Design and synthesis of systemically active metabotropic glutamate subtype-2 and -3 (mGlu2/3) receptor positive allosteric modulators (PAMs): Pharmacological characterization and assessment in a rat model of cocaine dependence

Article

pubs.acs.org/jmc

Design and Synthesis of Systemically Active Metabotropic Glutamate

Subtype‑2 and -3 (mGlu_2/3) Receptor Positive Allosteric Modulators

(PAMs): Pharmacological Characterization and Assessment in a Rat

Model of Cocaine Dependence

Raveendra-Panickar Dhanya,^†,#Douglas J. Sheﬄer,^†,#Russell Dahl,^†Melinda Davis,^†Pooi San Lee,^†

Li Yang,^†Hilary Highﬁeld Nickols,^§,∥Hyekyung P. Cho,^‡,∥Layton H. Smith,^†Manoranjan S. D’Souza,^⊥

P. Jeﬀrey Conn,^‡,∥Andre Der-Avakian,^⊥Athina Markou,^⊥and Nicholas D. P. Cosford*^,†

^†Cell Death and Survival Networks Program and Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research

Institute, 10901 N. Torrey Pines Road, La Jolla, California 92037, United States

§

∥

^‡Department of Pharmacology, Department of Pathology, and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt

University Medical Center, Nashville, Tennessee 37232, United States

^⊥Department of Psychiatry, School of Medicine, University of CaliforniaSan Diego, La Jolla, California 92093, United States

S

* Supporting Information

ABSTRACT: As part of our ongoing small-molecule

metabotropic glutamate (mGlu) receptor positive allosteric

modulator (PAM) research, we performed structure−activity

relationship (SAR) studies around a series of group II mGlu

PAMs. Initial analogues exhibited weak activity as mGlu₂

receptor PAMs and no activity at mGlu₃. Compound optimiza-

tion led to the identiﬁcation of potent mGlu_2/3selective PAMs

with no in vitro activity at mGlu_1,4−8or 45 other CNS receptors.

In vitro pharmacological characterization of representative

compound 44 indicated agonist-PAM activity toward mGlu₂

and PAM activity at mGlu₃. The most potent mGlu_2/3PAMs

were characterized in assays predictive of ADME/T and

pharmacokinetic (PK) properties, allowing the discovery of systemically active mGlu_2/3PAMs. On the basis of its overall proﬁle,

compound 74 was selected for behavioral studies and was shown to dose-dependently decrease cocaine self-administration in

rats after intraperitoneal administration. These mGlu_2/3receptor PAMs have signiﬁcant potential as small molecule tools for

investigating group II mGlu pharmacology.

neurotoxicity in mixed cultures of astrocytes and neurons,

whereas activation of mGlu₂receptors may be harmful.⁸

Various brain regions, including the cerebral cortex, hippo-

campus, striatum, amygdala, frontal cortex, and nucleus

accumbens, display high levels of mGlu₂and mGlu₃receptor

binding.^9,10This distribution pattern suggests a role for the

mGlu_2/3receptor subtypes in the pathology of neuropsychiatric

disorders such as anxiety,¹¹depression,^12,13schizophrenia,^14,15

drug dependence,¹⁶⁻²²neuroprotection,^23,24Alzheimer’s dis-

ease,²⁵and sleep/wake architecture.²⁶Thus, there is signiﬁcant

potential for the development of selective group II mGlu receptor

activators, including agonists and positive allosteric modulators

(PAMs), for the treatment of CNS diseases caused by aberrant

glutamatergic signaling.

INTRODUCTION

■

Glutamate is the major excitatory neurotransmitter in the

mammalian central nervous system (CNS), mediating fast

synaptic transmission through ion channels, primarily the

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

and kainate ionotropic glutamate receptor subtypes.¹The

metabotropic glutamate (mGlu) receptors are a family of eight

G protein-coupled receptors that are activated by glutamate and

perform a modulatory function in the nervous system.²⁻⁴The

group II mGlu receptors include the mGlu₂and mGlu₃receptor

subtypes, which couple with G_i/oproteins to negatively regulate

the activity of adenylyl cyclase.^3,5Localization studies suggest

that mGlu₂receptors act predominantly as presynaptic auto-

receptors to modulate the release of glutamate into the synaptic

cleft.⁶On the other hand, mGlu₃receptors exhibit a broad

distribution in the brain and have been shown to be present

on astrocytes.⁷In addition, it has been shown that activation

of mGlu₃receptors is required for the neuroprotective eﬀects

of mGlu_2/3agonists toward N-methyl-D-aspartate (NMDA)

Orthosteric (glutamate site) mGlu_2/3agonists such as

LY379268²⁷are constrained amino acid analogues that are

Received: January 10, 2014

Published: April 15, 2014

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Figure 1. Structures of mGlu₂receptor PAMs developed from BINA (1).

typically equipotent at both mGlu₂and mGlu₃, presumably

because of the high degree of sequence homology at the

glutamate-binding site for these two receptors.³Although

LY541850, an orthosteric ligand with mixed mGlu₂agonist/

mGlu₃antagonist activity, has been reported,^28,29currently

there are no truly mGlu₂selective orthosteric ligands available.

The systemically active mGlu_2/3receptor agonist LY379268

has been shown to reduce glutamate release from pre-

synaptic terminals in many brain regions and thus to decrease

glutamatergic neurotransmission.^30,31LY379268 is active in

several diﬀerent rodent models of CNS disorders including

anxiety,³²schizophrenia,³³Huntington’s disease,³⁴⁻³⁶and drug

dependence, where it attenuates cocaine self-administration both

in rats^20,37and in squirrel monkeys.¹⁸However, LY379268 also

inhibits responding for food and food-seeking behavior,^20,21,37,38

suggesting that mGlu_2/3receptor agonists exhibit nonselective

actions on responding for drug and nondrug reinforcers.

In addition to orthosteric agonists of Group II mGlu

receptors, multiple reports have recently described the synthesis

and characterization of selective mGlu₂receptor PAMs.^5,39−46

These compounds invariably resulted from the optimization of

hits obtained from high-throughput screening (HTS) of small

molecule libraries. PAMs, through their interaction at allosteric

sites on the mGlu receptor, positively modulate (i.e. potentiate)

the eﬀects of the endogenous orthosteric mGlu agonist

glutamate. The advantages of PAMs compared with orthosteric

agonists includes enhanced subtype selectivity, the potential for

spatial and temporal modulation of receptor activation, and

ease of optimization and ﬁne-tuning of druglike properties.⁴⁷

We recently reported the design, synthesis, and in vitro and

in vivo characterization of a series of potent and selective

mGlu₂receptor PAMs.^48,49The optimized compounds 2 and 3

(Figure 1), which were developed using the prototypical mGlu₂

receptor PAM BINA (1) as a starting point, were employed to

determine the eﬀects of selectively activating mGlu₂receptors

on cocaine or nicotine dependence.^48,49In these studies we

showed that compound 3, unlike the mGlu_2/3orthosteric

agonist LY379268, decreased cocaine self-administration in rats

at doses that did not aﬀect responding for food.⁴⁸We also

showed that compound 2 dose-dependently decreased nicotine

self-administration in rats following oral (po) administration.⁴⁹

Taken together, our data suggest that mGlu₂receptor PAMs

have the potential for therapeutic utility in the treatment of

drug dependence.

Figure 2. Structure of recently reported mGlu₂receptor NAM/mGlu₃

receptor PAM 4.

mGlu₂and mGlu₃receptors through an allosteric mechanism.

Considering the dearth of information on mixed mGlu_2/3

receptor PAMs, the development of such compounds would

provide valuable pharmacological tools. For example, a CNS

penetrant mGlu_2/3receptor PAM could facilitate investigations

into whether eﬀects on food responding in rats are due to

general activation of mGlu₃receptors or an eﬀect speciﬁc to

direct activation of the mGlu receptor by agonists that act at the

mGlu orthosteric binding site.

The strategy for the design and synthesis of mGlu_2/3receptor

PAMs grew out of our general approach to the development

of selective mGlu₂receptor PAMs. In addition to exploring the

structure−activity relationship (SAR) around compound 1,

which led to the series of selective mGlu₂receptor PAMs

exempliﬁed by 2 and 3,^48,49we also investigated compounds

such as 5−7 (Figure 3). The mGlu₂receptor PAMs 5−7

are representative of a series developed by Pinkerton and

co-workers at Merck.⁵¹The mGlu₂receptor PAMs in this

structural class were reported to exhibit varying degrees of in

vitro potency and eﬃcacy with little information provided

regarding subtype selectivity. Compound 8, a selective mGlu₂

receptor PAM that bears a resemblance to compounds 5−7,

was recently reported by researchers at Eli Lilly.⁵²With the

exception of the optimized compound 8, most of the

compounds in this series were reported to be modestly potent

at mGlu₂receptors in vitro and displayed suboptimal

pharmacokinetic (PK) proﬁles and brain penetration in vivo.

Our initial goal was to develop group II mGlu receptor PAMs

having the potential for systemic activity, with a focus on

maintaining or improving potency and eﬃcacy at mGlu₂while

in parallel investigating the PAM activity at mGlu₃receptors.

Herein we describe the design, synthesis, and pharmacological

characterization of a library of analogues from which a series of

mixed mGlu_2/3receptor PAMs with unique pharmacology were

discovered. Furthermore, because the selective mGlu₂receptor

PAM BINA decreased cocaine- but not food-maintained

responding,³⁷we investigated the in vivo eﬃcacy of one of

the synthesized mGlu_2/3receptor PAMs on cocaine- and food-

maintained responding in rats.

As noted above, there have been many accounts in the

literature describing selective mGlu₂receptor PAMs, whereas

very little has been reported on compounds that potentiate

the eﬀects of glutamate at mGlu₃receptors. This is somewhat

surprising given the signiﬁcant sequence homology (approx-

imately 75%) within the transmembrane regions of mGlu₂and

mGlu₃receptors. A single disclosure by Schann and co-workers

described compound 4 (Figure 2) which was reported to be a

mixed mGlu₂receptor negative allosteric modulator (NAM)/

mGlu₃receptor PAM.⁵⁰We hypothesized that it might be

possible to design and synthesize compounds that activate both

CHEMISTRY

■

Examination of compounds 5−7 suggested the presence of

common structural elements. Speciﬁcally, they consist of an aryl

ether connected via a butyl ether linker to an acetophenone

derivative. Thus, we envisioned a synthetic strategy wherein the

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Figure 3. Structures of mGlu₂receptor PAMs in the acetophenone series.

Scheme 1. General Synthesis of New Group II mGlu Receptor PAMs

key building blocks were synthesized and systematically

Our initial strategy for the design of the butyl ether derived

PAMs library was to systematically explore diﬀerent sub-

stitution patterns on the left-hand aryl ether moiety combined

with a set of four right-hand acetophenones. Speciﬁcally,

acetophenone derivatives were synthesized in which X was

either methyl (e.g., 14) or hydroxyl (e.g., 16) and where R³was

either isopropylmethylene (e.g., 14 and 16) or cyclopentyl-

methylene (e.g., 15 and 17). In the ﬁrst phase of library

synthesis, analogues in which R¹was a 4-carboxylate moiety

(14−21) were prepared and tested in vitro. Many of the

compounds were inactive or only very weakly active in the

assays. Known compounds 5 and 6 were synthesized and tested

as part of the library to provide a benchmark of activity for the

series. Some important observations were made during this

phase regarding the activity of the analogues tested. First,

benchmark compounds 5 and 6 exhibited PAM activity at

mGlu₂but not at mGlu₃receptors in the GIRK assay.

assembled to generate a compound library rapidly and

eﬃciently. This would allow the production of analogues with

suﬃcient diversity to investigate the requirements for optimal

potency, eﬃcacy, and pharmacokinetic properties. The structural

components were synthesized or purchased and incorporated

into the new group II mGlu receptor PAMs as shown in

Scheme 1. Brieﬂy, commercially available carboxylic acids were

converted to the corresponding acyl chloride derivatives (9)

using oxalyl chloride in CH₂Cl₂. After removal of solvents, the

acyl chlorides were employed in a Friedel−Crafts acylation of

substituted phenols (10) using aluminum chloride to provide the

key acetophenone derivatives 11. The phenol derivatives (12)

were coupled with 1,4-dibromobutane by heating with potassium

carbonate in acetonitrile to provide the corresponding bromo-

butoxybenzoate derivative (13). Finally, Finkelstein alkyla-

tion of intermediate 11 with 13 under microwave conditions

delivered the ester derivatives (14−17 and 18′−75′), which

were saponiﬁed with potassium hydroxide to provide the target

carboxylic acid derivatives 18−75.

