mGlu Sensor Assaya

Pharmacological Characterization. Lead structure 1d, Glu, and the four new analogues 2a−d were characterized as ligands at mGlu1,2,4−8 in an IP-One functional assay, which quantifies receptor signaling activity by inositol monophosphate (IP1) production34 (Table 2 and Figure 9), and at mGlu2,3 in a FRET-based mGlu sensor assay35 (Table 3). The latter assay measures changes in FRET occurring during receptor activation. First, the mGlu2 agonist selectivity profile was confirmed for 1d and with equipotency to that of Glu. Of the four newly synthesized analogues 2a−d, E-stereoisomer 2a most interestingly displayed selective agonist activity at mGlu2 equipotent to that of Glu/1d. In contrast, the other E-stereoisomer 2b showed no functional activity at any of the mGlu1−8 subtypes. This may be due to a steric clash between the cyclopropane methylene group and conserved residue Arg68, as pointed out in the docking study performed during the design phase. Z-stereoisomer 2c displayed no activity at groups I and II, full agonist activity at group III subtypes mGlu4,6,8 in the low to mid micromolar range, and no activity at mGlu7. Z-stereoisomer 2d also displayed selective agonist activity for group III over both groups I (Table 2) and II (Tables 2 and 3), mid-to-high nanomolar potency at mGlu4,6,8, and only mid-micromolar potency at mGlu7 (Table 2). Essentially, the pharmacological profiles of 2c and 2d are the same, but 2d is 20-fold more potent than 2c. This emphasizes the importance of the absolute stereochemistry of the Z-cyclopropane ring. In comparison with standard selective group III agonist L-AP4, the pharmacological profile of 2d is unique, as L-AP4 displays potent agonist activity at mGlu4 and mGlu8 and at least 30-fold lower potency at mGlu6 and mGlu7 (Table 2).

group II Glu DCG-IV 1d 2a 2b 2c 2d

mGlu2 EC50

mGlu3 EC50

16.09 ± 2.31 0.35 ± 006 120.30 ± 29.96 119.03 ± 26.03 >1000 >1000 >1000

3.40 ± 0.87 0.08 ± 0.01 >1000 >1000 >1000 >1000 >1000

a

All values are in micromolar. SEM calculated from n = 4−6 experiments.

In a radio ligand binding assay on rat synaptosomes,18 1d displayed negligible affinity for AMPA receptors (IC50 > 100 μM) and KA receptors (IC50 > 100 μM) and only midmicromolar affinity for the NMDA receptors (Ki = 52 [4.28 ± 0.06] μM). The two E-stereoisomers 2a and 2b displayed negligible affinity for AMPA receptors (IC50 > 100 μM), KA receptors (IC50 > 100 μM), and NMDA receptors (Ki > 100 μM), whereas the two Z-stereoisomers 2c and 2d displayed negligible affinity for AMPA receptors (IC50 > 100 μM) and KA receptors (IC50 > 100 μM) and low-micromolar affinity for NMDA receptors (Ki = 15 μM [4.82 ± 0.05] and Ki = 13 μM [4.89 ± 0.07], respectively). In an [3H]Asp uptake assay on cloned human excitatory amino acid transporter subtypes 1−3 (EAAT1−3), 1d and 2a−d displayed no substrate or inhibitory activity at concentrations up to 300 μM (IC50 > 300 μM).

Table 2. Agonist Activity of Glu, L-AP4, 1d, and New Conformational Restricted Analogues 2a−d at Rat mGlu1,2,4−8a group I Glu L-AP4 1d 2a 2b 2c 2d a

group II

group III

mGlu1 EC50

mGlu5 EC50

mGlu2 EC50

mGlu3 EC50

mGlu4 EC50

mGlu6 EC50

mGlu7 EC50

mGlu8 EC50

13.87 ± 4.93 ->1000 >1000 >1000 >1000 >1000

8.99 ± 3.44 ->1000 >1000 >1000 >1000 >1000

13.08 ± 3.09 -34.18 ± 2.77 33.69 ± 3.00 >1000 >1000 145.00 ± 43.97

--------

9.49 ± 1.19 0.03 ± 0.006 ≥100 >1000 >1000 2.48 ± 0.50 0.13 ± 0.05

65.09 ± 16.63 1.91 ± 0.29 >1000 >1000 >1000 17.82 ± 3.50 0.49 ± 0.06

-340.00 ± 26.9 >1000 >1000 >1000 >1000 119.70 ± 23.34

14.01 ± 4.45 0.07 ± 0.01 >1000 >1000 >1000 20.79 ± 1.50 0.68 ± 0.11

IP-One functional assay. All values are in micromolar. SEM calculated from n = 4−6 experiments. --, not tested. 919



