Synthesis and Pharmacological Characterization of C4Î²-Amide

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Synthesis and Pharmacological Characterization of C4#Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6dicarboxylates. Identification of (1S,2S,4S,5R,6S)-2-Amino-4-[(3methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist James A Monn, Steven S. Henry, Steven M. Massey, David K Clawson, Qi Chen, Benjamin A Diseroad, Rajni M. Bhardwaj, Shane Atwell, Frances Lu, Jing Wang, Marijane Russell, Beverly A. Heinz, Xu-Shan Wang, Joan H Carter, Brian G Getman, Kofi Adragni, Lisa M Broad, Helene E Sanger, Daniel Ursu, John T. Catlow, Steven Swanson, Bryan G Johnson, David B. Shaw, David L McKinzie, and Junliang Hao J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01481 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Synthesis

and

Pharmacological

Characterization

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of

C4β-Amide-Substituted

2-

Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1S,2S,4S,5R,6S)-2-Amino4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist

James A. Monn,† Steven S. Henry,† Steven M. Massey,† David K. Clawson,§ Qi Chen,† Benjamin A. Diseroad,# Rajni M. Bhardwaj,# Shane Atwell,§ Frances Lu,§ Jing Wang,§ Marijane Russell,§ Beverly A. Heinz,‡ Xu-shan Wang,‡ Joan H. Carter,‡ Brian G. Getman,‡ Kofi Adragni,# Lisa M. Broad,⊥ Helene E. Sanger,⊥ Daniel Ursu,⊥ John T. Catlow,∥ Steven Swanson,∥ Bryan G. Johnson,⊥ David B. Shaw,⊥ David L. McKinzie⊥ and Junliang Hao*,†

†

Discovery Chemistry Research and Technologies, ‡Quantitative Biology, §Structural Biology,

#

Small Molecule Design and Development, #Statistics, ∥Drug Disposition and ⊥Neuroscience

Research, Eli Lilly and Company, Indianapolis, IN 46285

ABSTRACT: Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4β-N-linked variants of (1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses.

From

this

investigation

we

identified

(1S,2S,4S,5R,6S)-2-amino-4-[(3-

methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule


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that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

■ INTRODUCTION Metabotropic glutamate (mGlu) receptors are members of the family C subgroup of the seventransmembrane (7-TM) domain receptor superfamily.

The mGlu receptors have attracted

considerable attention as targets for small molecule-based therapeutic interventions in

both

central nervous system (CNS) and non-CNS disorders.1 Of the known mGlu subtypes (mGlu1-8), mGlu2 and mGlu3 are among the most extensively studied. This has been driven by several factors, including the availability of potent, CNS-penetrant small molecule orthosteric agonists, orthosteric antagonists and allosteric modulators (both positive and negative), the robust physiologic effects produced by many of these ligands in preclinical animal models,2-4 and the demonstration of efficacy in generalized anxiety disorder5 and schizophrenia patients6,7 for oral prodrugs of mGlu2/3 agonists (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 1 (LY354740)8 and (1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2dioxide, 2 (LY404039),9 respectively (Figure 1). MGlu2 and mGlu3 receptors have historically been grouped together (i.e. group II mGluRs), a classification originally adopted owing to their high sequence homology, overlapping orthosteric agonist and antagonist pharmacology and common signaling mechanisms.

However, these

receptors are differentially localized in the CNS10 and are involved in distinct biological



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processes. While mGlu2 receptors are known to be responsible for the acute antipsychotic and analgesic effects of pharmacologically mixed mGlu2/3 agonists,11,12 single nucleotide polymorphisms in the gene encoding mGlu3 (GRM3) have been associated with increased risk for schizophrenia13 and mGlu3 receptors appear to preferentially regulate transmitter release in the spinal cord,14 suppressing aberrant release observed in this tissue in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis.15

Electrophysiological effects

associated with central mGlu3 receptor activation include suppression of GABA-mediated inhibition in the reticular nucleus of the thalamus,16 enhancement of network activity and induction of theta oscillations in the hippocampus,17 and post-synaptic responses in the prefrontal cortex including the initiation of long term depression and enhancement of working memory networks.18 MGlu3 receptors are also found on non-neuronal cells in the CNS as well as in the periphery.

Activation of astrocytic mGlu3 receptors promotes neuroprotection through

mechanisms involving increased glutamate transporter protein expression,19 enhanced release of neurotrophic factors20 increased glutathione synthesis and release,21 decreased production of the pro-apoptotic transcription factor BAD and increased production a pro-survival factor Bcl-xL.22 On the other hand, peripheral mGlu3 receptors play a role in immunological responses. Enrichment of GRM3 in peripheral blood mononuclear cells was observed as a long-term pharmacogenetic response to interferon-β in multiple sclerosis patients.23 In B-cells, mGlu3 receptors promote programmed cell death, suggesting therapeutic opportunity for treatment of disorders involving defective B-cell apoptosis such as systemic lupus erythematosus (SLE) and B-cell lymphoma.24,25 Consistent with this, systemic administration of either 1 or (1R,4S,5S,6S)-4amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid, 3 (LY379268, Figure 1)26 resulted in a reduction of autoimmune symptoms in a mouse model of SLE.24 Furthermore, in the AEA


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model, inhibition of glutamate carboxypeptidase II, the enzyme responsible for degrading the endogenous peptide mGlu3 receptor agonist N-acetylaspartylglutamate (NAAG), led to an improved clinical score, suppressed the proliferation and infiltration of CD4+ T-cells and CD11b+ macrophages into the CNS, and inhibited the production of IL17, TNFα and INF-γ.27 The primary amino acid sequences of both mGlu2 and mGlu3 receptors are known.28 Owing to their high degree of sequence homology, particularly within the orthosteric ligand binding site where glutamate binding residues are 100% identical (see Fig S1), the identification of orthosteric agonists or antagonists capable of differentiating these receptors has proved challenging. On the other hand, we have previously reported the identification of functionally selective mGlu2 receptor agonists (1S,2S,4R,5R,6S)-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 4,

(LY541850)29

and

(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-

ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 5, (LY2812223)30 (Figure 1).

These

compounds have the shared characteristic of displaying selectivity for mGlu2 over mGlu3 receptors as measured by second messenger changes in cellular assays, though not possessing appreciable mGlu2 selectivity based on affinity. Employing crystal structure determination and site-directed mutagenesis, we attributed their functional agonist selectivity to either direct or indirect interactions of the C4-substitutents present in these molecules (α-methyl in 4, αthiotriazolyl in 5) with amino acids residing at the periphery of the mGlu2 and mGlu3 glutamate binding sites.30,31 However, despite the successful identification of these functionally selective mGlu2 receptor agonists, the discovery of selective non-peptidergic mGlu3 receptor agonists has remained elusive. The neuropeptide NAAG has been reported to be a selective mGlu3 agonist,32 and while this mechanism has been challenged,33 the observation that NAAG inhibits evoked [3H]-glycine



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release from spinal synaptosomes in an mGlu2/3 antagonist-sensitive manner supports the hypothesis that it may indeed be an exquisitely potent mGlu3 agonist.14

More recently, patent

applications claiming mGlu3 positive allosteric modulators have appeared.34 In this account, we report the synthesis and characterization of a series of molecules in which the C4-position of 1 is appended with a variety of N-linked substituents, some of which have been found to bind with exceptionally high affinity and exhibit potent agonist activity at recombinant human mGlu3 (hmGlu3) receptors. Unexpectedly, certain members of this series were also observed to exhibit unprecedented selectivity ratios for hmGlu3 over hmGlu2.

Further characterization of one of

these molecules (1S,2S,4S,5R,6S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]-hexane2,6-dicarboxylic acid 8p (LY2794193), establishes it as a useful pharmacological tool for

Figure 1. Chemical structures of non-selective mGlu2/3 receptor agonists (1, 2, 3), functionally selective mGlu2 receptor agonists 4 and 5 and structural variants explored in this account.

1

4

2

3

5

Subject of current investigation

studying mGlu3 receptor function both in vitro and in vivo.


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■ CHEMISTRY A synthetic route leading to racemic variants of 8 has been described in the patent literature,35 while the preparation of non-racemic 8b has been reported by others.36 In this account, nonracemic C4β-alkylamino and amido analogs (8a-q) were readily prepared from the previously described non-racemic C4β-amine 637 (Scheme 1). Treatment of 6 with benzaldehyde under standard reductive amination conditions afforded the benzylamine intermediate 7a in 44% yield. Alternatively,

reaction

of

6

with

a

variety

of

carboxylic

acids

employing

dicyclohexylcarbodiimide (DCC) as the coupling reagent led to C4β-amido intermediates 7b-q. Yields were generally greater than 70%, ranging from 44% (3-chlorophenyl) to 99% (2chlorophenyl, see experimental section). Simultaneous removal of tert-butyloxycarbonyl and tert-butyl ester protecting groups from 7a-q was achieved in a single step using either HCl or AcOH.38

The final products 8a-q were isolated in 24-99% yield as the neutral zwitterions

directly from the reaction mixture or following chromatography (see experimental section for details). All final compounds were characterized by 1H-NMR,

13

C-NMR, HRMS and judged to

be >95% pure by LCMS or capillary electrophoresis. Additional confirmation of both relative and absolute stereochemistry for compounds of this series was obtained by inspection of a single crystal x-ray structure of 8p (Figure 2) as well as the co-crystalized structure of 8p with the hmGlu3 amino terminal domain (ATD) protein (vide infra).



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Scheme 1. Preparation of C4β-N-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates 7, 8

X

a b c d e f g h i j k l m n o p q

H,H O O O O O O O O O O O O O O O O

Figure 2. Single crystal x-ray structure of 8p.a

a

CIF file deposited with the Cambridge Crystallographic Data Center (Code: 1576904).


R C6H5 C6H5 C6H11 CH2C6H5 (CH2)2C6H5 (2-F)-C6H4 (3-F)-C6H4 (4-F)-C6H4 (2-Cl)-C6H4 (3-Cl)-C6H4 (4-Cl)-C6H4 (2-OH)-C6H4 (3-OH)-C6H4 (4-OH)-C6H4 (2-OMe)-C6H4 (3-OMe)-C6H4 (4-OMe)-C6H4

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■ RESULTS hmGlu2/3 Receptor Binding and Whole Cell cAMP Responses. Compounds 8a-q along with previously published comparators 1, 9 and 10 were assessed for their ability to displace [3H](1S,2S,4R,5R,6S)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid,

[3H]-11, ([3H]-

LY459477)37,39 from membranes expressing recombinant human mGlu2 (hmGlu2) or hmGlu3 receptors and for their effect on second messenger production in HEK cells stably expressing these proteins (Tables 1, 2). The minimum statistical ratios (MSRs)40 for these assays, defined as the smallest ratio between the potencies of two compounds that is statistically significant (p value < 0.05) were determined to be 2.28 (for hmGlu2 Ki), 2.27 (for hmGlu3 Ki), 2.31 (for hmGlu2 cAMP EC50) and 4.26 (for hmGlu3 EC50). Incorporation of a primary amine at the C4β-position of 1 resulted in a substantial (~60 fold) reduction in hmGlu2 receptor affinity for 9 (Ki = 4410 nM) compared to 1 (Ki = 71.7 nM), though binding affinity to hmGlu3 was largely unaffected (Ki = 284 nM for 9 vs. 107 nM for 1).

This provides affinity-based (molecular) mGlu3 selectivity

ratios (hmGlu2 Ki / hmGlu3 Ki) of 0.7 for 1 and 15 for 9. As previously reported, whole cell functional agonist responses elicited by 9 were largely unaffected compared to 1 in either hmGlu2 or hmGlu3 expressing cells (hmGlu2 and hmGlu3 EC50 values = 21.2 nM and 24.9 nM for 9 vs 7.0 nM and 27.9 nM for 1), leading to calculated functional agonist potency-based hmGlu3 selectivity ratios (hmGlu2 EC50 / hmGlu3 EC50) of 0.25 for 1 and 0.85 for 9. Acetylation of the C4-amine functionality in 9 led to a partial recovery of binding affinity for 10 (hmGlu2 Ki = 898 nM; hmGlu3 Ki = 177 nM, hmGlu3 molecular selectivity ~ 5), though whole cell agonist potency was negatively impacted compared to 9 (mGlu2 EC50 = 257 nM for 10 vs. 21.2 nM for 9; hmGlu3 EC50 = 193 nM for 10 vs. 24.9 nM for 9; mGlu3 functional selectivity of 10 = 1.3). Replacement of the acetyl group in 10 with benzoyl (8b) led to a slight (~3 fold) improvement in hmGlu2 affinity (Ki



= 294 nM for 8b vs. 898 nM for 10) but an unexpectedly robust (~26-fold) increase in affinity for hmGlu3 (Ki = 6.75 nM for 8b vs. Ki = 177 nM for 10), resulting in an mGlu3 molecular selectivity ratio of 43. The magnitude of this enhanced mGlu3 selectivity was not, however, maintained in the whole cell functional assays where 8b exhibited enhanced agonist potency in cells expressing both hmGlu2 (EC50 = 19.8 nM, 13-fold increase relative to 10) and hmGlu3 (EC50 = 4.96 nM, 39fold increase relative to 10) expressing cells, resulting in a functional agonist selectivity ratio of 4. The enhanced affinity, functional agonist potency and mGlu3 selectivity exhibited by 8b were not observed when the amide carbonyl of this molecule was replaced with a methylene group (8a: hmGlu2 Ki = 11200 nM, EC50 = 508 nM; hmGlu3 Ki = 1070 nM, EC50 = 356 nM). Additionally, when the phenyl ring of 8b was replaced with a cyclohexane (8c) or when either one (8d) or two (8e) methylene groups were introduced between the phenyl ring and amide carbonyl of 8b, not only was binding affinity to both mGlu2 and mGlu3 receptors substantially reduced, but agonist activity was either largely or completely abolished. Instead, low potency, submaximal efficacy antagonist pharmacology was observed. Furthermore, these structural changes (8c-e) led to the complete loss of molecular selectivity observed for 8b (Table 1). With these initial observations in hand, we sought to understand how substitution around the phenyl ring of 8b might affect mGlu2 and mGlu3 receptor affinity, whole cell agonist potency and mGlu2 vs. mGlu3 selectivity ratios (Table 2). As can be seen, the binding affinity and agonist potency at mGlu3 followed the general trend: m-substituted > p-substituted > o-substituted, with the exception of o-OHsubstituted analog 8l. Introduction of a fluorine atom at either the ortho-, meta- or para- positions of 8b had little impact on hmGlu2 or hmGlu3 affinity or agonist potency, though the small (3-fold) improvement in hmGlu3 receptor affinity for the meta-F derivative 8g compared to unsubstituted 8b (Ki = 2.11 nM for 8g vs. 6.75 nM for 8b) combined with the numeric, though not statistically


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significant, improvement in hmGlu2 affinity for 8g compared to 8b (Ki = 214 nM vs. 294 nM, respectively) resulted in an apparent two-fold change in the hmGlu3 molecular selectivity ratio for this analog (101-fold hmGlu3-selective for 8g vs 43-fold selective for 8b).

