Synthesis and Pharmacological Characterization of C4-Disubstituted

Article pubs.acs.org/jmc

Synthesis and Pharmacological Characterization of C4-Disubstituted Analogs of 1S,2S,5R,6S‑2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylate: Identification of a Potent, Selective Metabotropic Glutamate Receptor Agonist and Determination of Agonist-Bound Human mGlu2 and mGlu3 Amino Terminal Domain Structures James A. Monn,*,† Lourdes Prieto,† Lorena Taboada,† Concepcion Pedregal,†,# Junliang Hao,† Matt R. Reinhard,† Steven S. Henry,† Paul J. Goldsmith,† Christopher D. Beadle,† Lesley Walton,† Teresa Man,† Helene Rudyk,† Barry Clark,† David Tupper,† S. Richard Baker,† Carlos Lamas,† Carlos Montero,† Alicia Marcos,† Jaime Blanco,† Mark Bures,† David K. Clawson,§ Shane Atwell,§ Frances Lu,§ Jing Wang,§ Marijane Russell,§ Beverly A. Heinz,‡ Xushan Wang,‡ Joan H. Carter,‡ Chuanxi Xiang,‡ John T. Catlow,∥ Steven Swanson,∥ Helen Sanger,⊥ Lisa M. Broad,⊥ Michael P. Johnson,⊥ Kelly L. Knopp,⊥ Rosa M. A. Simmons,⊥ Bryan G. Johnson,⊥ David B. Shaw,⊥ and David L. McKinzie⊥ †

Discovery Chemistry Research and Technologies, ‡Quantitative Biology, §Structural Biology, ∥Drug Disposition, and ⊥Neuroscience Research, Eli Lilly and Company, Indianapolis, Indiana 46285, United States S Supporting Information *

ABSTRACT: As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure−activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein−ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

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INTRODUCTION

and common signal transduction mechanisms. However, these glutamate-responsive seven-transmembrane domain proteins are differentially expressed in the CNS5 and regulate distinct physiological processes. For instance, while both mGlu2 and mGlu3 appear to play a role in the antistress response observed for the potent, pharmacologically balanced mGlu2/3 agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 1 (LY354740, Figure 1),6 other physiological and behavioral effects of mGlu2/3 agonists appear to be subtype dependent. In this regard, mGlu2 activation, likely via presynaptic inhibition of neurotransmitter release, appears to be entirely responsible for the observed acute antipsychotic-like and analgesic effects of mGlu2/3 agonists in rodents,7,8 while

Metabotropic glutamate (mGlu) 2 and 3 receptors (mGlu2, mGlu3) have attracted considerable attention as potential therapeutic targets for diseases of the central nervous system (CNS). Owing to their roles in regulating glutamate neurotransmission in the brain and the widespread availability of small molecule tools such as orthosteric agonists, antagonists, and allosteric modulators (positive and negative), these receptors have been extensively studied over the past 2 decades.1 This body of work has considerably advanced our understanding of the role of mGlu2 and mGlu3 in psychiatric (e.g., anxiety, depression, schizophrenia, addiction),2 neurologic (e.g., neuropathic pain, neurodegeneration),3 and proliferative (e.g., glioma)4 disorders. Historically, mGlu2 and mGlu3 receptors have been clustered together (i.e., group II mGluRs) because of their high sequence homology, overlapping agonist and antagonist pharmacology, © XXXX American Chemical Society

Received: October 21, 2014

A

DOI: 10.1021/jm501612y J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry

(those possessing larger C4-substitutents, e.g., NHAc and SPh) were identified, this effort was not successful in generating any additional mGlu2 receptor-selective agonists such as 2.13 This exceedingly subtle effect of both substituent type (CH3) and stereochemistry (α not β) on pharmacological responses at these two highly homologous proteins inspired us to perform the current investigation in which we have prepared and characterized analogs of 2 and 3 in which both α- and β-methyl groups are incorporated at the C4-position of 1, either separately (4) or connected to each other in the form of small spirocyclic rings [5 (LY2934747)14 and 6].

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CHEMISTRY Previously described nonracemic bicyclic enone 715 served as the starting material for the preparation of C4-gem-dimethyl analog 4 (Scheme 1). Installation of the first methyl substituent was achieved by methyl cuprate addition to the convex face of enone 7.11 The resulting ketone 8 was obtained in 39% yield and was subsequently oxidized under Saegusa−Ito conditions to afford enone 9 in 77% yield. Enone 9 was again treated with methyl cuprate to give the desired C4-gem-dimethyl intermediate 10 (56%). Subjecting ketone 10 to Bucherer−Bergs conditions afforded a 5:1 mixture of diastereomeric C2-hydantoins in 58% yield. Following chromatographic separation, the minor (desired) diastereomer 11 was isolated in 9% yield and was subsequently hydrolyzed in refluxing sodium hydroxide to give amino acid analog 4. Purification by cation exchange chromatography afforded the zwitterion in quantitative yield. C4-spirocycles 5 and 6 were each prepared from nonracemic bicyclic ketone 1213 (Scheme 2). To avoid downstream issues in the synthesis of the spirocyclopropyl analog, alternative acidstable protecting groups were installed. Ketone 12 underwent transesterification with thionyl chloride in refluxing ethanol to give amino diethyl ester 13 in 89% yield. The amine was subsequently acylated to give fully protected 14 in 78% yield. By use of standard Wittig conditions, ketone 14 was converted to the exocyclic olefin 15 in 66% yield, which was subjected to Simmons−Smith cyclopropanation conditions to give 16 in 71% yield. This penultimate intermediate was hydrolyzed in refluxing sodium hydroxide to give amino acid analog 5. Purification by cation exchange chromatography afforded the zwitterion in 98%

Figure 1. Chemical structures of mGlu2/3 receptor agonists 1 and 3, mGlu2 agonist/mGlu3 antagonist 2, and compounds 4−6, prepared and evaluated in the current investigation.

neuroprotective effects of mGlu2/3 agonists have been demonstrated to depend on glial mGlu3 activation.9 In order to more firmly establish the role of each subtype in CNS disorders, it is of interest to identify potent pharmacological tools capable of differentiating them. While several positive allosteric modulators (PAMs) for mGlu2, acting downstream of the glutamate recognition site and enhancing the potency and/or efficacy of glutamate have been identified,10 few examples of orthosteric agonists capable of selective mGlu2-receptor activation have been described. Of these, compound 2 (LY541850, Figure 1), a C4α-methyl-substituted analog of 1, displayed a highly unusual mGlu2 agonist, mGlu3 antagonist profile both in cells expressing recombinant human mGlu receptor subtypes11 and in native tissue preparations.12 This distinct pharmacological profile was found to be diastereospecific, as the C4β-methyl (4S) epimer 3 exhibited, like 1, maximally efficacious agonist responses in cells expressing either hmGlu2 or hmGlu3 receptors.11 Recently, we reported on our attempt to identify additional mGlu2-selective agonists through the expansion of the structural diversity of compounds substituted at the C4-position of 1.13 Interestingly, while several potent mGlu2/3 agonists (those possessing relatively small C4α- and Cβ-substituents, e.g., F, N3, OH, and NH2) and a few weakly potent mGlu2/3 antagonists Scheme 1. Preparation of C4-gem-dimethyl Analog 4a

a

Reagents and Cconditions: (a) MeLi, CuI, Et2O, 39%; (b) (i) TMSI, Et3N, DCM; (ii) Pd(OAc)2, MeCN, 77%; (c) MeLi, CuI, Et2O, 56%; (d) (i) (NH4)2CO3, KCN, aq EtOH; (ii) chromatographic separation of diastereomers, 9%; (e) 2.5 M NaOH, reflux, 72 h, quantitative. B


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Journal of Medicinal Chemistry Scheme 2. Preparation of C4-spirocycles 5 and 6a

a

Reagents and conditions: (a) SOCl2, EtOH, 89%; (b) Ac2O, Et3N, DCM, 78%; (c) CH3(Ph)3PBr, NaHMDS, THF, 66%; (d) TFA, Et2Zn, CH2I2, DCM, 71%; (e) 2 M NaOH, reflux, 6 days, 98%; (f) (i) C3H5S(C6H5)2.BF4, KOH, DMSO, 30%; (g) p-CH3C6H4SO2NHNH2, MeOH, reflux; (ii) NaBH4, rt to reflux, 20%; (h) 50% aq HOAc, 160 °C, 6 min, 86%.

Table 1. Binding Affinities and Functional Effects of C4-Substituted 2- Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates at Recombinant Human Metabotropic Glutamate 2 and 3 Receptors

a

See Experimental Section for assay details.

in a microwave for 6 min gave 6 as the zwitterion in 86% yield.

yield. Treating ketone 12 with cyclopropyldiphenylsulfonium tetrafluoroborate provided the spirocyclobutanone 17 in 30% yield as a mixture of diastereomers. The mixture of cyclobutanone diastereomers was reduced using a modification of the Wolff−Kishner reaction. The ketone was converted to the tosylhydrazone which was reduced in situ with NaBH4 to give spirocyclobutane 18 in 20% yield.16 Subjecting the fully protected intermediate to 50% aqueous acetic acid at 160 °C

The purities of compounds 4 and 5 were determined to be >95% on the basis of elemental analysis, while 6 was judged to be 90% as determined by capillary electrophoresis (CE). The CE chromatogram for 6 and proton NMR spectra for each of these molecules are included in the Supporting Information. C


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Table 2. Functional Agonist or Antagonist Responses of 1 and 5 in Cells Expressing Recombinant Human mGlu Receptor Subtypes: EC50 ± SEM (nM) or IC50 ± SEM (nM) or Emax (%)a mGlu2

mGlu3

mGlu4

mGlu5

2+

2+

2+

2+

2+

mGlu6

mGlu7 2+

Glu8 2+

compd

Ca FLIPR

Ca FLIPR

Ca FLIPR

Ca FLIPR

Ca FLIPR

cAMP

Ca FLIPR

Ca FLIPR

1

>25000 >12500 >25000 >12500

34.4 ± 4.0 (94%) 39.0 ± 5.9 (94%)

140 ± 4.6 (103%) 285 ± 52.5 (55%)

>25000 >12500 >25000 >12500

>25000 >12500 >25000 >12500

3360 ± 706 (96%) 6980 ± 831 (100%)

>25000 >12500 >25000 >12500

>25000 >12500 >25000 >12500

5 a

mGlu1

IC50 values are in italic font, and Emax values are in parentheses. See Experimental Section for assay details.