Moreover, compound 6 was only very weakly active (EC₅₀

>

10 μM, 45% eﬃcacy) in the GIRK assay against mGlu₂. Second,

in general, compounds containing X = Me were less potent

than compounds in which X = OH. Third, whereas the methyl

4-benzoate derivatives 16 and 17 were inactive, the cor-

responding free carboxylic acid derivatives 20 and 21 were

active (20) or weakly active (21) as PAMs at both mGlu₂and

mGlu₃receptors. Furthermore, compound 20 was at least an

order of magnitude more potent as a PAM at mGlu₂than

benchmark compound 5 and had an EC₅₀of 2.7 μM (52%

eﬃcacy) against mGlu₃. Encouraged by the PAM activity found

for compounds 20 and 21, analogues containing free carboxylic

acid moieties at other positions on the aryl ether ring were

synthesized and tested (compounds 22−29). In the 3-carboxyl

series compounds 22 and 23 (X = Me) were inactive whereas

compounds 24 and 25 (X = OH) exhibited PAM activity

at both mGlu₂and mGlu₃receptors. Interestingly, in the

2-carboxyl series (compounds 26−29), all four compounds

were active as mGlu₂receptor PAMs with no activity at mGlu₃.

RESULTS AND DISCUSSION

■

The initial in vitro evaluation of analogues was performed in

thallium ﬂux assays in human embryonic kidney 293 (HEK)

cells expressing heteromeric G-protein-coupled inwardly

rectifying potassium (GIRK) 1/2 channels and rat mGlu₂or

rat mGlu₃receptors.⁴¹The potency and eﬃcacy of compounds

were established by measuring the concentration−response

relationship that potentiates the eﬀect of an EC₂₀concentra-

tion of glutamate. After determination of the concentration−

response relationship, the eﬀect of a maximally eﬀective

concentration (% Max) of each compound was determined.

For the allosteric potentiators, potency is expressed as the EC₅₀

for potentiation of the glutamate EC₂₀. Table 1 shows the

results of testing the compounds against rat mGlu₂or mGlu₃

receptors in vitro.

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a

Table 1. In Vitro Potency and Eﬃcacy Data at mGlu₂or mGlu₃Receptors for PAMs

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Table 1. continued

a

mGlu₂PAM EC₅₀(μM) data and glutamate Max (%) data represent the mean SEM for at least three independent experiments performed in

triplicate. ND = not determined.

These results prompted us to synthesize and test the next set

of analogues (compounds 30−51) containing carboxylic acid

moieties at the 3- or 4-position (R¹) in addition to substituents

at other positions around the aryl ether ring (i.e., R²= F, Cl,

Me, or OMe). In this series, the analogues containing X = Me

(30, 31, 34, 35) continued the trend of very weak (31,

EC₅₀> 10 μM, 36% eﬃcacy) or no mGlu₃activity (30, 34, 35,

12%−14% eﬃcacy), and therefore, all subsequent analogues

were synthesized with X = OH. Furthermore, several of the

analogues in this group (39, 41, 43, 47, and 49) were active as

PAMs at mGlu₂but were essentially inactive at mGlu₃receptors

(24%−31% eﬃcacy). However, in addition to being sub-

micromolar PAMs at mGlu₂, compounds 32, 33, 37, 40, 42, 45,

46, 48, and 51 were active as PAMs at mGlu₃receptors in the

micromolar range (EC₅₀≈ 1−4 μM, 40%−98% eﬃcacy). Most

promising of all, compounds 36, 44, and 50 possessed

submicromolar PAM activity at mGlu₃receptors as well as

good potency at mGlu₂. Especially noteworthy were com-

pounds 44 and 50, with EC₅₀values in the 600−700 nM range

at mGlu₃with excellent eﬃcacy as PAMs (74% and 100%,

respectively). These results suggested that our goal of

producing PAMs with potent activity at both mGlu₂and

mGlu₃receptors was attainable. Having identiﬁed favorable

substitution patterns for the left-hand aryl ether moiety, we next

designed a series of analogues (compounds 52−75) to

investigate which ketone alkyl group (R³) would impart the

best potency and eﬃcacy at both mGlu₂and mGlu₃receptors.

Thus, in this set of analogues, compounds were synthesized

with R³= methyl (52−56), ethyl (57−61), isopropyl (62−66),

n-propyl (67−71), or tert-butylmethylene (72−75). Interest-

ingly, none of the methyl ketone derivatives (52−56) had

activity at mGlu₃receptors, while in the ethyl ketone series

(57−61) only compound 58 (5-CO₂H, 2-OMe) had activity at

mGlu₃(EC₅₀= 6359 nM, 63% eﬃcacy). In the isopropyl

ketone series, only compound 63 (5-CO₂H, 2-OMe) had activ-

ity at mGlu₃(EC₅₀= 588 nM, 60% eﬃcacy). On the other

hand, in the n-propyl series, all compounds demonstrated some

level of activity. While 69 and 71 were weakly active at mGlu₃

(EC_50s> 10 μM, 53% and 41% eﬃcacy, respectively), 67 (4-

CO₂H, 2-OMe), 68 (5-CO₂H, 2-OMe), and 70 (3-CO₂H, 4-F)

demonstrated more potent mGlu₃activity (EC₅₀= 1830 nM,

62% eﬃcacy, EC₅₀= 496 nM, 64% eﬃcacy, EC₅₀= 438 nM,

57% eﬃcacy, respectively). It is noteworthy that compound 67

(4-CO₂H, 2-OMe) was the most potent PAM and selective

for mGlu₂with good eﬃcacy (EC₅₀= 34 nM, 86% eﬃcacy).

Finally, in the tert-butylmethylene series (compounds 72−75)

all four analogues exhibited good potency and excellent eﬃcacy

for both mGlu₂and mGlu₃receptors. Compound 72 was the

most potent against mGlu₃(EC₅₀= 151 nM, 106% eﬃcacy)

with similar potency against mGlu₂, while compounds 73−75

fell into the 258−310 nM potency range (102%−110% eﬃcacy)

against mGlu₃receptors. Thus, a combination of favored left-

hand aryl ether moieties with R³= tert-butylmethylene provides

compounds with unique PAM activity at both mGlu₂and mGlu₃

receptors.

With these results in hand, we tested some of the most

promising new PAMs to provide an estimation of their druglike

properties in vitro using absorption, distribution, metabolism,

and excretion (ADME) assays. The results of these studies are

shown in Table 2. The permeability across artiﬁcial membranes

was assessed via the parallel artiﬁcial membrane permeability

assay (PAMPA) assay and showed a range of results, with most

compounds showing some degree of permeability. Plasma and

microsomal stability was likewise acceptable, with only four of

the analogues showing less than 20% remaining after 1 h. In

addition, an algorithm was used to provide an estimate of plasma

protein binding for 11 of the analogues. The data, which are

provided in the Supporting Information (Table S1), suggest that

the compounds in this series are all >90% plasma protein bound.

Compounds 20, 25, 33, 44, 67, 72, and 73 were proﬁled

against the remaining mGlu receptor subtypes to determine

their selectivity relative to mGlu₂and mGlu₃(Table 3). With

the exception of 67, which displayed weak antagonist/NAM

activity (IC₅₀> 10 μM) at mGlu₁and mGlu₄, and compound

73, which showed weak PAM activity at mGlu₆(EC₅₀

>

10 μM), these compounds were found to be highly selective for

mGlu₂and mGlu₃. As a representative of this series, compound

72 was proﬁled against a representative panel of CNS receptors

through the NIMH Psychoactive Drug Screening Program

(PDSP; see http://pdsp.med.unc.edu/indexR.html for details).

As shown in Table S2 (see Supporting Information), at 10 μM,

no binding activity was detected for compound 72 at 45 CNS

receptors, suggesting that the new mGlu_2/3PAMs have a low

likelihood of oﬀ-target activity.

At a relatively early stage of the project and on the basis

of the overall in vitro proﬁle of the PAMs (Tables 1 and 2),

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Table 2. In Vitro ADME Data for Group II mGlu PAMs

a

Permeability is monitored by measuring the amount of compound that can diﬀuse through a polar brain lipid membrane to predict BBB

permeability.⁴²Percent remaining after incubation for 60 min at 37.5 °C.

b,c

we selected compounds 20, 36, 44, and 50 for in vivo

assessment of pharmacokinetic (PK) properties in rats. For this

initial evaluation we determined the PK properties of the com-

pounds by oral (po) and intravenous (iv) routes of administra-

tion as shown in Tables 4 and 5, respectively. The PAMs were

found to be systemically bioavailable with half-life (t_1/2) values

of greater than 90 min when dosed po and demonstrate a range

of maximal plasma levels from a low of 1.05 μM (50) to a high

of 12.46 μM (36) (Table 4). All compounds had moderate

volume of distribution at steady state (Vd_ss) and medium to

high clearance (CL) values, indicating moderate metabolism

with a primary distribution in plasma and extracellular ﬂuids,

suggesting that one or more of the PAMs might have promise

as candidates for in vivo studies (Table 5). The four com-

pounds exhibited low (20) to good oral bioavailability (50)

(% F) albeit at the relatively high oral dose of 20 mg/kg;

however, the brain levels of 20, 36, and 44 were low, resulting

in low brain/plasma ratios. Although the brain/plasma ratios

of these compounds are low, the total brain concentrations of

20 and 44 are 9-fold and 18-fold above the in vitro EC₅₀for

mGlu₂, respectively, and close to the in vitro EC_50sfor mGlu₃.

Since compound 44 had shown the best mix of in vitro and

in vivo properties at this stage, including potent activity

and eﬃcacy at mGlu₃receptors, this PAM was selected for

comprehensive in vitro pharmacological characterization. We

began with a more detailed investigation of the activity of 44 in

the mGlu₂and mGlu₃GIRK thallium ﬂux assays. The nature of

the GIRK assay requires that each compound is screened for

a single mode of pharmacology at a time, since activity is only

detected through the GIRK channel when thallium is added to

the assay. The data presented in Table 1 represent compounds

screened for activity in “PAM mode”, where a test compound

is added, followed 2.5 min later by an EC₂₀concentration of

glutamate in the presence of thallium. We had noted that the

response of 44 in “PAM mode” toward mGlu₂decreased

slightly at higher concentrations of test compound (Figure 4A).

