Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor

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DOI: 10.1002/cmdc.201402190

Exploring the Active Conformation of Cyclohexane

Carboxylate Positive Allosteric Modulators of the Type 4

Metabotropic Glutamate Receptor

Xavier Rovira,^{[b, c]}Youssef Harrak,^[a]Ana Trapero,^[a]Patricia Gonzꢀlez-Bulnes,^[a]

Fanny Malhaire,^[c]Jean-Philippe Pin,^[c]Cyril Goudet,^[c]Jesﬄs Giraldo,*^[b]and

Amadeu Llebaria*^[a]

The active conformation of a family of metabotropic glutamate

receptor subtype 4 (mGlu₄) positive allosteric modulators

(PAMs) with the cyclohexane 1,2-dicarboxylic scaffold present

tested in mGlu₄pharmacological assays. The results support

a VU0155041 active conformation, with the chair cyclohexane

having the aromatic amide substituent in an axial position and

the carboxylate in an equatorial position. Moreover, the recep-

tor displays enantiomeric discrimination of the chiral PAMs.

The constructed pharmacophore characterized a highly con-

strained mGlu₄allosteric binding site, thus providing a step for-

ward in structure-based drug design for mGlu₄PAMs.

in

cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic

acid (VU0155041) was investigated by testing structurally simi-

lar six-membered ring compounds that have a locked confor-

mation. The norbornane and cyclohexane molecules designed

as mGlu₄conformational probes and the enantiomers of the

trans diastereomer were computationally characterized and

Introduction

G protein-coupled receptors (GPCRs) represent the largest

family of cell-surface receptors. They recognize diverse messag-

es and transduce them into cellular responses through both

G protein-dependent and -independent pathways.^[1]These re-

ceptors constitute a valuable class of targets in therapeutic

drug development. Indeed, about 36% of all drugs approved

by the US Food and Drug Administration during the past three

decades target GPCRs.^[2]

induce a lower level of receptor densensitization, downregula-

tion, and internalization, resulting in decreased side effects.^[3,4]

However, orthosteric ligands also have their own therapeutic

advantages, such as better solubility, reduced interaction with

other brain proteins, and no turnover by CYP450.^[5]

Metabotropic glutamate (mGlu) receptors are a family of re-

ceptors belonging to class C GPCRs, for which research into al-

losteric compounds is particularly active.^[6]There are eight

mGlu receptors assembled into three groups (I, II, III). The

mGlu₄receptor, which belongs to group III, has recently at-

tracted much attention due to its involvement in several path-

ologies, such as Parkinson’s disease and chronic pain.^[7]There

are increasing evidences showing that orthosteric activation

and/or positive allosteric modulation of the mGlu₄receptor

may have positive therapeutic effects on these diseases.^[8]The

first selective mGlu₄PAM described was (À)-PHCCC.^[9]Some

years later, the Vanderbilt compound VU0155041 (rac-2,

Figure 1), the cis diastereomer of VU0003423 (Figure 1), was re-

ported as a selective mGlu₄PAM with a novel structure.^[10]

However, in spite of the good potency and selectivity of some

of its derivatives, efforts to improve the potency of

VU0155041, either by changing the size of the aliphatic ring or

the substituents, were of low success,^[11]suggesting that

VU0155041 accepts few, if any, structural changes. One of

these, the structurally similar cis-carboxamide Lu AF21934

(Figure 1) has been described as having improved PAM proper-

ties for mGlu₄pharmacology and in vivo activity.^[12]

In recent years, drug research on GPCRs has focused largely

on allosteric compounds.^[3]Allosteric modulators, either posi-

tive (PAMs) or negative (NAMs), have several advantages over

molecules competing with natural agonists for binding at or-

thosteric sites in the receptors. Allosteric ligands display high

subtype selectivity, modulate physiological conditions, and

[a] Dr. Y. Harrak, Dr. A. Trapero, Dr. P. Gonzꢀlez-Bulnes, Dr. A. Llebaria

Laboratory of Medicinal Chemistry, Department of Biomedicinal Chemistry

Institut de Quꢁmica AvanÅada de Catalunya (IQAC-CSIC)

Jordi Girona 18-26, 08034 Barcelona (Spain)

E-mail: amadeu.llebaria@cid.csic.es

[b] Dr. X. Rovira, Dr. J. Giraldo

Laboratory of Molecular Neuropharmacology & Bioinformatics

Institut de Neurociꢂncies and Unitat de Bioestadꢁstica

Universitat Autꢃnoma de Barcelona, 08193 Bellaterra (Spain)

E-mail: jesus.giraldo@uab.es

[c] Dr. X. Rovira, F. Malhaire, Dr. J.-P. Pin, Dr. C. Goudet

Institut de Gꢄnomique Fonctionnelle

CNRS UMR5203, Universitꢄ de Montpellier

and

With the aim of understanding the structural determinants

that make the VU0155041 molecular structure particularly suit-

able for PAM action, we found that, contrary to what was origi-

U661, INSERM, 141 Rue de la Cardonille, 34094 Montpellier (France)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201402190.

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interactions between the substituents and ring hydrogen

atoms. In the mGlu₄PAM trans-cyclohexane 3, we found that

diaxial conformer was energetically disfavored by 6.96 kcal

mol^{À1 [13]}

In contrast, the cis-1,2-cyclohexane arrangement de-

.

fines two chair conformers having one substituent in an axial

position and the other in an equatorial position. In our previ-

ous report,^[13]we found a low difference in energy (~0.9 kcal

mol^À1) between the two conformations of the compound

tested, both under vacuum and in the hydrophobic and hydro-

philic condensed media, which gives both conformations a sim-

ilar probability to compete for the receptor binding site. In the

present work, we studied some of the structural features of

the cyclohexane carboxylate PAMs binding to the mGlu₄recep-

tor by using different probe molecules. On one hand, we were

interested in the confirmation of the enantioselectivity in the

binding of this family of compounds by studying the activity

of the enantiomers of the weaker mGlu₄PAM trans-cyclohex-

ane 3 (Figure 1). On the other hand, we focused on an interest-

ing unresolved question related to the preferred active confor-

mation of the cis-cyclohexane 2 PAMs to the mGlu₄receptor,

which we addressed by testing cyclohexane molecules in

which the position of one substituent was frozen in an equato-

rial position while the other adopted an axial arrangement. A

differential activity in the mGlu₄receptor of these probe mole-

cules indicated the active conformation in (1R,2S)-2 binding to

this receptor. This information was employed to construct

a pharmacophore that may be useful both for virtual screening

and the characterization of the mGlu₄allosteric binding site,

thus providing a step forward in structure-based drug design

for mGlu₄PAMs.

Figure 1. Cyclohexane 1,2-dicarboxylic derivatives with positive allosteric

mGlu₄activity.

nally reported,^[10]the two VU0155041 enantiomers differed in

pharmacological activity, with (1R,2S)-2 being more potent

than (1S,2R)-2.^[13]In addition, two conformations for each of

the cis-VU0155041 and trans-VU0155040 diastereomers were

characterized by high-level quantum mechanical (QM) calcula- Results

tions. The enantioselective nature of recognition of this family

Compound design

in the mGlu₄receptor was consistently confirmed with the

structurally related cis-1,2-cyclohexanediamides,^[14]with the

(1S,2R) Lu AF21934 enantiomer more active than the (1R,2S)

Lu AF21935 enantiomer.^[12]

We envisaged two possible ways to address the question of

whether the active conformation of 2 could be determined by

obtaining and testing six-membered ring molecules to be used

as conformational probes. We used norbornane and cyclohex-

ene scaffolds with one of the substituents frozen in equatorial

position and the other forced to adopt an axial arrangement.

In the first approach, we proposed locking the cyclohexane

cis substituents by using a norbornane-type bicyclic scaffold

that defines a rigid arrangement of 1,2-substituents with the

endo position perpendicular to the cyclohexane plane and the

exo substituent located approximately in the cyclohexane

plane (Figure 2). The axial–equatorial arrangement found in

disubstituted cis-1,2-chair cyclohexanes, such as 2, is better

mimicked by one endo and one exo substituent in the bicyclic

scaffold. The main differences with active compound 2 are the

presence of a methano-1,4 carbon bridge, which forces the cy-

clohexane ring to adopt a skew-boat conformation, different

from the most stable cyclohexane chair conformation. Thus,

compound 4, with the dichlorophenylamide in an endo posi-

tion and the carboxylate in the exo position, would mimic the

1e2a cyclohexane conformer with an axial amide and equatori-

al carboxylate in (1R, 2S)-2. On the contrary, in carboxylic acid

Other potent and selective mGlu₄PAMs have been recently

[15]

described

that are structurally unrelated to the scaffold

present in VU0155041. However, the rich conformational flexi-

bility of the cyclohexane ring and the VU0155041 enantiomer

distinction at the receptor represent an attractive platform to

study the structural and functional features of allosteric poten-

tiation at the mGlu₄receptor. In this context, the different ac-

tivities between the cis-rac-2 (EC₅₀: 2.8 mm) and trans-rac-3

(EC₅₀: 22.0 mm) diastereomers, and the differential activity

found for the enantiomers^[13]of 2 (1R,2S)-2 (EC₅₀: 0.8 mm) and

(1S,2R)-2 (EC₅₀>100 mm) appear to define a very specific inter-

action of these ligands with the mGlu₄receptor. The presence

of 1,2-disubstitution in these compounds delineates different

conformations which, in the case of the more stable chair con-

formers, can place the groups in either axial or equatorial ar-

rangement. In the case of trans-1,2-substituted cyclohexanes,

the chair conformation having both substituents in equatorial

position is largely favored over the 1,2-diaxial conformation,

due to the destabilizing effects of the 1,3-diaxial nonbonding

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stituents at the carbon adjacent to the double bond in the

axial position is preferred (Figure 3). This minimizes the interac-

tion between allylic and double bond substituents, in a proto-

typical example of the 1,2-allylic strain effect (A^(1,2)strain).^[17]

Based on this stereochemical preference due to A^(1,2)strain, cy-

clohexenes 6 and 7 were proposed as conformational probes

to distinguish between the two 1e2a and 1a2e chair conforma-

tions of cis-1,2-cyclohexane 2, respectively. If A^(1,2)strain is op-

erating in cyclohexene 6, it is expected to result in the pre-

ferred conformation, with a pseudoaxial carboxamide and a car-

boxylate bonded to an olefinic carbon in the plane of the six-

membered ring, similar to an equatorial position in cyclohex-

ane. In an opposite fashion, in cyclohexene 7, the carboxylate

is favored in an axial position, whereas the amide is forced to

be in the plane of the six-membered ring by bonding to the

double bond. Finally, the enantiomers of trans compounds 3

could be mimicked by achiral cyclohexene compound 8 and o-

phthalamide 9, which display both substituents in the ring

plane as a locked approximation of the di-equatorial conforma-

tions of trans-3 (Figure 3).