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II selectivity may originate from a salt bridge interaction between the side chain carboxylate group and residue Arg64 (PDB code: 2E4U numbering), which is unique to mGlu2/3. Using 1d as a template, conformationally restricted analogues 2a−b were designed and synthesized in a stereoselective fashion. Most interestingly, E-stereoisomer 2a retained selective agonist activity at mGlu2 over mGlu3, whereas E-stereoisomer 2b was inactive at all of the mGlu1−8 subtypes. Most interestingly, the two Zstereoisomers, 2c and 2d, displayed selective agonist activity at group III subtypes mGlu4,6,8, with low potency at mGlu7. In general, 2d was 20-fold more potent than 2c, underlining the importance of the absolute stereochemistry of the trans cyclopropane ring. A preliminary modeling study of the most potent analogue, 2d, in mGlu4-hmdl suggested a Glu-like binding mode. Taken together, 2a is a valuable lead structure for the design and synthesis of potent mGlu2-selective agonists. Furthermore, 2c and 2d are attractive as new lead structures for the design and synthesis of new potent group III subtypeselective mGluR agonists. This work is ongoing in our laboratories.

To date, X-ray structures of an agonist bound to mGlu4 are not available. Thus, in order to investigate key ligand−receptor interactions underlying the observed selective group III agonist profile of 2c/2d, we constructed a homology model (hmdl) of mGlu4 based on the Glu-mGlu3 X-ray structure (PDB code: 2E4U). It is well-known that water molecules play an essential role in ligand−host binding, and docking of 2c/2d into mGlu4hmdl using any implicit solvation model did not result in meaningful ligand poses. We therefore decided to copy Glu and the full water matrix from the mGlu3 template X-ray structure into mGlu4-hmdl, followed by global protonation and energy minimization operations. Hereafter, the Glu ligand was deleted and three mGlu4-hmdls were created by removal of water molecules W872 (mGlu4-hmdl1), W872 + W810 (mGlu4hmdl2), and W872 + W810 + W843 (mGlu4-hmdl3). While docking 2d into mGlu4-hmdl3 gave a plethora of non-Glu-like binding modes, docking of 2d into mGlu4-hmdl1 and mGlu4hmdl2 both suggested a Glu-like binding mode in which the γcarboxylate group engages in salt bridge formation with Arg78 and Lys405 (conserved residues in mGlu1−8) and Lys74 (nonconserved residue; see Figure 2), whereas the δ-carboxylate group engages in salt bridge formation with Arg78 (Figure 10).

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EXPERIMENTAL SECTION

Chemistry. All reactions involving dry solvents or sensitive agents were performed under a nitrogen atmosphere, and glassware was dried prior to use. Solvents were dried according to standard procedures, and reactions were monitored by analytical thin-layer chromatography (TLC, Merck silica gel 60 F254 aluminum sheets). Flash chromatography was carried out using Merck silica gel 60A (35−70 μm). 1H and 13C NMR spectra were recorded on a Bruker Avance 400 MHz in CDCl3 using CHCl3 as internal standard unless otherwise noted. MS spectra were recorded using LC-MS performed using an Agilent 1200 solvent delivery system equipped with an autoinjector coupled to an Agilent 6400 triple quadrupole mass spectrometer equipped with an electrospray ionization source. Gradients of 5% aqueous acetonitrile containing 0.05% formic acid (eluent A) and 95% aqueous acetonitrile containing 0.05% formic acid (eluent B) were employed. Analytical HPLC was performed using a Dionex UltiMate 3000 pump and photodiode array detector (200 and 210 nm, respectively) installed with an XTerra MS C18 3.5 μm, 4.6 × 150 mm column, using a 5 → 95% MeCN gradient in H2O containing 0.1% TFA. Melting points were measured using a MPA 100 Optimelt automatic melting point system. Optical rotation was measured using a PerkinElmer 241 polarimeter with a Na lamp (589 nm). All commercial chemicals were used without further purification. The purity of all tested compounds was determined by HPLC to be >95%. For the synthesis of alkyl analogues 1a and 1b, see ref 36. For the synthesis of 1c−f, see ref 21. For the synthesis of 1g−k, see ref 18. General Procedure A: Synthesis of Diazomethane. An ethereal solution of diazomethane (0.31 M) was prepared from N-methyl-Nnitroso-4-toluenesulfonamide (Diazald) in the distillation set “DiazaldKit” (Aldrich). 1-((S)-2-Amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic Acid (2a/2b). (6S)-5-tert-Butyl 1,6-dimethyl 4-oxo-5azaspiro[2.4]heptane-1,5,6-tricarboxylate (5a/5b) (320 mg, 0.98 mmol) was dissolved in 4 M HCl (12 mL) and refluxed for 17 h. After concentration in vacuo, the diastereomeric mixture was separated by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to give 2a/2b (1:1 ratio) in 49% yield. Analytical data for (1R,2S)-1-((S)-2-amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic Acid (2a). White solid, 99.6% de. tR = 6.1 min (H2O/0.1% TFA). 1H NMR (400 MHz, D2O) δ 4.05 (dd, J = 7.6, 7.2 Hz, 1H), 2.55−2.46 (m, 2H), 2.25 (dd, J = 14.8, 7.6 Hz, 1H), 1.75 (ddd, J = 8.8, 5.2, 0.8 Hz, 1H), 1.47 (dd, J = 6.9, 5.2 Hz, 1H). 13C NMR (100 MHz, D2O) δ 176.0, 174.0, 172.5, 52.5, 28.9, 28.6, 28.1, 20.3. mp 140−142 °C (dec). [α]25 D = +92.9° (c 0.10, H2O). LC-MS (m/z) calcd for C8H11NO6 [M + H+], 218.1; found, 218.1. Analytical Data for (1S,2R)-1-((S)-2-amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic Acid (2b). White solid, 99.2% de. tR =