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RESULTS hmGlu2/3 Receptor Binding and Function. Newly prepared compounds 4−6, along with reference molecules 1− 3 were assessed for their ability to displace the potent, balanced mGlu2/3 receptor agonist radioligand [3H]-(1R,2S,4R,5R,6R)2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, [3H]19 ([3H]-LY459477; Supporting Information, Figure S1),17 from cell membranes expressing recombinant human mGlu2 or mGlu3 receptors and to inhibit forskolin-stimulated cAMP production in these cells (Table 1). Consistent with our previous findings,11 compounds 1−3 demonstrated competitive displacement of the orthosteric agonist radioligand, though compound 2 displayed somewhat lower apparent mGlu2 receptor affinity (Ki = 1040 nM) employing this radioligand compared to what was previously determined (Ki = 165 nM)11 using the orthosteric antagonist radioligand [3H]-2S-2-amino-2(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid ([3H]LY341495;18 Supporting Information, Figure S1). As previously reported, compounds 1 and 3 demonstrated potent and maximally efficacious agonist responses in both hmGlu2and hmGlu3-expressing cells, while compound 2 exhibited differential effects when tested against these two receptors, producing robust agonist activity in the hmGlu2 cells but predominant antagonist activity in hmGlu3-expressing cells.11,13 Incorporation of gem-dimethyl substitution at the C4-position of 1 as in 4 led to approximately a 38-fold and 19-fold decrease in affinity relative to 1 at mGlu2 and -3 receptors, respectively, and a 2- to 3-fold decrease in affinity compared to monomethyl substituted analogs 2 and 3 at both mGlu2 and mGlu3. In the whole cell functional assays, 4 produced a full, though weakly potent agonist response in mGlu2 expressing cells (EC50 = 599 nM, Emax = 92%) and antagonist activity with weak potency (IC50 = 12 900 nM) in cells expressing hmGlu3. Spirocyclobutane analog 6 behaved in a similar manner, displacing the orthosteric agonist radioligand with low affinity at both hmGlu2 (Ki = 2330 nM) and hmGlu3 (Ki = 1200 nM) receptors and producing a maximal, though relatively weak potency agonist response in the hmGlu2 expressing cells (EC50 = 726 nM, Emax = 88%), while in hmGlu3-expressing cells, 6 exhibited low potency antagonist pharmacology (IC50 = 7240 nM, Imax = 66%). In striking contrast to compounds 4 and 6, spirocyclopropane analog 5 exhibited relatively high affinity for both hmGlu2 (Ki = 260 nM) and hmGlu3 (Ki = 177 nM), as well as functional agonist responses comparable to that of 1 in cells expressing these receptors (EC50 = 8.4 nM and EC50 = 62.4 nM for 5 at hmGlu2 and hmGlu3, respectively). Compound 5 was further characterized to determine its molecular selectivity for mGlu2/3 receptors over other mGlu subtypes (Table 2). As in the case of other members of this chemotype such as 1, spirocyclopropane 5 exhibited a high degree of molecular selectivity for mGlu2 and mGlu3 subtypes

over other members of this gene family. It was also observed that when evaluated in the hmGlu3 FLIPR-based assay, 5 exhibited somewhat lower maximal efficacy (Emax = 55%) as compared to its effect in the hmGlu3 cAMP format (Emax = 88%) and in either hmGlu2 FLIPR or cAMP assays where it behaved as a maximal efficacy agonist (Emax = 94% and Emax = 99%, respectively). In addition to its selectivity over these closely related targets, compound 5 was evaluated against a panel of CNS receptors, transporters, and ion channels and against the hERG channel at a screening concentration of 10 μM. No significant effect of this molecule was observed for any of these targets (Supporting Information, Table S1). Cocrystallization Studies with the Amino Terminal Domain (ATD) of Recombinant hmGlu2 and hmGlu3. To better understand the molecular interactions influencing agonist binding to hmGlu2 and hmGlu3 receptors, cocrystallization of compounds 1 and 5 with the human mGlu2 ATD, as well as 1 with the human mGlu3 ATD, was performed.19,20 The crystal structures of hmGlu2-1 and hmGlu2-5 are shown in Figure 2. From a macroscopic perspective (Figure 2a and Figure 2c), it is evident that the densities for ligands 1 and 5 are associated with the hinge domain of the mGlu2 protein. This is in accord with previously published structures of other mGlu2/3 agonists with the rat mGlu3 ATD and an antagonist with the human mGlu3 ATD.21 The protein displays an overall closed topology, with the upper (LB1) and lower (LB2) lobes appearing to be in close contact with the bound ligand. More detailed views of the ligand−protein interactions for these molecules are depicted in Figure 2b and Figure 2d. For both 1 and 5, the C2-carboxylate functionalities are engaged in H-bonds with LB1 residues S145 (backbone NH) and T168 (backbone NH and side chain OH), the C2-ammonium groups are involved in H-bonds to LB1 A166 (backbone CO) and LB2 D295 (side chain CO2−), and the C6carboxylates are involved in salt bridges with the R61 and K377, each of which originates from LB1. Two tyrosine residues are evident within the agonist binding pocket, effectively bracketing two sides of the bicyclic ring systems of 1 and 5. The aromatic ring of Y216, a residue originating from LB2, occupies a space immediately adjacent to the C3-position of the bound agonists and may be involved in CH−π interactions with the C3β-H atoms of these ligands (distance between C3β-H atoms and the centroid of the tyrosine aromatic ring is 2.4 Å for 1 and 2.3 Å for 5).22 The aromatic ring Y144, a residue originating from LB1, is positioned above and extending out over C4- and C5-positions of these molecules, potentially interacting with the C5-hydrogen atoms of 1 and 5 (closest distance of 3.0 Å) as well as one of the C4-spirocyclopropane ring hydrogen atoms in 5 (closest distance of 2.3 Å). The binding pocket below the bicyclic ring system is made up of a LB2 loop containing two amino acids, D295 (previously mentioned) and G296. A relatively close contact between the pro-R methylene hydrogen of G296 with the C4α-H of 1 and one of the spirocyclopropane ring hydrogen D


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Figure 2. (a) 2.3 Å hmGlu2 ATD-1 cocrystal structure visualized in MOE.23 High level topology of the ligand−protein complex shows the overall closed conformation of upper and lower lobes of the ATD with the agonist ligand residing near the hinge region. LB1 and LB2 refer to upper and lower lobes of the ATD, respectively. (b) Site view of the hmGlu2 ATD-1 complex. Key contact points are noted with dotted lines as follows: black dotted lines with embedded cylinders represent hydrogen bonds and salt bridges; cyan dotted lines represent CH−π interactions. (c) 2.25 Å hmGlu2 ATD-5 cocrystal structure visualized in MOE.23 High level topology of the ligand−protein complex shows overall closed conformation of upper and lower lobes of the ATD with the agonist ligand residing near the hinge region. (d) Site view of the hmGlu2 ATD-5 complex. Key contact points are noted with dotted lines as described in (b). The CH−π interactions noted in (b) are present but not depicted for clarity. (e) Detailed view of hmGlu2 ATD residues in proximity to the C4αH of bound ligand 1. (f) Detailed view of hmGlu2 ATD residues in proximity to hydrogens on spirocyclopropane of bound ligand 5.

atoms in 5 is apparent (Figure 2e and Figure 2f). Given that the hydrogen atoms on the spirocyclopropane ring of 5 extend approximately 1.4 Å beyond the C4α-H of 1, it is interesting that the distances between these ligand-associated hydrogen atoms and the pro-R H of G296 are approximately equivalent (2.34 Å for 1, 2.26 Å for 5). This suggests that the mGlu2 binding pocket may have expanded slightly (∼1.3 Å) to accommodate 5. Also present in the LB2-associated space below the bound ligands 1

and 5 is the guanidinium terminus of R57, a residue that originates from LB1 and extends downward to LB2. This functionality appears to form H-bond interactions with the backbone CO atoms of both D295 and G296 and is relatively close to the C4α-H of 1 (3.41 Å) and to a spirocyclopropane C− H of 5 (3.42 Å) (Figure 2e and Figure 2f). Finally, a large loop in LB2 comprising four amino acids (T270, R271, S272, and E273) is positioned adjacent to the C4-position of the bound ligand 1, E


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Figure 3. (a) Macroscopic rendering of the 2.3 Å hmGlu3 ATD-1 cocrystal structure as visualized in MOE.23 (b) Site view of the hmGlu3 ATD cocrystallized with 1 (blue). (c) Overlay hmGlu2-1 (purple) and hmGlu3-1 (turquoise) cocrystal structures (protein alignment performed using LSQMAN program (see ref 24)). No discernible difference in macroscopic structures for these two agonist-bound complexes can be observed. (d) Site view of overlaid hmGlu2-1 (purple) and hmGlu3-1 (turquoise) structures. Q306 in mGlu3 may provide agonist binding site stabilization that is not possible in the case of mGlu2 (L300).

Q306. The positions of these LB2-associated amino acids in the hmGlu2-1 and hmGlu3-1 bound structures are shown in Figure 3d. There are a small number of additional amino acid differences between hmGlu2 and hmGlu3 within the ligand binding site. These are highlighted in Figure S2. Molecular Docking Studies. The strikingly different pharmacological effects of compounds 2 (antagonist) and 5 (agonist) in cells expressing mGlu3 receptors provided the impetus to examine how these two molecules might bind relative to each other at this target using the structure of mGlu3-1 as a template (Figure 4a and Figure 4b). Each of the molecules in Figure 4 was built in MOE23 directly from and superimposed onto compound 1 in its mGlu3-bound state. Specifically, after replacement of the C4α-H of 1 with a methyl group (to provide compound 2) and both C4 hydrogens of 1 with a spirocyclopropane ring (to provide compound 5), the resulting molecules 2 and 5 were individually minimized (MMFF94x force field in MOE) and molecular distances between these ligands and the mGlu3 protein subsequently determined. No additional cominimization of the resulting ligand−protein structure which would allow movement of either ligand or protein was performed. This was to ensure the integrity of the experimentally derived crystal structure coordinates. Comparison of the two modeled structures in Figure 4 reveals a significant difference in the predicted interatomic distance between these ligands and the methylene hydrogen atoms of G302, with a hydrogen atom on the methyl group of 2 making extremely close contact with the pro-R hydrogen atom of G302 (0.85 Å), while the closest

with the closest distance between 1 and this loop being 2.68 Å (C4α-H of 1 and Cα-H of R271, Figure 2e). The presence of the C4-spirocyclopropane ring in 5 results in a slightly decreased distance between the Cα-H of R271 and cyclopropane C−H of this ligand (2.36 Å, Figure 2f), but once again, on the basis of how much further (1.4 Å) this cyclopropane CH extends beyond the C4α-H of 1, this distance is greater (by approximately 1 Å) than what would be expected if the mGlu2 binding site had not expanded to accommodate 5. Human mGlu3 ATD Crystal Structure with 1. Attempts to obtain a suitably resolved cocrystal structure of the mGlu3 ATD with compound 5 have been unsuccessful to date. The crystal structure of compound 1 bound the ATD of hmGlu3 is shown in Figure 3. On both macroscopic (Figure 3a) and molecular (Figures 3b) levels, the hmGlu3-1 complex is highly analogous to that seen for this molecule with hmGlu2 (Figure 2a and Figure 2b). This result was not unexpected given the extremely high degree of homology in the glutamate binding region between these proteins. Key molecular interactions include the glutamate pharmacophore of 1 with LB1 residues S151, T174, and A172, LB2 acidic residue D301, and LB1 basic amino acids R68 and K389. An overlay24 of the crystal structures of 1 bound to hmGlu2 (purple) and hmGlu3 (turquoise) is depicted in Figure 3c, showing that these two structures are highly analogous in their overall topology. Two amino acids that reside in relatively close proximity to the agonist binding site are different when comparing hmGlu2 and hmGlu3. These are hmGlu2 residues E273 and L300, corresponding to hmGlu3 residues D279 and F


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Figure 4. (a) Energy minimized (MMFF94x, MOE)23 compound 2 built from 1 in hmGlu3 agonist binding site. Note the predicted close contact of one of the C4α-methyl hydrogen atoms with the pro-R H of G302. (b) Energy minimized (MMFF94x, MOE)23 compound 5 built from 1 in the hmGlu3 agonist binding site. The nearest contact between hydrogen atoms in ligand 5 and G302 methylene hydrogens is 0.9 Å more than what was observed for compound 2 (a). (c) Overlay of energy minimized forms of 2 (purple) and 5 (orange) highlights the distinct geometrical dispositions of C−H atoms on C4α-methyl vs spirocyclopropyl groups. (d) Conformation−energy relationship (MOE)23 for rotation of C4−CH3 bond in 2. The high energy (eclipsed) structures represent the conformation of the methyl substituent of 2 that would most closely overlap with spirocyclopropane of 5. A 600:1 ratio of staggered to eclipsed conformers is predicted to exist at 37 °C. (e) Energy minimized (MMFF94x, MOE)23 depiction of compound 2 built from 5 in hmGlu2 agonist binding site. The predicted distance between the closest hydrogen atom of 2 and the pro-R H of G296 (1.48 Å) is 0.6 Å greater than that found when 2 was docked in hmGlu3 (a).

relationship with the adjacent groups attached to the C4 carbon of the bicyclic ring system. A conformation−energy diagram representing potential energy changes (ΔG) with rotation about the C4−CH3 bond was generated in MOE23 and is shown in Figure 4d. The energy required to achieve a fully eclipsed conformation analogous to that of the spirocyclopropane (one in which steric interaction with G302 would be minimized) is estimated to be 4 kcal/mol above the minimum. The equilibrium constant that defines the relative population of the fully staggered and fully eclipsed conformations was determined using the

hydrogen atom on the spirocyclopropane of 5 resides significantly further away from both the pro-R (1.94 Å) and pro-S hydrogens (1.79 Å) of this residue. The reason for the shorter interatomic distance between the hydrogen atom on 2 and G302 as compared to the nearest hydrogen atoms on the spirocyclopropane of 5 is due to the distinct geometrical presentation of the hydrogen atoms associated with these C4substitutents as shown in Figures 4c. In the case of compound 2, the low energy conformation of the pendent methyl substituent is one in which the individual CH atoms reside in a staggered G