Figure 2. Norbornane derivatives 4 and 5, designed as conformational

probes for mGlu₄PAM cyclohexane carboxylic 2.

compound 5, the positions of the substituents were ex-

changed, and the resulting structure was similar to the 1a2e

conformer of 2, having an axial carboxylate and an equatorial

amide.

Synthesis

As the structural constraints imposed by the rigid bicyclic

scaffolds in 4 and 5 are of relevance and could preclude

a clear definition of the active conformation of 2, a second set

of conformationally defined six-membered ring molecules was

envisaged that was considered less intrusive than the expected

cyclohexane chair active conformation present in 1. The design

of these conformational probe molecules was based on the

well-known allylic strain effect, which is observed in double-

bond-containing molecules and defines a specific conforma-

tional selection, due to steric interactions between the olefin

substituents.^[16]This is especially well-studied in allylic-substi-

tuted cyclohexenes, where the ring conformation placing sub-

The bicyclic norbornane analogues were obtained from the

corresponding anhydrides following the sequence of reactions

described in Scheme 1. This consisted of the formation of endo

cis-monoester (Æ)-11 from anhydride (Æ)-10, which was selec-

tively epimerized^[18]at the ester carbon atom, followed by

amide coupling with the corresponding dichloroaniline to give

norbornene derivative (Æ)-13, which was subjected to ester hy-

drolysis to give (Æ)-14, followed by double bond hydrogena-

tion to give norbornane (Æ)-4. This compound displays an exo

carboxylate group and an endo carboxamide, as required. In

a similar sequence, the endo–endo benzyl ester (Æ)-15 was epi-

merized to norbornene (Æ)-16 and esterified to methyl benzyl

Figure 3. Conformational probes based on cyclohexene and aromatic analogues of mGlu₄PAMs 2 and 3.

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of cyclohexene (Æ)-7, we used

the monomethyl ester of the 2-

cyclohexene-1,2-dicarboxylic

acid (Æ)-25.^[20]After coupling

with 3,5-dichloroaniline, we ob-

tained the corresponding amido

ester (Æ)-26, which was not iso-

lated but instead was directly

hydrolyzed to the final carboxyl-

ic cyclohexene (Æ)-7. Finally,

achiral compounds 8 and 9 were

obtained from amidation of an-

hydrides 21 and 24, respectively.

Pharmacology

The functional activity of all

compounds was determined for

mGlu₄receptors transiently ex-

pressed in HEK293 cells. The pro-

duction of second messenger in-

ositol monophosphate (IP1) was

measured and correlated to re-

ceptor activation, as described in

the Experimental Section. Dose–

response curves were generated

in the presence of 10 nm of ago-

nist l-AP4 for each compound,

and their respective EC₅₀values

Scheme 1. Synthesis of norbornane derivatives. Reagents and conditions: a) MeOH, reflux, 16 h, 100%; b) NaOMe,

MeOH, reflux, 81%; c) 3,5-dichloroaniline, HATU, DIPEA, EtOAc, 508C, 67–83%; d) LiOH, THF/H₂O, RT, 61–88%;

e) Rh/C, THF, H₂(1 atm), 87%; f) BnOH, toluene, 708C, 90%; g) LDA, THF, À788C, 82%; h) MeI, K₂CO₃, THF, RT, 73%;

i) Pd/C, THF, H₂(2 atm), 94%.

diester (Æ)-17. Selective benzyl ester deprotection by catalytic

were estimated by fitting the data with the Hill equation. Data

are summarized in Table 1.

hydrogenation with concomitant double bond reduction pro-

duced the exo-carboxylic acid

norbornane (Æ)-18, which was

reacted with 3,5-dichloroaniline

to give (Æ)-19. After methyl

ester deprotection, norbornane

derivative (Æ)-5 was obtained,

having endo carboxylic and exo

amide functionalities, as re-

quired.

Compounds having a single

cyclohexane ring were mostly

prepared by reaction of the cor-

responding cyclic dicarboxylic

anhydrides with 3,5-dichloroani-

line (Scheme 2). In this way, the

enantiomers of the trans-cyclo-

hexane compounds (2) could be

readily obtained from the enan-

tiomeric C₂anhydrides (20)

which, in turn, were prepared

from the commercially available

diacids. Cyclohexene compound

(Æ)-6 was synthesized in the rac-

emic series from selective open-

Scheme 2. Synthesis of cyclohexane and cyclohexene derivatives. Reagents and conditions: a) 3,5-dichloroaniline,

THF, reflux, 60% for (1S,2S)-3, 60% for (1R,2R)-3, 63% for 8, 55% for 9, 41% for (Æ)-6; b) 3,5-dichloroaniline, Cl-

ing of anhydride (Æ)-23.^[19]On

the other hand, for the synthesis BOP, CH₂Cl₂; c) LiOH, THF/H2O, RT, 67%, two steps.

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Table 1. Activity of mGlu₄positive allosteric modulators (PAMs).

Compd

rac-2

rac-3

(1S,2S)-3

(1R,2R)-3

rac-4

EC₅₀[mm]^[a]

n

Compd

EC₅₀[mm]^[a]

n

1.3Æ0.1

14.5Æ3.6

7.9Æ1.8

16

4

3

rac-5

rac-6

rac-7

8

56.9Æ14.5

4.2Æ1.2

3

33.9Æ8.5

>100

12.2Æ2.6

9

>100

[a] Values correspond to the dose–response curves shown in Figure 4;

data represent the meanÆSEM of n independent experiments; where

EC₅₀>100 mm, the SEM was not calculated.

First, we tested the pharmacological activity of the probe

molecules, in which the position of one cyclohexane substitu-

ent was frozen in equatorial position, either by the norbornane

strategy that gave rise to compounds 4 and 5 or by the

double bond strategy that led to compounds 6–9. All com-

pounds were considered as PAMs, as they potentiated the

action of l-AP4 on mGlu₄receptor in a dose-dependent

manner (Figure 4). However, a difference in terms of potency

was noticeable in some cases (Table 1). The EC₅₀value of refer-

ence compound (Æ)-2 was found to be 1.3 mm, similar to what

was previously reported.^[10]The potency of both norbornane

derivatives–compounds (Æ)-4 and (Æ)-5–was decreased by 9-

and 44-fold, respectively, relative to compound (Æ)-2. With

regard to the double bonded derivatives, compound

6

showed a potency value of 4 mm and was thus within the low

micromolar range, similar to reference molecule (Æ)-2, while

the potency of the two other ligands was found to be signifi-

cantly decreased, resulting in an EC₅₀value of 34 mm for (Æ)-7

and more than 100 mm for compounds (Æ)-8 and the aromatic

derivative 9. With regard to the basal or maximal activation of

the dose–response curve, no major differences were observed

except for 8 and 9, for which the higher asymptote was not

determined, due to low potency and solubility issues. Thus,

each of the trans enantiomers displayed different pharmaco-

logical activity, with (1S, 2S)-3 being more active (EC₅₀: 8 mm)

than the racemic solution, and (1R, 2R)-3 being much less

active (EC₅₀>100 mm), similar to what was previously observed

for the two cis enantiomers.^[13]

Figure 4. PAM activities of norbornane and double-bonded derivatives on

the mGlu₄receptor in HEK293 cells. A) Normalized activity comparison of

racemic cis-(Æ)-2, trans-(Æ)-3, and norbornane compounds (Æ)-4 and (Æ)-5

as mGlu₄PAMs. B) Normalized activity comparison of racemic cis-(Æ)-2,

trans-(Æ)-3, and the enantiomers of trans-(Æ)-3 as mGlu₄PAMs. C) Normal-

ized activity comparison of the mGlu₄PAM activity of racemic cis-(Æ)-2 with

cyclohexene [(Æ)-6, (Æ)-7, and 8] as well as aromatic (9) probe compounds.

for both compounds. Axial and equatorial orientations for the

amide and carboxylate substituents, respectively, for com-

pound 4, and in reverse order for compound 5, were found. A

Conformational analysis

The geometries of all the com-

pounds were optimized by den-

sity functional theory (DFT) at

the level detailed in the Experi-

mental Section.