Figure 10. Docking of 2d in group III mGlu4-hmdl2 model built from mGlu3 (PDB code: 2E4U).

The cyclopropane ring of 2d is directed into the area in space occupied by W810. It is not clear if this would lead to exclusion of this water molecule from the binding pocket. The Lys74 residue in mGlu4 corresponds to Gln in mGlu6, Asn in mGlu7, and Lys in mGlu8 (Figure 2). These residue differences likely play a role in the observed agonist profile of 2c/2d among the mGluR group III subtypes and could be explored further by a detailed structure−activity relationship study.

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CONCLUSIONS On the basis of chemistry-driven discovery of novel ligands for the glutamate receptors, we synthesized 1d and showed that it quite unexpectedly was a selective mGlu2 agonist equipotent to Glu. In particular, its selectivity over mGlu3 is intriguing but difficult to explain based on available X-ray structures of mGlu2/ 3. On the other hand, our docking study suggests that the group 920



Article

165.4, 149.8, 136.5, 120.9, 83.8, 55.6, 52.6, 27.9, 27.8. mp 77−79 °C (dec). [α]25 D = −17.5° (c 0.15, dichloromethane). LC-MS (m/z) calcd for C12H17NO5 [M + H+], 256.1; found, 156.1 (-Boc). HPCL: purity210 > 95%. (6S)-5-tert-Butyl 1,6-Dimethyl 4-oxo-5-azaspiro[2.4]heptane1,5,6-tricarboxylate (5a/5b). DABCO (1.32 g, 11.77 mmol) was added to a solution of methyl bromoacetate (1.2 mL, 12.68 mmol) in dry MeCN (15 mL) at rt under a N2 atmosphere. The reaction mixture was stirred for 30 min, and then Cs2CO3 (3.83 g, 11.75 mmol) and a solution of 4 (1.00 g, 3.92 mmol) in dry MeCN (35 mL) were added. The reaction mixture was stirred at 80 °C for 22 h. The crude reaction was quenched with sat. aq. NH4Cl (200 mL) and extracted with Et2O (3 × 200 mL), and the combined organic phases were washed with brine (100 mL). The organic phase was dried over MgSO4. After concentration in vacuo, the crude product was purified by column chromatography on silica gel. This afforded the title compound as a yellow oil (633 mg, 1.92 mmol, 49%): Rf 0.38 (heptane/EtOAc 1:1). 1H NMR (400 MHz, MeOD) (∼3:4 ratio of diastereomers) δ 4.78 (dd, J = 5.6, 2.8 Hz, 0.5H), 4.75 (dd, J = 5.6, 2.8 Hz, 0.5H), 3.81 (s, 1.30H), 3.75 (s, 1.70H), 3.71 (s, 1.30H), 3.69 (s, 1.70H), 2.66−2.57 (m, 1H), 2.26 (dd, J = 8.8, 6.4 Hz, 0.5H), 2.18−2.09 (m, 1.5H), 1.62 (dd, J = 9.2, 4.8 Hz, 0.5H), 1.51 (dd, J = 9.2, 4.8 Hz, 0.5H), 1.48 (s, 9H), 1.43−1.37(m, 1H). 