This decrease could be caused by either receptor desensitiza-

tion or intrinsic agonist activity of 44 that was not detected

because of the mode in which the functional assay was

performed. To investigate this further, we carried out the same

experiments in the absence of an EC₂₀concentration of agonist

(agonist mode). For these experiments, test compounds were

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a

Table 3. mGlu Receptor Subtype Selectivity

compound

20

25

33

44

67

72

73

b

mGlu₁

mGlu₂

mGlu₃

mGlu₄

inactive

antagonist

FS = 0.3

inactive

E_min= 3%

E_max= 79%

Ago-PAM

FS = 3.2

Ago-PAM

FS = 12.5

E_min= 45%

E_max= 71%

Ago-PAM

FS = 13.3

E_min= 54%

E_max= 77%

Ago-PAM

FS = ND

Ago-PAM

FS = ND

Ago-PAM

FS = ND

Ago-PAM

FS = ND

E_min= 54%

E_max= 77%

E_min= 90%

E_max= 95%

E_min= 79%

E_max= 92%

E_min= 72%

E_max= 95%

E_min= 79%

E_max= 92%

PAM

Ago-PAM

FS = 7.1

Ago-PAM

FS = 8.9

FS = 2.7

E_min= 9%

E_max= 153%

FS = 4.5

E_min= 6%

E_max= 143%

FS = 2.5

E_min= 10%

E_max= 143%

FS = 3.9

E_min= 4%

E_max= 116%

FS = 2.9

E_min= 1%

E_max= 88%

E_min= 54%

E_max= 95%

E_min= 30%

E_max= 99%

b

inactive

antagonist

FS = 1.9

inactive

E_min= 0%

E_max= 74%

b

mGlu₅

mGlu₆

inactive

b

inactive

PAM

FS = 2.0

E_min= 8%

E_max= 93%

b

mGlu₇

mGlu₈

inactive

b

inactive

a

In these selectivity experiments, for all receptors a full concentration−response of agonist was performed once in triplicate in the presence and

absence of a 10 μM ﬁnal concentration of each compound. This allows determination of positive allosteric modulator (PAM) (left shift of the agonist

concentration response curve), antagonist (right shift in the agonist concentration response with a possible decrease in maximal agonist response),

and agonist (increase in baseline response) activity in a single experiment. General activity for each compound at each mGlu is listed (PAM,

antagonist, Ago-PAM, inactive) followed by the fold-shift (FS) of the agonist concentration−response obtained. Where tested compounds

demonstrate activity toward an mGlu receptor subtype, the maximal (E_max) and minimal (E_min) responses of the concentration−response of agonist

are indicated. Where 10 μM test compound induced greater than a 2-fold shift (FS) of the glutamate concentration−response curve (L-AP4 in the

case of mGlu₇), full compound concentration−response curves were performed in triplicate on 3 diﬀerent days to assess compound potency.

Compound 67 showed weak antagonist/NAM activity (IC₅₀> 10 μM) at mGlu₁and mGlu₄, and compound 73 showed weak PAM activity at mGlu₆

b

(EC₅₀> 10 μM). Inactive compounds show no ability to left- or right-shift the agonist concentration response curve at 10 μM. ND = not determined.

a

Table 4. In Vivo PK Data for mGlu₂/mGlu₃PAMs in Rats after po Administration (20 mg/kg)

compd

C_max(μM)

T_max(min)

AUC_(0→t)(μmol/L)·min

T_1/2(min)

F (%)

brain (μM)

plasma (μM)

brain/plasma

20

36

44

50

2.57 0.45

12.46 6.42

2.75 0.47

1.05 0.15

135 15

544.8 75.4

720.5 283.4

1274.6 246.3

466.2 105.2

303 55

89 17

23.5

58.2

41.9

60.4

1.12 0.43

0.23 0.06

0.81 0.17

ND

5.29 1.98

4.23 1.19

16.38 1.13

ND

0.20 0.03

0.06 0.03

0.05 0.01

ND

30

0

90 17

96 40

471 89

569 199

a

C_max: maximum concentration of the compound detected in plasma. T_max: time at C_max. AUC: area under the curve. t_1/2: terminal half-life. F: oral

bioavailability. Brains and plasma were harvested at or near the T_max. Compounds were dosed in a volume of 2 mL/kg po (n = 3−4) at 20 mg/kg in

0.6% Tween 80. ND = not determined.

added in the presence of thallium and GIRK activity was

immediately monitored. We found that 44 displayed intrinsic

agonist activity toward mGlu₂but not mGlu₃in the GIRK assay

(Figure 4B). Thus, this compound is best characterized as

having mGlu₂agonist-PAM activity and mGlu₃PAM activity in

the GIRK thallium ﬂux assays.

ligand glutamate (Figure 5). Fold-shift values are calculated

by determining the ratio of the potency of the orthosteric

agonist glutamate in the presence and absence of increasing

concentrations of an allosteric modulator. Increasing ﬁxed

concentrations of 44 dose-dependently shifted the glutamate

concentration−response of mGlu₂(Figure 5A) and mGlu₃

(Figure 5B) to the left, consistent with an enhancement of

glutamate responses. For these assays, the Ago-PAM activity

We next evaluated 44 in a fold-shift assay, another measure

of the potentiating activity of a PAM toward the orthosteric

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a

Table 5. In Vivo PK Data for mGlu₂/mGlu₃PAMs in Rats after iv Administration (2 mg/kg)

compd

C_max(μM)

CL (mL·min⁻¹·kg⁻¹

)

Vd_ss(L·kg⁻¹

)

AUC_(0→t)(μmol/L)·min

T_1/2(min)

20

36

44

50

5.00 0.60

3.52 0.13

6.43 0.39

2.15 0.14

21.09 3.66

38.78 2.55

14.58 1.18

58.29 7.98

0.81 0.14

0.89 0.03

0.55 0.04

2.87 0.60

231.7 42.12

123.7 7.7

304.6 25.8

77.2 8.0

30

18

28

2

3

0

57 12

a

C_max: maximum concentration of the compound detected in plasma. AUC: area under the curve. t_1/2: terminal half-life. CL: clearance. Vd_ss: steady

state volume of distribution. Compounds were injected in a volume of 1 mL/kg iv (n = 3−4) through an iv catheter at 2 mg/kg in 0.6% Tween 80 or

in 1 M NaOH. pH was adjusted to ∼7.

Figure 5. Compound 44 dose-dependently induces a leftward shift in

the glutamate concentration response at (A) mGlu₂and (B) mGlu₃in

GIRK thallium ﬂux assays. The increase in baseline in the mGlu₂GIRK

assay at higher concentrations of 44 is due to the Ago-PAM activity of

this compound. The leftward shifts induced by 44 indicate a

potentiation of the response of mGlu₂and mGlu₃to glutamate. The

maximal fold-shift at mGlu₂is 4.50 0.96 and was derived from the

test concentration of 44 (300 nM) due to Ago-PAM activity. The

Figure 4. Compound 44 displays Ago-PAM activity toward mGlu₂

and PAM activity toward mGlu₃in GIRK thallium-ﬂux assays. A

concentration−response of 44 was performed in the presence (A) and

absence (B) of an EC₂₀of glutamate in either the mGlu₂GIRK assay

(squares) or mGlu₃GIRK assay (triangles). In the mGlu₂assay, 44

displays both agonist and PAM activity and is characterized as an Ago-

PAM. In the mGlu₃assay, only PAM activity is detected. Data were

analyzed using nonlinear regression, providing EC₅₀values for each

curve. Data were obtained from three separate experiments performed

in triplicate, normalized to the response to 100 μM glutamate in each

experiment, and are expressed as the mean SEM.

maximal fold-shift at mGlu₃is 5.48

0.27 for the 10 μM test

concentration. Concentration−response relationships were generated

by adding a ﬁxed concentration of 44 to cells as indicated, followed by

increasing concentrations of glutamate. Data were analyzed using

nonlinear regression, providing EC₅₀values for each curve. Data were

obtained from three separate experiments performed in duplicate,

normalized to the response to 100 μM glutamate in each experiment,

and are expressed as the mean SEM.

toward mGlu₂is readily apparent as the increase in baseline at

low concentrations of glutamate (Figure 5A). These data are in

contrast with the results for mGlu₃(Figure 5B), which does not

show a change in baseline of the glutamate dose−response.

In the ﬁnal set of pharmacological characterization experi-

ments, we evaluated the potent PAM 44 in an orthogonal assay

of mGlu₃and mGlu₂activity. For mGlu₃we utilized the TREx

tetracycline-inducible system (Invitrogen). We developed a cell

line in which the expression of mGlu₃is dose-dependently

induced by tetracycline (Tet) and functionally coupled to calcium

mobilization by the promiscuous G protein G_α15(Figure 6). In

the absence of Tet, no measurable expression of mGlu₃is

detected either by Western blot (Figure 6A) or by functional

response to calcium mobilization (Figure 6B). The optimal

calcium mobilization response for this cell line was achieved at

20 ng/mL Tet for 20 h prior to assay. This concentration of

Tet was then utilized for further characterization of 44 in the

TREx293 mGlu₃G_α15calcium assay (Figure 7B), which shows

that 44 demonstrates mGlu₃PAM activity in this orthogonal

assay whereas the mGlu₂selective PAM BINA remains inactive.

These compounds were also evaluated in calcium assays utilizing

HEK293A mGlu₂G_α15cells as shown in Figure 7A. Unlike the

mGlu₂GIRK assay where it displays Ago-PAM activity,

compound 44 behaves as a PAM in the calcium assay. In future

experiments, it will be essential to determine the potency and

selectivity of 44 in native tissue preparations to determine

whether the observed proﬁle in vitro is replicated in vivo.

Following the in vitro pharmacological characterization of

44 as a representative analogue of this class of mGlu_2/3PAMs,

we wished to evaluate a member of this series in eﬃcacy studies

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Figure 6. Development of a cell line with inducible mGlu₃expression

coupled to calcium mobilization via the promiscuous G protein G_α15

.

(A) TREx293 mGlu₃G_α15cells were stimulated with the indicated

concentrations of tetracycline (Tet) for 20 h. Protein lysates were

prepared. Equivalent amounts of protein were loaded for all lanes, and

mGlu_2/3expression was detected by Western blot. Both monomeric

and dimeric mGlu₃were detected as indicated. (B) TREx293 mGlu₃

Figure 7. Compound 44 displays PAM activity toward mGlu₂and

mGlu₃in calcium assays utilizing the promiscuous G protein G_α15

.

G_α15cells were stimulated with the indicated concentrations of Tet for

A concentration−response of 44 (triangles) and the control mGlu2

selective PAM BINA (squares) was performed in the presence of an

EC₂₀of glutamate in either the (A) HEK293A mGlu₂G_α15calcium

assay or (B) TREx293 mGlu₃G_α15calcium assay. In both assays, 44

displays PAM activity. BINA displays PAM activity in the mGlu₂

calcium assay but is inactive in the mGlu₃calcium assay. For this assay,

mGlu₃expression was induced with 20 ng/mL Tet for 20 h prior to

assay. Data were analyzed using nonlinear regression, providing EC₅₀

values for each curve. Data were obtained from three separate experi-

ments performed in triplicate, normalized to the response to 100 μM

glutamate in each experiment, and are expressed as the mean SEM.

20 h, and a calcium mobilization assay was performed. Tet dose-

dependently induced a glutamate-simulated calcium response that was

maximal at 20 ng/mL Tet. Data were analyzed using nonlinear

regression. Data were obtained from three separate experiments

performed in triplicate, normalized to the response to 100 μM

glutamate in each experiment, and are expressed as the mean SEM.

in rats. Given the low brain levels achieved by po dosing for

compounds 20, 36, 44, and 50 (Table 4), we evaluated nine

compounds by intraperitoneal (ip) dosing in order to avoid

ﬁrst pass metabolism and to cast a wider net for a compound

suitable for rat eﬃcacy studies (Table 6). All compound plasma

levels were determined, but only those with the highest plasma

concentrations (i.e., 44, 73, 74, and 75) were evaluated for

brain levels. On the basis of its combination of potency, sel-

ectivity, and PK properties, compound 74 was selected for

eﬃcacy studies in rats.