Norbornenes

Optimized structures of bicyclic

compounds 4 and 5 are shown

in Figure 5. Conformations re-

sembling skew-boats for the six-

Figure 5. Optimized conformations for norbornane compounds A) (1R,2S,3S,4S)-3-((3,5-

dichlorophenyl)carbamoyl)bicyclo[2.2.1]heptane-2-carboxylic acid 4 and B) (1R,2R,3R,4S)-3-((3,5-

dichlorophenyl)carbamoyl)bicyclo[2.2.1]heptane-2-carboxylic acid 5. The enantiomers shown correspond to the

membered ring were obtained same substituent arrangement as reported for the (1R, 2S)-2 cis-active enantiomer.

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slight distortion of the ring geo-

metries from symmetry, which

was produced by an internal hy-

drogen bond between carboxyl-

ate and amide groups, was ob-

served. The planarity between

the central amide and the

phenyl ring was preserved. The

higher potency observed for

compound (Æ)-4, relative to

compound (Æ)-5 (Table 1), sug-

gests that the ligand–receptor

recognition process is better

suited for the axial aryl amide

and the equatorial carboxylate

than for those substituents in

the reverse order. As a conse-

quence, these findings indicate

that, of the two conformations

of cis compound 2 examined as

potential active conformations,

Figure 6. Optimized conformations for compounds A) (2S)-6-((3,5-dichlorophenyl)carbamoyl)cyclohex-1-enecarbox-

ylic acid 6, B) (1R)-2-((3,5-dichlorophenyl)carbamoyl)cyclohex-2-enecarboxylic acid 7, and C) 2-((3,5-dichlorophe-

nyl)carbamoyl)cyclohex-1-enecarboxylic acid 8. The enantiomers shown correspond to the same substituent ar-

rangement as reported for the (1R,2S)-2 cis-active enantiomer.

the chair conformation with the substituent arrangement pres-

ent in compound 4 is the most likely active conformation.

The internal hydrogen bond that is present in all of the com-

pounds analyzed (see below) fixes the carboxylate and aryl

amide moieties in a particular relative orientation whose ampli-

tude depends on the cyclohexane conformation. Considering

the very small size of the molecules studied and the existing

restrictions in the conformational flexibility of the substituents,

the relative orientation between the two cyclohexane substitu-

ents may represent a structural determinant of pharmacologi-

cal activity. Using the dihedral angle, the ends of which are the

carbons of the carbonyl groups (henceforth, a), as a measure

of the relative orientation between the carboxylate and the

aryl amide, a value of 95.28 was obtained for the putative

active conformation of compound 4 (equatorial carboxylate

and axial aryl amide).

These calculations confirmed that, according to our expecta-

tions based on the A^(1,2)strain effect, the allylic substituents in

the cyclohexene predominantly adopt an axial arrangement

and therefore can be considered bona fide conformational

probes for the two possible chair binding modes of cis-2. Final-

ly, a value of 78.68 was obtained for dihedral angle a (see defi-

nition in norbornanes section) for compound 6 under its pref-

erential conformation with the aryl amide group in an axial po-

sition.

An intermediate conformation for trans compound 3 as

a possible PAM conformational candidate

Pharmacologic experiments have shown that enantiomers of 3

differ in potency, with (1S,2S)-3 being more potent than

(1R,2R)-3. Two conformations were previously reported, with

1e2e (chair-like) being ~7 kcalmol^À1more stable than 1a2a

(half chair-like).^[13]However, it is known that a cyclohexane ring

can additionally adopt non-chair-like conformations,^[21]thereby

providing more candidate structures for receptor selection. To

examine whether cyclohexane flexibility could allow the trans

compound to adopt a conformation partly resembling that of

the active cis, we focused our attention on norbornane com-

pound 4, considering that the similarity between activities of

compounds 3 and 4 (Table 1) could be a reflection of the simi-

larity between their structures. The skew-boat conformation

obtained for norbornanes (Figure 5) has been characterized as

a secondary minimum between chair conformations in cyclo-

hexane and pyranosyl sugars by density functional studies,^[21]

thus suggesting that it is structurally and energetically attaina-

ble by trans compound 3. To obtain a conformational structure

for trans-3 resembling that of compound 4, the bridge methyl-

ene group was removed from norbornanes 4, and the resulting

structure was optimized (Figure 7). The cyclohexane ring

adopted a skew-boat conformation with the carboxylate group

and the aryl amide in pseudoequatorial and pseudoaxial posi-

Cyclohexenes

To computationally assess whether the conformation with axial

substituents in the allylic carbon adjacent to the double bond

was preferred in cyclohexenes 6 and 7, relative to the equatori-

al orientation, we calculated the differences in energy between

axial and equatorial substituent optimized conformations. The

calculations were done both in gas phase and in a continuum

model of water and produced the same trends, with the con-

formation with the axial substituent more stable both for com-

pounds 6 and 7 (Table S1, Supporting Information). In all con-

formations, the internal hydrogen bond between the carboxyl-

ate group and the nitrogen of the central amide was present.

Additionally, the dichlorophenyl ring and the amide group

formed a plane. Interestingly, the cyclohexene ring displayed

conformations in between skew-boat and half-chair of aliphatic

cyclohexane, due to the presence of the double bond

(Figure 6).

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does a carboxylate, and an additional structure with a hydrogen

bond between the NH₂of the cyclohexyl amide and the car-

bonyl of the central amide was found for each of the chair

conformations of the cyclohexyl ring (Figure 8). The conforma-

tion in panel A mimics the conformation considered active for

compound 2, displaying a value of 61.68 for dihedral angle a.

Figure 9A displays a plot of PAM potency of the compounds

analyzed in the study versus the dihedral angle a. The plot dis-

tinguishes three regions: I (compounds with carboxylate or

amide (Lu AF21934) in equatorial and arylamide in axial posi-

tions), II (compounds with both cyclohexyl substituents in

equatorial positions), and III (compounds with carboxylate in

axial and arylamide in equatorial positions). Interestingly,

a linear correlation (R=0.993 P<0.001) is found for com-

pounds in region I (the conformation we propose as the active

one), suggesting that potency depends on the fitting of cyclo-

hexyl substituents to the receptor binding site. Figure 9A also

includes the 1S,2R Lu AF21934 enantiomer to provide an exter-

nal confirmation of the enantioselective selection of the recep-

tor. The mGlu₄EC₅₀value for this compound was taken from

literature^[12]and was corrected relative to our data set. Howev-

er, this value is favored, relative to the other compounds of re-

gion I of the plot, because this corresponds to a single enan-

tiomer, whereas the other compounds of region I are all race-

mic.

Figure 7. Optimized skew-boat conformation for the (1S,2S)-3 trans-active

enantiomer.

tions, respectively. An internal hydrogen bond between the

carboxylate and amide groups was observed, as well as the

planarity of the aryl amide moiety. The conformation was

3.37 kcalmol^À1above the most stable diequatorial chair, and

could be considered to be of intermediate energy between

the diequatorial and diaxial half-chair conformations (6.96 kcal

mol^À1above the diequatorial conformation) that have been

previously determined.^[13]Finally, a value of 92.68, very close to

the one found for norbornane compound 4, was obtained for

dihedral angle a (see definition in norbornanes section) for

compound 3 in its secondary minimum state with cyclohexane,

displaying a skew-boat conformation. It can be reasonably hy-

pothesized that the similarity in pharmacological activities

found between compounds 3 and 4 could derive from their

similarities in substituent relative orientations.

In the conformational analysis we performed, we focused

our attention on the mutual orientation between substituents

of the cyclohexyl ring (1e2a, 1e2e, and 1a2e) and in the puck-

ered arrangement of the ring, for which chair, half-chair, and

skew-boat conformations have been obtained. However,

Active conformations and phar-

macophore modeling

In the present study, a collection

of

different

conformational

probe compounds was synthe-

sized and pharmacologically and

computationally characterized.

These molecules structurally

comprise a six-membered ring

(either

aliphatic,

including

a

double bond, or aromatic)

bearing 1,2-substitution with

a carboxylate and a dichloro-

substituted arylamide. In addi-

tion, and for comparison, the

conformational

structure

of

Lu AF21934, which results from

2 by replacing the carboxylate

with an amide, was calculated.

Similarly to 2, Lu AF21934 can

exhibit its cyclohexyl substitu-

ents as 1a2e or 1e2a. The amide

moiety in the cyclohexyl ring

opens more possibilities for in-

Figure 8. Optimized conformations of Lu AF21934. A,B) equatorial amide and axial arylamide; C,D) axial amide

and equatorial arylamide. An internal hydrogen bond is present in all conformations but involves the carbonyl of

the amide and the NH of the arylamide in (A) and (C) and the NH of the amide and the carbonyl group of the

arylamide in (B) and (D). The energies relative to the most stable conformation (C) are A: 2.24 kcalmol^À1, B:

ternal hydrogen bonds than 4.26 kcalmol^À1, C: 0 kcalmol^À1, and D: 1.54 kcalmol^À1

.

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result suggests that, for the limited data set studied,

ligand–receptor recognition may be affected by hy-

drophobic interactions involving the aliphatic ring,

either by the absence of the interaction or by the

steric hindrance resulting from inappropriate fitting

in the receptor binding pocket. This is in agreement

with previous results showing the sensitivity of the

cyclohexane ring in VU0155041 to changes in size.^[11]

Taken together, the three-point pharmacophore pro-

posed is consistent with the pharmacological data

shown by these mGlu₄PAMs and offers a way to de-

scribe the structural features necessary for the activi-

ty of these molecules in 3D space.