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Figure 5. (a) Agonist responses produced by L-glutamate in hmGlu2 wild type and mutant receptors transiently expressed in AV12 cells. (b) Agonist responses produced by compound 2 in hmGlu2 wild type and mutant receptors transiently expressed in AV12 cells. (c) Agonist responses produced by L-glutamate in hmGlu3 wild type and mutant receptors transiently expressed in AV12 cells. (d) Agonist responses produced by compound 2 in hmGlu3 wild type and mutant receptors transiently expressed in AV12 cells (note that the ordinate axis is expanded to show detail of this concentration response, which also required a higher concentration range to identify the top of the agonist curve). For (a)−(d), see Experimental Section for details.

equation K = e−ΔG/(RT). Using ΔG = 4 kcal/mol from Figure 4d and T = 310 K (body temperature) results in a K value of 634. That is, the population of the lowest energy, staggered conformation is enriched approximately 600-fold over the eclipsed one. Compound 2 was also built within the hmGlu2 ATD-5 crystal structure (Figure 4e), again with strict overlap of the atoms comprising the bicyclic ring system for 2 and 5. In this case, the interatomic distance between closest hydrogen atoms of 2 and G296 was determined to be 1.48 Å. If the mGlu3 agonist binding site is less flexible than that of mGlu2, it is possible that the predicted closer contact (0.85 Å) of a hydrogen atom of the C4α-methyl group present in 2 (specifically the H that is oriented downward in Figure 4c) with G302 of mGlu3 might impede effective closure of the lobes in this protein, resulting in the observed antagonist pharmacology. If indeed this is the basis for the observed mGlu3 antagonist pharmacology expressed by 2, it would indicate that the difference between the activation and blockade of activation of this 100 kDa molecular weight Gprotein-coupled receptor may have been determined by the rotational state about a single C−CH3 bond in 5. While this hypothesis is both intriguing and supported by the available structural data, it is important to note that (a) the crystal structures used in this analysis are of moderate resolution (2.25−

2.3 Å) and (b) our ligand modeling involved building compounds from the crystal structure bound ligands with minimization that did not allow for either protein or ligand movement. Site-Directed Mutagenesis. As noted above, there are two amino acid residues within approximately 8 Å of the bound mGlu2 agonist, mGlu3 antagonist 2 that are different between these receptors (mGlu2 E273 and L300 corresponding to mGlu3 D279 and Q306; Figure 3d). As shown, the mGlu2 E273/mGlu3 D279 residues do not appear to make contact with other receptor residues directly involved in the binding of 1. On the other hand, the amide terminus of mGlu3 Q306 may be involved in H-bond interactions with backbone amide carbonyls of W303 and G302, two residues that are situated on an LB2 loop that also contains the critical agonist-binding residue D301. The presence of the Q306 residue in mGlu3 could therefore provide additional stabilization to this loop which is not possible for mGlu2 (L300). Considering that this additional loop stabilization might underlie the observed mGlu2-agonist, mGlu3-antagonist pharmacology for compound 2, we generated site-directed mutants of both mGlu2 (E273D, L300Q, and E273D + L300Q) and mGlu3 (D279E, Q306L, D279E + Q306L) receptors and transiently expressed these into AV12 cells that stably express EAAT1 and H


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Journal of Medicinal Chemistry the “promiscuous” Gα15 subunit to allow G-protein mediated release of intracellular calcium.19 Compound 5, along with Lglutamate ( L -Glu) and the potent mGlu2/3 agonist (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, 20 (LY379268; Supporting Information, Figure S1),25 was then assessed for agonist responses in both transiently expressed wild type (WT) and mutant receptor-expressing cells. Neither the potency nor efficacy (defined by the response of 100 μM L-glutamate) of 20 was affected by the mutations in mGlu2 or mGlu3 (Supporting Information, Figure S3). Representative data (of two to three separate experiments) for L-glutamate and 2 are shown in Figure 5. The mGlu2 single (E273D, L300Q) and double (E273D + L300Q) mutants had no impact on the potency of glutamate when compared to WT mGlu2 expressing cells (Figure 5a). Similarly, no discernible effect of either the single (E273D) or double (E273D + L300Q) mutations on mGlu2 receptor potency of maximal efficacy for compound 2 was observed. However, the L300Q mutant had a modest negative impact on both potency (EC50 = 176 nM vs EC50 = 357 nM for WT and L300Q receptors, respectively) and maximal efficacy (Emax = 92% vs Emax = 75% for WT and L300Q receptors, respectively) of 2 (Figure 5b). In comparison, cells expressing mGlu3 receptor mutants D279E and D279E + Q306L (but not Q306L alone) negatively impacted the potency of glutamate (EC50 values of 1, 4.2, and 5.1 μM, for WT, D279E, and D279 + Q306L, respectively; Figure 5c). In contrast, the D279E mutation had no impact on compound 2 (i.e., less than a 6% agonist response up to 1 mM), but a small, yet discernible agonist response (EC50 = 34.8 μM, Emax = 16%) was observed for 2 in the Q306L mutant (Figure 5d), an effect that was lost in the double mutant (Q306L + D279E), suggesting a functional interplay between these two residues. The modest loss of agonist potency and efficacy of 2 in mGlu2 cells expressing the L300Q mutant (Figure 5b) combined with the apparent agonist response observed in the reciprocal mGlu3 receptor mutant (Figure 5d) is in general agreement with the hypothesis that this amino acid pair is involved in the pharmacological profile exhibited by 2 in cells expressing WT receptors. However, it is clear that these amino acids alone do not fully explain the observed mGlu2 receptor agonist, mGlu3 receptor antagonist pharmacological profile of this molecule. One limitation of this investigation is that transient transfections result in a population of cells expressing varying levels of receptor. It was therefore not possible to determine how, if at all, differing receptor expression levels in each transient cell line may have impacted ligand potency or efficacy. However, the fact that the potency of glutamate was unaffected in any of the mGlu2 mutants vs WT, and the potency of glutamate was not affected in the mGlu3 mutant (Q306L) which differentially responded to compound 2, provides some evidence that any differences in receptor expression levels were not the cause of the differential effects of 2 in these experiments. This is further supported by the lack of mutation effects on either the potency or efficacy of 20 (Supporting Information, Figure S3). Native Tissue Binding and Function of Compound 5. In order to establish the affinity and functional agonist potency of compound 5 in native tissue preparations, this molecule was evaluated for its ability to displace orthosteric agonist radioligand ([3H]19) binding from rat cortical membranes as well as for its ability to influence cAMP production in acutely isolated cerebrocortical nerve terminals (synaptosomes)26 and to suppress synaptically mediated responses (spontaneous Ca2+ oscillations) in cultured neurons.12a The results of these studies,

together with comparator analogs 1 and 2, are summarized in Table 3. Compounds 1, 2, and 5 inhibited [3H]19 binding to rat Table 3. Binding and Functional Agonist Responses for Compounds 1, 2, and 5 As Measured in Rat Cortical Tissuesa EC50 ± SD (nM) (Emax (%)) Ki ± SEM (nM)

compd

displacement of [3H]19 from rat forebrain membranes

inhibition of forskolinstimulated cAMP formation in rat cerebrocortical synaptosomes

inhibition of Ca2+ oscillations in cultured rat cortical neurons

1 2 5

476 ± 62 6331 ± 128 706 ± 103

19.8 ± 3.2 (112) 38.9 ± 11.1 (44) 14.2 ± 2.3 (87)

11.1 ± 16 (87) 127 ± 19 (21)b 31.4 ± 7.3 (94)

a

Maximum (100%) agonist response defined as the maximal inhibition of forskolin-stimulated cAMP achieved for 20 run at same time in this assay (data not shown). bData from ref 12a.

cortical membranes with Ki values of 476, 6,331, and 706 nM, respectively. These apparent affinities are lower than those determined in membranes expressing human mGlu2 and mGlu3 receptors (Table 1), though the same rank order of potency is maintained. As was observed in experiments involving the recombinant human receptors, compounds 1, 2, and 5 exhibited a considerable left shift in functional agonist potency relative to affinity in both the rat synaptosome (cAMP) and rat cultured neuron Ca2+ oscillation assays, a result that indicates that there is likely high mGlu2/3 receptor reserve in these tissues. Interestingly, while pharmacologically balanced mGlu2/3 agonists 1 and 5 exhibit high levels of efficacy in these functional assays, compound 2 elicited potent but partial agonist responses, achieving maximal efficacy levels only 20−30% of those produced by the other two. The molecular basis for this apparent partial agonist profile in native brain tissue for 2 is not understood and may suggest a functional interplay between mGlu2 (where 2 acts as agonist) and mGlu3 (where 2 acts as antagonist) in the rat cerebral cortex. Pharmacokinetic Attributes of Compound 5. In vitro evaluation of compound 5 (Table 4) revealed this molecule to Table 4. In Vitro Characteristics of 5 That Influence in Vivo Pharmacokinetic Properties assay

result

aqueous solubility (pH 7.4) DMSO solubility MDCK permeability (A−B transport)a % loss hepatic microsomes (mouse, rat, dog, human)b % inhibition human Cyp isoforms (3A4, 2D6, 2C9)c plasma unbound fraction (mouse, rat, dog, primate, human)d

>100 μM >10 mM 14% 100 μM at pH 7.4), no measurable microsomal metabolism across species, no significant inhibition of cytochrome P450 enzyme isoforms, and high (>95%) plasma unbound fraction. The in vivo pharmacokinetic attributes of 5 were assessed in the rat (Table 5). Intravenous administration of 5 (5 mg/kg) resulted in a maximal plasma drug concentration of approximately 77 μM. Compound 5 exhibited a slow rate of clearance (11.8 mL min−1 kg−1), a low volume of distribution (0.57 L/kg), and a 3.6 h plasma half-life. Administration of 5 by oral gavage (5 I


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Table 5. Mean Plasma Pharmacokinetic Parameters Associated with 5 Dosed by Oral, Intravenous and Intraperitoneal Routes of Administration in Rats19 dose groups parameter route AUC (0−24 h) C0 or Cmax Tmax CL Vdss T1/2 estimated bioavailability a

unit nM·h nM H mL min−1 kg−1 L/kg h %

5 mg/kg

5 mg/kg

1 mg/kg

3 mg/kg

10 mg/kg

iv, plasma (nM) ± SD 33661 ± 3811 76749 ± 5591

po, plasma (nM) ± SD 2826 ± 597 530 ± 165 2 nca nca 3.8 ± 2.0 8

ip, plasma (nM) ± SD 3050 ± 902 3580 ± 723 0.5 ± 0.0 nca nca 1.1 ± 1.1 45



11.8 ± 1.4 0.57 ± 0.05 3.6 ± 0.2

Not calculated.

Table 6. Mean Plasma and Cerebrospinal Fluid Concentrations of 5 Following Single Intraperitoneal Doses in Rats19 plasma (nM)

a

csf (nM) [fold over rat cortical synaptosomes EC50]

dose (mg/kg)

0.5 h

1h

4h

0.5 h

1h

4h

1 3 10

17120 ± 16216 13100 ± 3840 69600 ± 6950

9169 ± 6986 5530 ± 4820 32200 ± 12500

319 ± 188 103 ± 44.5 419 ± 78.8

17.5 ± 16.5 [1×] 200 ± 90.6 [14×] 1240 ± 74.5 [87×]

27.0 ± 11.0 [2×] 225 ± 60.8 [16×] 1112.5 ± 228 [78×]

BQLa 64.0 ± 68.6 [4×] 206 ± 44.7 [14×]

Below quantifiable levels.