Taking all the relevant data into account, we determined that

the mGlu_2/3receptor PAM 74 would be a good candidate for

evaluation in a rat model of cocaine dependence. When assessed

in vivo, compound 74 dose-dependently decreased cocaine- and

food-maintained responding [compound 74 dose main eﬀect:

F_3,51= 14.55; p < 0.0001] (Figure 8). Interestingly, however,

cocaine-maintained responding was decreased to a greater

extent than food-maintained responding at the highest dose

tested (40 mg/kg; p < 0.05). Because of the within-subjects

design of the dose response (i.e., each rat received each dose of

compound 74 using a Latin-square design), it was not possible

to collect brain samples to determine brain concentrations of

compound 74 at 40 mg/kg during behavioral testing. It is

unlikely that brain concentrations of compound 74 diﬀered

between cocaine- and food-maintained rats at this dose, sug-

gesting that the observed diﬀerences in behavior were not a

function of group diﬀerences in brain pharmacokinetic proper-

ties of compound 74, although this should be conﬁrmed in

future studies. We have previously shown that the mGlu_2/3

receptor agonist LY379268 similarly decreased both cocaine-

Table 6. In Vivo PK for mGlu_2/3PAMs in Rats after ip

a

Administration (10 mg/kg)

a

compd plasma (μM)

plasma t_1/2(min) brain (μM)

brain/plasma

44

50

60

65

68

72

73

74

75

10.57 2.52

43

20

0.23 0.10

0.03 0.01

ND

3.29 1.27

4.49 0.49

5.44 2.92

2.18 0.58

2.44 2.19

6.81 0.84

17.05 0.19

6.52 0.54

ND

22

ND

30

ND

28

ND

27

ND

132

106

80

0.47 0.35

0.56 0.10

0.22 0.04

0.02 0.01

0.03 0.01

0.01 0.01

a

Maximum concentration of the compound detected in plasma or

brain. t_1/2: terminal half-life. Compounds were dosed ip (n = 3) at

10 mg/kg in 10% EtOH/1% Tween 80. pH was adjusted to ∼7. Brains

and plasma were harvested at the T_max(30 min for all tested). ND =

not determined.

and food-maintained responding.^37,53Moreover, the selective

mGlu₂receptor PAM BINA decreased only cocaine-maintained

responding while having no eﬀect on food-maintained

responding.³⁷Consistent with our previous results, Morishima

et al. (2005) have demonstrated that mGlu₂receptor knockout

mice exhibited increased conditioned place preference for

cocaine.⁵⁴While these prior studies focused only on the role

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Companion system. H and ¹³C NMR spectra were obtained on a

Jeol 400 spectrometer at 400 and 100 MHz, respectively. Chemical

shifts are reported in δ (ppm) relative to residual solvent peaks or TMS

as internal standards. Coupling constants are reported in Hz. Melting

points were obtained using a capillary melting point apparatus (MEL-

TEMP) and are uncorrected. High-resolution ESI-TOF mass spectra

were acquired from the Mass Spectrometry Core at The Sanford-

Burnham Medical Research Institute (Orlando, FL). HPLC−MS

analyses were performed on a Shimadzu 2010EV LCMS instrument

using the following conditions: Kromisil C18 column (reverse phase,

4.6 mm × 50 mm); a linear gradient from 10% acetonitrile and 90%

water to 95% acetonitrile and 5% water over 4.5 min; ﬂow rate of

1 mL/min; UV photodiode array detection from 200 to 300 nm.

General Methods for the Synthesis of mGlu_2/3Receptor

PAMs. General Method A. To a stirred solution of methyl

4-hydroxybenzoate (1 mmol, 1 equiv) and 1,4-dibromobutane (3 mmol,

3 equiv) in ACN was added potassium carbonate (2 mmol, 2 equiv). The

reaction mixture was heated at reﬂux for 6 h, at which time it was cooled

to room temperature. The crude reaction mixture was diluted with

CH₂Cl₂and washed twice with 5% aqueous HCl (200 mL). The organic

layers were collected and washed twice with saturated NaHCO₃solution

(200 mL). The organic layers were collected, dried over Na₂SO₄, and

evaporated to dryness. To a stirred solution of AlCl₃(0.039 mol, 1 equiv)

in CH₂Cl₂at 0 °C under nitrogen, the acyl chloride (0.039 mol, 1 equiv)

was dissolved in CH₂Cl₂and added dropwise to the stirred solution. The

phenol (0.039 mol, 1 equiv) was added to the reaction mixture, and the

mixture was allowed to warm to room temperature over 12 h. The

reaction was quenched with HCl (5% aqueous), and CH₂Cl₂was added

(50 mL). The organic layer was separated and washed with saturated

NaHCO₃solution, then brine and dried over Na₂SO₄. The solvents were

removed by rotary evaporation, and the products were isolated by ﬂash

chromatography [SiO₂, hexanes/EtOAc (4:1)] and concentrated in vacuo.

To a crimp top microwave vial were added the phenol (1 mmol, 1 equiv),

bromobutoxy benzoate (1 mmol, 1 equiv), potassium carbonate (2 mmol,

2 equiv), and potassium iodide (0.1 mmol, 0.1 equiv), all dissolved in

ACN (0.2 M). The reaction mixture was heated in the microwave at

160 °C for 15 min. Following ﬁltration and evaporation of solvents, the

products were isolated by ﬂash chromatography or reverse phase HPLC

and lyophilized to provide the ﬁnal compounds which were determined to

1

Figure 8. The mGlu_2/3PAM, 74, decreased cocaine-maintained

responding, and to a lesser extent food-maintained responding, in rats.

At the highest dose tested (40 mg/kg), cocaine-maintained responding

was signiﬁcantly lower than food-maintained responding. The 40 mg/kg

dose decreased responding for food compared to vehicle only, whereas

the same dose decreased responding for cocaine compared to all other

doses tested. (∗) Responding was signiﬁcantly diﬀerent from 0 mg/kg.

(#) Responding was signiﬁcantly diﬀerent from cocaine. (@)

Responding was signiﬁcantly diﬀerent from 0, 10, and 20 mg/kg.

Data are expressed as the mean SEM of responding at baseline.

of mGlu₂receptors in responding for drug and natural rewards,

our present ﬁndings begin to delineate the individual roles

of mGlu₂and mGlu₃receptors in reward processing. These

patterns of results possibly suggest that activation of mGlu₂

receptors selectively modulates drug-reinforced behavior,

whereas activation of mGlu₃receptors either selectively

modulates responding for natural rewards or nonselectively

modulates responding for both drug and natural rewards. Use of an

mGlu_2/3receptor PAM, as reported here, provided an initial tool by

which to indirectly test these hypotheses. However, future develop-

ment and use of selective mGlu₃receptor PAMs are necessary

to test these hypotheses directly. Moreover, increasing mGlu_2/3

receptor activity using a PAM compared to a receptor agonist

may aﬀect behaviors reinforced by natural rewards to a lesser

extent relative to drug-reinforced behaviors. Thus, targeting mGlu₂

receptors with a PAM may be an eﬀective strategy for treating

drug dependence without aﬀecting other motivated behaviors.

In conclusion, a ligand−based rational design approach

utilizing certain previously reported selective mGlu₂receptor

PAMs as starting points led to a series of new mGlu_2/3PAMs.

A library of more than 60 analogues was synthesized and tested,

providing compounds with excellent potency and eﬃcacy at

mGlu₂and mGlu₃receptors in vitro. The most promising

mGlu_2/3PAMs were proﬁled in in vitro ADME assays, and on

the basis of these data, several compounds were selected for

PK studies in rats. The lead structures were found to be

essentially devoid of activity at other mGlu receptor subtypes

and 45 additional CNS receptors. Representative PAM 44 was

characterized extensively in vitro and was found to demonstrate

ago-PAM activity at mGlu₂and PAM activity at mGlu₃

receptors. Finally, the systemically active mGlu_2/3PAM 74

dose-dependently decreased cocaine self-administration in

rats following a single intraperitoneal dose. Future studies will

focus on additional characterization of the new mGlu_2/3PAMs

and further optimization of compounds with enhanced PAM

activity at mGlu₃receptors.

1

be >95% pure by HPLC−UV, HPLC−MS, and H NMR.

General Method B. To a stirred solution of the product from

“General Method A” (1 mmol, 1 equiv) in dioxane at room

temperature was added KOH (6 mmol, 6 equiv) in water (0.5 mL).

The mixture was stirred continuously for an additional 12 h. The

reaction was quenched with HCl (5% aqueous), and CH₂Cl₂(50 mL)

was added. The organic layer was separated and dried over Na₂SO₄.

The solvents were removed by rotary evaporation, and the products

were isolated by ﬂash chromatography [SiO₂, hexanes/EtOAc (1:1)] or

reverse phase HPLC and lyophilized to provide the ﬁnal compounds

which were determined to be >95% pure by HPLC−UV, HPLC−MS,

1

and H NMR. Compounds 4, 5, and 6 were synthesized according to

the published procedures.^50,51The following compounds were prepared

using the general procedures A and B from the appropriate starting

materials.

Methyl 4-(4-(2,3-Dimethyl-4-(3-methylbutanoyl)phenoxy)-

butoxy)benzoate (14). 14 was prepared according to general

1

procedure A. Colorless solid (0.070 g, 17%); mp 55−57 °C. H NMR

(CDCl₃): δ 7.86 (d, J = 7.8 Hz, 2H), 7.17−6.94 (m, 4H), 4.05 (t, J = 5.9

Hz, 2H), 3.88 (s, 3H), 3.80 (t, J = 6.0 Hz, 2H), 2.81 (d, J = 6.9 Hz, 2H),

2.29 (s, 3H), 2.20 (s, 3H), 2.10−2.01 (m, 1H), 1.88−1.84 (m, 4H), 0.93

(d, J = 6.4 Hz, 6H). ¹³C NMR (CDCl₃): δ 203.3, 166.8, 162.7, 155.5,

142.3, 132.2, 131.5, 130.6, 125.7, 125.4, 122.5, 114.0, 75.1, 67.7, 51.8,

51.7, 26.9, 25.9, 25.1, 22.63, 20.0, 12.3. ESI-MS m/z 413 [M + H]⁺.

HRMS m/z calcd for C₂₅H₃₂O₅[M + H]⁺: 413.2323. Found: 413.2256.

Methyl 4-(4-(4-(2-Cyclopentylacetyl)-2,3-dimethylphenoxy)-

butoxy)benzoate (15). 15 was prepared according to general

procedure A. Colorless solid (0.039 g, 9%). ¹H NMR (CDCl₃): δ 7.87

(d, J = 9.2 Hz, 2H), 7.17 (d, J = 7.8 Hz, 1H), 7.00−6.97 (m, 3H), 4.05

(t, J = 5.9 Hz, 2H), 3.88 (s, 3H), 3.80 (t, J = 5.9 Hz, 2H), 2.95 (d, J =

6.9 Hz, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 2.21−2.10 (m, 1H), 1.99−1.89

EXPERIMENTAL SECTION

■

General Chemistry. All reactions were performed in oven-dried

glassware under an atmosphere of argon with magnetic stirring. All

solvents and chemicals used were purchased from Sigma-Aldrich or

Acros, and were used as received without further puriﬁcation. Purity of

compounds was established by liquid chromatography−mass spec-

troscopy (HPLC−MS) and was >95% for all tested compounds. Silica

gel column chromatography was carried out using prepacked silica

cartridges from RediSep (ISCO Ltd.) and eluted using an Isco

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(m, 4H), 1.80−1.76 (m, 2H), 1.59−1.46 (m, 4H), 1.14−1.01 (m, 2H).

¹³C NMR (CDCl₃): δ 203.3, 166.8, 162.7, 155.5, 142.3, 132.2, 131.5,

130.6, 126.9, 126.2, 114.0, 74.7, 67.7,51.8, 49.0, 36.1, 32.7, 26.9, 25.9,

24.9, 20.0, 12.3. ESI-MS m/z 439 [M + H]⁺. HRMS m/z calcd for

C₂₇H₃₄O₅[M + H]⁺: 439.2479. Found: 439.2419.