Discussion

Allosteric modulation is an appealing concept in

drug discovery and promises interesting applications,

due to several advantages over targeting the orthos-

teric binding site of the receptors or enzymes.^[22]

Compounds binding to allosteric sites are of particu-

lar interest in GPCRs due to the high number of sub-

types, with higher subtype selectivity in allosteric

than in orthosteric sites, and the prospects for thera-

peutic interventions based in the pharmacological

regulation of the activity of these proteins.^[4b,23]Allo-

steric modulation at mGlu receptors is of interest,^[15]

and intense activity has been devoted to PAM devel-

opment for mGlu₄.^[8a,24]

Figure 9. A) Plot of PAM potency vs. the dihedral angle between cyclohexyl substituents

(a=C7ÀC1ÀC2ÀC11). Three regions were distinguished, depending on the a value:

I) axial arylamide and equatorial carboxylate or amide; II) equatorial arylamide and equa-

torial carboxylate or amide; and III) equatorial arylamide and axial carboxylate or amide.

The potency (log[EC₅₀]) value for Lu AF21934 was taken from literature^[12]and corrected

to our data set using the values of cis-2 from our work (À5.91) and from previously re-

ported^[12](À6) experiments. B) 3D pharmacophore model. Identification and location of

the three pharmacophoric points representing the selected compounds. The activity of

the molecules was related to fitting with the pharmacophore. In particular, in the less

potent compounds, the aliphatic ring did not match with the proposed hydrophobic

pharmacophoric feature.

The finding of VU0155041 (Æ)-2 as a selective

mGlu₄PAM with interesting activity in vivo has ex-

tended to the structural and conformational aspects

of its binding to mGlu₄receptor. We previously re-

ported that the PAM activity of this cis diastereomer

is associated with a single enantiomer (1R, 2S)-2.^[13]

Although less potent than the cis, the 1,2-cyclohex-

ane trans isomers (Æ)-3 are active PAMs at the mGlu₄

ligand structural analysis can only provide partial information,

as pharmacological activity depends on drug–receptor interac-

tions. In the absence of an mGlu₄receptor structure, a pharma-

cophore model can be constructed to go one step forward in

the understanding of drug action.

receptor, and in this paper, we disclosed that their activity is

also mainly associated with a single enantiomer, (1S,2S)-3, with

the other being essentially inactive. This corroborates the

enantiomeric discrimination observed at the allosteric mGlu₄

binding site, which is also confirmed in the case of the cyclo-

hexane-1,2-diamide derivatives, where the (1S,2R) isomer

Lu AF21934 is active as a mGlu₄PAM, but its (1R,2S) enantio-

mer, Lu AF21935, is inactive.^[12,14]Overall, this appears to define

a specific configuration at the substituted cyclohexane carbon

centers in the active compounds, which points to a restricted

binding geometry and a structurally rigid enantiomer-discrimi-

nating receptor site.

A 3D pharmacophore was constructed from the enantiomer

1R,2S-2 in the optimized 1e2a putative active conformation.

The model contains three pharmacophoric points, which repre-

sent the minimal number of features common to the set of VU

derivatives: an aromatic ring, a hydrogen bond acceptor (rep-

resenting either a carboxylate or and amide group), and a hy-

drophobic interaction associated with the six-membered ring

(Figure 9B). To properly compare all of the compounds, their

structures were superimposed using the arylamide group as

a reference frame. This allowed us to check whether the two

other pharmacophoric points were additionally fitted by each

of the molecules in the previously determined optimized con-

formations. Figure 9B shows that all compounds fulfilled the

hydrogen bond acceptor feature; however, the hydrophobic

region only matched with the most active molecules. This

The active conformation for the more potent cis isomers of

compound 1 has been addressed by two different types of

conformational probes. In consideration of the lack of activity

of one of the enantiomers in the 1,2-cyclohexanedicarboxylic

PAM compounds cis-(Æ)-2 and trans-(Æ)-3, and cis-cyclohex-

ane-1,2-dicarboxamides Lu AF21934 and Lu AF21935, we de-

cided to use racemic norbornane and cyclohexene compounds

to test the possible differential activities of mGlu₄PAMs. Nor-

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bornane compounds are PAMs of mGlu₄with a 4.7-fold lower

EC₅₀value for (Æ)-4 than (Æ)-5, favoring a binding mode with

an endo dichlorophenylamide substitution, perpendicular to

the six-membered ring, and an exo carboxylate that is posi-

tioned in the plane. This would indicate a preference for an

axial amide and an equatorial carboxylate in the active chair

conformation for cis compounds 2, as depicted in Figure 2,

and appears to define a very specific relative spatial disposition

of carboxylate and aryl amide substituents on the six-carbon

cycle for activity. The results obtained for the assay using bicy-

clic compound (Æ)-4 show an activity similar to that of trans-

(Æ)-3 at mGlu₄but lower than that found for cis-(Æ)-2. Interest-

ingly, these bicyclic compounds, having a trans arrangement of

substituents, seem to be more similar to the cis-(Æ)-2 active

conformation than cis endo–endo or exo–exo norbornanes.

These compounds have been reported to be inactive at mGlu₄

by Williams et al.^[11](Figure 10) and, remarkably, have a parallel

arrangement for the substituents which is structurally similar

to that of the cyclohexene in compound 8, which is inactive at

mGlu₄. The rigid norbornane system induces the cyclohexane

to adopt a skew-boat conformation, which is different from

the most stable cyclohexane chair conformations. This could

explain the lower potency of the bicyclic scaffold compounds

with respect to the most active compound, 2. In addition, a dif-

ference results in the relative orientations between carboxylate

and aryl amide cyclohexane substituents, where a value of

67.78 was obtained for the dihedral angle a of compound 2, in

good correspondence with its pharmacological activity

(Figure 9). In contrast to the norbornane scaffold, cyclohexenes

are found in half-chair conformations with allylic substituents

placed in pseudoaxial and pseudoequatorial positions, closer

to the axial–equatorial geometry found in chair cyclohexanes.

As mentioned before, the presence of substituents in the ole-

finic cyclohexene carbon atoms induces the allylic substituent

to prefer pseudoaxial positions due to A^(1,2)strain, as corrobo-

rated by the relative conformational energies obtained for (Æ)-

6 and (Æ)-7. The mGlu₄assay of cyclohexene conformational

probes revealed that (Æ)-6 is eightfold more active than (Æ)-7.

The more active cyclohexene (Æ)-6 has a pseudoaxial amide

substituent, and the carboxylate is located in the plane of the

six-membered ring. This preferred substituent arrangement is

in agreement with that obtained from the assay with norbor-

nane probes and indicate that the amide in an axial position

and an equatorial carboxylate substituent are likely present in

the active conformation of cis compound 2. Overall, the results

collected for these compounds support a very specific confor-

mational selection by the receptor for active cyclohexane car-

boxylic mGlu₄PAMs.

Figure 10. Summary of activities of molecules related to cyclohexanecarbox-

ylic mGlu₄PAM 2.

A question of interest is related to the activity of trans

isomer 3, which would not match a diequatorial arrangement

of substituents, as all compounds having both substituents

conformationally locked in the six-membered ring plane, such

as 8 or 9, have been found clearly inactive as mGlu₄PAMs. To

study if an energetically attainable conformation other than

a chair could be operative for binding to the receptor, we cal-

culated the energy of the optimized structure of the cyclohex-

ane skew-boat for trans compound 3 (Figure 7) and found that

a conformation at 3.37 kcalmol^À1above the most stable chair

conformation had an spatial arrangement of arylamide and car-

boxylate substituents similar to that present in the proposed

chair conformation of 2. This arrangement is similar to the half-

chair conformation of the most active cyclohexene, (Æ)-6, or

the skew-boat cyclohexane of the bicyclo[2.2.1]heptane-2-car-

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1

(for H) and 100 (for ¹³C), respectively, unless otherwise indicated.

¹H NMR experiments are reported in d units, parts per million

(ppm), and were measured relative to the residual solvent peak

(d=7.26 ppm for CDCl₃, d=3.31 ppm for CD₃OD, d=2.5 ppm for

[D₆]DMSO), and the coupling constants (J) are reported in hertz

(Hz). All ¹³C NMR spectra were reported in ppm relative to the sig-

nals for CDCl₃(77.16 ppm), CD₃OD (49.0 ppm), and [D₆]DMSO

(39.52 ppm). IR spectra were registered as film and were recorded

with an FT-IR Spectrometer. The purity of the compound was de-

termined by HPLC on an Alliance 2695 system with UV detection

at 254 nm using a Kinetex C18 (4.6 mm75 mm, 3.5 mm) column.

Unless otherwise stated, the following chromatography conditions

were used: mobile phase A, H₂O with 0.2% HCO₂H; mobile

phase B, CH₃CN with 0.2% HCO₂H; flow rate, 1.0 mLmin^À1; injec-

tion volume, 10 mL; elution gradient, 0–5 min, 5–90% B; 5–7 min,

90% B; 7–8 min, 90–100% B; 8–10 min, 100% B. The purity of

tested compounds was >95%, as determined by HPLC-UV. HRMS

were recorded in a time-of-flight (TOF) mass spectrometer with

electrospray ionization (ESI).

boxylic acid, (Æ)-4, which have similar EC₅₀values at the mGlu₄

receptor (Figure 10). This would qualitatively explain the activi-

ties found for the different compounds tested related to this in-

teresting scaffold, which seem to strongly depend on the cyclo-

hexane conformation and the resulting relative position of the

carboxylate and aryl amide substituents.