Figure 6. Mean plasma (A) and cerebrospinal fluid (B) levels of 5 following single intraperitoneal doses in rats:19 1 mg/kg (closed squares), 3 mg/kg (open diamonds), 10 mg/kg (closed triangles).

mg/kg) in the rat led to somewhat delayed access to plasma (Tmax = 2 h) and relatively low plasma drug levels which peaked near 0.5 μM at 2 h. Comparison of 24 h exposures following po and iv routes of administration in the rat revealed low (8%) oral bioavailability for this molecule. In contrast, intraperitoneal administration of 5 at doses of 1, 3, and 10 mg/kg led to its rapid appearance in plasma (Tmax ≈ 0.5 h) and high maximal drug levels (mean Cmax of approximately 4, 10, and 60 μM for 1, 3, and 10 mg/kg dose groups). A comparison of mean plasma exposures following ip and iv routes of administration suggests good overall bioavailability for 5 (36−72%) when given by the ip route. Evaluation of plasma and cerebrospinal fluid levels of 5 in the rat following ip dosing was performed in a separate experiment (Table 6, Figure 6). Plasma exposures at the 0.5 h time point in the 1 mg/kg dose group were approximately 5-fold higher than

those from the prior study (shown in Table 5), while plasma concentrations for the 3 and 10 mg/kg dose groups at 0.5 h were essentially identical across studies. Cerebrospinal fluid (csf) levels were apparent at the first time point (0.5 h) and showed dose-related increases at each of the assessed time points. The ratio of csf to plasma concentrations varied by dose and by time, being lowest at the earlier (0.5 and 1.0 h) time points but much greater at the later (4 h) sampling time, suggesting both a delay in entrance into the csf compartment and a slower rate of elimination from csf compared to plasma for this molecule. Finally, comparing the concentrations of compound 5 present in csf following these systemic doses to concentrations required for functional activation of mGlu2/3 receptors in native rat brain tissues (synaptosome cAMP EC50 = 14.2 nM, cultured neuron Ca2+ oscillation EC50 = 31.4 nM; Table 3), it is likely that effective J


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Figure 7. (a) Dose−response for 5 in the rat PCP locomotor activation assay:19 asterisk (∗) indicates statistically significant (p < 0.05, ANOVA followed by Dunnett’s test) compared to rats treated with PCP alone. (b) Dose−response for 5 in the rat formalin pain model:19 asterisk (∗) indicates statistically significant (p < 0.05, ANOVA followed by Dunnett’s test) compared to rats treated with formalin alone.

At a dose of 3 mg/kg, no impairment was evident over these time intervals.

target engagement has been achieved in rats over this dose range given that this molecule lacks appreciable protein binding and cell permeability, two properties which could otherwise reduce its effective concentration at the extracellular ATD binding sites. Behavioral Responses Elicited by Compound 5 in Rats. Given its potency at mGlu2/3 receptors and confirmed access to the CNS following systemic dosing, compound 5 was assessed in two animal models known to be sensitive to mGlu2/3 receptor agonists, the inhibition of phencyclidine (PCP) induced locomotor activation (an animal model of psychosis)2a−c and the inhibition of formalin-evoked paw licking (an animal model of pain).3a,b The activity of other mGlu2/3 receptor agonists in these models is known to be mGlu2-receptor-dependent.7,8 Because nonspecific (e.g., sedative) effects of drugs can confound the interpretation of efficacy in each of these assays, the effect of 5 on locomotor coordination was also evaluated utilizing the rotarod test. In each experiment, an aqueous (pH 7−8) solution of compound 5 was administered by the intraperitoneal route. Rats treated with PCP (5 mg/kg, sc) exhibited increased photobeam breaks over the 60 min observation period compared to vehicle-treated animals (Figure 7a). Administration of compound 5 (0.3, 1, 3, and 10 mg/kg, ip) 4 h prior to testing led to a dose-related reduction in horizontal ambulations, with statistical significance achieved at 3 mg/kg and a calculated ED50 value of 1.5 mg/kg. In the formalin pain assay, injection of a 5% formalin solution into the hindpaw of rats resulted in two phases of heightened nociceptive responses compared to vehicle treated animals (Figure 7b). Administration of compound 5 (0.3, 1, 3, and 10 mg/kg, ip) 60 min prior to formalin produced a doserelated decrease in nociceptive responses (paw licks) in both early and late phases which reached statistical significance at 1 mg/kg, resulting in a calculated ED50 of 0.7 mg/kg. In the rotarod test (Table 7), compound 5 produced locomotor impairment at 10 mg/kg when given by the ip route 1, 2, and 3 h prior to testing.

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DISCUSSION The therapeutic promise of molecules capable of effectively modulating mGlu2 and mGlu3 receptors in the CNS has inspired us to continue our investigation of compounds related to the potent, selective, and CNS-active compound 1. In this regard, we have previously reported that pharmacological potency as well as agonist vs antagonist activity can be modulated by the nature of substituents residing at the C4-position of 1, with relatively small substituents (e.g., F, OH, N3, NH2) providing potent mGlu2/3 agonist activity and larger ones (e.g., NHAc, SPh) exhibiting antagonist pharmacology.13 Among the numerous substituents explored within this SAR, C4α-methyl substitution is unique insofar as it results in a highly unexpected and unusual mGlu2 agonist/mGlu3 antagonist pharmacological profile for compound 2, whereas C4β-methyl substitution provides maximal efficacy, dual mGlu2/3 agonist pharmacololgy.11 In this account, we sought to better understand the molecular basis for this pharmacological profile given the extremely high homology near the glutamate recognition site of mGlu2 and mGlu3 receptors. To this end, we prepared molecules in which the C4-position of 1 was disubstituted either with methyl groups (4) or with methylene groups forming small spirocyclic rings (5 and 6). From this investigation, we found that compounds 4 and 6 maintained the unusual mGlu2 agonist/mGlu3 antagonist profile that had been observed for 2, though with a substantial (∼10fold) loss in potency. Conversely, compound 5 not only retained full agonist activity at both mGlu2 and mGlu3 receptors but surprisingly exhibited a nearly 8-fold mGlu2 agonist potency improvement over 2. Indeed, the overall mGlu2/3 agonist potency characteristics of 5 are comparable with those of two clinically tested mGlu2/3 receptor agonists, C4-unsubstituted compound 1 (the active component of LY544344), which has been clinically evaluated for the treatment of generalized anxiety disorder,27 and LY404039 (mGlu2 EC50 = 23 nM; hmGlu3 EC50 = 48 nM),28 the active component of an oral prodrug (LY2140023.H20, pomaglumetad methionil) which has been clinically evaluated for the treatment of schizophrenia.29 In an attempt to better understand the molecular basis for the potent and relatively balanced mGlu2/3 agonist activity of 5 in comparison to the distinct mGlu2 agonist/mGlu3 antagonist pharmacology of 2, we applied X-ray crystallography, molecular modeling, and site directed mutagenesis techniques. From the crystallographic analysis of both compound 1 (crystallized in the

Table 7. Effect of 5 in the Rat Rotarod Test for Motor Coordination time (s) maintained on rotarod (mean ± SEM) group

1h

2h

3h

vehicle 5 (3 mg/kg, ip) 5 (10 mg/kg, ip)

40.0 ± 0.0 34.4 ± 3.7 19.5 ± 6.2* a

40.0 ± 0.0 40.0 ± 0.0 17.4 ± 6.7* a

40.0 ± 0.0 40.0 ± 0.0 22.4 ± 5.3* a

a Asterisk (∗) indicates statistically significant (p < 0.05, product-limit survival fit test) compared to vehicle treated rats.

K


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bioavailable prodrug form of it will be disclosed in due course. Owing to the first moderate resolution hmGlu2- and hmGlu3agonist cocrystal structures (1 and 5 with mGlu2 ATD, 1 with the mGlu3 ATD), detailed insights into specific protein−agonist interactions have been revealed. These, in turn, have led to additional insights of the molecular interactions underpinning the highly unusual mGlu2 agonist−mGlu3 antagonist pharmacology for compound 2 which may be useful in future molecule design.

mGlu2 and mGlu3 ATDs) and compound 5 (crystallized in the mGlu2 ATD), it appears that the mGlu3 binding site may be somewhat more ridged compared to mGlu2, perhaps owing to additional LB2 loop stabilization by Q306 (L300 in mGlu2) such that the C4α-methyl substituent in 2 can be accommodated in the mGlu2 protein but within the mGlu3 binding site that results in a steric clash between one of the hydrogen atoms on the methyl group and methylene of G302. With this possible explanation in mind, we performed site directed mutagenesis studies to explore whether mutating L300 in mGlu2 to a glutamine, and Q306 in mGlu3 to a leucine might lead to a reduction in agonist activity for the former and the emergence of agonist activity in the latter for compound 2. The appearance of both lower maximal efficacy and right-shifted agonist potency for 2 in the mGlu2 L300Q mutant, combined with a discernible, albeit weak agonist response in the mGlu3 Q306L mutant, supports the hypothesis that this residue pair plays some role in the overall pharmacological profile of 2. It is acknowledged, however, that these residues alone cannot explain the overall in vitro potency and efficacy profile of 2, as neither agonist potency nor efficacy for this molecule in the mGlu3 Q306L mutant (EC50 = 34.8 μM, Emax = 16%) were restored to mGlu2 WT values (EC50 = 62.5 nM, Emax = 98%), and the reciprocal mutation in mGlu2 (L300Q) did not fully abolish its agonist activity. Owing to the mGlu2/3 agonist potency and selectivity characteristics of 5, we assessed this molecule in native rat brain tissue preparations, demonstrating potent binding affinity in cortical membranes and potent agonist responses both in a second-messenger assay performed in isolated cortical synaptosomes26 and in a neuronal culture assay which has been used to model synaptic activity.12a With confirmed native tissue agonist potency, we then determined the pharmacokinetic properties of 5 in the rat. As expected for a highly charged, polar molecule of this type, passive membrane permeability and oral bioavailability were low, while solubility, metabolic stability, and unbound fraction were high. Excellent plasma exposure was achieved following either iv or ip administration of 5 within a dose range of 1−10 mg/kg. Importantly, csf levels of 5 increased proportionately with dose over this range and were generally above the EC50 values determined for this molecule in both recombinant human and rat native tissue assays. Given that 5 has very low protein binding and does not penetrate effectively into cells, we posited that csf drug levels would be a reasonable estimate for drug concentrations at the extracellular mGlu2/3 receptor binding sites in the brain. In accordance with this hypothesis, compound 5, administered by ip route at 1, 3, and 10 mg/kg, provided efficacy in two mGlu2-driven behavioral tests, the reduction of PCP-evoked ambulations (3 and 10 mg/kg with p < 0.05) and of formalin-evoked pain behaviors (all doses p < 0.5). The lack of functional rotarod impairment at 1 and 3 mg/kg doses suggests that behavioral efficacy observed below 10 mg/kg was not confounded by nonspecific motor effects.

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EXPERIMENTAL SECTION

Chemical Methods. Synthesis. 1H and 13C NMR spectra were obtained on a Varian Unity INOVA 400 at 400 and 125 MHz, respectively, unless noted otherwise. TMS was used as an internal standard. LCMS data were obtained using an Agilent 1100 series HPLC on a Gemini-NX C18 110A, 50 mm × 2.00 mm column. HRMS data were obtained using an Agilent 1100 series LC and TOF mass spectrometer, with a Gemini-NX C18 110A, 50 mm × 2.00 mm column. Optical rotations were obtained on a PerkinElmer polarimeter 341. Melting points were obtained on a Laboratory Devices Mel-Temp 3.0. Microwave reactions were run in a Biotage initiator microwave. Capillary electrophoresis was performed on a Agilent Technologies 3D-CE equipped with a deuterium lamp, a photodiode array detector, and a 50 mm i.d., 363 mm o.d. fused-silica capillary, Ld = 40 cm, Lt = 48.5 cm. Separations were conducted at 20 °C at a voltage of 25 kV. Reactions were monitored by thin layer chromatography using silica gel 60 F254 plates from EMD and staining with ninhydrin. Unless otherwise noted, normal phase purifications were performed using RediSep prepacked columns from Teledyne Isco. Cation exchange chromatography was performed using Dowex 50W X8, 50−100 mesh, which was purchased from Aldrich. On the basis of elemental analysis, all final compounds were >95% pure unless otherwise noted. The numbering convention used in NMR assignments is provided in Figure 8.