Methyl 4-(4-(3-Hydroxy-2-methyl-4-(3-methylbutanoyl)-

phenoxy)butoxy)benzoate (16). 16 was prepared according to

general procedure A. Colorless solid (0.112 g, 27%); mp 72−74 °C.

¹H NMR (CDCl₃): δ 7.98−7.96 (m, 2H), 7.58 (d, J = 8.7 Hz, 1H),

6.90−6.88 (m, 2H), 6.41(d, J = 9.2 Hz, 1H), 4.09−4.03 (m, 4H), 3.86

(s, 3H), 2.76 (d, J = 7.3 Hz, 2H), 2.20−2.15 (m, 1H), 2.09 (s, 3H),

1.96−1.94 (m, 4H), 0.97 (d, J = 6.9 Hz, 6H). ¹³C NMR (CDCl₃): δ

205.1, 166.8, 162.6, 162.5, 162.3, 131.5, 129.3, 122.5, 114.0, 113.7,

102.4, 67.7, 67.5, 51.8, 46.8, 26.9, 26.8, 25.9, 22.7, 7.6. ESI-MS m/z

415 [M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₆[M + H]⁺: 415.2115.

Found: 415.2137.

1H), 6.73−6.69 (m, 1H), 4.04−4.03 (m, 4H), 2.74 (d, J = 6.9 Hz,

2H), 2.11 (s, 3H), 2.03 (s, 3H), 1.90−1.87 (m, 1H), 1.86−1.84 (m,

4H), 0.82 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ 205.6, 167.2,

162.9, 161.4, 158.6, 132.7, 131.4, 129.7, 124.7, 121.8, 119.8, 114.2,

109.8, 74.2, 67.6, 51.7, 25.6, 25.3, 22.4, 24.1, 19.9, 19.8, 12.0, 11.9. ESI-

MS m/z 399 [M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₅[M + H]⁺:

399.2166. Found: 399.2239.

3-(4-(4-(2-Cyclopentylacetyl)-2,3-dimethylphenoxy)butoxy)-

benzoic Acid (23). Colorless solid (0.114 g, 27%). ¹H NMR

(DMSO): δ 7.47−7.40 (m, 3H), 7.19−7.15 (m, 2H), 6.97 (d,

J = 7.8 Hz, 1H), 4.04 (t, J = 5.6 Hz, 2H), 3.73 (t, J = 5.9 Hz, 2H),

2.89 (d, J = 6.9 Hz, 2H), 2.21 (s, 3H), 2.11−2.04 (ovelapping

singlet and multiplets, 4H), 1.85−1.83 (m, 4H), 1.66−1.64 (m, 2H),

1.48−1.39 (m, 4H), 1.04−1.01 (m, 2H). ¹³C NMR (DMSO): δ 205.6,

167.2, 167.2, 158.2, 155.2, 141.9, 132.7, 131.4, 130.2, 129.7, 125.9,

125.2, 121.8, 119.3, 114.5, 74.4, 67.6, 48.4, 35.5, 32.0, 26.3, 25.4, 24.5,

20.0, 11.9. ESI-MS m/z 425 [M + H]⁺. HRMS m/z calcd for

C₂₆H₃₂O₅[M + H]⁺: 425.2323. Found: 425.2383.

3-(4-(3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy)-

butoxy)benzoic Acid (24). Colorless solid (0.148 g, 37%); mp 118−

120 °C. ¹H NMR (DMSO): δ 7.81 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 7.8

Hz, 1H), 7.39−7.34 (m, 2H), 7.14−7.13 (m, 1H), 6.58 (d, J = 9.2 Hz,

1H), 4.13 (t, J = 5.9 Hz, 2H), 4.07 (t, J = 5.9 Hz, 2H), 2.81 (d, J = 6.9

Hz, 2H), 2.11−2.08 (m, 1H), 1.95 (s, 3H), 1.88−1.86 (m, 4H), 0.91

(d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ 205.6, 167.2, 162.5, 161.4,

158.6, 132.3, 130.4, 129.7, 121.6, 119.4, 114.5, 113.5, 112.0, 103.4,

67.9, 67.3, 46.0, 25.6, 25.4, 22.5, 7.5. ESI-MS m/z 401 [M + H]⁺.

HRMS m/z calcd for C₂₃H₂₈O₆[M + H]⁺: 401.1959. Found:

401.1967.

3-(4-(4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy)-

butoxy)benzoic Acid (25). Colorless solid (0.230 g, 54%); mp 125−

127 °C. ¹H NMR (DMSO): δ 7.76 (d, J = 9.2 Hz, 1H), 7.46 (d, J = 7.8

Hz, 1H), 7.41−7.33 (m, 2H), 7.13−7.12 (m, 1H), 6.58 (d, J = 9.2 Hz,

1H), 4.08−4.03 (m, 4H), 2.94 (d, J = 7.3 Hz, 2H), 2.17−2.16 (m,

1H), 1.95 (s, 3H), 1.87−1.85 (m, 4H), 1.71−1.68 (m, 2H), 1.55−1.42

(m, 4H), 1.12−1.09 (m, 2H). ¹³C NMR (DMSO): δ 205.8, 162.5,

161.3, 158.6, 132.3, 130.3, 129.7, 121.6, 119.4, 114.6, 113.3, 112.0,

103.3, 67.9, 67.4, 43.6, 36.3, 32.1, 25.4, 24.5, 7.5. 167.2, ESI-MS m/z

427 [M + H]⁺. HRMS m/z calcd for C₂₅H₃₀O₆[M + H]⁺: 427.2115.

Found: 427.2122.

2-(4-(2,3-Dimethyl-4-(3-methylbutanoyl)phenoxy)butoxy)-

benzoic Acid (26). Colorless solid (0.207 g, 52%); mp 48−50 °C. ¹H

NMR (DMSO): δ 7.58 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H),

7.11−6.99 (m, 3H), 6.73−6.68 (m, 1H), 4.09−4.04 (m, 4H), 2.77 (d,

J = 7.3 Hz, 2H), 2.24 (s, 3H), 2.18 (s, 3H), 2.01−2.00 (m, 1H), 1.89−

1.86 (m, 4H), 0.86 (d, J = 6.8 Hz, 6H). ¹³C NMR (DMSO): δ 205.8,

167.4, 157.5, 156.5, 137.2, 133.0, 130.7, 125.9, 121.9, 120.1, 113.5,

109.3, 67.9, 67.4, 51.2, 48.7, 25.6, 24.6, 22.4, 19.8, 12.1, 11.5. ESI-MS

m/z 399 [M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₅[M + H]⁺:

399.2166. Found: 399.2284.

2-(4-(4-(2-Cyclopentylacetyl)-2,3-dimethylphenoxy)butoxy)-

benzoic Acid (27). Colorless solid (0.199 g, 47%); mp 90−92 °C. ¹H

NMR (DMSO): δ 7.59 (d, J = 7.9 Hz, 1H), 7.46−7.44 (m, 2H), 7.06

(d, J = 8.2 Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H),

4.05−4.04 (m, 4H), 2.79 (d, J = 6.9 Hz, 2H), 2.24 (s, 3H), 2.19 (s,

3H), 2.11−2.04 (m, 1H), 1.89−1.86 (m, 4H), 1.66−1.64 (m, 2H),

1.51−1.40 (m, 4H), 1.05−1.02 (m, 2H). ¹³C NMR (DMSO): δ 204.1,

167.5, 158.1, 157.4, 136.7, 132.8, 132.2, 130.6, 127.4, 125.5, 121.7,

120.0, 113.4, 107.9, 67.8, 67.5, 47.6, 36.1, 32.0, 25.5, 24.5, 16.5,

11.5. ESI-MS m/z 425 [M + H]⁺. HRMS m/z calcd for C₂₆H₃₂O₅

[M + H]⁺: 425.2323. Found: 425.2357.

Methyl 4-(4-(4-(2-Cyclopentylacetyl)-3-hydroxy-2-

methylphenoxy)butoxy)benzoate (17). 17 was prepared accord-

1

ing to general procedure A. Colorless solid (0.277 g, 63%). H NMR

(CDCl₃): δ 7.86 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 1H), 6.90 (d,

J = 9.2 Hz, 2H), 6.61 (d, J = 9.2 Hz, 1H), 4.11−4.09 (m, 4H), 3.87 (s,

3H), 2.90 (d, J = 7.3 Hz, 2H), 2.35−2.20 (m, 1H), 1.96 (s, 3H), 1.94−

1.92 (m, 4H), 1.85−1.76 (m, 2H), 1.59−1.47 (m, 4H), 1.87−1.19 (m,

2H). ¹³C NMR (CDCl₃): δ 205.3, 162.6, 162.5, 162.3, 131.6, 129.2,

122.5, 114.0, 113.8, 113.7, 102.4, 67.7, 67.5, 51.8, 44.0, 36.8, 32.6, 25.9,

24.8, 7.6. ESI-MS m/z 441 [M + H]⁺. HRMS m/z calcd for C₂₆H₃₂O₆

[M + H]⁺: 441.2272. Found: 441.2278.

4-(4-(2,3-Dimethyl-4-(3-methylbutanoyl)phenoxy)butoxy)-

benzoic Acid (18). Colorless solid (0.159 g, 40%). ¹H NMR

(DMSO): δ 7.84 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.2 Hz, 1H), 6.99 (d,

J = 8.7 Hz, 3H), 4.11 (t, J = 5.5 Hz, 2H), 3.75 (t, J = 5.5 Hz, 2H), 2.78

(d, J = 6.9 Hz, 2H), 2.24 (s, 3H), 2.15 (s, 3H), 2.02−1.98 (m, 1H),

1.87−1.80 (m, 4H), 0.86 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ

203.6, 167.0, 162.1, 155.1, 142.1, 131.7, 131.3, 130.4, 125.9, 125.2,

123.1, 114.2, 74.5, 67.5, 51.1, 27.2, 26.3, 25.3, 24.5, 22.3, 20.0, 12.0.

ESI-MS m/z 399 [M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₅[M + H]⁺:

399.2166. Found: 399.2191.

4-(4-(4-(2-Cyclopentylacetyl)-2,3-dimethylphenoxy)butoxy)-

benzoic Acid (19). Colorless solid (0.093 g, 22%). ¹H NMR

(DMSO): δ 7.83 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 7.8 Hz, 1H), 6.98 (d,

J = 8.7 Hz, 3H), 4.12 (t, J = Hz, 2H), 3.75 (t, J = Hz, 2H), 2.90 (d, J =

6.9 Hz, 2H), 2.25 (s, 3H), 2.14 (s, 3H), 2.09−2.07 (m, 1H), 1.86−1.84

(m, 4H), 1.69−1.62 (m, 2H), 1.51−1.37 (m, 4H), 1.04−0.98 (m, 2H).

¹³C NMR (DMSO): δ 207.8, 167.0, 162.3, 154.2, 142.1, 131.4, 130.4,

125.8, 125.6, 122.9, 114.3, 74.1, 67.9, 49.3, 35.6, 32.1, 25.6, 25.1, 24.5,

20.2, 12.1. ESI-MS m/z 425 [M + H]⁺. HRMS m/z calcd for

C₂₆H₃₂O₅[M + H]⁺: 425.2323. Found: 425.2366.