A comparable

doubling of hmGlu3 functional agonist selectivity for 8g (9-fold) compared to 8b (4-fold) was also observed. Introduction of a chlorine atom at the ortho-position of the phenyl ring (8i) had a slight, though non-statistically significant (1.3-fold) negative impact on hmGlu2 affinity, and a larger (4-fold) reduction in mGlu3 affinity when compared to 8b.

This decrease in hmGlu3

affinity relative to 8b was not, however, accompanied by a loss in functional agonist potency in mGlu3-expressing cells. In contrast, substitution at the meta-position with a chlorine atom led to an increase in both affinity and functional agonist potency at both hmGlu2 and hmGlu3 for 8j compared to 8b, the most notable impact being a 14-fold increase in hmGlu3 binding affinity (Ki = 0.47 nM for 8j vs. Ki = 6.75 nM for 8b) resulting in a calculated mGlu3 affinity-based selectivity ratio of 181 for this molecule. This improved molecular selectivity did not, however, result in an analogous improvement in functional mGlu3 agonist selectivity for 8j compared to 8b (10.7-fold for 8j vs. 4-fold for 8b) owing to similar increases in whole cell agonist potencies for 8j in cells expressing hmGlu2 (EC50 = 8.37 nM, 2-fold, not statistically significant improvement over 8b) and hmGlu3 (EC50 = 0.82 nM, 5-fold improvement over 8b). Substitution at the paraposition with a chlorine atom led to slight (2- to 3-fold) improvements in both mGlu2 and mGlu3 binding affinities and hmGlu2 agonist potency for 8k compared to 8b, but had no impact on either mGlu3 functional agonist potency or on mGlu2/3 selectivity ratios. Introduction of



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Table 1. Binding affinities and functional effects of C4β-N-substituted 2-aminobicyclo[3.1.0]hexane 2,6dicarboxylates at recombinant human metabotropic glutamate 2 and 3 receptorsa

[3H]-11 Ki (nM) [SEM]b

No. 1

X

R

-----------

Fold selectivity hmGlu3 vs. hmGlu2 based on binding affinity

hmGlu2e

hmGlu3f

71.7 [5.9]

107 [12.5]

0.7

cAMP EC50 (nM) [SEM], (Emax)c or IC50 (nM) [SEM] (Imax)d

Fold selectivity hmGlu3 vs. hmGlu2 based on agonist or antagonist potency

hmGlu2g

hmGlu3h

7.0 [1.2] (100%)

27.9 [3.0] (100%)

0.25

†

21.2 [2.17]* (99%)

24.9 [3.73] (98%)

0.85†

†

257 [16.2]* (93%)

193 [43.2]* (81%)

1.3

†

9i

H

--

4410 [598]*

284 [15.4]*

15

10i

C=O

CH3

898 [85]*

177 [8.6]

5.1

8a

CH2

C6H5

11200 [2420]*

1070 [172]*

10

†

508 [92]* (100%)

356 [87.9]* (94%)

1.4

†

8b

C=O

C6H5

294 [13]*

6.75 [1.82]*

43

†

19.8 [3.27]* (100%)

4.96 [1.1]* (92%)

4.0

†

3390 [1400] (29%)* 12000 [4350] (75%)

>25000* 9990 [4300] (78%)

>25000* 7660 [2430] (80%)

>25000* >25000 (20-30%)

n.c.

893 [351]* (57%) 5540 [2240] (40%)

>25000* 2050 [39] (57%)

2.7

8c

8d

8e

C=O

C6H11

C=O

CH2 C6H5

C=O

(CH2)2 C6H5

1520 [187]*

627 [60]*

595 [72]*

757 [155]*

425 [56]*

281 [33.9]*

2.0

†

1.4

†

2.1

†

a

1.2

Mean values from at least three independent experiments, see reference 30 for detailed biochemical methods. bKd values for [3H]-11 in membranes expressing hmGlu2 11.84 nM; hmGlu3: 0.45 nM. cAgonist activity assessed as an inhibition of forskolin-stimulated cAMP formation, Emax value is the maximal cellular agonist response as compared to that elicited by glutamate (100%). dAntagonist activity assessed as a reversal of DCG-IV-inhibited, forskolin-stimulated cAMP, Imax value is the maximal degree of inhibition of this agonist response. eAssay minimum statistical ratio (MSR)40 = 2.28. fAssay MSR = 2.27. gAssay MSR = 2.31. hAssay MSR=4.26. iOriginally prepared and characterized in reference 37. *statistically significant (p-value < 0.05) difference when compared to compound 1 employing the individual assay MSR values. †statistically significant (p-value < 0.05) difference in fold mGlu3 selectivity compared to compound 1. See supplementary section for methodology. n.c.: not calculated.


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Table 2. Binding affinities and functional effects of C4β-benzamide-substituted 2-aminobicyclo[3.1.0]hexane 2,6dicarboxylates at recombinant human metabotropic glutamate 2 and 3 receptorsa

Inhibition of [3H]-11 binding to human mGlu receptorsb Ki (nM) [SEM]

Fold selectivity hmGlu3 vs. hmGlu2 based on binding affinity

No.

R1

R2

R3

hmGlu2d

hmGlu3e

8b

H

H

H

294 [13]

6.75 [1.82]

43†

8f

F

H

H

218 [32.3]

5.79 [0.82]

38

8g

H

F

H

214 [31.8]

2.11 [0.33]*

8h

H

H

F

330 [38.8]

8i

Cl

H

H

8j

H

Cl

8k

H

8l

Functional agonist activity in cells expressing human mGlu receptors EC50 (nM) [SEM], (Emax)c

Fold selectivity hmGlu3 vs. hmGlu2 based on agonist potency

hmGlu2f

hmGlu3g

19.8 [3.27] (100%)

4.96 [1.1] (92%)

4.0†

17.1 [1.36] (99%)

5.1 [2.1] (83%)

3.4†

101†,#

18.1 [1.90] (100%)

2.0 [0.22] (90%)

9.0†,#

4.34 [0.49]

76†,#

19.1 [2.51] (100%)

6.6 [1.6] (83%)

2.9

391 [56.5]

27.4 [11.9]*

14

†,#

42.5 [8.79] (99%)

4.96 [2.0] (80%)

8.6

H

85.3 [6.65]

0.47 [0.08]*

181†,#

8.37 [0.69]* (100%)

0.82 [0.26]* (87%)

10†,#

H

Cl

137 [22.1]

2.29 [0.74]*

60

6.97 [0.88] (100%)

5.15 [1.9] (80%)

OH

H

H

58.2 [5.9]*

1.79 [1.01]*

32.5†

7.44 [0.80]* (98%)

0.17 [0.04]* (91%)

41†,#

8m

H

OH

H

31.5 [3.0]*

2.27 [1.10]*

13.9†,#

3.77 [1.09]* (98%)

0.71 [0.35]* (88%)

5.3†

8n

H

H

OH

54.7 [5.2]*

9.34 [4.06]

5.9†,#

6.32 [0.37]* (99%)

2.12 [0.96] (80%)

3.0

8o

OCH3

H

H

151 [15.6]

23.2 [7.15]*

6.5†,#

26.5 [1.59] (98%)

7.15 [3.3] (83%)

3.7†

8p

H

OCH3

H

412 [111]

0.927 [0.225]*

47.5 [3.55]* (98%)

0.47 [0.09]* (88%)

8q

H

H

OCH3

188 [11.5]

4.56 [1.64]

16.0 [2.16] (100%)

2.44 [1.07] (83%)

†,#

444

†

†,#

41.2†


1.4

†,#

†

†,#

100

†

†,#

6.5†


a

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Mean values from at least three independent experiments, see reference 30 for detailed biochemical methods. bKd values for [3H]-11 in membranes expressing hmGlu2: 11.84 nM; hmGlu3: 0.45 nM. cAgonist activity assessed as an inhibition of forskolinstimulated cAMP formation, Emax value is the maximal cellular agonist response as compared to that elicited by glutamate (100%). dAssay MSR = 2.28. eAssay MSR = 2.27. fAssay MSR = 2.31. gAssay MSR=4.26. *statistically significant (p-value < 0.05) difference compared to compound 8b employing the individual assay MSR values. †statistically significant (p-value < 0.05) difference in fold mGlu3 selectivity compared to compound 1. #statistically significant (p-value < 0.05) difference in fold mGlu3 selectivity compared to compound 8b. See supplementary section for details of this calculation.

a hydrophilic, electron donating hydroxyl group at the ortho-position of the aromatic ring in 8b resulted in an increase in binding affinity for 8l to hmGlu2 (Ki = 58.2 nM, 5-fold increase in affinity vs. 8b) and hmGlu3 (Ki = 1.79 nM, 4-fold increase compared to 8b), leading to an affinity-based mGlu3 selectivity preference (~32-fold) very similar to that of 8b itself (43-fold). The improvements in binding affinity for 8l were associated with a small increase in hmGlu2 agonist potency (EC50 = 7.44 nM, 2.5-fold improvement over 8b) and, more markedly, agonist potency at hmGlu3 (EC50 = 0.17 nM, 26-fold improvement over 8b). This differential effect on agonist potency resulted in a calculated hmGlu3 functional agonist selectivity ratio of approximately 41, an order of magnitude greater than observed for unsubstituted analog 8b. Incorporation of a hydroxyl group at the meta-position of the aryl ring led to nearly an order of magnitude improvement in hmGlu2 affinity for 8m (Ki = 31.5 nM) compared to 8b (Ki = 294 nM) but a smaller (3-fold) improvement in hmGlu3 binding affinity (Ki = 2.27 nM for 8m vs. 6.75 nM for 8b) leading to a diminished overall mGlu3 affinity-based selectivity preference (~14-fold) for this analog compared to that afforded by 8b. On the other hand, compared to 8b, the hmGlu2 agonist potency of 8m increased 5-fold compared while hmGlu3 agonist potency increased 10fold, resulting in comparable mGlu3 selectivity ratios for these compounds. Hydroxyl substitution at the para-position of the phenyl ring lead to a modest improvement in both hmGlu2 binding affinity (Ki = 54.7 nM, 5-fold improvement) and functional agonist potency (EC50 = 6.32 nM, 3fold improvement) for 8n compared to 8b with no substantial change in either hmGlu3 binding


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affinity (EC50 = 9.34 nM) or agonist potency (EC50 = 2.12 nM). In this instance, the calculated hmGlu3 molecular selectivity ratio was substantially diminished (~6-fold) relative to that for 8b (43-fold).

Incorporation of a methoxy-group at the ortho-position of 8b resulted in slightly

higher (2-fold) affinity for hmGlu2 (Ki = 151 nM) and slightly lower (3-fold) affinity for hmGlu3 (Ki = 23.2 nM) for 8o compared to 8b. As in the case of 8n, this had the overall effect of diminishing the hmGlu3 molecular selectivity ratio of 8o compared to 8b (6.5-fold vs. 43-fold). Functional agonist potencies for 8o at both hmGlu2 and hmGlu3 were only slightly diminished (less than 2-fold) compared to 8b, and the hmGlu3 functional agonist-based selectivity ratio was unaffected. The para-methoxy analog 8q had no notable impact on either affinity or agonist.

Figure 3. Graphical representation of hmGlu3 selectivity ratios (over hmGlu2) for compounds 1, 8b-q and 10 based on potency in displacing [3H]-11 from membranes expressing recombinant human mGlu2 and mGlu3 receptors (left panel) or potency in inhibiting forskolin-stimulated cAMP production in cells expressing recombinant human mGlu2 and mGlu3 receptor (right panel).a

a

Graphs prepared in Micosoft Excel from data in Tables 1 and 2. †statistically significant (p-value
25000 >12500

277 [41] (86%) n.d.

11.6 [2.0] (54%) n.d.

11757 (88%, n=1) >12500

>25000 >12500

1950 [219] (99%) n.d.

>25000 >12500

>25000 >12500

a

See references 30, 41a, and 41b for experimental methods; n.d., not determined.



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Figure 5. Agonist concentration-response curves for 8p in cells expressing hmGlu2 (■) or hmGlu3 (□) receptors employing cAMP (left panel) or Ca2+ / FLIPR (right) assay formats. Maximal (100%) activity in each assay is defined as the maximal agonist response produced by glutamate.

Native rat brain tissue binding and function. To establish the potency and efficacy of 8p in rodent brain tissue, its ability to displace specific binding of [3H]-11 to rat cortical membranes and to suppress electrically-stimulated neuronal excitation in primary cortical neurons was explored. Displacement of [3H]-11 from rat cortical membranes by 8p occurred over a concentration range of 10 nM – 10 µM, affording a Ki value of 106 ± 14 nM (Figure 6, upper left panel; Table 5), approximately 4-fold higher than that observed for 1 (Ki = 476 nM).