Conclusions

We designed and obtained several active compounds at the

mGlu₄receptor containing the cyclohexane carboxylate-aryla-

mide scaffold, which allowed us to identify the contributions

to pharmacological activity of the different combinations of

equatorial and axial substituents and to distinguish the active

disposition (that corresponding to 1e2a, as defined as com-

pound 2) from the inactive ones. The comparison between

compounds was made on the basis of their EC₅₀parameter es-

timates. This could be done because all analogues for which it

was possible to evaluate the maximal effect reached a similar

E_maxvalue to that of VU0155041, which was taken as a refer-

ence compound. Notably, because of the allosteric nature of

the ligands, the EC₅₀parameter results from a combination of

effects that, under the operational model of allosterism,^[25]in-

clude: the affinity and operational efficacy of the orthosteric

ligand, the affinity and operational efficacy of the allosteric

ligand, and the binding and functional cooperativities between

the orthosteric and the allosteric ligands.^[26]In addition, the

pharmacophore model illustrates the importance that the flexi-

bility of the aliphatic ring may require to properly fit the mole-

cule within the receptor cavity. The data obtained, together

with literature concerning mGlu₄receptor PAM activity of VU

derivatives,^[10,11]suggest that ligand–receptor recognition in-

volves a geometrically constrained region of the receptor bind-

ing site, with functional activity depending on the flexibility of

the ligand to fit the receptor pocket. Further work at the re-

ceptor level will be needed to confirm this hypothesis. Indeed,

in the absence of the crystal structure of the receptor–ligand

complex, site-directed mutagenesis and modeling studies may

shed light on mGlu₄receptor enantioselectivity for compound

VU0155041 and the specific conformation within the receptor

cavity. These studies would ultimately lead to a detailed knowl-

edge of the receptor–ligand interactions, which would aid in

the development of new compounds with improved activities.

(1SR,2RS,3SR,4RS)-3-(methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-

2-carboxylic acid (11): A solution of 10 (1.0 g, 6.09 mmol) in MeOH

(4 mL) was heated at reflux and stirred for 16 h. The reaction mix-

ture was then cooled to room temperature, and the solvent was re-

moved under reduced pressure to afford 1.19 g (6.09 mmol, 100%)

1

of 11 as a white solid: H NMR (400 MHz, CDCl₃): d=1.33 (dd, 1H,

J=8.6, 0.5 Hz), 1.47 (dt, 1H, J=8.6, 1.8 Hz), 3.12–3.21 (m, 2H), 3.27

(dd, 1H, J=10.2, 3.1 Hz), 3.32 (dd, 1H, J=10.2, 3.2 Hz), 3.58 (s, 3H),

6.20 (dd, 1H, J=5.5, 2.7 Hz), 6.30 (dd, 1H, J=5.3, 2.9 Hz),

10.88 ppm (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): d=46.2, 46.7, 48.2,

48.4, 48.9, 51.6, 134.4, 135.7, 173.0, 178.7 ppm. The ¹H NMR and

¹³C NMR spectra were identical to those previously reported.^[18]

(1SR,2RS,3RS,4RS)-3-(methoxycarbonyl)bicyclo[2.2.1]hept-5-ene-

2-carboxylic acid (12): A solution of 10 (1.1 g, 5.61 mmol) in

MeOH (20 mL) was added to a solution of NaOMe (6.41 mL, 25% in

MeOH, 28 mmol) at 508C, and the resulting solution was heated at

reflux for 6 h. The solvent was removed under reduced pressure,

and the remaining white solid was cooled to 08C, dissolved in

CH₂Cl₂, and acidified to pH 2 using a 1n HCl solution. The mixture

was extracted with CH₂Cl₂(390 mL), and the combined organic

layers were washed with brine, dried over MgSO₄, filtered, and con-

centrated under reduced pressure. The residue was purified by

column chromatography (100:2 to 95:5, CH₂Cl₂/MeOH gradient) to

give 0.89 g (4.54 mmol, 81%) of 12 as a white solid: mp: 108–

110 8C; lit.^[18]mp: 95.58C; ¹H NMR (400 MHz, CDCl₃): d=1.46 (dd,

1H, J=8.8, 1.4 Hz), 1.61 (d, 1H, J=8.8 Hz), 2.64 (d, 1H, J=3.8 Hz),

3.12 (brs, 1H), 3.28 (brs, 1H), 3.41 (dd, 1H, J=4.4, 3.8 Hz), 3.71 (s,

3H), 6.12 (dd, 1H, J=5.2, 2.3 Hz), 6.27 (dd, 1H, J=4.7, 3.3 Hz),

11.28 ppm (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): d=45.7, 47.0, 47.6,

47.7, 48.1, 52.3, 135.3, 137.8, 174.9, 179.6 ppm; IR (film): n˜ =3400–

2400, 1731, 1707, 1269, 1180 cm^À1; HRMS calcd for C₁₀H₁₁O₄:

195.0657 [MÀH]^À, found: 195.0648; the spectroscopic data were

identical to those previously reported.^[18]

Experimental Section

Chemistry

General: All moisture-sensitive reactions were carried out under ni-

trogen. All materials were obtained commercially and used without

further purification. Solvents were dried prior to use with alumina

in a solvent purification system or distilled and dried by standard

methods. Thin-layer chromatography (TLC) was performed on silica

gel (Alugram Sil G/UV), and flash chromatography was done using

silica gel 60 (40–63 mm, Panreac). Analytical samples were homoge-

neous, as confirmed by TLC, and afforded spectroscopic results

consistent with the assigned structures. ¹H and ¹³C NMR spectra

were obtained in CDCl₃, CD₃OD, or [D₆]DMSO solutions at 400 MHz

(1RS,2RS,3RS,4SR)-methyl

3-((3,5-dichlorophenyl)carbamoyl)-

bicyclo[2.2.1]hept-5-ene-2-carboxylate (13): HATU (0.93 g,

2.45 mmol) was added to a solution of 12 (0.32 g, 1.63 mmol) in

EtOAc (8 mL) containing DIPEA (0.57 mL, 3.26 mmol), and the reac-

tion mixture was stirred for 10 min. Consequently, 3,5-dichloroani-

line (0.26 g, 1.63 mmol) was added, and the resulting solution was

stirred at 508C. After 18 h, the reaction mixture was cooled to

room temperature and diluted with EtOAc (50 mL). The organic

layer was washed with aqueous saturated NaHCO₃(40 mL) and

brine (40 mL), dried over MgSO₄, and filtered. The solvent was

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evaporated in vacuo, and the resulting residue was purified by

flash chromatography (200:1 to 100:1 CH₂Cl₂/EtOAc gradient) to

give 0.46 g (1.35 mmol, 83%) of 13 as a white solid: mp: 120–

(m, 2H), 3.30–3.36 (m, 2H), 4.93 (AB, 1H, J=12.3 Hz), 5.10 (AB, 1H,

J=12.3 Hz), 6.22 (dd, 1H, J=5.6, 2.9 Hz), 6.29 (dd, 1H, J=5.6,

2.9 Hz), 7.28–7.36 ppm (m, 5H); ¹³C NMR (100 MHz, CDCl₃): d=46.4,

46.8, 48.2, 48.5, 48.9, 66.6, 128.3, 128.5, 128.6, 134.6, 135.6, 136.1,

172.4, 177.9 ppm; the spectroscopic data were identical to those

previously reported.^[27]IR (film): n˜ =3400–2400, 1745, 1700, 1548,

1336, 1224, 1172, 1143, 1029 cm^À1; HRMS calcd for C₁₆H₁₆O₄Na:

295.0946 [M+Na]⁺, found: 295.0964.

1

1238C; H NMR (400 MHz, CDCl₃): d=1.52 (d, 1H, J=8.9 Hz), 1.57

(ddd, 1H, J=8.8, 3.3, 1.6 Hz), 2.61 (dd, 1H, J=5.2, 1.6 Hz), 3.20–

3.29 (m, 3H), 3.81 (s, 3H), 6.24–6.28 (m, 2H), 7.05 (t, 1H, J=1.8 Hz),

7.47 (d, 2H, J=1.8 Hz), 8.21 ppm (brs, 1H); ¹³C NMR (100 MHz,

CDCl₃): d=45.3, 46.2, 48.0, 49.0, 51.4, 52.8, 117.8, 124.0, 135.3,

135.9, 136.7, 140.0, 171.4, 176.0 ppm; IR (film): n˜ =3319, 3080,

2983, 2951, 1731, 1705, 1672, 1588, 1533, 1442, 1270, 1179 cm^À1

;

(1SR,2RS,3RS,4RS)-3-((benzyloxy)carbonyl)bicyclo[2.2.1]hept-5-

ene-2-carboxylic acid (16): A solution of 15 (1.1 g, 4.04 mmol) in

THF (10 mL) was cooled to À788C, and LDA (6.06 mL, 2m in THF,

12.12 mmol) was added over 30 min. After the addition was com-

plete, the reaction mixture was stirred for 5 h at À788C. Then, 1n

HCl solution was added until the solution became acidic and

clear. The mixture was extracted with CH₂Cl₂(350 mL), and the

combined organic layers were dried over MgSO₄, filtered, and

concentrated under reduced pressure. The residue was purified

by column chromatography (3:1, hexane/EtOAc and 1% acetic

acid) to give 0.90 g (3.31 mmol, 82%) of 16 as a colorless oil:

¹H NMR (400 MHz, CDCl₃): d=1.47 (dd, 1H, J=8.9, 1.7 Hz), 1.62 (d,

1H, J=8.9 Hz), 2.72 (dd, 1H, J=4.5, 1.5 Hz), 3.16 (brs, 1H), 3.30

(brs, 1H), 3.47 (dd, 1H, J=4.4, 3.8 Hz), 5.12–5.21 (m, 2H), 6.14

(dd, 1H, J=5.6, 2.8 Hz), 6.29 (dd, 1H, J=5.6, 3.1 Hz), 7.28–

7.43 ppm (m, 5H); ¹³C NMR (100 MHz, CDCl₃): d=45.8, 47.4, 47.5,

47.9, 47.9, 66.9, 128.2, 128.4, 128.7, 135.4, 136.0, 137.9, 174.2,

178.8 ppm; IR (film): n˜ =3400–2400, 1729, 1705, 1456, 1333, 1268,

1209, 1174 cm^À1; HRMS calcd for C₁₆H₁₆O₄Na: 295.0946 [M+Na]⁺,

found: 295.0959.