13C NMR (100 MHz, CDCl3) δ 172.4, 172.3, 171.7, 171.5, 171.0, 170.9, 149.2, 149.1, 84.0, 83.9, 56.7, 56.5, 52.9, 52.6, 52.1, 31.9, 31.1, 30.9, 27.9, 27.3, 26.9, 25.9, 25.8, 22.7, 21.5, 18.5. LC-MS (m/z) calcd for C15H21NO7 [M + H+], 328.1; found, 228.1 (- Boc). HPLC: purity210 > 95%. (6S)-4-Oxo-5-azaspiro[2.4]heptane-1,6-dicarboxylic Acid (6a/6b). (6S)-5-tert-Butyl 1,6-dimethyl 4-oxo-5-azaspiro[2.4]heptane1,5,6-tricarboxylate (5a/5b) (110 mg, 0.33 mmol) was stirred in 4 M HCl (12 mL) at rt for 4 h. After concentration in vacuo, the diastereoisomic mixture was separated by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to give 6a and 6b in 48% total yield. Analytical Data for (1R,3S,6S)-4-Oxo-5-azaspiro[2.4]heptane-1,6dicarboxylic Acid (6a). White solid, 99.8% d.e. tR = 10.9 min (H2O/ 0.1% TFA). 1H NMR (400 MHz, D2O) δ 4.45 (dd, J = 10.0, 3.6 Hz, 1H), 2.75 (dd, J = 14.0, 10.0 Hz, 1H), 2.35 (dd, J = 14.0, 3.6 Hz, 1H), 2.17 (dd, J = 8.8, 6.4 Hz, 1H), 1.50 (dd, J = 8.8, 4.8 Hz, 1H), 1.44 (dd, J = 6.4, 4.8 Hz, 1H). 13C NMR (100 MHz, D2O) δ 179.1, 176.7, 174.7, 53.4, 30.3, 29.2, 24.9, 18.9. mp 210−212 °C (dec). [α]25 D = −66.6° (c 0.14, H2O). LC-MS (m/z) calcd for C8H9NO5 [M + H+], 200.1; found, 200.1. Analytical Data for (1S,3R,6S)-4-Oxo-5-azaspiro[2.4]heptane-1,6dicarboxylic Acid (6b). White solid, 99.9% d.e. tR = 17.2 min (H2O/ 0.1% TFA). 1H NMR (400 MHz, D2O) δ 4.48 (dd, J = 9.6, 4.0 Hz, 1H), 2.77 (dd, J = 14.4, 9.6 Hz, 1H), 2.32 (dd, J = 14.4, 4.0 Hz, 1H), 2.12 (dd, J = 8.8, 6.4 Hz, 1H), 1.55 (dd, J = 8.8, 5.2 Hz, 1H), 1.45 (dd, J = 6.4, 5.2 Hz, 1H). 13C NMR (100 MHz, D2O) δ 179.0, 176.3, 174.6, 53.4, 30.6, 28.8, 25.8, 17.2. mp 176−178 °C (dec). [α]25 D = +135.5° (c 0.18, H2O). LC-MS (m/z) calcd for C8H9NO5 [M + H+], 200.1; found, 200.1. 6a was fully deprotected by reflux in 4 M HCl for 17 h, concentrated, and analyzed by HPLC. tR = 7.01 min (unequivocally assigns (1S,2R) stereochemistry to 2b). 6b was fully deprotected by reflux in 4 M HCl for 17 h, concentrated, and analyzed by HPLC. tR = 6.1 min (unequivocally assigns (1R,2S) stereochemistry to 2a). (6S)-4-Oxo-5-azaspiro[2.4]heptane-1,6-dicarboxylic Acid (6c/6d). Thionyl chloride (0.19 mL, 2.58 mmol) was added to a solution of 1-((S)-2-amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic acid (2c/2d) (160 mg, 0.74 mmol) in dry MeOH (10 mL) at 0 °C under a N2 atmosphere. The reaction mixture was stirred at rt for 1 h and refluxed for 30 min at 70 °C. The MeOH was removed under reduced pressure, and the crude mixture was stirred in 4 M HCl (12 mL) at rt for 3 h. After concentration in vacuo, the diastereomeric mixture was separated by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to give 6c and 6d (1:3) in 48% yield. Analytical Data for 6c. Clear foam, 15 mg, 10% yield. tR = 9.69 min (H2O/0.1% TFA). 1H NMR (600 MHz, D2O) δ 4.55 (ddd, J = 9.5, 5.2, 1.1, 1H), 2.78 (dd, J = 13.8, 9.5, 1H), 2.35−2.31 (m, 2H), 1.71 (dd, J = 8.4, 5.4, 1H), 1.46 (dd, J = 9.4, 5.4, 1H). 13C NMR (150 MHz, D2O) δ