As

previously noted, functional mGlu2/3 agonist potency in rat cortical tissues is significantly leftshifted relative to affinity, presumably owing to high receptor reserve.31 In the rat cortical neuron Ca2+ oscillation assay, 8p exhibited a biphasic inhibition of spontaneous Ca2+ transients (high affinity EC50 = 0.44 nM; 48% of the total agonist response; low affinity EC50 = 43.6 nM; 52% of the total agonist response) with combined maximal agonist efficacy (Emax) of 66% (Figure 6, lower left panel; Table 5). Both high and low potency sites for 8p were sensitive to 1 µM of the mGlu2/3 receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)


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propanoic acid, 12 (LY34149542 Figure 6, lower right panel).

For reference, compound 1

displayed an EC50 = 9.8 nM and Emax = 99% (Figure 6, upper right panel; Table 5).



Figure 6. Native tissue affinitya and and functional agonist responsesb of 1 and 8p. Top left: displacement of [3H]-11 binding from rat cortical membranes by 1 (open squares) and 8p (closed squares). Top right: Inhibition of spontaneous Ca2+ oscillations in cultured rat embryonic cortical neurons by 1. Bottom left: Biphasic inhibition of cortical Ca2+ oscillations by 8p. Bottom right: Inhibition of cortical Ca2+ oscillations neurons by 8p in the absence (closed circles) and presence (open circles) of 1 µM 12.

a

See reference 31 for experimental details. bSee experimental section for details.


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Table 5. Binding and functional responses of 1 and 8p in rat brain preparations Displacement of [3H]-11 from rat forebrain membranes

Inhibition of spontaneous Ca2+ oscillations in cultured rat cortical neurons

Ki (nM) [SEM]

EC50 [SEM] (nM)

Emax [SEM] (%)

1

476 [62]c

9.8 [0.8]

99 [1.2]

0.44 [0.2]

32 [0.1]

8p

106 [14] 43.6 [7.9]

34 [0.1]

Cocrystallization of 8p with the hmGlu3 Amino Terminal Domain (ATD). To better understand the molecular basis for the remarkable mGlu3 receptor potency and selectivity exhibited by 8p, this compound was co-crystallized with recombinant human mGlu3 ATD protein. The resulting crystal structure of compound 8p bound to the ATD of human mGlu3 (2.82 Å resolution) is shown in Figure 7a. Macroscopically, this ligand-protein complex is highly analogous to those seen for other agonist-binding mGlu3 structures, such as that with compound 1 (pdb code: 4XAR) and glutamate (pdb code: 5CNK). The overall topology of the complex displays an open angle of 27.0o, comparable to two previously published hmGlu3-agonist structures 4XAR (hmGlu3-1, 25.1o), and 5CNK (hmGlu3-glutamate, 23.9-24.4o)43 and the hmGlu2-5 structure 5CNJ (27.5-28.2o).30 At the atomic level, the key interactions between the αamino acid and C6-carboxylic acid of 8p and the mGlu3 protein are nearly identical to those observed in the mGlu3 complex with 1. The α-amino acid associates with the hinge region of the protein and interacts through H-bonds with S151, A172, T174 from LB1, Y122, D301 from LB2, and the distal acid is involved in salt bridge interactions with R68 and K389 from LB-1 and an additional H-bond interaction with S149 (Figure 7b). The distance between the C6-carboxylate of 8p and the guanidinium of R64 is also within 3.2 Å, suggesting a possible interaction. These



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eight residues are identical between human mGlu2 and mGlu3. On the other hand, a unique interaction between 8p and the mGlu3 ATD was observed for the aryl ring of the C4β-benzamide functionality. In this case, the m-methoxyphenyl ring was observed to be intercalated between the sides chains of Y150 from the upper lobe (LB1) and R277 from the lower lobe (LB2) (Figure 7c). The centroid-centroid distance between the aryl ring of 8p and Y150 is within the typical range observed for the π-π stacking interactions.44a Although the geometry is not quite parallel, the distance between the guanidinium carbon atom of R277 and the centroid of benzamide aryl ring of 8p is also well within the distance limit of acceptable π-cation interaction.44b This spatial relationship of this triad is further stabilized by two residues adjacent to R277, with the hydroxyl and NH of S278 stabilizing the conformation of the the benzamide of 8p via H-bonds with the benzamide carbonyl, and the carboxylate of D279 stabilizing the conformation of the guanidine side chain of R277 through a planar bidentate salt bridge (Figure 7c).

A two dimensional

representation of mGlu3 residues either proximal to or interacting with 8p is shown in Figure 7d (generated from MOE45). The conformations of the Y150 and R277 sidechains present in the 8pmGlu3 structure have not been observed in other agonist-bound mGlu3 crystal structures and are therefore likely to be ligand-induced. In the published mGlu3 structures with glutamate (5CNK) and 1 (4XAR), the side chains of Y150 from LB1 and R277 from LB2 are in close proximity to each other, engaged in a π-cation interaction.

Manual positioning of 8p into the 1-mGlu3

binding site in MOE resulted in steric clashes between the C4β-benzamide and this Y150-R277 pair (Figure 8a).

In order for 8p to be accommodated, the side chain of Y150 appears to have

rotated about the Cβ-CAr bond by approximately 46o from its position in 5CNK and 4XAR, and the R277 sidechain shifted away from its position in these structures (Figure 8b). This latter movement was accompanied by a positional change in the D279 sidechain and stabilized by the


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planar divalent guanidinium-carboxylate salt bridge to this acidic residue. While the original direct π-cation interaction between the Y150 and R277 pair observed in the 4XAR and 5CNK structures was lost on binding of 8p, this negative impact on the stability of the closed lobe conformation appears to have been offset by new interactions of these side chains with the intercalated m-methoxyphenyl ring of 8p (vide infra).



Figure 7. (a) The structure of 8p-mGlu3 ATD complex (2.82 Å resolution). The ligand 8p is situated between the upper lobe (LB1) and the low lobe (LB2). (b) The key residues involved in binding with the α-amino acid and the C6-carboxylic acid of 8p. These residues are identical between human mGlu2 and mGlu3 receptors. (c) The unique intercalated arrangement of C4β benzamide of 8p between Y150 and R277 residues is highlighted, noting the distances between interacting groups. Note the conformational stabilization of 8p by sidechain by S278, while the R277 sidechain is conformationally rigidified due to a planar divalent salt-bridge interaction with D279. (d) Two dimensional map indicating amino acids in proximity of and/or interacting with 8p when bound to the hmGlu3 ATD.


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Figure 8. (a) Docked model of 8p (red) in the 1-mGlu3 structure (4XAR, blue). The crystal structure of compound 8p from the 8p-mGlu3 complex was manually superimposed onto 1 in the 1-mGlu3 structure. The resulting structure predicts severe steric clashes (orange disc) between the ligand 8p and the side chains of residues Y150 and R277. (b) Rotation of Y150 and shift of R277 in the 8p-mGlu3 complex (red) from the positions observed in the 1-mGlu3 structure (blue) results in an expanded ligand binding pocket that can both accommodate and productively engage the C4-benzamide group present in 8p.



Binding energy of R277-8p-Y150 triad. As revealed by the comparison of X-ray crystal structures of 1-mGlu3 (4XAR) and 8p-mGlu3 (PDB code: 6B7H) ATD complexes, significant conformational changes in R277/Y150 region of mGlu3 were required in order to accommodate the large group of 8p (Figure 7 and 8). Specifically, the binding of 8p appears to have disrupted a direct cation-π interaction between R277-Y150 observed in the 1-bound structure, replacing it with a sandwich-like cation-π-π triad involving R277, the aromatic ring of 8p and Y150. Cationπ interactions between positively charged residues (arginine or lysine) and aromatic residues (tyrosine, phenylalanine, or tryptophan) are frequently observed in protein systems. For example, over 70% of arginine sidechains were found to be within 6 Å of aromatic sidechains based on a survey in Protein Database Bank (PDB).44 The interaction energies between cation and π systems were sensitive to both the relative orientation and distance between the interacting groups. Strongest arginine-π interactions occurred when the guanidinium group was oriented either parallel (i.e. stacked or face-to-face) or perpendicular (i.e. T-shaped or edge-to-face) to the aromatic ring, with binding energies estimated to be as high as -14.9 kcal/mol computed at M06/6-31G(d,p) level without basis set superposition error (BSSE) corrections.46 To assess energy changes associated with the binding of the aromatic ring fragment of 8p, we estimated the energies using model compounds containing only the side chains of Y150 and R277 (starting from beta carbon atom of these residues), and the truncated 3-methoxybenzamide fragment of 8p in the geometry observed in the corresponding X-ray crystal structures (1-hmGlu3, 4XAR and 8phmGlu3 PDB code: 6B7H). The binding energy of the sandwiched R277-8p-Y150 triad (-12.91 kcal/mol) was calculated to be 2.49 kcal/mol greater than that of R277-Y150 interaction observed in the mGlu3-1 structure (10.42 kcal/mol, Table 6). This energy difference is consistent with, and may largely explain, the


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115-fold (i.e., 2.81 kcal/mol in free energy change calculated using the Gibbs free energy equation) increase in hmGlu3 affinity for 8p (Ki = 0.93 nM) vs. 1 (Ki = 107 nM; see Tables 1 and 2). While the geometry of the guanidinium functionality of R277 with the aromatic ring of 8p was not as optimal for cation-π interactions as that observed for the paired R277-Y150 interaction in the 1-mGlu3 structure (i.e., 3.9 Å vs. 3.7 Å in distance, and 30.3° vs. 6.2° in plane orientation, respectively), the additional π-π interaction between the aryl ring of 8p and Y150 appears to have more than compensated for loss of this direct residue-residue interaction. On the other hand, the observed geometry of the 8p aryl ring-Y150 interaction was indeed favorable for parallel π-π stacking (3.7 Å and 10.8°) with this interaction energy estimated to be -4 kcal/mol when the 8p aryl ring-Y150 interaction was calculated in isolation. Finally, it is noted that the aromatic ring of 8p rotated approximately 180° when bound to mGlu3 compared to its orientation in the isolated small molecule X-ray crystal structure (Figures 2, 9). The conformational search results suggested that the mGlu3-bound conformation of the arylamide of 8p had the lowest gas phase energy while the conformation of 8p observed in the single crystal X-ray (Figure 2) was only slightly higher in energy (0.76 kcal/mol). However, when this conformation of 8p (Figure 2) was docked into the mGlu3 ATD structure, the binding energy was estimated to be 2.39 kcal/mol higher than that found for the observed 8p-mGlu3 complex.



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Table 6. Geometrya, conformation and binding energiesb of 1-mGlu3 and 8p-mGlu3 complexes. Model Compound Dimeric Y150-R277 interaction from mGlu3 ATD complex with compound 1 (4XAR)

Parameter

Value

centroid distance (Å)

3.7

plane relative orientation (°)

6.2

binding energy of dimer Y150-R277 (kcal/mol)

-10.42

Y150 to 8p

3.7

R277 to 8p

3.9

Y150 to 8p

10.8

R277 to 8p

30.3

centroid distance (Å) Trimeric Y150-8p-R277 interaction based on (PDB code: 6B7H)

plane relative orientation (°) binding energy of Y150-8pR277 triad (kcal/mol)

a

-12.91

The centroid and plane of Y150 and 8p were defined from the phenyl ring, and those of R277 from the guanidinium group. bThe calculations were performed at M06-2X/cc-pVTZ(-f)+ level using Jaguar software.47a, b, c The counterpoise method (CP) using dimer basis set or trimer basis set was applied for the correction of basis set superposition error (BSSE) in the binding energy calculations of dimer or trimers, respectively.47d,e The geometries of monomer, dimer and trimer model compound systems were optimized at the same level with constrains on the heavy atoms of the phenyl ring, guanidinium group and beta carbon atoms in the case of dimer and trimer, before the energies were calculated. The binding energy of a trimer A-B-C was calculated as ∆EABC (binding) = EABC,1ABC - (EA,0A + EB,0B + EC,0C) + (EA,1A - EA,1ABC) + (EB,1B - EB,1ABC) + (EB,1B EB,1ABC), where the superscripts refer to the systems used for the generation of basis sets, the letters in the subscripts refer to the systems, and the numbers in the subscripts refer to the geometry of the systems, i.e., 0 from optimized geometry of individual monomers alone, and 1 from optimized geometry of the trimer complex.


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Figure 9. Overlay of 8p conformations observed in small molecule X-ray and in 8p-mGlu3 complex.

Pharmacokinetic Attributes of 8p. In order to identify appropriate doses for use in vivo rodent pharmacodynamics and/or behavioral studies, we assessed both in vitro and in vivo drug disposition-associated characteristics of 8p. As has been generally observed for other members of the 2-aminobicyclohexane-2,6-dicarboxylic acid scaffold,30,31 8p was found to possess high aqueous solubility (>100 µM at pH 7.4), low ( 50% in cAMP assay format from references 8,9, 26, 29-31 and 37. bKi values obtained from displacement of [3H]-11 from membranes expressing recombinant human mGlu2 or mGlu3 receptors. (data for previously published agonists is included in supporting information section, Table S2). See reference 30 for assay details.