HRMS calcd for C₁₆H₁₅Cl₂NO₃Na: 362.0327 [M+Na]⁺, found:

362.0321.

(1RS,2RS,3RS,4SR)-3-((3,5-dichlorophenyl)carbamoyl)bicyclo-

[2.2.1]hept-5-ene-2-carboxylic acid (14): LiOH (63 mg, 2.65 mmol)

was added to a solution of 13 (0.30 g, 0.88 mmol) in THF (3 mL)

and water (1 mL) at 08C. The mixture was stirred at room tempera-

ture for 1 h and then acidified to pH 2 using a 1n HCl solution.

The mixture was extracted with EtOAc (320 mL), and the com-

bined organic layers were dried over MgSO₄, filtered, and concen-

trated under reduced pressure. The residue was purified by

column chromatography (100:1 to 95:5, CH₂Cl₂/MeOH gradient) to

give 0.18 g (0.54 mmol, 61%) of 14 as a white solid: mp: 214–

1

2168C; H NMR (400 MHz, CD₃OD): d=1.49 (dd, 1H, J=8.5, 1.3 Hz),

1.66 (d, 1H, J=8.5 Hz), 2.72 (d, 1H, J=3.8 Hz), 3.16 (brs, 1H), 3.28

(brs, 1H), 3.38 (t, 1H, J=3.9 Hz), 6.03 (dd, 1H, J=5.6, 2.8 Hz), 6.31

(dd, 1H, J=5.5, 3.1 Hz), 7.09 (t, 1H, J=1.8 Hz), 7.57 ppm (d, 2H, J=

1.9 Hz); ¹³C NMR (100 MHz, CD₃OD): d=47.9, 48.4, 48.7, 49.1, 51.0,

118.9, 124.1, 134.9, 136.1, 138.8, 142.4, 174.0, 178.9 ppm; IR (film):

n˜ =3400–2400, 1708, 1657, 1587, 1538, 1445, 1404, 1269,

1181 cm^À1; HRMS calcd for C₁₅H₁₃Cl₂NO₃Na: 348.0170 [M+Na]⁺,

found: 348.0152.

(1RS,2RS,3RS,4SR)-2-benzyl 3-methyl bicyclo[2.2.1]hept-5-ene-

2,3-dicarboxylate (17): K₂CO₃(0.60 g, 4.37 mmol) was added to

a solution of 15 (0.70 g, 2.57 mmol) in THF (10 mL), and the mix-

ture was stirred for 30 min at room temperature. Then, methyl

iodide (0.19 mL, 3.08 mmol) was added, and the mixture was

stirred at room temperature for an additional 24 h. The reaction

mixture was diluted with 50 mL of CH₂Cl₂and 40 mL of a 1n HCl

solution. The organic layer was separated, and the aqueous layer

was extracted with CH₂Cl₂(250 mL). The combined organic layers

were dried over MgSO₄, filtered, and concentrated under reduced

pressure to afford 0.54 g (1.89 mmol, 73%) of 17 as a colorless oil:

¹H NMR (400 MHz, CDCl₃): d=1.46 (dd, 1H, J=8.8, 1.8 Hz), 1.61 (dt,

1H, J=8.8, 1.4 Hz), 2.75 (dd, 1H, J=4.5, 1.7 Hz), 3.15 (brs, 1H), 3.27

(brs, 1H), 3.42 (t, 1H, J=4.3 Hz), 3.64 (s, 3H), 5.09–5.21 (m, 2H),

6.08 (dd, 1H, J=5.6, 2.8 Hz), 6.27 (dd, 1H, J=5.6, 3.2 Hz), 7.29–

7.40 ppm (m, 5H); ¹³C NMR (100 MHz, CDCl₃): d=45.8, 47.4, 47.5,

47.9, 48.0, 52.0, 66.8, 128.2, 128.4, 128.7, 135.4, 136.1, 137.7, 173.8,

174.4 ppm; IR (film): n˜ =3067, 3034, 2988, 2951, 2870, 1732, 1310,

1266, 1207, 1165, 1164, 1013 cm^À1; HRMS calcd for C₁₇H₁₈O₄Na:

309.1103 [M+Na]⁺, found: 309.1092.

(1SR,2RS,3RS,4RS)-3-((3,5-dichlorophenyl)carbamoyl)bicyclo-

[2.2.1]heptane-2-carboxylic acid (4): In a glass pressure flask, com-

pound 14 (0.04 g, 0.12 mmol) was dissolved in THF (8 mL), and

rhodium (0.013 g, 5% Rh on activated C) was then added. The

flask was repeatedly filled and evacuated with H₂and vigorously

stirred at room temperature for 4 h under an atmosphere of H₂

(balloon). The reaction mixture was next filtered through a plug of

Celite to separate the catalyst, and the filter was washed three

times with MeOH. The combined filtrates and washings were con-

centrated under reduced pressure. The residue was purified by

column chromatography (100:1 to 100:5, CH₂Cl₂/MeOH gradient)

to give 0.035 g (0.11 mmol, 87%) of 4 as a white solid: mp: 220–

1

2238C; H NMR (400 MHz, CD₃OD): d=1.33–1.47 (m, 4H), 1.56–1.67

(m, 2H), 2.61 (d, 1H, J=4.0 Hz), 2.65 (brs, 1H), 2.86 (d, 1H, J=

4.9 Hz), 3.23 (t, 1H, J=4.4 Hz), 7.10 (t, 1H, J=1.6 Hz), 7.61 ppm (d,

2H, J=1.8 Hz); ¹³C NMR (100 MHz, CD₃OD): d=24.5, 29.8, 39.7,

42.4, 42.9, 49.6, 52.9, 118.9, 124.1, 136.1, 142.5, 173.9, 179.2 ppm; IR

(film): n˜ =3400–2400, 1711, 1655, 1587, 1538, 1445, 1404,

1184 cm^À1; HRMS calcd for C₁₅H₁₅Cl₂NO₃Na: 350.0327 [M+Na]⁺,

found: 350.0332.

(1SR,2RS,3RS,4RS)-3-(methoxycarbonyl)bicyclo[2.2.1]heptane-2-

carboxylic acid (18): In a glass pressure flask, compound 16

(0.44 g, 1.54 mmol) was dissolved in THF (20 mL), and Pd/C (0.06 g,

5–15% Pd on activated C, water-wet) was then added. The flask

was repeatedly filled and evacuated with H₂and vigorously stirred

at room temperature for 24 h under H₂(2 atm). The reaction mix-

ture was next filtered through a plug of Celite to separate the cata-

lyst, and the filter was washed three times with MeOH. The com-

bined filtrates and washings were concentrated under reduced

pressure to afford 0.29 g (1.44 mmol, 94%) of 18 as a white solid;

mp: 97–998C; ¹H NMR (400 MHz, CD₃OD): d=1.18–1.27 (m, 1H),

1.27–1.37 (m, 2H), 1.41–1.51 (m, 1H), 1.51–1.57 (m, 1H), 1.63 (tt,

1H, J=11.8, 4.3 Hz), 2.57 (d, 1H, J=3.9 Hz), 2.60 (brs, 1H), 2.73

(dd, 1H, J=5.4, 1.2 Hz), 3.14–3.20 (m, 1H), 3.69 ppm (s, 3H);

(1SR,2RS,3SR,4RS)-3-((benzyloxy)carbonyl)bicyclo[2.2.1]hept-5-

ene-2-carboxylic acid (15): Benzyl alcohol (1.0 mL, 9.75 mmol) was

added dropwise to a stirred suspension of 10 (0.8 g, 4.87 mmol) in

toluene (6 mL). The resulting reaction mixture was heated at 708C

for 8 h. During this period, the material gradually dissolved. Subse-

quently, the resulting clear solution was concentrated in vacuo to

dryness. The residue obtained was triturated with cold Et₂O

(20 mL) and washed with cold Et₂O (310 mL). The resulting white

solid was dried in vacuo to give 1.2 g (4.41 mmol, 90% yield) of

15: mp: 125–1278C; lit.^[27]mp: 1208C; ¹H NMR (400 MHz, CDCl₃):

d=1.33 (d, 1H, J=8.6 Hz), 1.49 (dt, 1H, J=8.7, 1.8 Hz), 3.18–3.22

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¹³C NMR (100 MHz, CD₃OD): d=25.1, 29.8, 38.9, 41.5, 43.1, 50.1,

50.7, 52.3, 175.6, 177.9 ppm; IR (film): n˜ =3400–2400, 1735, 1703,

1437, 1296, 1261, 1197 cm^À1; HRMS calcd for C₁₀H₁₃O₄: 197.0814

[MÀH]^À, found: 197.0813.

was stirred at room temperature for 3 h. The solvent was removed

in vacuo, and a quantitative yield of anhydride (1S,2S)-20 was ob-

tained. Without further purification, this compound was allowed to

react with 3,5-dichloroaniline (136 mg, 0.84 mmol) in dry THF

(15 mL) under nitrogen atmosphere. The reaction mixture was

stirred at room temperature overnight, and the solvent was re-

moved in vacuo. The residue was washed with CH₂Cl₂to give the

product (160 mg, 60%): ee: 100%; conditions: Daicel CHIRALPA-

K IA, eluent: hexane (0.1% TFA)/iPrOH (0.1% TFA) (92:8); flow

0.5 mLmin^À1; detection l=254 nm; t_R: 19.6 min; HRMS: calcd mass

for C₁₄H₁₆NO₃Cl₂[M+H]: 316.0507, found: 316.0505.