7.01 min (H2O/0.1% TFA). 1H NMR (400 MHz, D2O) δ 4.25 (dd, J = 7.6, 7.2 Hz, 1H), 2.56−2.49 (m, 2H), 2.41 (dd, J = 15.2, 7.6 Hz, 1H), 1.78 (dd, J = 8.8, 5.2 Hz, 1H), 1.48 (dd, J = 6.8, 4.8 Hz, 1H). 13C NMR (100 MHz, D2O) δ 175.8, 173.8, 171.8, 51.7, 28.6, 28.1, 27.7, 20.6. mp 130−132 °C (dec). [α]25 D = −37.4° (c 0.12, H2O). LC-MS (m/z) calcd for C8H11NO6 [M + H+], 218.1; found, 218.1. 1-((S)-2-Amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic Acid (2c/2d). tert-Butyl (2S)-2-((tert-butoxycarbonyl)amino)-3(3-methyl-2,4-dioxo-3-azabicyclo[3.1.0]-hexan-1-yl)propanoate (11a/ 11b) (860 mg, 2.33 mmol) was stirred in conc. HCl (20 mL) and acetic acid (99%, 20 mL) at 120 °C. After 13 days, additional conc. HCl (5 mL) and acetic acid (99%, 5 mL) were added, and the reaction mixture was stirred for a further 6 days. After concentration in vacuo, the crude mixture was purified by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to afford title compound 2c/2d as a white solid (177 mg, 0.81 mmol, 35%). tR = 3.42 min (H2O/0.1% TFA, 1:1). 1H NMR (600 MHz, D2O) (1:3 ratio of diastereomers) δ 4.23−4.27 (m, 1H), 2.62 (dd, J = 15.0, 6.6 Hz, 0.67H), 2.50 (dd, J = 15.0, 6.6 Hz, 0.33H), 2.32−2.27 (m, 1H), 2.11 (dd, J = 15.0, 7.2 Hz, 0.31H), 2.01 (dd, J = 15.0, 7.2 Hz, 0.69H), 1.89−1.83 (m, 1H), 1.46−1.41 (m, 1H). 13C NMR (150 MHz, D2O) δ 174.6, 174.5, 173.5, 171.8, 171.7, 52.0, 51.7, 34.9, 34.8, 29.9, 29.8, 29.5, 29.1, 18.9, 18.8. LCMS (m/z) calcd for C8H11NO6 [M + H+], 218.1; found, 218.0. (1S,2S)-1-((S)-2-Amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic Acid (2c). Pyroglutamate 6c (35 mg, 0.18 mmol) was refluxed in 6 M HCl for 19 h. After concentration in vacuo, the crude mixture was purified by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to afford 2c (white solid, 20 mg, 53%). tR = 3.51 min (H2O/0.1% TFA). 1H NMR (600 MHz, D2O) δ 4.15 (dd, J = 12.0, 9.6 Hz, 1H), 2.34 (dd, J = 22.8, 10.2 Hz, 1H), 2.24 (dd, J = 13.2, 10.2 Hz, 1H), 2.18 (dd, J = 22.8, 12.0 Hz, 1H), 1.86 (dd, J = 10.2, 8.4 Hz, 1H), 1.45 (dd, J = 13.2, 7.8 Hz, 1H). 13C NMR (150 MHz, D2O) δ 175.1, 174.1, 172.7, 52.7, 35.4, 30.2, 29.5, 19.3. mp 96−98 °C (dec). LC-MS (m/z) calcd for C8H11NO6 [M + H+], 218.1; found, 218.0. (1R,2R)-1-((S)-2-Amino-2-carboxyethyl)cyclopropane-1,2-dicarboxylic acid (2d). Pyroglutamate 6d (60 mg, 0.30 mmol) wase refluxed in 6 M HCl for 19 h. After concentration in vacuo, the crude mixture was purified by HPLC using a Gemini-NX 5 μ, C18 110A, AXI 250 × 21.20 mm column (H2O/0.1% TFA) to afford 2d (white solid, 52 mg, 80%). tR = 3.48 min (H2O/0.1% TFA). 1H NMR (600 MHz, D2O) δ 4.21 (m, 1H), 2.59 (dd, J = 15.0, 6.6 Hz, 1H), 2.29 (dd, J = 8.4, 6.6 Hz, 1H), 2.01 (dd, J = 15.0, 7.8 Hz, 1H), 1.84 (m, 1H), 1.42 (dd, J = 9.0, 5.4 Hz, 1H). 13C NMR (150 MHz, D2O) δ 174.7, 173.6, 171.8, 51.8, 34.8, 29.9, 29.2, 81.9. mp 88−90 °C (dec). LC-MS (m/z) calcd for C8H11NO6 [M + H+], 218.1; found, 218.0. (S)-1-tert-Butyl-2-methyl-4-methylene-5-oxopyrrolidine-1,2dicarboxylate (4). A precooled solution of (S)-1-tert-butyl 2-methyl 5oxopyrrolidine-1,2-dicarboxylate (3) (5.00 g, 20.56 mmol) in dry THF (100 mL) was added dropwise to a solution of 1.0 M LiHMDS in dry THF (22.7 mL, 22.61 mmol) over 25 min at −78 °C under a N2 atmosphere. The reaction mixture was stirred for 40 min, and then Eschenmoser’s salt (7.61 g, 41.11 mmol) was added. The reaction mixture was stirred at −78 °C for 1 h and at −30 °C for 2 h. The crude reaction was quenched with H2O (100 mL) and extracted with EtOAc (3 × 100 mL), and the combined organic phases were washed with brine (100 mL). The organic phase was dried over MgSO 4. After concentration in vacuo, the crude product was directly used for the next step without purification. Methyl iodide (31 mL, 498 mmol) was added to a solution of the crude (3.251 g, 10.82 mmol) in MeOH (42 mL) and stirred at rt for 3 days. After concentration in vacuo, the crude product was suspended in sat. aq. NaHCO3 (70 mL) and extracted with EtOAc (3 × 120 mL), and the combined organic phases were washed with brine (120 mL). After concentration in vacuo, the crude product was purified by column chromatography on silica gel. This afforded the title compound as a white solid (1.66 g, 6.82 mmol, 63%): Rf 0.73 (heptane/EtOAc 1:4). 1H NMR (400 MHz, CDCl3) δ 6.25 (t, J = 4.0 Hz, 1H), 5.52 (t, J = 4.0 Hz, 1H), 4.64 (dd, J = 12.0, 4.0 Hz, 1H), 3.78 (s, 3H), 3.12−3.04 (m, 1H), 2.76−2.70 (m, 1H), 1.53 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.5, 921