While the molecular basis for the selectivity exhibited by 8p is not completely understood, we were able to obtain a moderately resolved (2.82 Å) co-crystal structure of 8p with the recombinant human mGlu3 ATD in the closed (agonist) conformation. We have previously disclosed a C4α-substituted thiotriazole analog 5 (LY2812223) which exhibits marked mGlu2 agonist activity, and noticed two distinct orientations of the Y144 sidechain in the two protomers of the mGlu2 crystal structure with this molecule (5CNJ).30 Specifically, the aromatic ring of Y144 rotated either clockwise along the Cα-Cβ bond in one protomer (ca. 100o relative to the


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Y144 position in the 1-mGlu2 structure) or along the Cβ-CAr bond in the other protomer (ca. 25o relative to the Y144 position in the 1-mGlu2 structure, Figure 15a) and noted that these rotations allowed the otherwise sterically imposing thiotriazole group to fit into and stabilize a closed (agonist state) conformation of mGlu2 but not mGlu3. We ascribed this to the ability of the thiotriazole ring of 5 to form a direct hydrogen bond with E273 in mGlu2, an interaction which is less likely to occur with the shorter D279 residue in mGlu3.30 Strikingly, in the mGlu3 structure with 8p, we noticed a similar large movement of Y150 (corresponds to Y144 in mGlu2), in this case rotating counter-clockwise along Cβ-CAr bond (ca. 46o relative to the Y150 position in the mGlu3 structure with 1, 4XAR)31 in order to accommodate the large C4β-benzamide substituent (Figure 15b). This Y150 rotation provides an expanded pocket for the arylamide, allowing it to simultaneously H-bond to S278 and productively intercalate between the aromatic ring of Y150 and the guanidinium functionality of R277, the latter residue being held in a conformationallyrestricted state owing to a planar guanidinium-carboxylate salt bridge with D279 (Figure 7c). It is noted that 8p itself may be preorganized for efficient mGlu3 binding owing to a hydrogen bond between the C4 amide NH and C2 carboxylate observed in the isolated unbound structure (see Figure 2 and Figure 9), thereby requiring less energy to adopt the observed mGlu3-bound conformation. We posit that the highly stabilized R277-D279 complex underlies the remarkable 400-fold difference in affinity of 8p for mGlu3 relative to mGlu2. Thus, while movement of Y144 to accommodate the benzamide of 8p in a closed form of mGlu2 must occur (evidenced by the fact that 8p produced a maximal agonist response in cells expressing this receptor), the stability of the closed mGlu2-8p complex is proposed to be lower compared to mGlu3-8p owing to the presence of the longer E273 sidechain that cannot orient the distal carboxylate to form a similar planar salt bridge with R271. In support of this idea, an mGlu3 associated D279-R277 planar



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bidentate salt-bridge has now been observed in two of the three human mGlu3-agonist structures (those ligated with 8p or 1) as well as in five other rat mGlu3-agonist structures (2E4U, 2E4V, 2E4W, 2E4X and 2E4Y). In contrast, a corresponding carboxylate-guanidinium interaction completely absent for the E273-R271 pair in three of the four published hmGlu2-agonist structures (4XAQ, 5CNJ, 4XAS). Thus, for distinct reasons, the E273 (mGlu2) / D279 (mGlu3) residues likely underlie the observed agonist selectivity profiles of both 530 (mGlu2-selective) and 8p (mGlu3-selective). The 8p-hmGlu3 ATD structure may also explain the observed general trend of the effects of substitution patterns on mGlu3 binding affinity and agonist potency: m-substituted > p-substituted > o-substituted, with the exception of o-OH-substituted analog 8l. Specifically, substitution at the ortho-position with F, Cl, or OMe groups is predicted to significantly distort the co-planarity of the benzamide, resulting lower binding affinity and potency at mGlu3.

However, o-OH

substitution as in 8l could potentially form internal H-bonding with the adjacent carbonyl oxygen, re-enforcing the co-planarity of the benzamide and afford improved mGlu3 affinity and potency relative to the other o-substituted analogs. For the p-substituted derivatives, potential steric clashes with the backbone of S100 and side chains of S152 and R277 may occur, leading to diminished mGlu3 binding affinity and potency. Based on the 8p-mGlu3 complex, it appears msubstitution is sterically allowed and, as evident from Table 2, each of the m-substituted analogs exhibit similar affinities for mGlu3 receptors (0.47 nM to 2.27 nM). The observed mGlu3 vs. mGlu2 selectivity profiles for these analogs may be attributed to differences in their affinities for mGlu2 which exhibit a wider variance (31.5 nM to 412 nM), with compound 8p exhibiting the largest negative impact on mGlu2 affinity within this series. The molecular basis for the reduced mGlu2 affinity of 8p is currently unknown.


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Figure 15. (a) Rotation of Y144 in two protomers of mGlu2 complex with 5 (5CNJ, orange) relative to that in mGlu2 complex with 1 (4XAQ, cyan). (b) Opposite directional rotation of Y150 (8p-mGlu3) compared to Y144 (5-mGlu2); Y150 rotates counter-clockwise in 8p-mGlu3 (PDB code: 6B7H, red) relative to that in 1-mGlu3 (4XAR, blue). For comparison purpose, the Y150 residue of 8p-mGlu3 protein (red) was overlaid with Y144 of 1-mGlu2 (4XAQ, cyan), and Y144 in 5-mGlu2 (5CNJ, orange).

To establish the agonist potency of 8p in rodent brain, we employed both radioligand binding and functional response (neuronal Ca2+ oscillations) assays that have been employed for other members of this SAR.65,68

Compound 8p completely displaced [3H]-11 from rat cortical

membranes with a Ki= 106 nM, approximately 4-fold lower than the affinity exhibited by 1 (Ki= 479 nM). Interestingly, when examined in the neuronal Ca2+ oscillation assay, 8p suppressed Ca2+ transients in a biphasic manner, with clearly discernable high potency (EC50 = 0.44 nM) and low potency (EC50 = 43.6 nM) sites, each accounting for approximately half of the entire agonist response (Emax = 66%).

The close concordance of these native tissue-derived potencies with

those observed for 8p in the hmGlu3 cAMP (EC50 = 0.47 nM) and hmGlu2 cAMP (EC50 = 47.5 nM) assays is provocative, particularly in light of prior experiments employing cultured cortical neurons derived from WT, mGlu2 and mGlu3 knock out mice demonstrating that both mGlu2 and



mGlu3 contribute about equally to the inhibition of Ca2+ oscillations by pharmacologically balanced mGlu2/3 receptor agonists.65 We therefore posit that the high affinity component of the Ca2+ oscillation response to 8p is mGlu3, while the low affinity component is mGlu2. In order to guide future in vivo studies designed to assess selective mGlu3 receptor activation, compound 8p was assessed for its ability to access the cerebrospinal fluid of rats. Given that 8p has negligible protein binding and does not effectively penetrate into cells, we hypothesized that csf levels of this agent would be a reasonable estimate for those present at the extracellular mGlu2/3 receptor binding sites in the brain.

This hypothesis is supported by pharmacokinetic-

pharmacodynamic response data for other compounds in this chemical class.30,31 From the current studies, it is concluded that based on the mGlu3 potency established in the cAMP assay format, mGlu3-selective levels of 8p in the CNS can be achieved with acute subcutaneous doses as low as 0.02 mg/kg and not achieve mGlu2 receptor-active levels until doses at or above 2 mg/kg. Consistent with the latter prediction, 8p (s.c. administration) was found to inhibit PCP-evoked ambulations in rats in a dose-dependent manner with statistically significant effects only observed at doses at and above 10 mg/kg. Moreover, spontaneous locomotor activity was significantly affected by 8p following a 30 mg/kg dose, an effect observed with other mGlu2/3 agonists in acute rodent behavioral paradigms.50 CSF levels at a dose of 10 mg/kg are predicted to be over 500-fold the EC50 value for mGlu3 (~10 nM; estimated from Figure 6) but within 6-fold of the EC50 for mGlu2 (~40 nM; Figure 6, Table 5). CSF exposures of 8p following chronic infusion were similarly determined and should help to guide dose selections for future studies requiring longer durations of mGlu2 or mGlu3 target engagement.20c In this case, mGlu3 target engagement is predicted for s.c. infusion regimens of 0.1 mg/kg/day while mGlu2 engagement is predicted for dose levels of 10 mg/kg/day or higher. Assay format-associated differences in maximal mGlu3


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agonist efficacy achieved by 8p were also observed (Emax values of 88% and 54% for cAMP and FLIPR formats, respectively). It is unclear how this attribute might relate to in vivo responses. For instance, it is conceivable that 8p could exert near-maximal agonist responses in tissues possessing high mGlu3 receptor reserve while in tissues where reserve is lower, partial agonist / antagonist characteristics could be expressed. Additional experiments, both in vitro and in vivo will be required to understand the impact of this apparent submaximal agonist efficacy attribute.

■ CONCLUSION In summary, we have expanded our exploration of substituents appended to the C4-position of the pharmacologically balanced mGlu2/3 receptor agonist 1, a venture which has previously led to the discovery of highly potent mGlu2-selective agonists such as 5.

Incorporation of appropriately

substituted benzamides at the C4β-position of 1 has now provided highly potent, mGlu3 receptorselective agonists, exemplified by 8p. Systemic dosing of 8p in rats resulted in csf levels of this compound predicted to selectively engage and activate mGlu3 receptors in the brain.

It is

therefore anticipated that 8p will be a useful tool for studying the effects of mGlu3 receptor activation both in vitro and in vivo.



■ EXPERIMENTAL SECTION General. 1H- and

13

C-NMR spectra were obtained on a Varian Unity INOVA 400 at 400 MHz

and 125 MHz respectively, unless noted otherwise. NMR spectra of finals were taken in D2O with 1 drop of KOD added to solubilize the substrate. TMS was used as an internal standard. LCMS data were obtained using an Agilent 1100 Series HPLC on a Gemini-NX C18 110A, 50 X 2.00 mm column. HRMS were obtained using an Agilent 1100 Series LC and TOF mass spectrometer, on a Gemini-NX C18 110A, 50 X 2.00 mm column. 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 oC 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® pre-packed columns from Teledyne Isco. Reversed-phase purifications were performed using RediSep Rf Gold® pre-packed columns from Teledyne Isco. Cation exchange chromatography was performed using Dowex 50W X8; 50-100 mesh, which was purchased from Aldrich. Unless otherwise noted all compounds were >95% pure as judged by HPLC or CE analysis. Numbering convention used in NMR assignments is provided in Figure 15.

Active compounds are confirmed to not be pan assay interference

compounds. They have been profiled across proteins within (see manuscript text and Table 4) and outside (see supporting information section Table S1) the mGlu gene family and are shown to be highly selective in their activities at the specified targets.


Page 48 of 94

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Figure 16. Numbering convention used in NMR peak assignments.

General Procedure A: carboxylic

acid

(1.1-1.2

Preparation of amides 7b-7q via DCC coupling.

equiv.),

dicyclohexylcarbodiimide

(1.5-1.6

equiv.)

and

The 1-

hydroxylbenzotriazole hydrate (1.4-1.5 equiv.) were combined in DMF (0.15-0.1 M) and stirred for 10-30 min at which time amine 6 (1.0 equiv.) was added and the reaction allowed to stir overnight. The reaction was diluted with organic (typically ethyl acetate) and washed with water. The aqueous phase was back-extracted with organic. The combined organic washes were washed with brine, dried over Na2SO4, filtered and concentrated. Unless otherwise stated, the crude material was purified via reversed-phased chromatography eluting with a gradient from 70/30 to 0/100 0.1% aq. formic acid/ACN. General Procedure B: Deprotection conditions for compounds 8a-k, 8m, 8p using aqueous acetic acid.38 A suspension of fully protected amino acid in 50% aq. acetic acid (0.15 M – 0.1 M) was heated to 160 oC in a microwave for 6 min. The crude reaction was concentrated to dryness. The solid was taken up in water and concentrated three times to remove excess acetic acid. Unless otherwise noted, the amino acids final products were of >95% purity and no additional purification was required. General Procedure C: Deprotection conditions for compounds 8l, 8n, 8o, 8q using HCl in dioxane. The fully protected amino acid was taken up in dioxane (0.2 M) and treated



with 4 M HCl in dioxane (15 equiv.). The reaction was heated to 75 oC overnight, unless otherwise noted, then concentrated in vacuo to afford the HCl salt. General Procedure D: Purification using anion exchange chromatography. The resin (BioRad AG 1-X8; OH Form) was prepared by first washing in a fritted funnel with water. The resin was soaked in 1N NaOH for 5 min then the solvent was removed by filtration. This soaking in NaOH was repeated. The resin was washed with H2O until pH = 7 was achieved. The freshly prepared resin was added to a glass column. Approximate resin bed was 1" diameter x 3" high. The crude material was dissolved in a minimal amount of H2O and adjust to pH = 8-10. The material was carefully loaded to the top of the resin bed. The eluent drip rate was maintained at 1 drop every 2-3 seconds. After initial loading volume dropped to the resin surface, 5 mL 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. After the pH raised to 14 then returned to pH = 7 the flow rate was increased. The column was washed with 5 column volumes of water, 10 column volumes of 1/1 H2O /THF, 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 3N acetic acid. The eluent containing the product was concentrated in vacuo. Water was added and concentrated in vacuo 4 times to remove excess acetic acid. The product was dried under vacuum. General Procedure E: Purification using cation exchange chromatography. The resin (Dowex 50X8 – 100) was prepared by first washing in a fritted funnel with water, THF, and water. The resin was soaked in 3N NH4OH for 5 min 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 1N HCl for 5 min then the solvent was removed by


Page 50 of 94

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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 resin bed was 1" diameter x 3" high. The crude material was dissolved in 4 mL 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 seconds. After initial loading volume dropped to the resin surface, 5 mL 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 then returned to pH = 7 the flow rate was increased. The column was washed with 5 column volumes of water, 10 column volumes of 1/1 H2O /THF, 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 10% aq. pyridine. The eluent containing the product was concentrated in vacuo. Water was added and concentrated in vacuo 4 times to remove excess pyridine. The product was dried under vacuum. Di-tert-butyl

(1S,2S,4S,5R,6S)-4-(benzylamino)-2-((tert-

butoxycarbonyl)amino)bicyclo[3.1.0]hexane-2,6-dicarboxylate (7a). A solution of 6 (211 mg, 0.511 mmol, 1 equiv.) and benzaldehyde (0.06 mL, 0.6 mmol, 1 equiv.) in 1,2-dichloroethane (3.4 mL, 0.15 M) was treated with sodium triacetoxyborohydride (129 mg, 0.609 mmol, 1.2 equiv). Allowed to stir at ambient temperature overnight. The crude reaction was purified on a C18 reverse-phase column eluting with a gradient from 90/10 to 30/70 0.1% formic acid in water/ACN.