(1RS,2RS,3RS,4SR)-methyl

bicyclo[2.2.1]heptane-2-carboxylate

3-((3,5-dichlorophenyl)carbamoyl)-

(19): HATU (0.57 g,

1.51 mmol) was added to a solution of 18 (0.20 g, 1.01 mmol) in

EtOAc (10 mL) containing DIPEA (0.35 mL, 2.02 mmol), and the re-

action mixture was stirred for 10 min at room temperature. Conse-

quently, 3,5-dichloroaniline (0.16 g, 1.01 mmol) was added, and the

resulting solution was stirred at 508C. After 18 h, the reaction mix-

ture was cooled to room temperature and diluted with EtOAc

(50 mL). The organic layer was washed with an aqueous saturated

NaHCO₃(40 mL) and brine (40 mL), dried over MgSO₄, and filtered.

The solvent was evaporated in vacuo, and the resulting residue

was purified by flash chromatography (10:1 to 6:1, hexane/EtOAc

gradient) to give 0.23 g (0.67 mmol, 67%) of 19 as a colorless oil:

¹H NMR (400 MHz, CDCl₃): d=1.22–1.30 (m, 2H), 1.33–1.39 (m, 1H),

1.48–1.66 (m, 2H), 1.69–1.77 (m, 1H), 2.58–2.70 (m, 3H), 3.06–3.13

(m, 1H), 3.76 (s, 3H), 7.04 (t, 1H, J=1.7 Hz), 7.48 (d, 2H, J=1.8 Hz),

8.03 ppm (brs, 1H); ¹³C NMR (100 MHz, CDCl₃): d=24.5, 29.4, 38.5,

39.4, 40.8, 51.1, 51.5, 52.4, 117.8, 123.9, 135.3, 140.2, 172.8,

175.3 ppm; IR (film): n˜ =3327, 3123, 3082, 2956, 2878, 1788, 1733,

1697, 1671, 1588, 1533, 1446, 1409, 1262, 1179 cm^À1; HRMS calcd

for C₁₆H₁₈Cl₂NO₃: 342.0664 [M+H]⁺, found: 342.0656.

(1R,2R)-2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarboxylic

acid (1R,2R)-3: The experimental procedure followed was identical

to that for enantiomer (1S,2S). The starting material in this case

was (1R,2R)-cyclohexane-1,2-dicarboxylic acid (145 mg, 0.84 mmol),

giving intermediate anhydride (1R,2R)-20 which, after reaction with

3,5-dichloroaniline, afforded 162 mg of (1R,2R)-3 (60% yield): ee:

98%; conditions: Daicel CHIRALPAK IA, eluent: hexane (0.1% TFA)/

iPrOH (0.1% TFA) (92:8); flow 0.5 mLmin^À1; detection l=254 nm;

t_R: 16.9 min (major enantiomer) and 19.6 min (minor enantiomer);

HRMS: calcd mass for C₁₄H₁₆NO₃Cl₂[M+H]: 316.0507, found:

316.0520.

2-((3,5-dichlorophenyl)carbamoyl)cyclohex-2-enecarboxylic acid

(6): 3,5-Dichloroaniline (40 mg, 0.25 mmol, 1 equiv) was added to

a stirred solution of anhydride 23^[19](38 mg, 0.25 mmol) in dry THF

(5 mL). The reaction mixture was stirred at reflux for 24 h. Then the

solvent was evaporated in vacuo, giving the crude product, which

was purified by column chromatography (hexanes/EtOAc, 3:1) to

(1RS,2RS,3RS,4SR)-3-((3,5-dichlorophenyl)carbamoyl)bicyclo-

[2.2.1]heptane-2-carboxylic acid (5): LiOH (0.04 g, 1.59 mmol) was

added to a solution of 19 (0.18 g, 0.53 mmol) in THF (6 mL) and

water (2 mL) at 08C. The mixture was stirred at room temperature

for 18 h and then acidified to pH 2 using a 1n HCl solution. The

mixture was extracted with EtOAc (320 mL), and the combined

organic layers were dried over MgSO₄, filtered, and concentrated

under reduced pressure. The residue was purified by column chro-

matography (3:1 to 1:1, hexane/EtOAc gradient) to give 0.15 g

1

give 6 (32 mg, 41%) as a white solid H NMR (400 MHz, [D₆]DMSO):

d=12.18 (br, 1H), 10.31 (s, 1H), 7.63 (d, J=1.8 Hz, 2H), 7.21 (s, 1H),

7.05 (t, J=3.4 Hz, 1H), 3.38 (t, J=4.0 Hz, 1H), 2.17 (m, 2H), 1.85 (m,

1H), 1.75 (m, 1H), 1.61 (m, 1H), 1.50 ppm (m, 1H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=173.9, 168.0, 142.30, 142.1, 134.5 (2C),

129.4, 122.5, 117.4 (2C), 42.5, 27.1, 25.4, 19.0 ppm; IR (film): n˜ =

3319, 3191, 2931, 2859, 1724, 1680, 1509, 1437, 1125, 1109, 857,

677 cm^À1; HPLC: C18 OSD2, 5 mm, 1504.6 mm, eluent: H₂O (0.1%

TFA)/acetonitrile (0.1% TFA) (35:65), flow 1 mLmin^À1, detection l=

254 nm; t_R: 8.3 min; HRMS calcd for C₁₄H₁₃Cl₂NO₃: 314.0361 [M+

H]⁺, found: 314.069.

1

(0.46 mmol, 88%) of 5 as a white solid: mp: 201–2038C; H NMR

(400 MHz, CD₃OD): d=1.28–1.34 (m, 1H), 1.37–1.47 (m, 2H), 1.48–

1.58 (m, 1H), 1.60–1.70 (m, 1H), 1.70–1.76 (m, 1H), 2.46 (d, 1H, J=

3.9 Hz), 2.65 (t, 1H, J=3.5 Hz), 2.80 (dd, 1H, J=5.3, 0.8 Hz), 3.35

(ddd, 1H, J=6.1, 4.4, 1.9 Hz), 7.10 (t, 1H, J=1.8 Hz), 7.62 ppm (d,

2H, J=1.9 Hz); ¹³C NMR (100 MHz, CD₃OD): d=25.2, 30.4, 38.4,

41.2, 45.0, 49.6, 51.8, 118.9, 124.1, 136.1, 142.7, 175.3, 177.0 ppm; IR

(film): n˜ =3400–2400, 1699, 1660, 1587, 1539, 1445, 1409,

1195 cm^À1; HRMS calcd for C₁₅H₁₆Cl₂NO₃: 328.0507 [M+H]⁺, found:

328.0335.

6-((3,5-dichlorophenyl)carbamoyl)cyclohex-1-enecarboxylic acid

(7): BOP-Cl (70 mg, 0.275 mmol, 1.1 equiv) was added to a stirred

solution of ester-acid 25^[20](46 mg, 0.25 mmol, 1 equiv), and DIPEA

(0.047 mL, 0.275 mmol, 1.1 equiv) in CH₂Cl₂(5 mL) was added at

08C. After addition, the reaction was stirred for 30 min then, 3,5-di-

chloroaniline (44 mg, 0.275 mmol, 1.1 equiv) and DIPEA (0.047 mL,

0.275 mmol, 1.1 equiv) were added. After completion (16 h), the re-

action was diluted with CH₂Cl₂, then quenched with aqueous satu-

rated NaHCO₃solution. The resulting aqueous layer was extracted

with CH₂Cl₂. The combined organic layers were washed with brine,

dried over anhydrous Na₂SO₄, filtered. and evaporated in vacuo to

give crude rac-26 as a yellow oil, which was directly used without

purification. This material was dissolved in THF/H₂O (1/1) (5 mL)

and LiOH (28.5 mg, 1.25 mmol, 5 equiv) was added at room tem-

perature. After completion (TLC, 12 h), the reaction was quenched

with aqueous 1n HCl and diluted with EtOAc. The resulting aque-

ous layer was extracted with EtOAc. The combined organic layers

were washed with brine, dried over anhydrous Na₂SO₄, filtered,

and evaporated in vacuo to give an oil. Purification was achieved

by flash column chromatography on silica gel (hexane/EtOAc gra-

dient (3:1) to give 7(51 mg, 67%) as a white solid: ¹H NMR

(400 MHz, [D₆]DMSO): d=12.23 (br, 1H), 10.17 (s, 1H), 7.75 (d, J=

1.8 Hz, 2H), 7.19 (m, 1H), 6.73 (t, J=3.3 Hz, 1H), 3.47 (t, J=4.0 Hz,

(1RS,2RS)-2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarbox-

ylic acid (rac-3): Racemic trans-cyclohexane-1,2-dicarboxylic anhy-

dride (100 mg, 0.65 mmol) and 3,5-dichloroaniline (105 mg,

0.65 mmol) were dissolved in THF (4 mL) and stirred at 658C over-

night. The solvent was removed in vacuo, and the residue was

washed with CH₂Cl₂to give the product as a white solid (175 mg,

85%): ¹H NMR (500 MHz, DMSO): d=1.27 (m, 6H), 1.74 (m, 2H),

1.92 (m, 1H), 2.02 (m, 1H), 7.23 (s, 1H), 7.65 (s, 2H), 10.31 (s, 1H),

12.11 ppm (brs, 1H); ¹³C NMR (75 MHz, DMSO): d=25.0, 28.7, 29.3,

44.1, 46.6, 116.9, 122.1, 134.1, 141.7, 174.3, 176.0 ppm; IR (film): n˜ =

3327, 3123, 3082, 2956, 2878, 1788, 1733, 1697, 1671, 1588, 1533,

1446, 1409, 1262, 1179 cm^À1; HRMS: calcd mass for C₁₄H₁₄NO₃Cl₂

[MÀH]: 314.0351, found: 314.0345.