Figure 8. Numbering convention used in NMR peak assignments. (1S,2S,5R,6S)-2-Amino-4,4-dimethylbicyclo[3.1.0]hexane2,6-dicarboxylic Acid (4). Hydantoin 11 (61 mg, 0.23 mmol, 1.0 equiv) was treated with 2.5 M NaOH (4.0 mL, 4.0 mmol, 17 equiv) and heated to reflux for 3 d. The reaction was cooled to ambient temperature and the pH adjusted to approximately 8 with 5 N HCl. The material was filtered washing with water (solids discarded). The filtrate was concentrated in vacuo. The crude material was purified by cation exchange chromatography. The Dowex resin was prepared by first washing in a fritted funnel with water, THF, and water. The resin was soaked in 3 N NH4OH for 5 min, and then the solvent was removed by filtration. This soaking in NH4OH was repeated. The resin was washed with H2O until pH = 7 was achieved. Next the resin was soaked in 1 N HCl for 5 min, and then the solvent was removed by filtration. This HCl soaking was also repeated. The resin was washed with H2O until pH = 7 was reached. The freshly prepared resin was added to a glass column. Approximate bed was 1 in. diameter × 3 in. high. The crude material was dissolved in 4 mL of H2O and adjust to pH = 4. The material was carefully loaded to the top of the resin bed. The eluent drip rate was maintained at 1 drop every 2−3 s. After initial loading volume dropped to the resin surface, 5 mL of H2O was added. After the solvent dropped to the resin bed the column was filled with water and the drip rate maintained. The pH of the eluent was closely monitored. Once the pH dropped to 0 and then returned to pH = 7, the flow rate was increased. The column was washed with 5 column volumes of water, 10 column

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CONCLUSION The current investigation has led to the identification of compound 5, a highly potent mGlu2/3 receptor agonist as measured both in cells expressing recombinant human receptors and in native rat cortical tissues (synaptosomes and cultured neurons). Further characterization of 5 has established its pharmacokinetic behavior in plasma and csf of rats following systemic dosing, leading to experiments that have established its robust efficacy in animal models of psychosis and pain. Additional in vivo characterization of this molecule and an orally L


Article


addition. After complete addition the reaction was warmed to −10 °C and stirred for 1 h. The reaction was quenched with about 200 mL of saturated aq NH4Cl, slowly warmed to ambient temperature, and stirred for 1.5 h. Et2O was added (500 mL), and the mixture was filtered through Celite washing with saturated aq NH4Cl and Et2O. The filtrate layers were separated, and the aq layer was back-extracted with Et2O. The combined organics were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography, eluting with 85/15 to 70/30 hexanes/ethyl acetate to give 8 as a white solid (1.80 g, 39%). Mp = 62.0−63.5 °C. [α]D20 30.54 (c 0.999, CHCl3). MS (ES+) 183.0 [M + H]+. 1H NMR (400 MHz, CDCl3, δ): 4.17 (q, J = 7.0 Hz, 2H, CH2 ester), 2.53 (pentet, J = 7.5 Hz, 1H, H4), 2.35 (m, 1H, H5), 2.31 (m, 1H, H1), 2.27 (dd, J = 18, 7.5 Hz, H3α), 2.06 (t, J = 2.9 Hz, H6), 1.73 (d, J = 18 Hz, H3β), 1.29 (t, J = 7.0 Hz, 3H, CH3 ester), 1.17 (d, J = 7.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3, δ): 211.2 (C2), 170.9 (CO ester), 61.26 (CH2 ester), 40.61 (C3), 36.21 (C5) 35.10 (C1), 30.08 (C4), 27.08 (C6), 21.94, (CH3), 14.17 (CH3 ester). Ethyl (1S,5R,6S)-4-Methyl-2-oxobicyclo[3.1.0]hex-3-ene-6carboxylate (9). A solution of 8 (1.65 g, 9.05 mmol, 1.00 equiv) in dichloromethane (45 mL) was cooled to 0 °C and treated with triethylamine (3.0 mL, 22 mmol, 2.4 equiv) and iodotrimethylsilane (1.55 mL, 10.9 mmol, 1.20 equiv). The TMS-I was added at such a rate that the internal temperature did not rise above 5.0 °C. The reaction was stirred at 0 °C for 2.5 h. The reaction was diluted with Et2O and washed with saturated NH4Cl (3×) and brine. The organic was dried over Na2SO4, filtered, and concentrated in vacuo. The resultant silyl enol ether was dissolved in acetonitrile (45 mL), cooled to 0 °C, and treated with palladium(II) acetate (2.25 g, 10.0 mmol, 1.11 equiv) in a single portion. The cooling bath was removed, and the reaction was stirred at ambient temperature overnight. The reaction was diluted with ether and filtered through Celite/silica gel washing with ether. The filtrate was concentrated in vacuo to give an orange oil which was purified via silica chromatography, eluting with 9:1 hexanes/acetone to give 9 (1.25 g, 77%) as a white crystalline solid. Mp = 54−56 °C. [α]D20 −244 (c 1.00, CHCl3). MS (ES+) 181.0 [M + H]+. 1 H NMR (400 MHz, CDCl3, δ): 5.44 (s, 1H, H3), 4.16 (m, 2H, CH2 ester), 2.79 (m, 1H, H5), 2.60 (m, 1H, H1), 2.25 (t, J = 2.6 Hz, 1H, H6), 2.20 (d, J = 1.2 Hz, 3H, CH3), 1.29 (t, J = 7.1 Hz, 3H, CH3 ester). 13C NMR (125 MHz, CDCl3, δ): 203.2 (C2), 174.2 (C4), 168.2 (CO ester), 124.6 (C3), 61.29 (CH2 ester), 44.38 (C6), 31.53 (C1/5) 31.34 (C1/5), 18.68, (CH3), 14.11 (CH3 ester). Ethyl (1S,5R,6S)-4,4-Dimethyl-2-oxobicyclo[3.1.0]hexane-6carboxylate (10). A vigorously stirred suspension of cuprous iodide (5.34 g, 28.0 mmol, 5.00 equiv) in diethyl ether (10 mL) was cooled to an internal temperature of 2 °C. 1.6 M MeLi in Et2O (35 mL, 56 mmol, 10 equiv) was added slowly maintaining the temperature below 10 °C. The suspension was stirred for 30 min and then it was cooled to −75 °C. A solution of enone 9 (1.01 g, 5.60 mmol, 1.00 equiv) in diethyl ether (10 mL) was added dropwise. Internal temperature was kept below −50 °C during the addition. After complete addition the reaction was warmed to −10 °C and stirred for 1 h and then warmed to 0 °C for 1.5 h. The reaction was quenched slowly with about 75 mL of saturated NH4Cl. The mixture was allowed to slowly warm to ambient temperature and was stirred for 1 h. Et2O (200 mL) was added, and the mixture was filtered through Celite washing with both saturated NH4Cl and Et2O. The filtrate layers were separated. The aqueous layer was back-extracted with Et2O. The combined organics were washed with saturated NH4Cl (3×), brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified on silica eluting with 9:1 hexanes/ethyl acetate to give 10 (0.62 g, 56%) as a colorless oil. [α]D20 61.5 (c 0.999, CHCl3). MS (ES+) 197.0 [M + H]+, 214.0 [M + NH4]+. 1H NMR (400 MHz, CDCl3, δ): 4.17 (m, 2H, CH2 ester), 2.32 (m, 2H, H1/H5), 2.10 (t, J = 3.0 Hz, 1H, H6), 1.91, 1.82 (ABq, J = 18 Hz, 2H, H3α/β), 1.29 (t, J = 7.1 Hz, 3H, CH3 ester), 1.22 (s, 3H, CH3), 1.21 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3, δ): 210.4 (C2), 170.3 (CO ester), 61.30 (CH2 ester), 46.70 (C3), 40.81 (C1/5), 36.71 (C5/ 1), 35.61 (C4) 29.59 (CH3), 26.27 (C6), 25.91 (CH3), 14.15 (CH3 ester).

volumes of 1/1 H2O/THF, and then 10 column volumes H2O. All eluent up to this point was discarded. The compound was displaced from the resin by washing with 15 column volumes of 15% aq pyridine. The eluent containing the product was concentrated in vacuo. The material was dissolved in water and freeze-dried to give 4 (42 mg, 86%) as a white solid. [α]D20 −31.0 (c 0.200, 0.1 N NaOH). MS (ES+) 214.2 [M + H]+. 1H NMR (400 MHz, D2O/1 drop pyridine-d5, δ): 2.11 (dd, J = 6.0, 2.8 Hz, 1H, H5), 1.92 (d, J = 14.3 Hz, 1H, H3β), 1.73 (dd, J = 6.2, 3.1 Hz, 1H, H1), 1.53 (t, J = 3.0 Hz, 1H, H6), 1.32 (d, J = 14.4 Hz, 1H, H3α), 1.12 (s, 3H, CH3), 1.00 (s, 3H, CH3). A copy of the H NMR is available in the Supporting Information (Figure S5). 13C NMR (125 MHz, D2O/1 drop of pyridine-d5, δ): 180.5 (CO2H), 176.3 (CO2H), 68.06 (C2), 46.17 (C3), 40.10 (C1/5), 39.69 (C1/5), 32.05 (CH3), 27.60 (CH3), 26.33 (C6), 24.26 (C4). Anal. Calcd for C10H15NO4· 0.2NH4Cl·0.2NaCl: C, H, N. HRMS ESI (m/z) calcd for C10H15NO4: 213.1001. Found: 213.1008. (1S,2S,5R,6S)-2-Aminospiro[bicyclo[3.1.0]hexane-4,1′-cyclopropane]-2,6-dicarboxylic Acid (5). To 16 (1.2 g, 3.8 mmol) was added 2 M NaOH (11.5 mL, 23.1 mmol). The reaction mixture was heated at reflux under a blanket of nitrogen for 21 h. The reaction was allowed to cool to rt at which time additional 2 M NaOH (5.8 mL, 12 mmol) was added and the mixture returned to reflux for a further 120 h. After cooling, the reaction mixture was filtered to remove any insoluble particles and then concentrated to half volume. Purification by cation exchange chromatography (Dowex 50X8-100) was then undertaken as follows. The solution was loaded onto the resin and allowed to flow through the column at a drip rate of about 1 drop every 1−2 s. After the initial loading volume had dropped to the resin surface, the resin was washed with HPLC grade water. The pH of the effluent was monitored, and rinsing with HPLC grade water continued until pH had dropped to pH < 1 and returned to pH = 7. Once the complete pH cycle had been observed, the column was washed sequentially with one column volume each of HPLC grade water, HPLC grade water/THF (1:1), and further HPLC grade water. Finally, the resin was washed with 10% v/v pyridine in HPLC grade water until no further product was detected by TLC. The effluent was concentrated under reduced pressure to give a white solid. The solid was dissolved in 90/10 HPLC grade water/acetonitrile and lyophilized to yield 5 (800 mg, 3.8 mmol, 98%) as a white solid. [α]D20 −19.5 (c 0.200, 0.1 N NaOH). MS (ES+) 212.1 [M + H]+. 1H NMR (400 MHz, 5% v/v pyridine-d5 in D2O, δ): 2.12 (dd, J = 6.4, 3.0 Hz, 1H, H1 or H5), 1.78 (t, J = 3.0 Hz, 1H, H6), 1.78, (d, J = 14 Hz, 1H, H3), 1.63 (d, J = 14 Hz, 1H, H3), 1.51 (dd, J = 6.4, 3.0 Hz, 1H, H1 or H5), 0.70 (m, 1H, c-PrCH2), 0.56 (m, 1H, c-PrCH2), 0.46 (m, 1H, cPrCH2), 0.38 (m, 1H, c-PrCH2). A copy of the H NMR is available in the Supporting Information (Figure S6). Anal. Calcd for C10H13NO4· 0.2H2O: C, H, N. HRMS ESI (m/z) calcd for C10H13NO4: 211.0845. Found: 211.0847. (1S,2S,5R,6S)-2-Aminospiro[bicyclo[3.1.0]hexane-4,1′-cyclobutane]-2,6-dicarboxylic Acid (6). Fully protected amino acid 18 (83 mg, 0.18 mmol) in a microwave vial was dissolved in acetic acid (2 mL). To the solution was added water (2 mL). The resulting suspension was heated in a Biotage microwave at 160 °C for 10 min. The resultant solution was concentrated under reduced pressure to yield crude reaction product. Material was suspended in water (10 mL) and lyophilized to yield 6 (35 mg, 0.155 mmol, 86%) as a white solid. Capillary electrophoresis: 90% purity. MS (ES+) 226.0 [M + H]+. 1H NMR (400 MHz, 2.5% w/v KOD in D2O, δ): 2.04 (d, J = 13.9 Hz, 1H), 1.94−1.86 (m, 4H), 1.77−1.67 (m, 4H), 1.29 (m, 1H), 1.07 (d, J = 13.9 Hz, 1H). A copy of the CE trace and H NMR are available in the Supporting Information (Figures S4 and S7, respectively). HRMS ESI (m/z) calcd for C11H16NO4 [M + H]+: 225.1074. Found: 226.1071. Ethyl (1S,4S,5R,6S)-4-Methyl-2-oxobicyclo[3.1.0]hexane-6carboxylate (8). To suspension of cuprous iodide (21.3 g, 112 mmol, 4.45 equiv) in diethyl ether (20 mL) cooled to an internal temperature of 2 °C was added 1.6 M MeLi in Et2O (140 mL, 220 mmol, 8.9 equiv) so that the temperature did not rise above 15 °C (approximate addition time 1 h). The suspension was stirred for 30 min and then cooled to −75 °C. A solution of enone 715 (4.65 g, 25.1 mmol, 1.00 equiv) in diethyl ether (35 mL) and THF (7 mL) was added dropwise. Internal temperature was kept below −50 °C during the M