LCMS indicated the presence of an impurity that was not removed during

chromatography. The material was repurified on a C18 reverse-phase column eluting with a gradient from 75/25 to 5/95 10 mM ammonium bicarbonate/ACN, which afforded 112 mg (44%) of 7a. MS (ES+) 503 [M+H]+.

1

H NMR (400 MHz, CDCl3, δ): 7.40-7.23 (m, 6H), 5.23 (bs,



Page 52 of 94

1H), 3.84 (m, 2H, CH2Ph), 3.35 (d, J = 7.3 Hz, 1H, H4), 2.77 (bm, 1H, H3β), 2.32 (m, 1H, H1/5), 2.11 (bm, 2H, H1/5, H3α), 1.48 (s, 10H, H6 and t-Bu), 1.47 (s, 9H, t-Bu), 1.45 (s, 9H, t-Bu). Di-tert-butyl

(1S,2S,4S,5R,6S)-4-benzamido-2-((tert-

butoxycarbonyl)amino)bicyclo[3.1.0]hexane-2,6-dicarboxylate (7b).

Following general

procedure A using benzoic acid (100 mg, 0.816 mmol, 1.1 equiv.), dicyclohexylcarbodiimide (226 mg, 1.10 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (144 mg, 1.04 mmol, 1.4 equiv.), 6 (295 mg, 0.715 mmol, 1.0 equiv.) and DMF (8.0 mL) the title compound, 7b (198 mg, 0.383 mmol), was obtained in 53.6% yield. MS (ES+) 461 [M-tBu+H]+, 405 [M-2tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.27 (bd, 1H), 7.89 (m, 2H), 7.54-7.45 (m, 3H), 5.39 (bs, 1H), 4.83 (t, J = 7.6 Hz, 1H, H4), 2.77 (bd, J = 15 Hz, 1H H3β), 2.23 (m, 1H, H1/5), 2.10 (dd, J = 5.8, 3.2 Hz, 1H, H1/5), 1.73 (dd, J = 15, 7.7 Hz, 1H, H3α), 1.69 (t, J = 3.0 Hz, 1H, H6), 1.53 (s, 9H), 1.48 (s, 9H), 1.47 (s, 9H). Di-tert-butyl

(1S,2S,4S,5R,6S)-2-((tert-butoxycarbonyl)amino)-4-

(cyclohexanecarbonylamino)bicyclo[3.1.0]hexane-2,6-dicarboxylate (7c). Following general procedure A using cyclohexylcarboxylic acid (105 mg, 0.807 mmol, 1.1 equiv.), dicyclohexylcarbodiimide (231 mg, 1.12 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (142 mg, 1.03 mmol, 1.4 equiv.), 6 (302 mg, 0.732 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7c (316 mg, 0.605 mmol), was obtained in 82.6% yield. MS (ES+) 523 [M+H], 467 [M-tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 7.36(bs, 1H), 5.33 (bs, 1H), 4.62 (t, J = 7.9 Hz, 1H, H4), 2.59 (bd, J = 15 Hz, 1H H3β), 2.19 (m, 1H, H1/5), 2.10 (tt, J = 12, 3.5 Hz, 1H, CH of cyclohexyl), 1.94 (dd, J = 6.0, 3.2 Hz, 1H, H1/5), 1.89 (m, 2H, CH2 of cyclohexyl), 1.79 (m, 2H,


Page 53 of 94 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CH2 of cyclohexyl), 1.68 (m, 2H, CH2 of cyclohexyl), 1.62 (m, 1H, H3α), 1.60 (t, J = 2.9 Hz, 1H, H6), 1.52 (s, 9H), 1.462 (s, 9H), 1.455 (s, 9H), 1.39-1.22 (m, 4H, CH2 of cyclohexyl). Di-tert-butyl

(1S,2S,4S,5R,6S)-2-((tert-butoxycarbonyl)amino)-4-[(2-

phenylacetyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate

(7d).

Following

general

procedure A using phenylacetic acid (110 mg, 0.81 mmol, 1.1 equiv.), dicyclohexylcarbodiimide (230 mg, 1.1 mmol, 1.6 equiv.) 1-hydroxybenzotriazole hydrate (150 mg, 1.1 mmol, 1.5 equiv.), 6 (300 mg, 0.73 mmol, 1.0 equiv.) and DMF (8.0 mL). The crude material was purified via normalphased chromatography eluting with a gradient from 10-30% acetone in hexanes to give the title compound, 7d (330 mg, 0.63 mmol), in 87% yield. MS (ES+) 475 [M-tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 7.41 (bd, 1H), 7.36-7.26 (m, 5H), 5.29 (bs, 1H), 4.61 (t, J = 8.1 Hz, 1H, H4), 3.57 (s, 2H, CH2Ph), 2.51 (bd, J = 15 Hz, 1H H3β), 2.09 (m, 1H, H1/5), 1.89 (dd, J = 5.8, 3.1 Hz, 1H, H1/5), 1.61 (m, 1H, H3α), 1.58 (t, J = 2.9 Hz, 1H, H6), 1.45 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu), 1.42 (s, 9H, t-Bu). Di-tert-butyl

(1S,2S,4S,5R,6S)-2-((tert-butoxycarbonyl)amino)-4-(3-

phenylpropanoylamino)bicyclo[3.1.0]hexane-2,6-dicarboxylate (7e). procedure

A

using

3-phenylpropionic

acid

(132

mg,

0.861

Following general mmol,

1.2

equiv.),

dicyclohexylcarbodiimide (226 mg, 1.10 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (143 mg, 1.04 mmol, 1.4 equiv.), 6 (298 mg, 0.722 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7e (310 mg, 0.569 mmol), was obtained in 78.8% yield. MS (ES+) 545 [M+H]+, 489 [M-tBu+H]+.

1

H NMR (400 MHz, CDCl3, δ): 7.36 (bs, 1H), 7.31-7.19 (m, 5H), 5.32 (bs, 1H),

4.61 (t, J = 7.9 Hz, 1H, H4), 2.99 (t, J = 8.2 Hz, 2H, CH2Ph), 2.52 (m, 2H, C(O)CH2 and H3β),



Page 54 of 94

2.14 (dd, J = 5.9, 2.8 Hz, 1H, H1/5), 1.88 (dd, J = 5.8, 3.1 Hz, 1H, H1/5), 1.63 (m, 1H, H3α), 1.59 (t, J = 3.0 Hz, 1H, H6), 1.50 (s, 9H, t-Bu), 1.462 (s, 9H, t-Bu), 1.459 (s, 9H, t-Bu). Di-tert-butyl


fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate

(7f).

procedure

0.805

A

using

2-fluorobenzoid

acid

(114

mg,


1.1

equiv.),

dicyclohexylcarbodiimide (222 mg, 1.08 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (145 mg, 1.05 mmol, 1.4 equiv.), 6 (302 mg, 0.732 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7f (226 mg, 0.423 mmol), was obtained in 57.7% yield. MS (ES+) 557 [M+Na]+, 367 [M-3tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.40 (bs, 1H), 8.05 (dt, J = 7.3, 1.7 Hz, 1H), 7.47 (m, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.14 (dd, J = 11, 8.3 Hz, 1H), 5.36 (bs, 1H), 4.89 (t, J = 7.6 Hz, 1H, H4), 2.77 (bd, J = 15 Hz, 1H, H3β), 2.26 (m, 1H, H1/5), 2.11 (dd, J = 5.7, 3.0 Hz, 1H, H1/5), 1.72 (dd, J = 15, 8.1 Hz, 1H, H3α), 1.67 (t, J = 2.8 Hz, 1H, H6), 1.52 (s, 9H, t-Bu), 1.47 (s, 18H, tBu). Di-tert-butyl


fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7g). procedure

A

using

3-fluorobenzoic

acid

(115

mg,

0.813


1.1

equiv.),

dicyclohexylcarbodiimide (223 mg, 1.13 mmol, 1.6 equiv.) 1-hydroxybenzotriazole hydrate (141 mg, 1.02 mmol, 1.4 equiv.), 6 (299 mg, 0.725 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7g (288 mg, 0.539 mmol), was obtained in 74.3% yield. MS (ES+) 557 [M+Na]+, 367 [M-3tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.34 (bs, 1H), 7.63 (m, 2H), 7.44 (m, 1H), 7.21 (dt, J = 8.1, 2.0 Hz, 1H), 5.41 (bs, 1H), 4.80 (t, J = 7.6 Hz, 1H, H4), 2.77 (bd, J = 15 Hz, 1H,


Page 55 of 94 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H3β), 2.22 (m, 1H, H1/5), 2.09 (m, 1H, H1/5), 1.76-1.69 (m, 2H, H3α and H6), 1.54 (s, 9H, t-Bu), 1.48 (s, 9H, t-Bu), 1.47 (s, 9H, t-Bu). Di-tert-butyl

(1S,2S,4S,5R,6S)-2-((tert-butoxycarbonyl)amino)-4-[(4Following general

fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7h). procedure

A

using

4-fluorobenzoic

acid

(114

mg,

0.805

mmol,

1.1

equiv.),

dicyclohexylcarbodiimide (229 mg, 1.11 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (143 mg, 1.04 mmol, 1.4 equiv.), 6 (299 mg, 0.725 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7h (300 mg, 0.561 mmol), was obtained in 77.4% yield. MS (ES+) 557 [M+Na]+, 367 [M-3tBu+H]+.

1

H NMR (400 MHz, CDCl3, δ): 8.40 (bs, 1H), 8.27 (dd, J = 7.89 m, 2H),

7.14 (t, J = 8.6 Hz, 2H), 5.40 (bs, 1H), 4.80 (t, J = 7.7 Hz, 1H, H4), 2.76 (bd, J = 15 Hz, 1H, H3β), 2.21 (m, 1H, H1/5), 2.09 (dd, J = 5.8, 3.0 Hz, 1H, H1/5), 1.72 (dd, J = 15, 7.7 Hz, 1H, H3α), 1.69 (t, J = 3.0 Hz, 1H, H6), 1.53 (s, 9H, t-Bu), 1.48 (s, 9H, t-Bu), 1.47 (s, 9H, t-Bu). Di-tert-butyl


chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7i). procedure

A

using

2-chlorobenzoic

acid

(170

mg,

1.1


1.1

equiv.),

dicyclohexylcarbodiimide (300 mg, 1.4 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (210 mg, 1.4 mmol, 1.4 equiv.), 6 (400 mg, 0.97 mmol, 1.0 equiv.) and DMF (6.5 mL) the title compound, 7i (530 mg, 0.96 mmol), was obtained in 99% yield. MS (ES-) 549 [M-H]-. 1H NMR (400 MHz, CDCl3, δ): 7.84 (bs, 1H), 7.56 (dd, J = 7.0, 2.2 Hz, 1H), 7.39 (dd, J = 7.5, 1.8 Hz, 1H), 7.35-7.27 (m, 2H), 5.32 (bs, 1H), 4.84 (t, J = 8.4 Hz, 1H), 2.72 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H), 1.70 (dd, J = 16, 8.4 Hz, 1H), 1.64 (t, J = 3.1 Hz, 1H), 1.45 (s, 9H), 1.44 (s, 9H), 1.43 (s, 9H).



Di-tert-butyl

Page 56 of 94


chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7j). procedure

A

using

3-chlorobenzoic

acid

(128

mg,

0.793


1.1

equiv.),

dicyclohexylcarbodiimide (228 mg, 1.11 mmol, 1.6 equiv.) 1-hydroxybenzotriazole hydrate (141 mg, 1.02 mmol, 1.4 equiv.), 6 (295 mg, 0.715 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7j (175 mg, 0.318 mmol), was obtained in 44.4% yield. MS (ES+) 573 [M+Na]+, 383 [M-3tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.35 (bs, 1H), 7.90 (s, 1H), 7.74 (d, J = 7.4 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 5.40 (bs, 1H), 4.79 (t, J = 7.8 Hz, 1H, H4), 2.76 (bd, J = 15 Hz, 1H, H3β), 2.22 (m, 1H, H1/5), 2.09 (dd, J = 5.7, 3.1 Hz, 1H, H1/5), 1.72 (dd, J = 15, 7.4 Hz, 1H, H3α), 1.70 (t, J = 2.9 Hz, 1H, H6), 1.54 (s, 9H, t-Bu), 1.48 (s, 18H, t-Bu), 1.47 (s, 18H, t-Bu). Di-tert-butyl


chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7k). procedure

A

using

4-chlorobenzoic

acid

(132

mg,

0.826


1.2

equiv.),

dicyclohexylcarbodiimide (230 mg, 1.11 mmol, 1.6 equiv.) 1-hydroxybenzotriazole hydrate (142 mg, 1.03 mmol, 1.4 equiv.), 6 (296 mg, 0.718 mmol, 1.0 equiv.) and DMF (7.0 mL) the title compound, 7k (324 mg, 0.588 mmol), was obtained in 81.9% yield. MS (ES+) 573 [M+Na]+, 383 [M-3tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.31 (bs, 1H), 7.83 (d, J = 8.4, 1H), 7.45 (d, J = 8.5 Hz, 1H), 5.40 (bs, 1H), 4.80 (t, J = 7.4 Hz, 1H, H4), 2.76 (bd, J = 15 Hz, 1H, H3β), 2.21 (m, 1H, H1/5), 2.09 (dd, J = 5.7, 3.0 Hz, 1H, H1/5), 1.75-1.69 (m, 2H, H3α and H6), 1.53 (s, 9H, tBu), 1.48 (s, 18H, t-Bu), 1.47 (s, 18H, t-Bu).