(1S,2S)-2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarboxylic

acid (1S,2S)-3: (1S,2S)-cyclohexane-1,2-dicarboxylic acid (145 mg,

0.84 mmol) was dissolved in trifluoroacetic anhydride (3 mL,

21 mmol) under nitrogen atmosphere, and the reaction mixture

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2H), 2.19 (m, 2H), 1.88 (m, 1H), 1.77 (m, 1H), 1.54 ppm (m, 2H);

¹³C NMR (100 MHz, [D₆]DMSO): d=175.4, 167.7, 142.3, 135.7, 134.3

(2C), 133.2, 122.6, 117.9 (2C), 40.2, 25.9, 25.2, 19.5 ppm; HPLC:

OSD2, 5 mm, 1504.6 mm, eluent: H₂O (0.1% TFA)/acetonitrile

(0.1% TFA) (35:65), flow 1 mLmin^À1, detection l=254 nm; IR (film):

n˜ =3329, 3193, 2938, 2857, 1720, 1668, 1527, 1431, 1131, 1128,

853, 671 cm^À1; t_R: 6.8 min; HRMS calcd for C₁₄H₁₃Cl₂NO₃: 314.0361

[M+H]⁺. found: 314.0367.

10 nm as an orthosteric agonist. The IP-One stimulation buffer, pro-

vided with the IP-One kit (Cisbio Bioassays, France), contains LiCl at

50 mm, which inhibits the degradation of IP1 to myoinositol, which

is then accumulated in the cell. HTRF reagents, monoclonal anti-

body specific to IP1 labeled with europium cryptate and IP1 cou-

pled with the dye d2, diluted in a lysis buffer, were added. After

1 h incubation time, resolved fluorescence at 620 nm and 665 nm

was measured by Rubistar (BMG Labtech, Germany). The specific

signal, which resulted from the energy transfer between europium

cryptate and the donor d2, was proportional to the ratio between

the fluorescence emitted at 620 nm and 665 nm, and, consequent-

ly, to the concentration of IP1 in the lysate. All points were deter-

mined in triplicate. The dose-response curves were fitted using

GraphPad Prism software and the following equation: y=

[(y_maxÀy_min)/(1+10(logEC₅₀Àx))]+y_min, in which EC₅₀is the concen-

tration of the compound necessary to obtain the half-maximal

effect. VU0155041 was used as a reference compound for the

dose–response curve normalization.

2-((3,5-dichlorophenyl)carbamoyl)cyclohex-1-enecarboxylic acid

(8): 3,5-Dichloroaniline (40 mg, 0.25 mmol, 1 equiv) was added to

a stirred solution of anhydride 21 (38 mg, 0.25 mmol) in dry THF

(5 mL). The reaction mixture was stirred at reflux for 24 h. Then,

the solvent was evaporated in vacuo to give the crude product,

which was purified by column chromatography (hexanes/EtOAc,

3:1) to give (49 mg, 63%) as a white solid: ¹H NMR (400 MHz,

[D₆]DMSO): d=12.52 (br, 1H), 10.29 (s, 1H), 7.63 (d, J=1.7 Hz, 2H),

7.23 (s, 1H), 2.29 (m, 2H), 2.25(m, 2H), 1.60 ppm (m, 4H); ¹³C NMR

(100 MHz [D₆]DMSO): d=170.2, 167.9, 144.1, 142.2, 134.4 (2C),

128.2, 122.7, 117.8 (2C), 28.3, 25.2, 21.7, 21.4 ppm; IR (film): n˜ =

3323, 3187, 2927, 2862, 1712, 1676, 1513, 1435, 1122, 1117, 863,

666 cm^À1; HPLC: C18 OSD2, 5 mm, 1504.6 mm, eluent: H₂O (0.1%

TFA)/acetonitrile (0.1% TFA) (35:65), flow 1 mLmin^À1, detection l=

254 nm; t_R: 7.8 min; HRMS calcd for C₁₄H₁₃Cl₂NO₃: 314.0361 [M+

H]⁺, found: 314.0357.

Computational details

All geometry optimization calculations were performed with Gaus-

sian 03.^[30]Density functional theory (DFT) at the B3LYP/6-31+G(d)

level was performed in all cases. To account for environmental ef-

fects, geometry optimizations at the same QM level were per-

formed using the polarizable continuum solvent model (PCM)^[31]

for water environment.

2-((3,5-dichlorophenyl)carbamoyl)benzoic acid (9): 3,5-Dichloroa-

niline (115 mg, 0.71 mmol) was added to a stirred solution of

phthalic anhydride 24 (100 mg, 0.68 mmol) in dry THF (5 mL). The

reaction mixture was stirred at reflux for 24 h, then the solvent was

evaporated in vacuo to give the crude product, which was washed

with CH₂Cl₂to give 9 (115 mg, 55%) as a brown solid: ¹H NMR

(500 MHz, [D₆]DMSO): d=7.32 (s, 1H), 7.57 (d, 1H, J=7.5 Hz), 7.61

(t, 1H, J=7.5 Hz), 7.69 (t, 1H, J=7.5 Hz), 7.75 (s, 2H), 7.92 (d, 1H,

J=7.5 Hz), 10.69 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d=

117.7, 122.9, 129.7, 130.0, 130.2, 132.4, 134.4, 138.3, 141.8, 167.1,

168.2 ppm; HPLC: C18 OSD2, 5 mm, 1504.6 mm, eluent: H₂O

(0.1% TFA)/acetonitrile (0.1% TFA) (65:35), flow 1 mLmin^À1, detec-

tion l=254 nm; t_R: 9.1 min, >96%; HRMS calcd for C₁₄H₉NO₃NaCl₂:

331.9857 [M+Na]⁺; found 331.9846.

Pharmacophore modeling was carried out using Discovery Studio

version 3.5.0. The optimized conformations of all molecules were

superimposed based on the molecular overlay method, mainly

using their steric properties in order to align the arylamide group

well, which is a common feature in all molecules. Then, all features

of the reference compound (1Re,2Sa)-2 were mapped. The minimal

features that fit all active molecules, an aromatic ring, a hydrogen

bond acceptor, and a hydrophobic interaction, were kept for the

following analysis of the molecules. The pharmacophoric points

were provided with location constraints of 1.8 and 1.4 radii for

the hydrophobic and hydrogen bond acceptor features, respective-

ly.

Biological methods

Pharmacological experiments were performed in HEK293 human

embryonic kidney cells cultured in Dulbecco’s modified Eagle’s

medium (GIBCO-BRL Life Technologies Inc., Cergy Pontoise, France) Acknowledgements

supplemented with 10% fetal calf serum (FCS, GIBCO-BRL Life

Technologies) and antibiotics (penicillin and streptomycin,

This study was supported in part by the Ministerio de Economꢁa

100 UmL^À1each final, GIBCO-BRL Life Technologies). The com-

pounds were tested on rat mGlu₄, which was transiently transfect-

ed in HEK293 cells by electroporation, as described elsewhere,^[28]

and plated in 96-well microplates. The receptor was co-transfected

with a chimeric Gq/i-protein, which couples activation of the re-

ceptor to the phospholipase C pathway, thus allowing monitoring

of receptor activity by measurement of IP1 production using the

kit IP-One HTRF Assay from Cisbio Bioassays (France).^[29]Additional-

ly, a plasmid encoding the high-affinity glutamate transporter

EAAC1 was co-transfected to prevent the influence of the gluta-

mate released by the cells in the medium. For IP1 accumulation

measurements, cells were seeded after transfection in polyorni-

thine-coated, black-walled 96-well plates and cultured for 24 h in

glutamate/glutamine-free medium. The media was removed, and

cells were incubated for 30 min in a solution containing the drugs

at the tested concentrations plus l-AP4 at a concentration of

y Competitividad (Spain) (SAF2010-19257) and the Fundaciꢅ La

Maratꢅ de TV3 (Spain) (110230, 110231, and 110232). The authors

acknowledge support from the Generalitat de Catalunya (Spain)

(2009SGR-1072) and Consejo Superior de Investigaciones Cientifi-

cas (CSIC, Spain). Computations were performed in part at the

Center for Scientific and Academic Services of Catalonia (CESCA,

Spain). Cell-based pharmacological assays were performed on

the ARPEGE (Pharmacology Screening-Interactome) platform fa-

cility at the Institut de Gꢄnomique Fonctionnelle (France). X.R.

was supported by a Federation of European Biochemical Societies

(FEBS) long-term fellowship and the Beatriu de Pinꢅs program of

Agꢂncia de Gestiꢅ d’Ajuts Universitaris i de Recerca (AGAUR,

Spain). X.R., C.G., F.M., and J.P.P. were supported by the Fonda-

tion Recherche Mꢄdicale (FRM team DEQ20130326522).

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should keep in mind that the numbering for the carbons bearing the

arylcarbamoyl substituents is 1 in the cyclohexane dicarboxamides and

2 in the cyclohexane carboxylic acid compounds. Correspondingly, the

numbering is 1 for the carbon bearing the carboxylic acid group,

whereas the numbering is 2 for the ring carbon-bonded to the CONH₂

group, as indicated in Figure 1.

Keywords: allosteric modulators · conformational probes ·

metabotropic glutamate receptors · GPCRs · norbornanes

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presence of a carboxamide in Lu AF21934 and Lu AF21935 in place of

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ents is changed. Therefore, when these compounds are compared, one

Received: July 24, 2014

Published online on September 5, 2014

ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2014, 9, 2685 – 2698 2698

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Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor

DOI: 10.1002/cmdc.201402190

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