4-(4-(3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy)-

butoxy)benzoic Acid (20). Colorless solid (0.208 g, 52%); mp 153−

155 °C. ¹H NMR (DMSO): δ 7.84 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.7

Hz, 1H), 6.96 (d, J = 9.2 Hz, 2H), 6.60 (d, J = 9.2 Hz, 1H), 4.11−4.06

(m, 4H), 2.79 (d, J = 6.9 Hz, 2H), 2.10−2.08 (m, 1H), 1.93 (s, 3H),

1.89−1.86 (m, 4H), 0.89 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ

206.2, 167.0, 162.5, 162.3, 161.4, 131.4, 130.4, 122.9, 114.3, 113.5,

112.0, 103.4, 67.7, 67.5, 51.8, 44.0, 36.8, 32.6, 25.4, 22.4, 7.6. ESI-MS

m/z 401 [M + H]⁺. HRMS m/z calcd for C₂₃H₂₈O₆[M + H]⁺:

401.1956. Found: 401.1966.

4-(4-(4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy)-

butoxy)benzoic Acid (21). Colorless solid (0.192 g, 45%); mp 158−

160 °C. ¹H NMR (DMSO): δ 7.83−7.81 (m, 3H), 6.69 (d, J = 8.7 Hz,

2H), 6.59 (d, J = 8.7 Hz, 1H), 4.11−4.08 (m, 4H), 2.97 (d, J = 6.9 Hz,

2H), 2.22−2.01 (m, 1H), 1.96 (s, 3H), 1.89−1.86 (m, 4H), 1.72−1.71

(m, 2H), 1.59−1.55 (m, 2H), 1.45−1.53 (m, 2H), 1.15−1.12 (m, 2H).

¹³C NMR (DMSO): δ 207.8, 167.0, 162.5, 162.3, 161.4, 131.6, 131.3,

2-(4-(3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy)-

butoxy)benzoic Acid (28). Colorless solid (0.164 g, 41%); mp 101−

1

103 °C. H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz, 1H), 7.59 (d, J =

9.6 Hz, 1H), 7.42−7.41 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.95 (t, J =

7.4 Hz, 1H), 6.60 (d, J = 9.2 Hz, 1H), 4.11−4.07 (m, 4H), 2.79 (d, J =

6.9 Hz, 2H), 2.09−2.02 (m, 1H), 1.95 (s, 3H), 1.89−1.86 (m, 4H),

0.90 (d, J = 6.4 Hz, 6H). ¹³C NMR (CDCl₃): δ 205.6, 167.4, 162.5,

161.3, 157.4, 132.9, 130.6, 130.3, 121.6, 120.0, 113.4, 111.9, 103.3,

67.9, 67.8, 45.8, 36.3, 25.4, 22.4, 7.5. ESI-MS m/z 401 [M + H]⁺.

122.9, 114.2, 112.8, 111.9, 103.8, 69.0, 68.9, 43.8, 36.2, 31.1, 24.5,

22.4, 8.9. ESI-MS m/z 427 [M + H]⁺. HRMS m/z calcd for C₂₅H₃₀O₆

[M + H]⁺: 427.2115. Found: 427.2120.

3-(4-(2,3-Dimethyl-4-(3-methylbutanoyl)phenoxy)butoxy)-

benzoic Acid (22). Colorless solid (0.167 g, 42%). ¹H NMR

(DMSO): δ 7.49−7.46 (m, 2H), 7.41−7.39 (m, 2H), 7.16−7.14 (m,

4164

dx.doi.org/10.1021/jm5000563 | J. Med. Chem. 2014, 57, 4154−4172

Journal of Medicinal Chemistry

Article

HRMS m/z calcd for C₂₃H₂₈O₆[M + H]⁺: 401.1959. Found:

401.1990.

127.3, 125.6, 117.8, 117.6, 116.0, 107.9, 67.9, 67.4, 47.6, 36.1, 32.0,

25.6, 24.5, 16.5, 11.4. ESI-MS m/z 443 [M + H]⁺. HRMS m/z calcd

for C₂₆H₃₁FO₅[M + H]⁺: 443.2228. Found: 443.2214.

2-(4-(4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy)-

butoxy)benzoic Acid (29). Colorless solid (0.158 g, 37%); mp 93−

95 °C. ¹H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 9.6

Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 6.69 (t, J =

7.3 Hz, 1H), 6.63 (d, J = 9.2 Hz, 1H), 4.14−4.11 (m, 4H), 2.98 (d, J =

6.8 Hz, 2H), 2.23−2.18 (m, 1H), 1.97 (s, 3H), 1.92−1.87 (m, 4H),

1.75−1.71 (m, 2H), 1.58−1.47 (m, 4H), 1.15−1.11 (m, 2H). ¹³C

NMR (DMSO): δ 205.8, 167.4, 162.5, 161.3, 157.4, 132.9, 130.6,

130.3, 121.6, 120.0, 113.4, 113.2, 103.3, 111.9, 67.8, 67.9, 43.9, 36.3,

32.0, 25.6, 24.4, 7.5. ESI-MS m/z 427 [M + H]⁺. HRMS m/z calcd for

C₂₅H₃₀O₆[M + H]⁺: 427.2115. Found: 427.2235.

2-Fluoro-5-(4-(3-hydroxy-2-methyl-4-(3-methylbutanoyl)-

phenoxy)butoxy)benzoic Acid (36). Colorless solid (0.188 g,

45%); mp 99−101 °C. ¹H NMR (DMSO): δ 7.76 (d, J = 9.2 Hz, 1H),

7.28−7.15 (m, 3H), 6.58 (d, J = 9.2 Hz, 1H), 4.10−4.03 (m, 4H),

2.78 (d, J = 7.3 Hz, 2H), 2.10−2.01 (m, 1H), 1.94 (s, 3H), 1.87−1.84

(m, 4H), 0.89 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ 205.7,

165.0, 162.5, 161.4, 154.4, 130.4, 121.3, 117.8, 116.0, 113.5, 112.0,

103.4, 67.9, 67.8, 46.0, 25.6, 25.5, 22.5, 7.5. ESI-MS m/z 419

[M + H]⁺. HRMS m/z calcd for C₂₃H₂₇FO₆[M + H]⁺: 419.1864.

Found: 419.1863.

2-Chloro-4-(4-(2,3-dimethyl-4-(3-methylbutanoyl)phenoxy)-

butoxy)benzoic Acid (30). Colorless solid (0.078 g, 18%); mp 77−

79 °C. ¹H NMR (DMSO): δ 7.72−7.68 (m, 1H), 7.09 (d, J = 7.8 Hz,

1H), 6.91−4−6.91(m, 3H), 4.06−4.03 (m, 4H), 2.62 (d, J = 6.9 Hz,

2H), 2.11 (s, 3H), 2.01 (s, 3H), 1.88−1.86 (m, 4H), 1.74−1.72 (m,

1H), 0.78 (d, J = 6.4 Hz, 6H). ¹³C NMR (DMSO): δ 204.1, 167.5,

162.0, 155.6, 143.3, 134.1, 133.9, 131.8, 131.7, 126.0, 125.9, 122.3,

117.1, 113.8, 75.8, 69.9, 51.7, 26.1, 25.8, 25.2, 22.8, 20.6, 12.5. ESI-MS

m/z 433 [M + H]⁺. HRMS m/z calcd for C₂₄H₂₉ClO₅[M + H]⁺:

433.1776. Found: 433.1799.

5-(4-(4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy)-

butoxy)-2-ﬂuorobenzoic Acid (37). Colorless solid (0.208 g, 47%);

mp 110−112 °C. ¹H NMR (DMSO): δ 7.76 (d, J = 9.2 Hz, 1H), 7.28

(d, J = 8.7 Hz, 1H), 7.15−7.13 (m, 2H), 6.59 (d, J = 9.2 Hz, 1H),

4.10−4.03 (m, 4H), 2.94 (d, J = 7.3 Hz, 2H), 2.20−2.10 (m, 1H), 1.94

(s, 3H), 1.87−1.84 (m, 4H), 1.72−1.70 (m, 2H), 1.56−1.44 (m, 4H),

1.12−1.01 (m, 2H). ¹³C NMR (DMSO): δ 206.5, 165.0, 162.5, 161.3,

154.4, 130.3, 121.3, 118.0, 117.8, 116.0, 113.3, 112.0, 103.3, 67.9, 67.8,

44.0, 36.0, 32.1, 25.5, 25.4, 24.5, 7.5. ESI-MS m/z 445 [M + H]⁺.

HRMS m/z calcd for C₂₅H₂₉FO₆[M + H]⁺: 445.2021. Found:

445.2027.

2-Chloro-4-(4-(4-(2-cyclopentylacetyl)-2,3-dimethylphenoxy)-

butoxy)benzoic Acid (31). Colorless solid (0.115 g, 25%); mp 97−

3-{4-[3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]-

1

99 °C. H NMR (DMSO): δ 7.68 (d, J = 8.2 Hz, 1H), 7.38 (d, J =

butoxy}-4-methylbenzoic Acid (38). Colorless solid (0.277 g,

1

8.7 Hz, 1H), 6.88 (s, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.74 (d, J = 8.7 Hz,

1H), 4.07−4.03 (m, 4H), 2.71 (d, J = 6.9 Hz, 2H), 2.10 (s, 3H), 1.98−

1.94 (m, 1H), 1.95 (s, 3H), 1.79−1.77 (m, 4H), 1.66−1.64 (m, 2H),

1.51−1.40 (m, 4H), 1.05−1.02 (m, 2H). ¹³C NMR (DMSO): δ 205.8,

166.0, 162.5, 161.4, 133.2, 130.4, 122.3, 116.6, 113.5, 111.7, 103.4,

68.0, 67.8, 46.0, 26.1, 25.6, 25.3, 25.1, 22.5, 7.5. ESI-MS m/z 459

[M + H]⁺. HRMS m/z calcd for C₂₆H₃₁ClO₅[M + H]⁺: 459.1933.

Found: 459.1942.

67%); mp 151−153 °C. H NMR (DMSO): δ 7.79 (d, J = 9.2 Hz,

1H), 7.43−7.41 (m, 2H), 7.20 (d, J = 7.8 Hz, 1H), 6.60 (d, J = 8.7 Hz,

1H), 4.14−4.10 (m, 4H), 2.81 (d, J = 6.9 Hz, 2H), 2.15 (s, 3H), 2.13−

2.12 (m, 1H), 1.97 (s, 3H), 1.91−1.89 (m, 4H), 0.90 (d, J = 6.9 Hz,

6H). ¹³C NMR (DMSO): δ 205.8, 167.4, 162.5, 161.3, 157.4, 132.9,

130.6, 130.3, 121.6, 120.0, 113.4, 113.2, 111.9, 103.3, 67.9, 67.8, 43.9,

36.3, 32.0, 25.6, 24.4, 7.5. ESI-MS m/z 415 [M + H]⁺. HRMS m/z

calcd for C₂₄H₃₀O₆[M + H]⁺: 415.2115. Found: 415.2009.

2-Chloro-4-(4-(3-hydroxy-2-methyl-4-(3-methylbutanoyl)-

3-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

phenoxy)butoxy)benzoic Acid (32). Colorless solid (0.191 g,

butoxy}-4-methylbenzoic Acid (39). Colorless solid (0.277 g,

1

44%); mp 113−115 °C. H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz,

63%). H NMR (DMSO): δ 7.79 (d, J = 9.2 Hz, 1H), 7.42−7.40 (m,

2H), 7.00 (s, 1H), 6.94 (d, J = 8.7 Hz, 1H), 6.60 (d, J = 9.2 Hz, 1H),

4.10−4.08 (m, 4H), 2.80 (d, J = 6.9 Hz, 2H), 2.13−2.01 (m, 1H), 1.92

(s, 3H), 1.89−1.86 (m, 4H), 0.90 (d, J = 6.4 Hz, 6H). ¹³C NMR

(DMSO): δ 205.8, 166.0, 162.5, 161.4, 133.2, 130.4, 122.3, 116.6,

113.5, 111.7, 103.4, 68.0, 67.8, 46.0, 26.1, 25.6, 25.3, 25.1, 22.5,

7.5. ESI-MS m/z 435 [M + H]⁺. HRMS m/z calcd for C₂₃H₂₇ClO₆

[M + H]⁺: 435.1569. Found: 435.1563.