Page 57 of 94 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Di-tert-butyl


hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7l).

Following general

procedure A using salicylic acid (46.2 mg, 0.334 mmol, 1.1 equiv.), dicyclohexylcarbodiimide (95.2 mg, 0.461 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (57.7 mg, 0.418 mmol, 1.4 equiv.), 6 (125 mg, 0.303 mmol, 1.0 equiv.) and DMF (3.0 mL). The crude material was purified via normal-phased chromatography eluting with a gradient from 10-15% acetone in hexanes to give the title compound, 7l (100 mg, 0.188 mmol), in 62.0% yield. MS (ES+) 533 [M+H]+, 477 [M-tBu+H]+. 1H NMR (400 MHz, CDCl3, δ): 12.58 (s, 1H), 8.60 (bs, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.41 (m, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1H), 5.42 (bs, 1H), 4.78 (t, J = 7.2 Hz, 1H, H4), 2.79 (bd, J = 15 Hz, 1H, H3β), 2.21 (m, 1H, H1/5), 2.09 (dd, J = 5.7, 3.0 Hz, 1H, H1/5), 1.73 (m, 1H, H3α), 1.71 (t, J = 3.0 Hz, 1H, H6), 1.55 (s, 9H, t-Bu), 1.48 (s, 18H, t-Bu), 1.47 (s, 18H, t-Bu). Di-tert-butyl


hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7m). procedure

A

using

3-hydroxybenzoic

acid

(150

mg,

1.1


1.1

equiv.),

dicyclohexylcarbodiimide (300 mg, 1.4 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (210 mg, 1.4 mmol, 1.4 equiv.), 6 (400 mg, 0.97 mmol, 1.0 equiv.) and DMF (6.5 mL) the title compound, 7m (480 mg, 0.90 mmol), was obtained in 93% yield. MS (ES-) 531 [M-H]-.

1

H

NMR (400 MHz, CDCl3, δ): 8.28 (bs, 1H), 7.61 (bs, 1H), 7.28 (m, 2H), 7.08 (bs, 1H), 6.99 (m, 1H), 5.37 (bs, 1H), 4.78 (t, J = 6.6 Hz, 1H), 2.72 (m, 1H), 2.19 (m, 1H), 2.06 (m, 1H), 1.92 (m, 1H), 1.69 (m, 1H), 1.49 (s, 9H), 1.44 (s, 18H).



Di-tert-butyl

Page 58 of 94


hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7n). procedure

A

using

4-hydroxybenzoic

acid

(190

mg,

1.3


1.1

equiv.),

dicyclohexylcarbodiimide (380 mg, 1.8 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (260 mg, 1.7 mmol, 1.4 equiv.), 6 (500 mg, 1.2 mmol, 1.0 equiv.) and DMF (8.1 mL) the title compound, 7n (460 mg, 0.86 mmol), was obtained in 71% yield. MS (ES+) 533 [M+H]+.

1

H

NMR (400 MHz, CDCl3, δ): 8.18 (bs, 1H), 7.75 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 5.45 (bs, 1H), 4.75 (t, J = 7.5 Hz, 1H), 3.39 (m, 1H), 2.18 (m, 1H), 2.06 (t, J = 4.4 Hz, 1H), 1.71 (m, 2H), 1.59 (m, 1H), 1.50 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H). Di-tert-butyl


methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7o). procedure

A

using

2-methoxybenzoic

acid

(160

mg,

1.1


1.1

equiv.),

dicyclohexylcarbodiimide (300 mg, 1.4 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (210 mg, 1.4 mmol, 1.4 equiv.), 6 (400 mg, 0.97 mmol, 1.0 equiv.) and DMF (6.5 mL). The crude material was purified via normal-phased chromatography eluting with a gradient from 20-40% ethyl acetate in hexanes affording the title compound, 7o (500 mg, 0.92 mmol), in 95% yield. MS (ES+) 547 [M+H]+.

1

H NMR (400 MHz, CDCl3, δ): 9.26 (bs, 1H), 8.21 (dd, J = 8.4, 1.8 Hz,

1H), 7.42 (m, 1H), 7.03 (m, 1H), 6.95 (dd, J = 8.4, 0.4 Hz, 1H), 5.33 (bs, 1H), 4.89 (t, J = 7.5 Hz, 1H), 4.04 (s, 3H), 2.78 (bd, J = 15 Hz, 1H), 2.23 (m, 1H), 2.07 (dd, J = 6.2, 3.1 Hz, 1H), 1.69, (dd, J = 15, 7.9 Hz, 1H), 1.61 (t, J = 2.6 Hz, 1H), 1.49 (s, 9H), 1.44 (s, 9H), 1.43 (s, 9H). Di-tert-butyl


methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7p).


Following general

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procedure

A

using

3-methoxybenzoic

acid

(331

mg,

2.14

mmol,

1.1

equiv.),

dicyclohexylcarbodiimide (614 mg, 2.95 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (417 mg, 2.72 mmol, 1.4 equiv.), 6 (804 mg, 1.95 mmol, 1.0 equiv.) and DMF (12 mL). The crude material was purified via normal-phased chromatography eluting with a gradient from 20-30% ethyl acetate in hexanes affording the title compound, 7p (685 mg, 1.25 mmol), in 64.3% yield. MS (ES+) 569 [M+Na]+. 1H NMR (400 MHz, CDCl3, δ): 8.27 (bs, 1H), 7.44-7.40 (m, 2H), 7.34 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.1, 2.3 Hz, 1H), 5.37 (bs, 1H), 4.78 (t, J = 8.1 Hz, 1H, H4), 3.86 (s, 3H, -OCH3), 2.74 (bd, J = 15 Hz, 1H, H3β), 2.19 (m, 1H, H1/5), 2.07 (dd, J = 6.1, 3.4 Hz, 1H, H1/5), 1.70, (dd, J = 15, 7.7 Hz, 1H, H3α), 1.66 (t, J = 3.0 Hz, 1H, H6), 1.50 (s, 9H, t-Bu), 1.45 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu). Di-tert-butyl


methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylate (7q). procedure

A

using

4-methoxybenzoic

acid

(160

mg,

1.07


1.1

equiv.),

dicyclohexylcarbodiimide (300 mg, 1.45 mmol, 1.5 equiv.) 1-hydroxybenzotriazole hydrate (210 mg, 1.36 mmol, 1.4 equiv.), 6 (400 mg, 0.97 mmol, 1.0 equiv.) and DMF (6.5 mL). The crude material was purified via normal-phased chromatography eluting with a gradient from 20-40% ethyl acetate in hexanes giving the title compound, 7q (440 mg, 0.81 mmol), in 83% yield. MS (ES+) 547 [M+H]+. 1H NMR (400 MHz, CDCl3, δ): 8.29 (bs, 1H), 7.81 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.8 Hz, 1H), 5.37 (bs, 1H), 4.77 (t, J = 7.9 Hz, 1H, H4), 3.84 (s, 3H, OCH3), 2.73 (bd, J = 16 Hz, 1H, H3β), 2.19 (m, 1H, H1/5), 2.06 (dd, J = 5.7, 3.0 Hz, 1H, H1/5), 1.69 (dd, J = 16, 7.5 Hz, 1H, H3α), 1.66 (t, J = 3.5 Hz, 1H, H6), 1.50 (s, 9H, t-Bu), 1.44 (s, 9H, t-Bu), 1.43 (s, 9H, t-Bu).



Page 60 of 94

(1S,2S,4S,5R,6S)-2-Amino-4-(benzylamino)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (8a). Fully protected amino acid 7a (33 mg, 0.066 mmol and 82 mg, 0.16 mmol) was deprotected according to general procedure B. Each batch was combined and purified on C18 eluting with a gradient from 100% 0.1% aq. acetic acid to 60/40 0.1% aq. acetic acid/ACN . The purified product was concentrated and dried under vacuum at 50 oC giving 8a (40 mg, 61%) as a white solid. Capillary Electrophoresis: > 99% purity.

1

H NMR (400 MHz, D2O/KOD, δ): 7.42 (m,

5H), 4.27, 4.16 (ABq, J = 13 Hz, 2H, -CH2Ph), 3.98 (d, J = 6.4 Hz, 1H, H4), 2.49 (d, J = 16 Hz, 1H, H3β), 2.22 (m, 2H, H1 & H/5), 1.92 (dd, J = 16, 6.6 Hz, 1H, H3α), 1.77 (m, 1H, H6).

13

C NMR

(125 MHz, D2O/KOD, δ): 177.4 (CO), 174.8 (CO), 131.03 (Ph), 129.6 (Ph), 129.5 (Ph), 129.4 (Ph), 65.74 (C2), 59.52 (C4), 48.69 (CH2Ph), 32.58 (C3), 32.20 (C1/5), 28.35 (C1/5), 23.65 (C6). HRMS ESI (m/z): calcd. for C15H18N2O4 [M+H]+: 291.1345. Found: 291.1345. (1S,2S,4S,5R,6S)-2-Amino-4-benzamido-bicyclo[3.1.0]hexane-2,6-dicarboxylic

acid

(8b). Fully protected amino acid 7b (190 mg, 0.368 mmol) was deprotected according to general procedure B giving 8b (81 mg, 72%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.71 (d, J = 7.3 Hz, 2H, ortho), 7.54 (t, J = 7.1 Hz, 1H, para), 7.46 (t, J = 7.1 Hz, 2H, meta), 4.51 (d, J = 7.1 Hz, 1H, H4), 1.99-1.95 (m, 2H, H3β & H1/5), 1.83 (dd, J = 5.8, 2.9 Hz 1H, H1/5), 1.60 (dd, J = 15, 7.1 Hz, 1H, H3α), 1.52 (t, J = 2.9 Hz, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 183.0

(CO) 180.8 (CO), 168.5 (CO), 133.4 (Ph), 132.2 (Ph), 128.9 (Ph), 126.8 (Ph), 66.06 (C2), 52.28 (C4), 40.31 (C3), 37.11 (C1/5), 32.31 (C1/5), 23.74 (C6).

HRMS ESI (m/z):

calcd. for

C15H16N2O5 [M+H]+: 305.1137. Found: 305.1129. (1S,2S,4S,5R,6S)-2-Amino-4-(cyclohexanecarbonylamino)bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8c). Fully protected amino acid 7c (270 mg, 0.517 mmol) was deprotected


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according to general procedure B giving 8c (158 mg, 98.6%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 4.27 (d, J = 7.2 Hz, 1H, H4), 2.07 (m, 1H, H1/5), 1.92 (dd, J = 5.9, 3.0 Hz, 1H, H1/5), 1.81 (d, J = 15 Hz, 1H, H3β), 1.74 (m, 2H, cHex), 1.67 (m, 3H, cHex), 1.56 (m, 1H, cHex), 1.49 (dd, J = 15, 7.1 Hz, 1H, H3α), 1.42 (t, J = 2.7 Hz, 1H, H6), 1.27-1.06 (m, 5H, cHex). 13

C NMR (125 MHz, D2O/KOD, δ): 183.0 (CO), 180.8 (CO), 178.4 (CO), 65.90 (C2), 51.51

(C4), 45.16 (C1/5), 40.59 (C3), 36.99 (C1/5), 32.25 (cHex methine), 29.04, 29.00, 25.30, 25.18, 23.70 (C6). HRMS ESI (m/z): calcd. for C15H22N2O5 [M+H]+: 311.1607. Found: 33.1606. (1S,2S,4S,5R,6S)-2-Amino-4-[(2-phenylacetyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8d). Fully protected amino acid 7d (320 mg, 0.603 mmol) was deprotected according to general procedure B. The material was purified on C18 eluting with water. The purified product was concentrated and dried under vacuum at 50 oC giving 8d (97 mg, 51%) as a white solid. MS (ES+) 319 [M+H]+.

1

H NMR (400 MHz, D2O/KOD, δ): 7.35-7.24 (m, 5H),

4.26 (d, J = 7.1 Hz, 1H, H4), 3.49 (s, 2H, -CH2Ph), 1.94 (dd, J = 5.7, 2.8 Hz, 1H, H1/5), 1.83 (d, J = 15 Hz, 1H, H3β), 1.70 (m, 1H, H1/5), 1.49 (dd, J = 15, 7.1 Hz, 1H, H3α), 1.42 (t, J = 2.8 Hz, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 182.8 (CO), 180.8 (CO), 173.0 (CO), 135.0 (Ph), 129.1

(Ph), 129.0 (Ph), 127.3 (Ph), 65.85 (C2), 52.02 (C4), 42.90 (CH2Ph), 40.61 (C3), 36.91 (C1/5), 32.02 (C1/5), 22.72 (C6). HRMS ESI (m/z): calcd. for C16H18N2O5 [M+H]+: 319.1294. Found: 319.1286. (1S,2S,4S,5R,6S)-2-Amino-4-(3-phenylpropanoylamino)bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8e). Fully protected amino acid 7e (275 mg, 0.505 mmol) was deprotected according to general procedure B giving 8e (132 mg, 78.7%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 7.27-7.14 (m, 5H), 4.12 (d, J = 7.2 Hz, 1H, H4), 2.82 (t, J = 7.2 Hz, 2H,



Page 62 of 94

NC(O)CH2-), 2.43 (t, J = 7.2 Hz, 2H, -CH2Ph), 1.82 (dd, J = 5.8, 3.1 Hz, 1H, H1/5), 1.59 (d, J = 15 Hz, 1H, H3β), 1.47 (dd, J = 5.5, 2.5 Hz, 1H, H1/5), 1.36 (dd, J = 15, 7.3 Hz, 1H, H3α), 1.33 (t, J 13

= 2.8 Hz, 1H, H6).