Article

Journal of Medicinal Chemistry Ethyl (1S,2S,5R,6S)-4,4-Dimethyl-2′,5′-dioxospiro[bicyclo[3.1.0]hexane-2,4′-imidazolidine]-6-carboxylate (11). Ketone 10 (590 mg, 3.01 mmol, 1.00 equiv), ammonium carbamate (711 mg, 9.11 mmol, 3.03 equiv), and potassium cyanide (300 mg, 4.61 mmol, 1.53 equiv) were combined in ethanol (18 mL)/water (12 mL). The reaction vessel was capped and heated to 40 °C for 2 days. The reaction was removed from the heat and allowed to stand at rt for an hour prior to cooling to 0 °C for 2 h. The solid was collected via vacuum filtration washing with a small amount of cold water. The solid was a 95:5 mixture of the undesired 2R hydantoin diastereomer. The filtrate was concentrated to remove ethanol and then extracted twice with ethyl acetate. The combined organics were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a 3:2 mixture of diastereomers favoring the desired 2S hydantoin. The mixture was dissolved in 3:1 MeOH/DCM (4 mL) and purified through five injections on a Chiralpak AD-H, 2.1 cm × 15 cm × 5 μm column, eluting with 15% EtOH/CO2 at a flow rate of 70 mL/min. The separation afforded 11 (61 mg, 7%) as a white foam. MS (ES+) 267.0 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ): 10.61 (s, 1H, NH), 7.88 (s, 1H, NH), 4.05 (m, 2H, CH3 ester), 2.02 (m, 1H, H1), 1.87 (t, J = 3.1 Hz, 1H, H6), 1.79 (d, J = 14.6 Hz, 1H, H3β), 1.75 (m, 1H, H5), 1.31 (s, 3H, CH3β), 1.29 (d, J = 14.6 Hz, 1H, H3α), 1.20 (t, J = 7.1 Hz, 3H, CH3 ester), 1.02 (s, 3H, CH3α). 13C NMR (125 MHz, DMSO-d6, δ): 177.6 [C(O)N, 172.1 (CO2), 156.2 [NC(O)N], 70.16 (C2), 60.72 (CH2 ester), 45.56 (C3), 40.85 (C5), 35.36 (C1), 28.33 (CH3), 27.56 (CH3), 20.74 (C6), 14.54 (CH3 ester), C2 signal obscured by DMSO. Diethyl (1S,2S,5R,6R)-2-Amino-4-oxobicyclo[3.1.0]hexane2,6-dicarboxylate (13). To a stirred solution of ketone 12 (15 g, 36 mmol) in ethanol (365 mL) was added thionyl chloride (13.3 mL, 182 mmol). Following addition the mixture was heated to reflux. After 24 h the reaction was allowed to cool to rt and then concentrated under vacuum. The mixture was concentrated from DCM three times. The residue was then partitioned between 200 mL of ethyl acetate and 150 mL of saturated aqueous sodium hydrogen carbonate. The phases were separated and the organic layer washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum to obtain 13 (8.24 g, 32.3 mmol, 89%) as a colorless oil. MS (ES+) 256.1 [M + H]+. 1H NMR (400 MHz, CDCl3, δ): 4.19 (m, 4H), 2.68 (m, 1H), 2.54 (d, J = 18 Hz, 1H), 2.48 (m, 1H), 2.32 (m, 1H), 2.07 (d, J = 18 Hz, 1H), 1.29 (m, 6H). Diethyl (1S,2S,5R,6R)-2-Acetamido-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylate (14). To a stirred solution of 13 (7.2 g, 28 mmol) in DCM (72 mL) was added triethylamine (7.9 mL, 56 mmol) followed by acetic acid anhydride (5 mL, 50 mmol). The mixture was stirred at ambient temperature for 2 h before addition of water (100 mL). The mixture was stirred vigorously for 20 min. The organic phase was separated using a hydrophobic frit and evaporated. The crude material was purified on silica, eluting with a gradient of 70/30 to 90/10 ethyl acetate/hexanes to yield 14 (6.53g, 22 mmol, 78%) as a pale yellow amorphous solid. MS (ES+) 298.2.1 [M + H]+. 1H NMR (400 MHz, CDCl3, δ): 6.29 (br, 1H), 4.23 (m, 4H), 2.97 (d, J = 19 Hz, 1H), 2.82 (dd, J = 5.6, 3.2 Hz, 1H), 2.49 (dd, J = 5.6, 2.7 Hz, 1H), 2.43 (t, J = 3.2 Hz, 1H), 2.39 (d, J = 19 Hz, 1H), 2.05 (s, 3H), 1.28 (m, 6H). Diethyl (1S,2S,5R,6S)-2-Acetamido-4-methylenebicyclo[3.1.0]hexane-2,6-dicarboxylate (15). An oven-dried 250 mL round-bottom flask was charged with (methyl)triphenylphosphonium bromide (9.72 g, 26.7 mmol) and dry T (122 mL). The suspension was cooled to 0 to −5 °C and treated with 2 M sodium bis(trimethylsilyl)amide in THF (13.3 mL, 26.7 mmol) in a dropwise manner. The resultant bright yellow mixture was stirred at 0 °C for 20 min before addition of 14 (6.1 g, 20 mmol) as a solution in THF (30 mL). The mixture was allowed to warm slowly over 3 h to rt before stirring for a further 20 h. The mixture was quenched with ice−water (200 mL) and extracted with ethyl acetate (200 mL). The organic phase was separated and washed sequentially with water (100 mL) and brine (100 mL) before drying over MgSO4, filtering, and concentrating under reduced pressure. To the crude material was added diethyl ether (70 mL) and hexanes (20 mL), and the resulting solution was seeded with triphenylphosphine oxide. After standing for 2 h, the solution was decanted and concentrated onto silica. Material was then purified on silica, eluting with a gradient of 60/40 to 80/20 ethyl acetate/hexanes to

give 6.8 g of material still contaminated with triphenylphosphine oxide. Repurification on silica eluting with 60/40 ethyl acetate/hexanes yielded clean 15 (3.98g, 13.5 mmol, 66%) as a viscous yellow oil. MS (ES+) 296.2 [M + H]+. 1H NMR (400 MHz, CDCl3, δ): 6.07 (br, 1H), 5.07 (d, J = 2.4 Hz, 1H), 4.90 (d, J = 2.4 Hz, 1H), 4.17 (m, 4H), 3.13 (d, J = 17 Hz, 1H), 2.68 (dd, J = 6.1, 3.2 Hz, 1H), 2.53 (dd, J = 6.1, 2.7 Hz, 1H), 2.01 (m, 4H), 1.99−1.97 (m, 0H), 1.94 (t, J = 2.9 Hz, 1H), 1.26 (m, 6H). Diethyl (1S,2S,5R,6S)-2-Acetamidospiro[bicyclo[3.1.0]hexane-4,1′-cyclopropane]-2,6-dicarboxylate (16). A solution of TFA (1.9 mL, 25 mmol) in DCM (12.5 mL) was added dropwise to a stirred mixture of DCM (12.5 mL) and 1 M diethylzinc in heptanes (25 mL, 25 mmol) while maintaining the mixture between −5 to 0 °C. After 10 min, a solution of diiodomethane (2.0 mL, 25 mmol) in DCM (12.5 mL) was added. After a further 10 min, a solution of 15 (2.5 g, 8.3 mmol) in DCM (12.5 mL) was added. After 60 min the ice bath was removed and the reaction mixture left to stir overnight at rt. The clear reaction solution was quenched by dropwise addition of the reaction mixture into 0.2 M aq HCl (200 mL) under vigorous stirring. After 1 h the DCM layer was separated, washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to yield crude product contaminated with unreacted starting material. The material was purified on silica, eluting with a gradient of 50/50 to 100/0 ethyl acetate/hexanes to give 3.1 g of a colorless oil. Additional purification by SFC (tR = 2.48 min (UV, 200 nm); HPLC column, AD-H 30 mm × 250 mm, 5 μm; CO2 gradient, 5% isopropyl alcohol with 0.2% dimethylethylamine for 0.5 min, then 5%−27% in 2.2 min; column temperature, 35 °C; pressure, 100 000 kPa; flow rate, 210 mL/min) yielded 16 (1.8 g, 5.9 mmol, 71%) as a colorless oil. MS (ES+) 310.1 [M + H]+. 1H NMR (400 MHz, CDCl3, δ): 6.27 (br, 1H), 4.24 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 2.56 (dd, J = 6.6, 2.7 Hz, 1H), 2.18 (d, J = 14 Hz, 1H), 1.99 (s, 3H), 1.92 (t, J = 2.9 Hz, 1H), 1.62 (d, J = 14 Hz, 1H), 1.58 (dd, J = 6.6, 3.2 Hz, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H), 0.67 (m, 3H), 0.48 (m, 1H). Di-tert-butyl (1S,2S,5R,6S)-2-(tert-Butoxycarbonylamino)-2′oxospiro[bicyclo[3.1.0]hexane-4,1′-cyclobutane]-2,6-dicarboxylate (17). To a stirred solution of 12 (2.5 g, 6.1 mmol) and cyclopropyldiphenylsulfonium tetrafluoroborate (2.67 g, 8.51 mmol) in anhydrous DMSO (30 mL) at room temperature was added powdered potassium hydroxide (682 mg, 12.2 mmol) in one portion. The resulting orange mixture was stirred at room temperature under nitrogen for 2 h. Reaction mixture was poured into 0.5 M aqueous tetrafluoroboric acid (100 mL) and extracted into ethyl acetate (3 × 80 mL). Combined organics were dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified on silica, eluting with a gradient of isohexanes to 60/40 isohexanes/ethyl acetate to yield 17 (831 mg, 1.84 mmol, 30%) as a white solid (mixture of diastereomers). MS (ES+) 474.0 [M + Na]+. 1H NMR (400 MHz, CDCl3, δ): 5.14 (br, 1H), 2.99 (m, 4H), 2.54 (m, 1H), 2.19 (m, 1H), 2.15 (m, 1H), 1.85 (m, 1H), 1.62 (m, 1H), 1.50 (s, 9H), 1.44 (s, 9H), 1.43 (s, 9H). Di-tert-butyl (1S,2S,5R,6S)-2-(tert-Butoxycarbonylamino)spiro[bicyclo[3.1.0]hexane-4,1′-cyclobutane]-2,6-dicarboxylate (18). A stirred solution of ketone 17 (400 mg, 0.89 mmol) and ptoluenesulfonyl hydrazide (198 mg, 1.06 mmol) in methanol (20 mL) was heated at reflux for 16 h. The reaction mixture was allowed to cool to rt before portionwise addition of sodium borohydride (670 mg, 17.7 mmol) over 1 h, maintaining the internal temperature below 40 °C. Once addition was complete, the reaction mixture was heated at reflux for a further 16 h. The reaction mixture was allowed to cool to rt, and additional sodium borohydride (335 mg, 8.86 mmol) was added portionwise, keeping the temperature below 40 °C. Reaction was then heated at reflux for 24 h. The reaction mixture was poured into a mixture of ethyl acetate (250 mL) and 0.5 M aq HCl (100 mL) and stirred vigorously for 5 min. The organic phase was separated, washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material was purified initially on silica, eluting with a gradient of isohexanes to 50/50 isohexanes/ethyl acetate, followed by secondary reverse phase purification using C18 modified silica and a gradient of 25/75 (0.1% v/v formic acid in water)/(0.1% v/v formic acid in acetonitrile) to 0.1% v/v formic acid in acetonitrile to yield 18 (83 mg, 0.18 mmol, 20%) as a colorless oil. MS (ES+) 460.2 [M + Na]+. 1H N