2H), 7.20 (d, J = 7.3 Hz, 1H), 6.59 (d, J = 9.2 Hz, 1H), 4.16−4.08 (m,

4H), 2.95 (d, J = 7.3 Hz, 2H), 2.23−2.21 (m, 1H), 2.15 (s, 3H), 1.96

(s, 3H), 1.94−1.90 (m, 4H), 1.73−1.71 (m, 2H), 1.57−1.45 (m, 4H),

1.17−1.14 (m, 2H). ¹³C NMR (DMSO): δ 205.8, 167.3, 162.4, 161.2,

156.4, 131.3, 130.3, 129.6, 121.4, 113.2, 111.9, 111.2, 103.3, 67.8, 67.2,

43.2, 36.3, 32.0, 25.4, 24.4, 16.1, 7.5. ESI-MS m/z 441 [M + H]⁺.

HRMS m/z calcd for C₂₆H₃₂O₆[M + H]⁺: 441.2272. Found:

441.2245.

2-Chloro-4-(4-(4-(2-cyclopentylacetyl)-3-hydroxy-2-

methylphenoxy)butoxy)benzoic Acid (33). Colorless solid (0.168

4-{4-[3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]-

1

g, 30%); mp 130−132 °C. H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz,

butoxy}-3-methylbenzoic Acid (40). Colorless solid (0.282 g,

1

2H), 7.03 (s, 1H), 7.00 (d, J = 9.2 Hz, 1H), 6.58 (d, J = 9.2 Hz, 1H),

4.10−4.08 (m, 4H), 2.95 (d, J = 7.3 Hz, 2H), 2.21−2.19 (m, 1H), 1.94

(s, 3H), 1.89−1.86 (m, 4H), 1.77−1.71 (m, 2H), 1.56−1.45 (m, 4H),

1.13.1.10 (m, 2H). ¹³C NMR (DMSO): δ 205.8, 166.0, 162.5, 161.4,

133.2, 130.4, 122.3, 116.6, 113.5, 111.7, 103.4, 68.0, 67.8, 46.0, 26.1,

25.6, 25.3, 25.1, 22.5, 7.5. ESI-MS m/z 461 [M + H]⁺. HRMS m/z

calcd for C₂₅H₂₉ClO₆[M + H]⁺: 461.1725. Found: 461.1721.

5-(4-(2,3-Dimethyl-4-(3-methylbutanoyl)phenoxy)butoxy)-

2-ﬂuorobenzoic Acid (34). Colorless solid (0.133 g, 32%). ¹H NMR

(DMSO): δ 7.27−7.15 (m, 4H), 6.94 (d, J = 7.8 Hz, 1H), 3.98−3.68

(m, 4H), 2.71 (d, J = 6.9 Hz, 2H), 2.19 (s, 3H), 2.09 (s, 3H), 2.05−

1.95 (m, 1H), 1.89−1.81 (m, 4H), 0.80 (d, J = 6.4 Hz, 6H). ¹³C NMR

(DMSO): δ 202.9, 155.2, 154.4, 142.2, 131.8, 130.5, 126.0, 125.3,

120.6, 118.0, 117.7, 116.1, 74.5, 68.1, 51.1, 26.3, 25.5, 24.6, 22.4, 20.1,

12.0. ESI-MS m/z 417 [M + H]⁺. HRMS m/z calcd for C₂₅H₂₉FO₅

[M + H]⁺: 417.2072. Found: 417.2116.

68%); mp 125−127 °C. H NMR (DMSO): δ 7.78 (d, J = 9.2 Hz,

1H), 7.73−7.71 (m, 1H), 7.67 (d, J = 1.4 Hz, 1H), 6.96 (d, J = 8.7 Hz,

1H), 6.59 (d, J = 9.2 Hz, 1H), 4.13−4.10 (m, 4H), 2.80 (d, J = 6.9 Hz,

2H), 2.11 (s, 3H), 2.10−2.07 (m, 1H), 1.93 (s, 3H), 1.92−1.89 (m,

4H), 0.89 (d, J = 6.4 Hz, 6H). ¹³C NMR (DMSO): δ 205.7, 167.2,

162.4, 161.3, 160.2, 131.5, 130.3, 129.2, 125.7, 122.3, 113.4, 111.9,

110.6, 103.3, 67.8, 67.4, 45.9, 25.4, 22.4, 15.8, 7.5. ESI-MS m/z 415

[M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₆[M + H]⁺: 415.2115.

Found: 415.2084.

4-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

butoxy}-3-methylbenzoic Acid (41). Colorless solid (0.273 g,

62%). ¹H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 8.7

Hz, 1H), 7.68 (s, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.58 (d, J = 8.7 Hz,

1H), 4.12−4.08 (m, 4H), 2.94 (d, J = 6.9 Hz, 2H), 2.20−2.01 (m,

1H), 2.12 (s, 3H), 1.93 (s, 3H), 1.90−1.89 (m, 4H), 1.75−1.73 (m,

2H), 1.55−1.44 (m, 4H), 1.13−1.01 (m, 2H). ¹³C NMR (DMSO): δ

205.8, 167.2, 162.4, 161.2, 160.2, 131.5, 130.3, 129.2, 125.7, 122.3,

113.2, 111.9, 110.6, 103.3, 67.8, 67.4, 43.2, 36.3, 32.0, 25.4, 24.4,

15.8, 7.4. ESI-MS m/z 441 [M + H]⁺. HRMS m/z calcd for C₂₆H₃₂O₆

[M + H]⁺: 441.2272. Found: 441.2270.

5-(4-(4-(2-Cyclopentylacetyl)-2,3-dimethylphenoxy)butoxy)-

2-ﬂuorobenzoic Acid (35). Colorless solid (0.177 g, 40%); mp

1

118−120 °C. H NMR (DMSO): δ 7.44 (d, J = 8.2 Hz, 1H), 7.27−

7.11 (m, 3H), 6.77 (d, J = 8.7 Hz, 1H), 3.98−3.70 (m, 4H), 2.77 (d,

J = 7.3 Hz, 2H), 2.18 (s, 3H), 2.10−2.01 (m, 1H), 2.02 (s, 3H), 1.89−

1.83 (m, 4H), 1.67−1.64 (m, 2H), 1.48−1.38 (m, 4H), 1.05−1.03 (m,

2H). ¹³C NMR (DMSO): δ 204.0, 165.0, 158.1, 154.3, 136.8, 132.2,

3-Fluoro-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)-

phenoxy]butoxy}benzoic Acid (42). Colorless solid (0.188 g,

45%). ¹H NMR (DMSO): δ 7.80 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 9.2 Hz,

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1H), 7.61−7.59 (m, 1H), 7.23 (t, J = 8.5 Hz, 1H), 6.60 (d, J = 9.2 Hz,

1H), 4.18−4.13 (m, 4H), 2.81 (d, J = 6.9 Hz, 2H), 2.20−2.10 (m,

1H), 1.93 (s, 3H), 1.89−1.87 (m, 4H), 0.90 (d, J = 6.4 Hz, 6H). ¹³C

NMR (DMSO): δ 206.3, 166.7, 163.0, 161.8, 150.8, 130.9, 127.2,

117.1, 114.7, 114.0, 112.5, 103.9, 69.0, 68.2, 46.5, 26.0, 25.7, 22.9,

8.0. ESI-MS m/z 419 [M + H]⁺. HRMS m/z calcd for C₂₃H₂₇FO₆

[M + H]⁺: 419.1864. Found: 419.1848.

4-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

butoxy}-2-methylbenzoic Acid (49). Colorless solid (0.273 g,

1

62%). H NMR (DMSO): δ 7.78 (d, J = 9.2 Hz, 2H), 6.77−6.76 (m,

2H), 6.58 (d, J = 8.7 Hz, 1H), 4.11−4.05 (m, 4H), 2.94 (d, J = 7.3 Hz,

2H), 2.46 (s, 3H), 2.20−2.01 (m, 1H), 1.94 (s, 3H), 1.86−1.84 (m,

4H), 1.74−1.71 (m, 2H), 1.56−1.45 (m, 4H), 1.13−1.01 (m, 2H). ¹³C

NMR (DMSO): δ 205.8, 168.0, 162.4, 161.1, 142.1, 132.8, 130.3,

129.6, 122.0, 117.2, 113.2, 111.9, 111.5, 103.3, 67.7, 67.2, 43.2, 36.3,

32.0, 25.2, 24.4, 21.8, 7.5. ESI-MS m/z 441 [M + H]⁺. HRMS m/z

calcd for C₂₆H₃₂O₆[M + H]⁺: 441.2272. Found: 441.2248.

4-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

butoxy}-3-ﬂuorobenzoic Acid (43). Colorless solid (0.213 g, 48%);

1

mp 160−162 °C. H NMR (DMSO): δ 7.79 (d, J = 9.2 Hz, 1H),

3-{4-[3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]-

butoxy}-4-methoxybenzoic Acid (50). Colorless solid (0.254 g,

59%); mp 143−145 °C. ¹H NMR (DMSO): δ 7.80 (d, J = 8.7 Hz, 1H),

7.51 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 7.00 (d, J = 8.7 Hz,

1H), 6.60 (d, J = 9.2 Hz, 1H), 4.14−4.04 (m, 4H), 3.77 (s, 3H), 2.81

(d, J = 6.9 Hz, 2H), 2.10−2.01 (m, 1H), 1.95 (s, 3H), 1.88−1.84 (m,

4H), 0.90 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ 205.7, 167.1,

162.5, 161.3, 152.7, 147.5, 130.3, 123.0, 113.4, 111.9, 111.1, 103.3, 67.9,

55.7, 45.9, 25.5, 22.4, 7.5. ESI-MS m/z 431 [M + H]⁺. HRMS m/z

calcd for C₂₄H₃₀O₇[M + H]⁺: 431.2064. Found: 431.2060.

3-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

butoxy}-4-methoxybenzoic Acid (51). Colorless solid (0.273 g,

60%). ¹H NMR (DMSO): δ 7.80 (d, J = 9.2 Hz, 1H), 7.51 (d, J = 8.2

Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.60 (d,

J = 9.2 Hz, 1H), 4.14−4.04 (m, 4H), 3.77 (s, 3H), 2.95 (d, J = 6.9 Hz,

2H), 2.21−2.10 (m, 1H), 1.94 (s, 3H), 1.88−1.84 (m, 4H), 1.72−1.69

(m, 2H), 1.56−1.45 (m, 4H), 1.14−1.12 (m, 2H). ¹³C NMR

(DMSO): δ 205.8, 167.1, 162.4, 161.2, 152.7, 147.5, 130.3,123.1,

113.2, 111.9, 111.1, 103.3, 67.9, 55.7, 43.2, 36.3, 32.0, 25.4, 24.4,

7.5. ESI-MS m/z 457 [M + H]⁺. HRMS m/z calcd for C₂₆H₃₂O₇

[M + H]⁺: 457.2221. Found: 457.2224.

4-(4-(4-Acetyl-3-hydroxy-2-methylphenoxy)butoxy)-3-

methoxybenzoic Acid (52). Colorless solid (0.255 g, 58%); mp

138−140 °C. ¹H NMR (DMSO): δ 7.75 (d, J = 9.2 Hz, 1H), 7.48 (d,

J = 8.2 Hz, 1H), 7.39 (s, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 8.7

Hz, 1H), 4.13−4.07 (m, 4H), 3.75 (s, 3H), 2.53 (s, 3H), 1.93 (s, 3H),

1.89−1.87 (m, 4H). ¹³C NMR (DMSO): δ 204.4, 167.7, 163.1, 161.5,

152.3, 148.9, 131.4, 123.6, 11.0, 112.6, 112.4, 112.3, 103.9, 68.5,

68.3, 55.9, 26.9, 26.0, 25.7, 8.0. LC−MS (ESI) calcd for C₂₁H₂₄O₇

[M + H]⁺: 389.15. Found: 389.05. HRMS (ESI) calcd for C₂₁H₂₄O₇

[M + H]⁺: 389.1595. Found: 389.1587.