Article

NMR (150 MHz, CDCl3) δ 176.9, 176.6, 175.0, 173.8, 170.9, 170.8, 155.5, 155.3, 82.6, 82.5, 80.1, 80.0, 52.7, 52.5, 31.3, 30.7, 28.3, 28.2, 28.0, 27.9, 26.2, 25.5, 24.5, 24.4. LC-MS (m/z) calcd for C18H28N2O6 [M + H+], 369.2; found, 213.1 (-Boc and -tert-butyl). 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 glutamate concentration, receptors were cotransfected with the high-affinity glutamate transporter EAAC1 and medium was replaced by Glutamax (Life Technologies) at least 2 h before stimulation. IP-One Functional Assay. To monitor receptor signaling 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 mGlu2 and mGlu3 single-dose tests, cells were incubated with 3 μM LY341495 for 20 min at 37 °C. Then, cells were washed two times and stimulated with 1 mM test compound. Experiments were performed at least in duplicates, and data were analyzed using Prism software (GraphPad, La Jolla, CA, USA). FRET-Based mGlu Sensor Assay. To measure FRET changes occurring during mGluR activation, the 20 kDa SNAP-tag enzyme was fused to the receptor’s N-terminus. This allows covalent labeling with fluorophores as described previously.35 Twenty four hours after transfection, cells were incubated with 100 nM SNAP-Lumi4-Tb (trFRET donor) and 60 nM SNAP Green (acceptor) in Tag-Lite buffer (Cisbio Biossays, Codolet, France) for 1 h. Donor and acceptor concentrations were chosen in order to label surface receptors equivalently with both one donor and one acceptor molecule. Then, cells were washed three times with buffer before drug addition. The FRET measurements were recorded at 520 nm using PHERAstar FS microplate reader. Experiments were performed at least in duplicates, and data were analyzed using Prism software (GraphPad, La Jolla, CA, USA). In Silico Study. The in silico study was performed using the software package MOE 2014 (Molecular Operating Environment, Chemical Computing Group) using the built-in mmff94x force field and the GB/ SA continuum solvation model. Docking. The receptor proteins were first prepared for docking by running the algorithm Protonate 3D. For mGlu3 (PDB code: 2E4U), water no. 872 was then deleted. Docking of 2a−d was carried out using the Induced Fit algorithm under the standard setup with the mmff94x force field, solvation was set to distance, and ligand atoms was selected as docking site. Receptor + solvent was selected as the target protein. Highest scoring binding modes of 2a−d were calculated as S (kcal/mol) = −8.688, −9.128, −9.849, and −9.276, respectively. Stochastic Conformational Search. The three carboxylate groups of 1d were protonated prior to the run. Collapsed structures (intramolecular salt bridge formation) were dismissed before lowenergy conformations of 1d were identified. Homology Models. The complete human mGlu4 amino acid sequence was aligned with mGlu3 (PDB code: 2E4U), and homology models were constructed using the built-in function with a standard setup (amber99 force field, solvation as distance). To the highest scoring mGlu4 homology model, Glu and the complete water matrix from mGlu3 were imported, and a global 3D protonation algorithm was run (standard setup). Thereafter, Glu was deleted and three homology models were created by deleting the following specific water molecules: W872 (mGlu4-hmdl1), W872 + W810 (mGlu4-hmdl2), and W872 + W810 + W843 (mGlu4-hmdl3).