C NMR (125 MHz, D2O/KOD, δ): 182.7 (CO), 180.7 (CO), 173.8 (CO),

140.3 (Ph), 128.7 (Ph), 128.4 (Ph), 126.4 (Ph), 65.78 (C2), 51.54 (C4), 40.41 (C3), 37.87 (CH2CH2Ph), 36.78 (C1/5), 32.00 (C1/5), 31.20 (CH2CH2Ph), 23.64 (C6). HRMS ESI (m/z): calcd. for C17H20N2O5 [M+H]+: 333.1450. Found: 333.1442. (1S,2S,4S,5R,6S)-2-Amino-4-[(2-fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8f). Fully protected amino acid 7f (210 mg, 0.393 mmol) was deprotected according to general procedure B giving 8f (118 mg, 93.2%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 7.68 (dt, J = 7.7, 1.6 Hz, 1H), 7.50 (m, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.18 (dd, J = 12, 8.4 Hz, 1H), 4.54 (d, J = 7.3 Hz, 1H, H4), 1.98-1.95 (m, 2H, H3β & H1/5), 1.83 (m, 1H, H1/5), 1.59 (dd, J = 15, 7.5 Hz, 1H, H3α), 1.49 (t, J = 3.1 Hz, 1H, H6).

13

C NMR (125 MHz,

D2O/KOD, δ): 182.9 (CO), 180.8 (CO), 165.1 (CO), 160.0 (d, J = 251 Hz, CF), 133.8 (d, J = 9.5 Hz), 130.0 (d, J = 1.5 Hz), 124.7 (d, J = 3.7 Hz), 121.3 (d, J = 12 Hz), 116.4 (d, J = 23 Hz), 65.98 (C2), 52.39 (C4), 40.41 (C3), 37.28 (C1/5), 32.26 (C1/5), 23.77. HRMS ESI (m/z): calcd. for C15H15FN2O5 [M+H]+: 323.1043. Found: 323.1038. (1S,2S,4S,5R,6S)-2-Amino-4-[(3-fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8g). Fully protected amino acid 7g (230 mg, 0.430 mmol) was deprotected according to general procedure B giving 8g (137 mg, 98.8%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 7.52-7.42 (m, 3H), 7.27 (t, J = 8.0 Hz, 1H), 4.49 (d, J = 7.0 Hz, 1H, H4), 1.99-1.95 (m, 2H, H3β & H1/5), 1.82 (m, 1H, H1/5), 1.60 (dd, J = 15, 7.3 Hz, 1H, H3α), 1.52 (t, J = 2.5 Hz, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 183.0 (CO), 180.8 (CO), 167.1 (CO),


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162.6 (d, J = 243 Hz, CF), 135.8 (d, J = 7.3 Hz), 130.8 (d, J = 7.3 Hz), 122.6 (d, J = 2.9 Hz), 118.9 (d, J = 21 Hz), 113.9 (d, J = 23 Hz), 66.05 (C2), 52.36 (C4), 40.21 (C3), 37.10 (C1/5), 32.22 (C1/5), 23.72. HRMS ESI (m/z): calcd. for C15H15FN2O5 [M+H]+: 323.1043. Found: 323.1038. (1S,2S,4S,5R,6S)-2-Amino-4-[(4-fluorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8h). Fully protected amino acid 7h (230 mg, 0.430 mmol) was deprotected according to general procedure B giving 8h (135 mg, 97.4%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 7.72 (dd, J = 8.7, 5.4 Hz, 2H), 7.16 (t, J = 8.7 Hz, 2H), 4.48 (d, J = 7.2 Hz, 1H, H4), 1.97-1.93 (m, 2H, H3β & H1/5), 1.81 (m, 1H, H1/5), 1.58 (dd, J = 15, 7.3 Hz, 1H, H3α), 1.50 (t, J = 2.8 Hz, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 183.0 (CO), 180.8 (CO), 167.5

(CO), 164.8 (d, J = 249 Hz, CF), 129.8 (d, J = 2.9 Hz), 129.4 (d, J = 9.5 Hz), 115.7 (d, J = 22 Hz), 66.04 (C2), 52.31 (C4), 40.28 (C3), 37.07 (C1/5), 32.27 (C1/5), 23.72. HRMS ESI (m/z): calcd. for C15H15FN2O5 [M+H]+: 323.1043. Found: 323.1038. (1S,2S,4S,5R,6S)-2-Amino-4-[(2-chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8i). Fully protected amino acid 7i (180 mg, 0.327 mmol) was deprotected according to general procedure B giving 8i (94 mg, 85%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.46-7.32 (m, 4H), 4.50 (d, J = 7.0 Hz, 1H, H4), 2.02 -1.97 (m, 2H, H3β & H1/5), 1.88 (m, 1H, H1/5), 1.60 (dd, J = 15, 7.2 Hz, 1H, H3α), 1.51 (t, J = 2.8 Hz, 1H, H6).

13

C NMR

(125 MHz, D2O/KOD, δ): 182.7 (CO), 180.7 (CO), 168.5 (CO), 134.5, 131.7, 130.2, 130.1, 128.5, 127.4, 65.93 (C2), 52.47 (C4), 40.45 (C3), 36.88 (C1/5), 31.92 (C1/5), 23.74. HRMS ESI (m/z): calcd. for C15H15ClN2O5 [M+H]+: 339.0748. Found: 339.0749.



Page 64 of 94

(1S,2S,4S,5R,6S)-2-Amino-4-[(3-chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8j). Fully protected amino acid 7j (175 mg, 0.318 mmol) was deprotected according to general procedure B. The crude material was purified on a 50 g C18 reverse-phase column eluting with a gradient from 100/0 to 40/60 0.1% acetic acid in water/ACN giving 8j (42 mg, 39%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.71 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 4.49 (d, J = 6.8 Hz, 1H, H4), 1.99-1.96 (m, 2H, H3β & H1/5), 1.83 (m, 1H, H1/5), 1.61 (dd, J = 15, 7.1 Hz, 1H, H3α), 1.52 (m, 1H, H6).

13

C

NMR (125 MHz, D2O/KOD, δ): 183.0 (CO), 180.1 (CO), 167.1 (CO), 135.3, 134.2, 131.9, 130.4, 127.0, 125.1, 65.99 (C2), 52.39 (C4), 40.22 (C3), 37.16 (C1/5), 32.25 (C1/5), 23.75. HRMS ESI (m/z): calcd. for C15H15ClN2O5 [M+H]+: 339.0748. Found: 339.0741. (1S,2S,4S,5R,6S)-2-Amino-4-[(4-chlorobenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8k). Fully protected amino acid 7k (300 mg, 0.544 mmol) was deprotected according to general procedure B giving 8k (180 mg, 95.7%) as a white solid.

1

H NMR (400

MHz, D2O/KOD, δ): 7.62 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 4.45 (d, J = 7.1 Hz, 1H, H4), 1.93 (d, J = 15, 1H, H3β), 1.93 (dd, J = 6.2, 2.9 Hz, 1H, H1/5), 1.78 (dd, J = 5.8, 2.9 Hz, 1H, H1/5), 1.56 (dd, J = 15, 7.2 Hz, 1H, H3α), 1.48 (t, J = 3.1 Hz, 1H, H6).

13

C NMR (125 MHz,

D2O/KOD, δ): 183.0 (CO), 180.7 (CO), 167.4 (CO), 137.5, 132.0, 128.9, 128.4, 66.02 (C2), 52.31 (C4), 40.25 (C3), 37.04 (C1/5), 32.22 (C1/5), 23.72.

HRMS ESI (m/z):

calcd. for

C15H15ClN2O5 [M+H]+: 339.0748. Found: 339.0743. (1S,2S,4S,5R,6S)-2-Amino-4-[(2-hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8l). Fully protected amino acid 7l (220 mg, 0.41 mmol) was deprotected according to general procedure C. The product was isolated by isoelectric precipitation by


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dissolving 8l·HCl in water and adjusting the pH to 3. The solid collected via vacuum filtration was dried under vacuum giving 8l (87 mg, 66%) as a white solid.

1

H NMR (400 MHz,

D2O/KOD, δ): 7.67 (d, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.52 (t, J = 7.3 Hz, 1H), 4.42 (d, J = 7.2 Hz, 1H, H4), 2.14-2.10 (m, 2H, H3β & H1/5), 1.89 (m, 1H, H1/5), 1.61 (dd, J = 15, 7.5 Hz, 1H, H3α), 1.45 (m, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ):

182.5 (CO), 181.3 (CO), 169.8 (CO), 169.0, 133.6, 129.6, 122.0, 118.5, 113.4, 65.91 (C2), 52.04 (C4), 43.16 (C3), 36.29 (C1/5), 31.98 (C1/5), 24.18. HRMS ESI (m/z): calcd. for C15H16N2O6 [M+H]+: 321.1087. Found: 321.1084. (1S,2S,4S,5R,6S)-2-Amino-4-[(3-hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8m). Fully protected amino acid 7m (125 mg, 0.235 mmol) was deprotected according to general procedure B giving 8m (74 mg, 98%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.16 (t, J = 8.0 Hz, 1H), 6.86 (s, 1H), 6.85 (d, J = 7.9 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 4.48 (d, J = 6.8 Hz, 1H, H4), 1.98-1.94 (m, 2H, H3β & H1/5), 1.82 (m, 1H, H1/5), 1.59 (dd, J = 15, 6.8 Hz, 1H, H3α), 1.50 (m, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 183.1 (CO),

180.8 (CO), 169.5 (CO), 166.6, 135.0, 130.0, 122.8, 116.8, 112.6, 66.04 (C2), 52.18 (C4), 40.45 (C3), 37.12 (C1/5), 32.36 (C1/5), 23.76. HRMS ESI (m/z): calcd. for C15H16N2O6 [M+H]+: 321.1087. Found: 321.1082. (1S,2S,4S,5R,6S)-2-Amino-4-[(4-hydroxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8n). Fully protected amino acid 7n (415 mg, 0.779 mmol) was deprotected according to general procedure C except the reaction was only heated to 70 oC for 5 h. The crude material was purified according to general procedure D. This did not afford analytically pure material. The partially purified material was further purified on cation exchange according to



Page 66 of 94

general procedure E giving 8n (60 mg, 24%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.53 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 4.48 (d, J = 7.0 Hz, 1H, H4), 1.99-1.94 (m, 2H, H3β & H1/5), 1.81 (m, 1H, H1/5), 1.60 (m, 1H, H3α), 1.51 (m, 1H, H6).

13

C NMR (125 MHz,

D2O/KOD, δ): 180.9 (CO), 171.4 (CO), 168.7, 129.3, 118.8, 118.0, 66.10 (C2), 52.06 (C4), 40.60 (C3), 37.19 (C1/5), 32.63 (C1/5), 23.82. HRMS ESI (m/z): calcd. for C15H16N2O6 [M+H]+: 321.1087. Found: 321.1085. (1S,2S,4S,5R,6S)-2-Amino-4-[(2-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8o). Fully protected amino acid 7o (433 mg, 0.872 mmol) was deprotected according to general procedure C except the reaction was stirred at 75 oC for 2 h then rt for 72 h. An additional 2.5 mL of 4 M HCl in dioxane was added and the reaction heated to 75 ⁰C for an additional 4h.

The reaction was concentrated in vacuo.

The crude material was purified

according to general procedure D giving 8o (230 mg, 68%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.70 (m, 1H), 7.48 (m, 1H), 7.10-7.02 (m, 2H), 4.55 (m, 1H, H4), 3.89 (s, 3H), 2.02 -1.97 (m, 2H, H3β & H1/5), 1.84 (m, 1H, H1/5), 1.62 (m, 1H, H3α), 1.49 (m, 1H, H6).

13

C NMR

(125 MHz, D2O/KOD, δ): 182.8 (CO), 181.0 (CO), 167.2 (CO), 157.3, 133.4, 130.1, 121.4, 120.9, 112.2, 65.96 (C2), 55.90 (OCH3), 52.29 (C4), 41.05 (C3), 37.20 (C1/5), 32.33 (C1/5), 23.91. HRMS ESI (m/z): calcd. for C16H18N2O6 [M+H]+: 335.1243. Found: 335.1240. (1S,2S,4S,5R,6S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8p). Fully protected amino acid 7p (8.9 g, 16 mmol) was deprotected according to general procedure B giving 8p (5.1 g, 94%) as a white solid. [α]D20 -24.64 (c = 1.00, 0.1 N NaOH). 1H NMR (400 MHz, D2O/KOD, δ): 7.40 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.26 (s, 1H), 7.11 (dd, J = 8.3, 2.6 Hz, 1H), 4.50 (d, J = 7.0 Hz, 1H, H4), 3.81 (s, 3H), 2.00 -


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1.96 (m, 2H, H3β & H1/5), 1.83 (m, 1H, H1/5), 1.61 (dd, J = 15, 7.1 Hz, 1H, H3α), 1.53 (t, J = 3.0 Hz, 1H, H6).

13

C NMR (125 MHz, D2O/KOD, δ): 183.0 (CO), 180.8 (CO), 168.0 (CO), 159.1,

135.1, 130.2, 119.4, 118.0, 112.2, 66.06 (C2), 55.23 (OCH3), 52.32 (C4), 40.27 (C3), 37.10 (C1/5), 32.28 (C1/5), 23.74. HRMS ESI (m/z): calcd. for C16H18N2O6 [M+H]+: 335.1243. Found: 335.1240. (1S,2S,4S,5R,6S)-2-Amino-4-[(4-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6dicarboxylic acid (8q). Fully protected amino acid 7q (280 mg, 0.51 mmol) was deprotected according to general procedure C giving and purified according to general procedure D affording 8q (120 mg, 64%) as a white solid. 1H NMR (400 MHz, D2O/KOD, δ): 7.64 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 4.44 (d, J = 6.9 Hz, 1H, H4), 3.76 (s, 3H, OCH3), 1.96-1.90 (m, 2H, H3β & H1/5), 1.78 (m, 1H, H1/5), 1.57 (dd, J = 15, 6.8 Hz, 1H, H3α), 1.48 (m, 1H, H6).