Article

Journal of Medicinal Chemistry NMR (400.13 MHz, CDCl3, δ): 5.09 (br, 1H), 2.57 (br, 1H), 2.36 (d, J = 3.5 Hz, 1H), 2.07 (m, 4H), 1.90 (m, 2H), 1.77 (m, 1H), 1.47 (s, 9H), 1.45 (m, 1H), 1.44 (s, 9H), 1.43 (s, 9H), 1.20 (d, J = 14 Hz, 1H). Biological Methods. Binding Assays. Membranes were prepared either from AV12 cells stably expressing rat glutamate transporter EAAT1 and mGlu2 or mGlu3 or from rat cortex. Compounds were solubilized in 0.1 N NaOH as 10 mM stocks, stored at −20 °C, and serially diluted in assay buffer (10 mM potassium phosphate, 100 mM potassium bromide buffer, pH 7.6) supplemented with 20 mM HEPES buffer at the start of each experiment. Compounds were tested in a 96well format in a 3× dilution series to generate a 10-point curve. Diluted compound was incubated with mGlu2 or mGlu3 membranes for 90 min at 25 °C in the presence of 2 nM [3H]1917 (18 Ci/mmol). Membranes were harvested by filtration and quantified by liquid scintillation counting. Specific binding was calculated as % binding in absence of any inhibitor, corrected for binding in the presence of unlabeled 19. IC50 values were calculated with a four-parameter logistic curve fitting program (ActivityBase, version 5.3.1.22). Ki values were calculated using standard methods from IC50, Kd, and ligand concentrations. cAMP Assays. Agonists. Inhibition of forskolin-stimulated cAMP production in cells expressing recombinant mGlu2 (Bmax = 21.8 pmol/ mg protein) or mGlu3 (Bmax = 5.3 pmol/mg protein) receptors was assessed using HTRF in AV12 cells stably expressing rat glutamate transporter EAAT1 and mGlu2 or mGlu3. Twenty-four hours before the assay, cells were plated at a density of 8000−10000 cells per well (mGlu2) or 6000−8000 cells per well (mGlu3) in tissue culture treated, 96-well, half-area black plates and incubated in medium containing 250 μM (mGlu2) or 125 μM (mGlu3) glutamine. Compounds were solubilized in 0.1 N NaOH as 10 mM stocks, stored at −20 °C, and serially diluted in assay buffer (Hanks buffered salt solution (HBSS) with 0.1% BSA and 0.5 mM IBMX) supplemented with 20 mM HEPES buffer at the start of each experiment. Compounds were tested in 10point concentration−response curves using 3-fold serial dilution. The final reaction mixture contained 1 μM forskolin (Sigma F6886) and up to 25 μM test compound. Reactions were incubated at 37 °C for 20 min. DCG-IV was employed as the positive control. After lysis, Cisbio detection reagents were incubated at room temp for 1 h. The HTRF signal (ratio of fluorescence at 665 to 620 nm) was detected with an EnVision plate reader. Raw data were converted to pmol/well of cAMP with a cAMP standard curve generated for each experiment. Relative EC50 values were calculated from the top−bottom range of the concentration−response curve using a four-parameter logistic curve fitting program (ActivityBase, version 5.3.1.22). Antagonists. Reversal of the inhibition of forskolin-stimulated cAMP production by test compounds is measured using homogeneous timeresolved fluorescence technology (HTRF; Cisbio catalog no. 62AM4PEB). The medium is removed, and the cells are incubated with 100 μL of cAMP stimulation buffer (STIM) for 30 min at 37 °C. STIM buffer contains 500 mL of HBSS, 1000 mL of DPBS, 0.034% BSA, 1.67 mM HEPES, and 500 μM IBMX (Sigma I5879). Compounds are tested in 10-point concentration−response curves using 3× serial dilution followed by further 40-fold dilution into STIM buffer. DCG IV (Tocris 0975) serves as the reference agonist. The final reaction mixture contains 1 μM (for mGlu2) or 3 μM (for mGlu3) forskolin (Sigma F6886), DCG IV at its EC90, and up to 25 μM test compound. Cells are incubated at 37 °C for 20 min. To measure the cAMP levels, cAMP-d2 conjugate and anti-cAMP-cryptate conjugate in lysis buffer are incubated with the treated cells at room temperature for 1 h (mGlu2) or 1.5 h (mGlu3). The HTRF signal is detected using an EnVision plate reader (PerkinElmer) to calculate the ratio of fluorescence at 665 to 620 nm. The raw data are converted to cAMP amount (pmol/well) using a cAMP standard curve generated for each experiment. Relative IC50 values are calculated from the top−bottom range of the concentration− response curve using a four-parameter logistic curve fitting program (ActivityBase, version 5.3.1.22). hmGlu2 and hmGlu3 Ca2+ FLIPR Assays. AV12 cells stably expressed rat glutamate transporter EAAT1, Gα15 subunit, and mGlu2 (Bmax = 1.35 pmol/mg protein) or mGlu3 (Bmax = 6.1 pmol/mg protein). The coexpression of Gα15 expression allows these Gi/o-coupled receptors to signal through the phospholipase C pathway, resulting in

the ability to measure receptor activation via transient increases in Ca2+ flux. Twenty-four hours before assay, cells were plated at 85K (mGlu2) or 115K (mGlu3) cells/well into 96-well, black-walled, poly-D-lysinecoated plates and incubated in medium containing 250 μM (for mGlu2) or 125 μM (for mGlu3) of L-glutamine. Compounds were solubilized in 0.1 N NaOH as 10 mM stocks, stored at −20 °C, and serially diluted in assay buffer supplemented with 20 mM HEPES buffer at the start of each experiment. Compounds were tested in 10-point concentration− response curves using 3-fold serial dilution. Intracellular calcium levels were monitored before and after the addition of compounds using Fluo3 AM dye in a FLIPR instrument. The maximal response (ECmax) was defined using 100 μM glutamate. Compound effect was measured as max − min peak heights in RFUs corrected for basal fluorescence in the absence of glutamate. Agonist effects were quantified as percent stimulation induced by compound alone relative to the maximal glutamate response. All data were calculated as relative EC50 values using a four-parameter logistic curve fitting program (ActivityBase, version 5.3.1.22). Antagonist effects were quantified by calculating the percent inhibition of a response elicited by an EC90 concentration of glutamate. Recombinant mGlu Receptor Selectivity Assays. The activity of test compounds for the other human mGlu receptors was assessed in either FLIPR30 or cAMP31 modes using methods analogous to those developed for mGlu2 and mGlu3. Potentiator and antagonist FLIPR assays used a glutamate agonist at levels generating an EC10 or EC90 response. Antagonist effects were quantified by calculating the % inhibition of the EC90 response; potentiation effects were quantified as % increase in the presence of an EC10 response relative to the ECmax response. The mGlu6 cAMP assays used L-AP4 (Tocris) as the reference agonist. All data were calculated as relative IC50 or EC50 values using a four-parameter logistic curve fitting program (ActivityBase, version 5.3.1.22). Site-Directed Mutants of mGlu2 and mGlu3. Wild-type and mutant mGluRs were generated by PCR, cloned into the pcDNA3.1 vector, and transiently transfected using Fugene HD into AV-12 cells stably expressing the rat glutamate transporter EAAT-1 and the Gα15 subunit. Transfected cells were cultured in DMEM with high glucose supplemented with 5% heat inactivated, dialyzed fetal bovine serum, 1 mM sodium pyruvate, 20 mM HEPES, and 1 mM L-glutamine at 37 °C in an atmosphere containing 6.5% CO2 for 48 h. Cells were harvested and suspended in freeze media (FBS with 6% DMSO) at 107 cells/mL, and aliquots were stored in liquid nitrogen. Twenty-four hours before the assay, cells were plated at a density of 10 000 cells per well in tissue culture treated, 384-well, black plates in 50 μL of DMEM with high glucose supplemented as above except that only 250 μM L-glutamine was used. Intracellular calcium levels were monitored before and after the addition of compounds using a fluorometric imaging plate reader (FLIPR, Molecular Devices). The assay buffer comprised Hank’s buffered salt solution (HBSS) supplemented with 20 mM HEPES. The medium was removed, and cells were incubated with 25 μL of assay buffer containing calcium-4 dye (Molecular Probes, R8141) for 2 h at 25 °C. Compounds were prepared as a 3× dilution series and tested in 10point concentration curves. A total of 50 fluorescent images were collected. The maximal response was defined as that induced by ECmax (100 μM glutamate). The compound effect was measured as maximal minus minimal peak heights in relative fluorescent units (RFUs) corrected for basal fluorescence measured in the absence of glutamate. Agonist effects were quantified as percent stimulation induced by compound alone relative to the maximal glutamate response. All data were calculated as relative EC50 values using a four-parameter logistic curve fitting programs (ActivityBase, version 5.3.1.22, and GraphPad Prism, version 6.03). Rat Cortical Synaptosomes cAMP Assay. Rat cortical synaptosomes were prepared essentially as described by Dunkley et al.26 Ten-week-old Sprague−Dawley rats were sacrificed by decapitation, the brains surgically removed, and the cortex was collected and rinsed several times in ice-cold homogenizing buffer (320 mM sucrose, 1 mM EDTA, 5 mM Tris, pH 7.4). The tissue was cut into small pieces, homogenized using 10 strokes, and centrifuged at 1600g for 10 min at 4 °C. The supernatant was layered over the top of a three-step Percoll gradient (3%, 15%, 23%) and spun at 48000g for 5 min at 4 °C. Fractions O


Article

Journal of Medicinal Chemistry F2−F4 were collected and pooled, diluted to 35−40 mL with ice-cold homogenizing buffer, and centrifuged at 31000g for 30 min at 4 °C. The pellet was resuspended in 35−40 mL of washing buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 10 mM glucose, and 10 mM HEPES, pH 7.4), pelleted at 27000g for 10 min at 4 °C, and resuspended in 1−2 mL of washing buffer. Protein concentration was determined using the Pierce Coomassie Plus assay. Synaptosomes were diluted to 200 μg/mL in washing buffer and stored on ice. Compounds were solubilized in 0.1 N NaOH as 10 mM stocks, stored at −20 °C, and serially diluted in assay buffer supplemented with 20 mM HEPES buffer at the start of each experiment. The reversal of forskolin-stimulated cAMP production was conducted using the same HTRF methodology as described for recombinant cells. In this case, each well contained 5 μg of purified synaptosomes, the reaction was conducted for 1 h at 37 °C, and the incubation with cAMP-d2 conjugate and anti-cAMP cryptate conjugate was conducted for 2 h prior to signal detection. Cultured Neuron Ca2+ Oscillation Assay.12a Primary cortical neuronal cultures were prepared from embryonic day 18 Sprague− Dawley rats (Harlan, Bicester, Oxon) in accordance with the Animals (Scientific Procedures) Act 1986. In brief, dissociated RCN cultures were prepared in serum-free Neurobasal medium containing B27 supplement and 0.5 mM L-glutamine (Invitrogen). The volume was adjusted to give a final cell density of 2.06 × 106 cells/cm2. Cells were then plated into 96-well poly-D-lysine (PDL) coated plates (Biocoat) at 100 μL/well and incubated at 37 °C. Experiments were run between day 7 and day 9 in magnesium (Mg2+) free buffer containing the following (mM): NaCl, 135; KCl, 5; CaCl2, 2.5; glucose, 10; HEPES, 10; final pH 7.2. Cells were loaded with the Ca2+ fluorescent probe Fluo-3 AM (Molecular Probes) at room temperature for 1 h. Robust and synchronized Ca2+ oscillations were measured on the FLIPR (Molecular Devices). Cells were excited at 488 nm by an argon-ion laser, and images were captured by a cooled CCD camera. The frequency of Ca2+ oscillations was taken over a 200 s period, and experiments were run for a maximum of 10 min. Compounds were solubilized in 0.1 N NaOH as 10 mM stocks, stored at −20 °C, and serially diluted in assay buffer supplemented with 20 mM HEPES buffer at the start of each experiment. Effects of compound additions were normalized to the frequency of Ca2+ oscillations before compound addition (baseline) within each well. All data were normalized to the maximal agonist control ((2R,4R)-APDC, 30 μM, Tocris), and concentration−response curves were created using GraphPad Prism, version 3.02. The EC50 and/ or IC50 (half the maximal effect of a drug) and the Emax (maximal drug effect) are expressed as the mean ± SEM. Cloning, Expression, Purification, and Crystallization of mGlu2 and mGlu3 ATDs. All clones were generated by PCR and TOPO-cloned into custom TOPO adapted pFastBac vectors (Life Technologies) and sequence verified. Expression in Sf 9 cells was done with an optimized Bac to Bac expression protocol. mGlu2 and mGlu3 are secreted proteins, and natural leaders were used. For mGlu2 and mGlu3 secreted media, 1 mL of Ni-NTA resin was added per liter of supernatant and stirred for 2−4 h at 4 °C. The resin was collected and washed with 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 25 mM imidazole, and 10% glycerol. Protein was then eluted with 250 mM imidazole in the same buffer. For mGlu2, samples were further purified by Ni-NTA, size exclusion chromatography, or anion exchange chromatography (depending on sample purity and amount). For mGlu3, samples were further cleaned by only Ni-NTA. Final samples were concentrated and buffer exchanged with 50 mM Tris, pH 8.0/150 mM NaCl. The molecular masses of protein samples were determined using matrix assisted laser desorption/ionization (MALDI)-MS (Voyager, DE-RP, Applied Biosystems, Foster City, CA) and a liquid chromatography (LC)−electrospray ionization (ESI) mass spectrometer equipped with a quadrupole time-of-flight mass analyzer (Xevo-QTOF, Waters Technologies). This information was used to assess both purity and chemical homogeneity and to compare measured molecular masses to the calculated molecular masses derived from the expected protein sequence. Cocrystals were grown at room temperature using the sitting-drop vapor diffusion method with ∼10 mg/mL protein concentration with 5 mM compound in the protein solution and a