178.5, 176.2, 173.5, 53.2, 32.9, 29.3, 28.8, 17.3. LC-MS (m/z) calcd for C8H9NO5 [M + H+], 200.1; found, 200.1. Analytical Data for 6d. White solid, 34 mg, 23% yield. tR = 11.90 min (H2O/0.1% TFA). 1H NMR (600 MHz, D2O) δ 4.47 (ddd, J = 9.6, 4.0, 0.7, 1H), 2.76 (dd, J = 13.7, 9.6, 1H), 2.35 (dd, J = 13.7, 4.0, 1H), 2.31− 2.24 (m, 1H), 1.74 (dd, J = 7.8, 5.5, 1H), 1.48 (dd, J = 8.6, 5.5, 1H). 13C NMR (150 MHz, D2O) δ 178.3, 176.7, 173.3, 53.4, 33.4, 29.6, 29.5, 16.3. mp 188−190 °C (dec). LC-MS (m/z) calcd for C8H9NO5 [M + H+], 200.1; found, 200.1. tert-Butyl (S)-2-((tert-Butoxycarbonyl)amino)-3-(1-methyl2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)propanoate (9). N-Methyl morpholine (2.28 mL, 20.74 mmol) and isobutyl chloroformate (2.71 mL, 20.74 mmol) were added to a stirred solution of commercially available α-tert-butyl-N-Boc-L-aspartate (7) (6.01 g, 20.74 mmol) in dry THF (110 mL) at −20 °C under a N2 atmosphere in the dark. After 40 min, a precooled solution of 2-mercaptopyridine N-oxide (3.16 g, 24.89 mmol) and Et3N (3.56 mL, 25.51 mmol) in THF (60 mL) was added. The reaction mixture was stirred with the exclusion of light for 4 h. The precipitate was removed by filtration (rapidly and dry), and the filtrate was irradiated with two tungsten lamps (2 × 100 W) in the presence of N-methylmaleimide (11.52 g, 103.69 mmol) at 0 °C (ice bath) for 1 h under a N2 atmosphere. After removal of THF under reduced pressure, the residue was partitioned between dichloromethane (400 mL) and sat. aq. NaHCO3 (300 mL). The layers were separated, and the organic phase was washed with 0.5 N HCl (400 mL) and brine (300 mL). The organic phase was dried over MgSO4. After concentration in vacuo, the crude NMR indicated that the 2-thiopyridyl moiety was present (intermediate 8). Signals in the 1H NMR spectrum (400 MHz, CDCl3) at 8.28−8.26 (m, 1H), 7.60−7.52 (m, 1H), 7.29−7.25 (m, 1H), and 7.07−7.03 (m, 1H) ppm. LC-MS (m/z) calcd for C22H31N3O6S [M + H+], 466.2; found, 466.2. The yellow solid was dissolved in dry dichloromethane (100 mL), cooled to 0 °C, and treated with a solution of mCPBA (3.94 g, 22.80 mmol) in dichloromethane (100 mL). After 2 h at rt, another portion of mCPBA (0.72 g, 4.20 mmol) was added, and the reaction mixture was stirred for an additional 1 h. The reaction mixture was diluted with dichloromethane (200 mL) and washed with sat. aq. NaHCO3 (100 mL), 0.5 N HCl (100 mL), and brine (100 mL). The organic phase was dried over MgSO4. After concentration in vacuo and drying under high vacuum for minimum 1 h, the crude product was dissolved in dry toluene (150 mL) and heated at reflux for 1.5 h. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel. This afforded title compound 9 as a yellow solid (3.67 g, 10.36 mmol, 50%): Rf 0.62 (dichloromethane/EtOAc 5:1). 1H NMR (600 MHz, CDCl3) δ 6.43 (s, 1H), 5.23 (d, J = 6.6 Hz, 1H), 4.46 (q, J = 5.4 Hz, 1H), 3.00−2.97 (m, 4H), 2.78 (dd, J = 15.6, 4.8 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 171.3, 170.6, 170.1, 155.2, 145.0, 128.7, 83.1, 80.2, 52.4, 29.5, 28.3, 28.0, 23.8. mp 92−94 °C (dec). LC-MS (m/ z) calcd for C17H26N2O6 [M + H+], 355.2; found, 199.1(-Boc and tertbutyl moieties). tert-Butyl (2S)-2-((tert-Butoxycarbonyl)amino)-3-(3-methyl2,4-dioxo-3-azabicyclo[3.1.0]-hexan-1-yl)propanoate (11a/ 11b). An ethereal solution of diazomethane (45 mL, 16.6 mmol) was added to a stirred solution of 9 (1.00 g, 2.82 mmol) in diethyl ether (40 mL) at 0 °C under a N2 atmosphere. After 15 min at rt, anhydrous CaCl2 (2.39 g, 19.75 mmol) was added to destroy the excess diazomethane. After filtration and evaporation under high vacuum to give a white foam, the unstable intermediate pyrazoline 10a/10b was dissolved in dry acetonitrile (40 mL) in a round-bottomed quartz flash. Benzophenone (1.53 g, 8.40 mmol) was added, and the solution was deoxygenated with nitrogen. The solution was irradiated with mercury lamps (6 × 24W, UVC light, 100 nm ≤ λ ≤ 280 nm) at 21−48 °C for 1.5 h. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel. This afforded title compound 11a/11b as a white foam (891 mg, 2.42 mmol, 86%). Rf 0.37 (EtOAc/ heptane 1:1). 1 H NMR (600 MHz, CDCl 3 ) (∼5:6 ratio of diastereomers) δ 5.39 (d, J = 7.8 Hz, 0.55H), 5.29 (d, J = 9.6 Hz, 0.45H), 4.34−4.28 (m, 1H), 2.86 (s, 1.46H), 2.84 (s, 1.54H), 2.79 (dd, J = 15.0, 4.8 Hz, 0.42H), 2.57 (dd, J = 15.0, 9.6 Hz, 0.58H), 2.43−2.38 (m, 1H), 1.97 (dd, J = 15.0, 4.8 Hz, 0.53H), 1.50−1.32 (m, 19.47 H). 13C 922