13

C NMR (125

MHz, D2O/KOD, δ): 183.0 (CO), 180.8 (CO), 167.8 (CO), 161.9, 128.8, 125.8, 114.1, 66.10 (C2), 55.48 (OCH3), 52.20 (C4), 40.46 (C3), 37.10 (C1/5), 32.38 (C1/5), 23.76. HRMS ESI (m/z): calcd. for C16H18N2O6 [M+H]+: 335.1243. Found: 335.1237.

Crystal structure of 8p. A prismatic-like specimen of 8p (obtained directly from the material isolated in the experimental above) approximate dimensions 0.020 mm x 0.050 mm x 0.060 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured. A total of 4992 frames were collected. The total exposure time was 27.73 hours. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 11725 reflections to a maximum θ angle of 75.123° (0.80 Å resolution), of which 2981 were independent (average redundancy 3.933, completeness = 99.3%, Rint = 8.67%, Rsig = 7.02%) and 2594 (87.02%) were



greater than 2σ(F2). The final cell constants of a = 19.056(14) Å, b = 11.274(1) Å, c = 7.054(4) Å, β = 100.689(6)°, volume = 1489 (2) Å3, are based upon the refinement of the XYZ-centroids of 6294 reflections above 20 σ(I) with 9.161° < 2θ < 148.6°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.781. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.943 and 0.981. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group C2, with Z = 4 for the formula unit, C16H18N2O6. The final anisotropic full-matrix least-squares refinement on F2 with 263 variables converged at R1 = 4.6%, for the observed data and wR2 = 10.5% for all data. The goodness-of-fit was 1.07. The largest peak in the final difference electron density synthesis was 0.23 e-/Å3 and the largest hole was -0.24 e-/Å3 with an RMS deviation of 0.073 e-/Å3. On the basis of the final model, the calculated density was 1.492 g/cm3 and F(000), 704 e-. Absolute structure parameter69 Flack x = 0.010(192) from 1016 selected quotients.70

Co-crystallization of Human mGlu3 ATD with 8p. Co-crystals of compound 8p and the purified amino terminal domain of hmGlu331 were grown at room temperature using the sittingdrop vapor diffusion method with ~10 mg/mL protein concentration, 5 mM 8p 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 angstrom 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 structure of 8p-hmGlu3 was solved using the separate domains of rat


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mGlu3 as search models in PHASER. The resulting structure was built using Coot, refined using Buster and validated using MolProbity. Biological Methods. Preparation of Cultured Rat Neurons and Measurement of [Ca2+]i using FLIPR. Inhibition of spontaneous Ca2+ oscillations in cortical neurons by 1 and 8p was assessed employing the method described in reference 65.

Briefly, rat cortical neurons (RCN) were prepared from the

excised brains of embryonic day 18 Sprague-Dawley (SD) rats, obtained from Charles River (Margate, UK). Pregnant rats were first anesthetized in a CO2 atmosphere then euthanized by cervical dislocation. Embryos were removed under sterile conditions. The neocortices were prepared according published methods71 with one minor difference, MACS Neurobrew-21 (Miltenyl Biotec GmbH) was used as the growth supplement. Neocortices were plated into 96 well poly-D lysine coated plates (Biocoat, BD Oxford UK), 100 µl at a cell density of 70 X 104 cells per ml and incubated at 37 oC for 7-8 days prior to the experiment (time in culture determined by optimal calcium oscillation frequency).Media was removed and cells were incubated in HEPES buffered tyrodes solution + Ca2+ & Mg2+ (Gibco) and 4 µM Fluo 3-AM/0.05 % pluronic F127 (Invitrogen) for 1 h in the dark. The solution was then replaced with HEPES buffered tyrodes solution minus Ca2+ & Mg2+ (Gibco) plus 2.5 mM CaCl2+

(Mg2+ free buffer)

immediately before transferring the plate onto the FLIPR (Molecular Devices, UK). Compound addition was automated; in a 1 addition protocol, the agonist was added after a baseline read of 300 sec . Fluorescence signals were recorded at 1 sec intervals with a 0.4 sec exposure time. Peak Pro software (Molecular devices) was used to detect peaks in individual wells, and the change in oscillation frequency/amplitude was determined by counting the number/max-min of calcium peaks in a defined time interval (300 sec) after compound addition and comparing to the same



time interval before compound addition (baseline). Data were then normalized to give a % response compared to vehicle control wells. To obtain EC50 and Emax values, curves were fitted to a 4-parameter logistic curve fit model, (two site fit for 8p) using GraphPad Prism software (San Diego, CA). Data from each culture preparation were averaged over 2-6 cell plates and repeated on at least 3 independent preparations. Data represent mean values ± SEM. Determination of 8p in Plasma and Cerebrospinal Fluid Following Subcutaneous Dosing in Rat. 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 8p was prepared by dissolution in water and adjusted to approximately pH 7 with NaOH. 8p was administered by the subcutaneous route at the indicated doses. Blood samples were collected from a femoral artery catheter into EDTA tubes containing phenylmethane sulfonyl fluoride (PMSF) to inhibit postcollection amide hydrolysis, centrifuged, and the plasma stored at -70 ºC until analyzed. Cerebrospinal fluid (csf) samples were collected via syringe from the cisterna magna and also stored at -70 ºC until analyzed. 25 µL aliquots of thawed plasma or csf were mixed with an equal volume of methanol:water (1:1, for csf samples) or plasma and 180 µL of internal standard solution (1:1 acetonitrile:methanol for csf samples or 9:1 acetonitrile:water for plasma samples). After mixing, the samples were centrifuged to pellet the precipitated proteins, and the resulting csf supernatants were diluted 2 fold with water. The plasma supernatants were concentrated and reconstituted in 40 µL of 2% formic acid in water. Ten microliter aliquots were analyzed by LCMS/ MS using two Shimadzu LC-10ADvp pumps units with a SCL-10A controller (Kyoto, Japan), a Gilson 215 liquid handler (Middleton, WI), 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 of csf samples was


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accomplished on a 2.1 mm × 50 mm, 5 µm Betasil C18 Javelin HPLC column (Thermo Electron Corp, Waltham, MA) using a binary step gradient. The mobile phase system for csf samples was composed of Water:TFA:1M NH4HCO3 (2000:8:2, v/v/v; mobile phase A) and Acetonitrile:TFA: 1M NH4HCO3 (2000:8:2, v/v/v; mobile phase B). The step gradient profile changed from 0.1% B at 0.0 min, 10% B at 0.1 to 0.2 min, 35% B at 0.30−0.40 min, and 98% B at 0.41min, and returning to 0.1% B at 0.73 min. Chromatographic separation of plasma samples was accomplished on a 2.1 mm × 50 mm, 5 µm BetaBasic C8 HPLC column (Thermo Electron Corp, Waltham, MA) using a binary step gradient. The mobile phase system for plasma samples was composed of Water:1M NH4HCO3 (2000:10, v/v; mobile phase A) and Acetonitrile (mobile phase B). The step gradient profile changed from 1% B at 0.0 to 0.2 min, 38% B at 0.3 to 0.4 min, and 98% B at 0.41min, and returning to 1% B at 0.61 min. The flow rate was 1.5 mL/min and the column was at ambient temperature, with flow directed to the mass spectrometer between 0.25 and 0.5 min for all analysis. The selected reaction monitoring for Compound 8p (M + H)+ transition was m/z 335.1 > 134.8. Pharmacokinetic parameters were calculated by noncompartmental analysis using Watson 7.4 (Thermo Fischer Scientific). Pharmacokinetics of 8p Employing Osmotic Minipump Infusions in Rat. Male Sprague Dawley rats (Harlan Industries, Indianapolis, IN) had free access to food and water at all times. Compound 8p was prepared by dissolution in water and adjusted to approximately pH 7 with NaOH. The compound was administered at doses of 0.5, 1.5, 5, and 15 mg/kg/day by subcutaneous infusion utilizing an Alzet 2ML1 osmotic pump (Cupertino, CA). Blood was collected from all animals (N=5/dose grp) at 6, 12, 24, 48, and 72 h post dosing via tail clip. CSF was collected from the cisterna magna by syringe at 72 h post dosing and if the CSF sample was visually pink then the CSF and corresponding 72 h blood sample were discarded. Blood was



centrifuged and the plasma and CSF were stored at -70 C until analyzed. Calibration standards were prepared by serial dilution of a 1 mg/mL analyte stock solution (80:20 N-methyl-2pyrrolidone:water) with methanol/water (1:1, v/v) which were then used to fortify control plasma or artificial CSF (Harvard Apparatus, Holliston, MA) to yield analyte concentrations of 2.5, 5, 25, 125, 250, 1250, 5000 ng/mL. A 25 uL aliquot of each study sample, appropriate calibration standard, and control matrix were transferred to a 96-well plate. The study samples, calibration standards, and control matrix sample were then mixed with 180 uL of acetonitrile/methanol (1:1, v/v) containing internal standard. After mixing, the samples were centrifuged to pellet the precipitated proteins, and the resulting supernatants were diluted 2-fold with water. Ten microliter aliquots were analyzed by LC-MS/MS using 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. The analytes were chromatographically separated using a Betasil C18 5µm 20x2.1mm Javelin (Thermo Electron Corp, Waltham, MA). The pumps were Shimadzu LC-10ADvp units with a SCL-10A controller (Kyoto, Japan), and a Gilson 215 liquid handler (Middleton, WI) was used as the autosampler. The mobile phase system was composed of Water/TFA/1M NH4HCO3 (2000:8:2 v/v) (Mobile Phase A) and ACN/TFA/1M NH4HCO3 (2000:8:2 v/v) (Mobile Phase B). The gradient profile changed from 0.1% B at 0 min, 10% B at 0.10 min to 0.20 min, 35% B at 0.30 to 0.40 min, and 98% at 0.41 to 0.72 min. The flow rate was 1.5 mL/min, chromatography was performed at ambient temperature, and the flow was directed to the mass spectrometer between 0.25 and 0.50 min. For compound 8p the selected reaction monitoring (M+H) transition m/z was 335.1 > 134.8. The TurboIonSpray temperature was maintained at 700 ºC, with collision, curtain, nebulizing, and desolvation gas (nitrogen)


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settings of 12, 10, 70, and 50, respectively. The ionspray voltage was set to 1500 V, while the respective declustering, entrance, and exit potentials were 80, 10, and 10.

■ ANIMAL STUDIES All studies involving the use of laboratory animals in the United States 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. All studies involving use of laboratory animals in the United Kingdom were in accordance with the U.K. Animal Scientific Procedures Act of 1986, and all procedures were approved through the British Home Office Inspectorate. Rat Phencyclidine (PCP)-Induced and Spontaneous Locomotor Activity Naïve, male Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) weighing 200-250 g were used in both the PCP-induced and spontaneous locomotor activity studies. Rats were free-fed and tested during the light phase of their circadian cycle. For the PCP blockade study, rats received a s.c. administration of vehicle, 1, 3, or 10 mg/kg 8p (N=8/group) and then placed in the locomotor test arena (Kinder Scientific, Poway, CA) for 30 min prior to s.c. administration of 5 mg/kg PCP. Locomotor activity was assessed for 60 minutes. For the spontaneous locomotor study, rats received vehicle, 3, 10, or 30 mg/kg 8p (N=8/group) and placed in the test arena. Activity was recorded for 90 minutes; total activity after 30 min and 90 min were separately analyzed using ANOVA and a Dunnett Multiple Comparison Test (alpha set at 0.05).

■ ASSOCIATED CONTENT Supporting Information



Molecular formula strings (CSV) Amino acid differences in hmGlu2 and hmGlu3 proximal to the bound agonist 1 (Figure S1) Extended selectivity assessment for compound 8p (Table S1) Binding affinity and functional agonist potency data for previously published agonists possessing the aminobicyclo[3.1.0]hexane dicarboxylate substructure and methodology used to provide 95% confidence intervals in order to define statistical differences in selectivity ratios between compounds are included (Table S2) Determination of confidence intervals for potency ratios (page S6)


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Accession Codes hmGlu3 ATD with LY2794193 (8p), (PDB code: 6B7H).

Authors will release the atomic

coordinates and experimental data upon article publication

■ AUTHOR INFORMATION Corresponding Author *Phone: 317-276-9101. E-mail: [email protected]

Notes All authors were employed by Eli Lilly and Company during time this research was conducted, and all funding for the work was provided by Eli Lilly and Company. The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS The authors gratefully acknowledge Manuel Molina for generating capillary electrophoresis data used in the determination of compound purity; This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC0206CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility.




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■ ABBREVIATIONS USED SLE, systemic lupus erythematosus; ATD, amino terminal domain; cAMP, 3-5-cyclic adenosine monophosphate; FLIPR, fluorescence imaging plate reader; mGlu, metabotropic glutamate; PCP, 1-phenylcyclohexylpiperidine

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Presented

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52nd

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Meeting

Hollywood, FL, Dec 8-12, 2013 (c) Liu, W.; Downing, A. C. M.; Munsie, L. M.; Chen, P.; Reed, M. R.; Ruble, C. L.; Landschulz, K. T.; Kinon, B. J.; Nisenbaum, L. K. Pharmacogenetic analysis of the mGlu2/3 agonist LY2140023 monohydrate in the treatment of schizophrenia. Pharmacogenomics J. 2012, 12, 246-254. (d) Kinon, B. J.;



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Schoepp, D. D.

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Synthesis and Pharmacological Characterization of C4Î²-Amide

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