reservoir solution containing various PEGs and salts. Harvested crystals were cryoprotected using well solution supplemented with 20−25% glycerol, ethylene glycol, or PEG400 and flash-frozen in liquid nitrogen. Data were collected at Lilly Research Laboratories CAT, sector 31 of the Advanced Photon Source of Argonne National Laboratory. 0.9793 Å radiation, using a diamond (111) monchrometer, was used for the data set collected on a Rayonix 225-HE CCD detector. These data were indexed with xds and scaled and truncated using scala and truncate (CCP4). The crystal structures of ligand bound human mGlu2 and human mGlu3 were solved using the separate domains of rat mGlu3 as search models in PHASER. The resulting structure was built using Coot, refined using Buster, and validated using MolProbity. Rat Pharmacokinetics. Compound 5 was dissolved in water, adjusted to pH of 7−7.5 with NaOH, and administered to fasted male Sprague−Dawley rats (approximately 250 g, Harlan Industries) at the indicated doses by oral gavage, intravenous, or intraperitoneal route. Serial blood samples (11−12 samples per rat) were collected from a jugular vein catheter into EDTA tubes, centrifuged, and stored frozen until analyzed. Then 25 μL aliquots of thawed plasma were mixed with an equal volume of methanol/water (1:1) containing an analogue internal standard. The mixture was diluted with 300 μL of water and added to a Waters MAX 10 mg SPE plate. The plate was washed with 300 μL of water followed by 300 μL of methanol, and the analyte eluted with 400 μL of methanol/formic acid (96:4). Extracts were concentrated, reconstituted in 50 μL of water, and analyzed by LCMS/MS using two Shimadzu LC-20AD pumps (Kyoto, Japan), a Leap PAL autosampler (Carrboro, NC), and a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS; Foster City, CA) equipped with a TurboIonSpray interface and operated in positive ion mode. Chromatographic separation was accomplished on a 2.1 mm × 50 mm, 5 μm BioBasic AX HPLC column (Thermo Scientific, Pittsburgh, PA) using a binary step gradient. The initial mobile phase system was composed of methanol/water (40:60, v/v; mobile phase A) and glacial acetic acid/water (25:75, v/v; mobile phase B). The step gradient profile changed from 1% B for 0.2 min, 35% B at 0.30−0.40 min, 60% B at 0.50 min and returning to 1% B at 0.76 min. The flow rate was 1.0 mL/min and the column was at ambient temperature, with flow directed to the mass spectrometer between 0.27 and 0.5 min. The selected reaction monitoring (M + H)+ transition was m/z 204.1 → 77.1. Pharmacokinetic parameters were calculated by noncompartmental analysis using Watson 7.4 (Thermo Fischer Scientific). Rat csf Pharmacokinetics. Male Sprague−Dawley rats (Harlan Industries, Indianapolis, IN) were used for all experiments. Rats had free access to food and water at all times. Compound 5 was prepared by dissolution in 1 μL of 5 N NaOH per milligram followed by addition of water for a dose volume of 1 mL/kg. The pH was checked for neutrality prior to injection. All drugs were administered by the intraperitoneal route at doses of 1, 3, and 10 mg/kg. Plasma and csf were collected at 0.5, 1, and 4 h postdosing. The csf was collected via syringe from the cisterna magna. Plasma and csf were stored at −70 °C until analyzed as described above. Rat Phencyclidine (PCP) Induced Locomotor Assay. Male Sprague−Dawley rats (175−250 g, Harlan Industries, Indianapolis, IN) were used for all experiments (approximately 48 animals are used per experiment). Motor activity is monitored by placing an individual rat in a transparent, plastic shoe-box cage of the dimensions 45 cm × 25 cm × 20 cm, with 1 cm depth of wood chips as bedding, and a metal grill or plastic lid is on top of the cage. Motor monitors (Kinder Scientific) consist of a rectangular rack of 12 photobeams arranged in an 8 × 4 formation, (or a high density grouping of 22 in a 15 × 7 pattern) at a height of 5 cm, with a second rack (for measuring rearing behaviors) at a height of 15 cm. The shoe-box cage is placed inside these racks, with the racks on a 3 foot high tabletop in an isolated room. Test compounds are dissolved in deionized water with dropwise addition of 5 M NaOH to achieve a pH 7−8 with sonication and administered by intraperitoneal route to rats that had been fasted overnight, then placed in the test cage and allowed to acclimate for 30 min prior to the PCP challenge. Two hours after test compound dosing and acclimation, PCP-HCl (5 mg/kg, sc) is administered and the subject is placed back in the cage for an additional 60 min while activity is monitored as photobeam breaks. All data analysis P


Article


Animal Studies. All studies involving the use of laboratory animals were consistent with the Guidelines for Ethical Treatment of Research Animals published by the American Veterinary Medical Association and approved by the Institutional Animal Care and Use Committee (IACUC) at Lilly Research Laboratories.

is performed using GraphPad Prism (San Diego, CA, USA). To obtain statistical significance using a one-way analysis of variance (ANOVA) with a post hoc Dunnetts multiple comparison test, 8 animals per treatment group are required to obtain meaningful data because of variation of behavioral responses in animals and 6 treatment groups can be run per experiment (includes controls and up to 5 test substances or a dose response curve); this is the usual maximum though some studies require fewer animals. When performing ED50 calculations of dose− response generated data, dose levels are converted to log values and responses are calculated to percent reversals of stated doses. A nonlinear regression curve fit with a sigmoidal dose response variable slope analysis is then performed. Rat Formalin Pain Assay. Male Sprague−Dawley rats (200−240 g; Harlan Industries, Indianapolis, IN) were used for all experiments. Animals were housed in group cages and maintained at a constant temperature and a 12 h light/12 h dark cycle for at least 72 h before the studies were performed. Rats had free access to food and water at all times except during the collection of data. Animals were randomized into treatment groups. Injection solutions of test molecules were prepared by dissolution in 1 μL of 5 N NaOH per milligram of drug followed by addition of water for a dose volume of 1 mL/kg. The pH was checked for neutrality prior to injection. All drugs were administered by the intraperitoneal route. The administration of formalin into plantar surface of the rat hind paw results in two phases of pain behavior, an early phase during the first 10 min after formalin administration and a late phase during the subsequent 40 min. The scoring for these studies was automated using commercially available startle chambers (model SR-Lab, San Diego Instruments, San Diego, CA) which detected movements of the rats by means of an accelerometer. Rats were injected with vehicle or a dose of drug and individually placed in holding cylinders. At specified time points, the rats were removed from the cylinders and administered formalin (50 μL of a 5% solution in saline) subcutaneously into the plantar surface of the right hind paw and immediately placed back into the cylinders. The cylinders were then placed on the load cells of the detection system, and the response was monitored continuously for 60 min in 1 s bins. The number of pain behavior events [the number of 1 s bins with >20 load units (baseline sniffing and breathing)] was totaled in 5 min intervals. Data were converted determining the number of events over threshold (>20 load units) in each 1 s time bin over the 60 min of data collection. The early phase score is the sum of events greater than 20 load units from time 0 to 5 min. The late-phase score was obtained by adding the total number events greater than 20 load units from minute 11 to minute 40 of the data collection period. Formalin data were evaluated by one-way analysis of variance (ANOVA), and the appropriate contrasts were analyzed by Dunnett’s t test for two-sided comparisons using JMP (version 6.0) statistical analysis program (SAS Institute Inc., Cary, NC). Differences are considered to be significant if the p-value was less than 0.05. Data are presented as mean values with standard errors of the mean values (±SEM). Rat Rotarod Performance Assay. The ability of the compound to induce sedation/ataxia or motor impairment was examined using an accelerating rotarod test. All rats were given three training trials to maintain posture on the rotarod accelerating to 17 rpm in 5 s and maintaining speed for 40 s (Omnitech Electronics Inc., Columbus, OH) prior to the actual day of drug testing. On the following day, rotarod testing was conducted on eight male Sprague−Dawley rats per dose at different time points (1, 2, and 3 h) following intraperitoneal injection of drug or vehicle. Animals that did not fall off the rotarod were given a maximum score of 40 s. All rats were given up to three trials (single trial = 40 s at 17 rpm) to maintain posture on the rod. The best time achieved after three trials was recorded for that particular rat/time point and used in data calculations. Rotarod data were evaluated by calculation of the mean and standard error for each treatment as well as experimental design-appropriate statistics (product-limit survival fit test) which are then used to determine statistical significance of outcome (Wilcoxon χ2 ) using JMP (version 6.0) statistical analysis program (SAS Institute Inc., Cary, NC). Differences are considered to be significant if the p-value was less than 0.05. Data are presented as mean values with standard errors of the mean values (±SEM).

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ASSOCIATED CONTENT

* Supporting Information S

Effects of 5 against a broad selectivity panel; chemical structures of 19, 20, and LY341495; effect of LY379268 in cells expressing WT and mutant hmGlu2 and hmGlu3 receptors; overlay of hmGlu2 and hmGlu3 ATDs showing amino acid differences; combustion analyses, capillary electrophoresis traces, and H NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Protein Data Bank accession codes are the following: 4XAQ, hmGlu2 ATD with LY354740 (1); 4XAR, hmGlu3 ATD with LY354740 (1); 4XAS, hmGlu2 ATD with LY2934747 (5).

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AUTHOR INFORMATION

Corresponding Author

*Phone: 317-276-9101. E-mail: [email protected]. Notes

The authors declare no competing financial interest. # C.P.: Deceased Nov 1, 2013.

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DEDICATION This paper is dedicated to our dear colleague and friend, Conchi Pedregal. Gone but not forgotten. ABBREVIATIONS USED ATD, amino terminal domain; cAMP, 3′,5′-cyclic adenosine monophosphate; C0, plasma concentration after iv dosing extrapolated to t = 0; Cmax, peak plasma concentration after oral dosing; CL, iv clearance; csf, cerebrospinal fluid; FLIPR, fluorescence imaging plate reader; HTRF, homogeneous timeresolved fluorescence; mGlu, metabotropic glutamate; PCP, 1phenylcyclohexylpiperidine; Vdss, volume of distribution at steady state

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REFERENCES

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