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01333. 1 H NMR spectra and analyzed, annotated ROESY spectra of 6a−d (PDF) SMILES strings for 1d, 2a−d, 6a, and 6b (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +45 35 33 62 44. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for the design and synthesis work was obtained from the Danish Medical Research Council, and the pharmacology part was supported by grants from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Cisbio Bioassays, the Fondation pour la Recherche Médicale (Equipe FRM DEQ20130326522) to J.-P.P. and the Agence Nationale pour la Recherche (ANR-13-BSV10006, antares) to C.G. A.S.T. was supported by a Ph.D. fellowship from the Ministère de l’Education Nationale et de la Recherche and by the Fondation Recherche Médicale (FRM FDT20140931071). We thank Professor Matthew Stanley Johnson, Department of Chemistry, University of Copenhagen, for allowing us to use his personalized mercury lamp reactor. The authors also thank Isabelle Brabet and Laurent Prézeau and the ARPEGE Pharmacology Screening-Interactome platform facility at the Institute for Functional Genomics (Montpellier, France).

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ABBREVIATIONS USED L-CCG-I, (2S,1′S,2′S)-2-(2′-carboxycyclo-propyl)glycine; DCG-IV, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine; CNS, central nervous system; EAAT2, excitatory amino acid transporter subtype 2; Glu, (S)-glutamate; iGluRs, ionotropic Glu receptors; IP1, inositol monophosphate; mGluRs, metabotropic Glu receptors; AMPA, α-amino-3-hydroxy-5-methyl-4isoxazole propionic acid; KA, kainic acid; NMDA, N-methyl-Daspartate

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REFERENCES

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