3-(4-(4-Acetyl-3-hydroxy-2-methylphenoxy)butoxy)-4-

methoxybenzoic Acid (53). Colorless solid (0.264 g, 68%); mp

135−136 °C. ¹H NMR (DMSO): δ 7.76 (d, J = 9.2 Hz, 1H), 7.50 (d,

J = 10.0 Hz, 1H), 7.42 (s, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.60 (d, J =

9.2 Hz, 1H), 4.13−4.03 (m, 4H), 3.77 (s, 3H), 2.53 (s, 3H), 1.94

(s, 3H), 1.92−1.88 (m, 4H). ¹³C NMR (DMSO): δ 204.3, 167.6,

163.1, 161.5, 153.3, 148.0, 131.4, 123.7, 123.5, 114.0, 113.7, 112.3,

111.7, 103.9, 68.4, 56.2, 26.8, 25.9, 8.0. LC−MS (ESI) calcd for

C₂₁H₂₄O₇[M + H]⁺: 389.15. Found: 389.00. HRMS (ESI) calcd for

C₂₁H₂₄O₇[M + H]⁺: 389.1595. Found: 389.1585.

7.70−7.76 (m, 1H), 7.60−7.58 (m, 1H), 7.22 (t, J = 8.7 Hz, 1H), 6.58

(d, J = 9.2 Hz, 1H), 4.17−4.12 (m, 4H), 2.94 (d, J = 7.3 Hz, 2H),

2.20−2.18 (m, 1H), 1.92 (s, 3H), 1.89−1.87 (m, 4H), 1.72−1.69 (m, 2H),

1.55−1.44 (m, 4H), 1.12−1.10 (m, 2H). ¹³C NMR (DMSO): δ 206.3,

166.7, 162.9, 161.8, 150.8, 130.8, 127.2, 123.8, 117.0, 114.7, 113.8,

112.5, 103.8, 69.0, 68.2, 43.8, 36.8, 32.6, 25.7, 25.0, 8.0. ESI-MS m/z 445

[M + H]⁺. HRMS m/z calcd for C₂₅H₂₉FO₆[M + H]⁺: 445.2021.

Found:.445.2014.

4-{4-[3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]-

butoxy}-3-methoxybenzoic Acid (44). Colorless solid (0.301 g,

1

70%); mp 133−135 °C. H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz,

1H), 7.53−7.51 (m, 1H), 7.40 (d, J = 1.8 Hz, 1H), 7.00 (d, J = 8.2 Hz,

1H), 6.59 (d, J = 9.2 Hz, 1H), 4.13−4.07 (m, 4H), 3.76 (s, 3H), 2.83

(d, J = 6.9 Hz, 2H), 2.12−2.10 (m, 1H), 1.94 (s, 3H), 1.90−1.88 (m,

4H), 0.89 (d, J = 6.9 Hz, 6H). ¹³C NMR (DMSO): δ 205.7, 167.1,

162.5, 161.3, 151.9, 148.4, 130.3, 122.9, 113.4, 111.9, 103.3, 67.9, 67.8,

55.4, 45.9, 25.4, 22.4, 7.5. ESI-MS m/z 431 [M + H]⁺. HRMS m/z

calcd for C₂₄H₃₀O₇[M + H]⁺: 431.2064. Found: 431.2061.

4-{4-[4-(2-Cyclopentylacetyl)-3-hydroxy-2-methylphenoxy]-

butoxy}-methoxybenzoic Acid (45). Colorless solid (0.292 g,

1

64%). H NMR (DMSO): δ 7.80 (d, J = 9.2 Hz, 1H), 7.50−7.49 (m,

1H), 7.39 (d, J = 1.8 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.60 (d, J = 9.2

Hz, 1H), 4.14−4.07 (m, 4H), 3.75 (s, 3H), 2.95 (d, J = 6.9 Hz, 2H),

2.21−2.19 (m, 1H), 1.94 (s, 3H), 1.89−1.87 (m, 4H), 1.74−1.72 (m,

2H), 1.56−1.45 (m, 4H), 1.14−1.12 (m, 2H). ¹³C NMR (DMSO): δ

205.8, 167.1, 162.4, 161.2, 151.8, 148.4, 130.3, 123.1, 113.2, 112.1,

111.9, 103.3, 67.9, 67.8, 55.5, 43.2, 36.3, 32.0, 25.5, 25.2, 24.4, 7.5. ESI-

MS m/z 457 [M + H]⁺. HRMS m/z calcd for C₂₆H₃₂O₇[M + H]⁺:

457.2221. Found: 457.2218.

3-Chloro-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)-

phenoxy]butoxy}benzoic Acid (46). Colorless solid (0.252 g,

1

58%); mp 147−149 °C. H NMR (DMSO): δ 7.86−7.84 (m, 3H),

7.21 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 9.2 Hz, 1H), 4.20−4.15 (m, 4H),

2.81 (d, J = 6.9 Hz, 2H), 2.21−2.10 (m, 1H), 1.93 (s, 3H), 1.92−1.89

(m, 4H), 0.90 (d, J = 6.4 Hz, 6H). ¹³C NMR (DMSO): δ 206.0, 166.6,

163.0, 161.8, 150.7, 130.9, 127.2, 117.1, 114.9, 114.0, 112.5, 103.8,

69.0, 68.1, 46.3, 26.0, 25.8, 22.4, 8.0. ESI-MS m/z 435 [M + H]⁺.

HRMS m/z calcd for C₂₃H₂₇ClO₆[M + H]⁺: 435.1569. Found:

435.1557.

3-Chloro-4-{4-[4-(2-cyclopentylacetyl)-3-hydroxy-2-

methylphenoxy]butoxy}benzoic Acid (47). Colorless solid

3-(4-(4-Acetyl-3-hydroxy-2-methylphenoxy)butoxy)-2-

methylbenzoic Acid (54). Colorless solid (0.204 g, 55%); mp 140−

142 °C. ¹H NMR (DMSO): δ 7.73 (d, J = 9.2 Hz, 1H), 7.27 (d, J = 8.7

Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 6.58 (d, J =

8.7 Hz, 1H), 4.11−4.02 (m, 4H), 2.52 (s, 3H), 2.28 (s, 3H), 1.93 (s,

3H), 1.89−1.87 (m, 4H). ¹³C NMR (DMSO): δ 204.3, 169.7, 163.0,

161.5, 157.4, 133.2, 131.4, 127.1, 126.7, 121.9, 114.8, 114.0, 112.3,

103.8, 68.3, 68.1, 26.8, 25.9, 13.1, 8.0. LC−MS (ESI) calcd for

C₂₁H₂₄FO₆[M + H]⁺: 373.16. Found: 373.00. HRMS (ESI) calcd for

C₂₁H₂₄O₆[M + Na]⁺: 395.1465. Found: 395.1464.

1

(0.253 g, 55%). H NMR (DMSO): δ 7.87−7.82 (m, 3H)), 7.23 (d,

J = 8.2 Hz, 1H), 6.62 (d, J = 9.2 Hz, 1H), 4.22−4.17 (m, 4H), 2.98 (d,

J = 6.9 Hz, 2H), 2.23−2.21 (m, 1H), 1.95 (s, 3H), 1.94−1.91 (m, 4H),

1.76−1.74 (m, 2H), 1.56−1.44 (m, 4H), 1.18−1.15 (m, 2H). ¹³C

NMR (DMSO): δ 206.0, 166.7, 162.9, 161.8, 151.0, 130.8, 127.2,

123.7, 117.0, 114.6, 113.9, 112.5, 103.8, 69.1, 68.2, 43.7, 36.8, 32.7,

25.7, 25.1, 8.0. ESI-MS m/z 461 [M + H]⁺. HRMS m/z calcd for

C₂₅H₂₉ClO₆[M + H]⁺: 461.1725. Found: 461.1720.

4-{4-[3-Hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]-

butoxy}-2-methylbenzoic Acid (48). Colorless solid (0.269 g,

5-(4-(4-Acetyl-3-hydroxy-2-methylphenoxy)butoxy)-2-ﬂuo-

1

65%): mp 136−138 °C. H NMR (DMSO): δ 7.79 (d, J = 9.6 Hz,

robenzoic Acid (55). Colorless solid (0.229 g, 0.253 g, 61%); mp

1

2H), 6.78−6.76 (m, 2H), 6.59 (d, J = 9.2 Hz, 1H), 4.11−4.06 (m,

4H), 2.80 (d, J = 7.3 Hz, 2H), 2.45 (s, 3H), 2.10−2.01 (m, 1H), 1.94

(s, 3H), 1.86−1.84 (m, 4H), 0.89 (d, J = 6.9 Hz, 6H). ¹³C NMR

(DMSO): δ 205.7, 168.0, 162.4, 161.3, 161.1, 142.1, 132.8, 130.3,

122.0, 117.2, 113.4, 111.9, 111.5, 103.3, 67.7, 67.2, 45.9, 25.3, 22.4,

21.8, 7.5. ESI-MS m/z 415 [M + H]⁺. HRMS m/z calcd for C₂₄H₃₀O₆

[M + H]⁺: 415.2115. Found: 415.2071.

102−104 °C. H NMR (DMSO): δ 7.75 (d, J = 8.7 Hz, 1H), 7.26−

7.13 (m, 3H), 6.59 (d, J = 9.2 Hz, 1H), 4.11−4.03 (m, 4H), 2.52 (s,

3H), 1.94 (s, 3H), 1.92−1.86 (m, 4H). ¹³C NMR (DMSO): δ 204.3,

165.4, 163.0, 161.5, 154.8, 154.6, 131.4, 118.4, 118.2, 116.5, 114.0,

112.3, 103.8, 68.3, 26.9, 25.8, 8.0. LC−MS (ESI) calcd for C₂₀H₂₁FO₆

[M + H]⁺: 377.13. Found: 377.00. HRMS (ESI) calcd for C₂₀H₂₁FO₆

[M + H]⁺: 377.1395. Found: 377.1395.

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4-(4-(4-Acetyl-3-hydroxy-2-methylphenoxy)butoxy)-3-

20.0, 8.1. LC−MS (ESI) calcd for C₂₃H₂₈O₇[M + H]⁺: 417.18.

Found: 417.05. HRMS (ESI) calcd for C₂₃H₂₈O₇[M + Na]⁺:

439.1727. Found: 439.1725.

methylbenzoic Acid (56). Colorless solid (0.201 g, 54%); mp 188−

1

190 °C. H NMR (DMSO): δ 7.70−7.65 (m, 2H), 7.64 (s, 1H),

6.93 (d, J = 8.7 Hz, 1H), 6.57 (d, J = 9.2 Hz, 1H), 4.09−4.04 (m, 4H),

2.49 (s, 3H), 2.08 (s, 3H), 1.89 (s, 3H), 1.88−1.86 (m, 4H). ¹³C NMR

(DMSO): δ 204.4, 167.7, 163.0, 161.5, 160.8, 132.0, 131.4, 129.7,

126.2, 122.8, 114.0, 112.3, 112.2, 108.8, 68.3, 67.9, 26.8, 25.9, 25.8,

16.4, 8.0. LC−MS (ESI) calcd for C₂₁H₂₄O₆[M + H]⁺: 373.16. Found:

373.00. HRMS (ESI) calcd for C₂₁H₂₄O₆[M + Na]⁺: 395.1465. Found:

395.1463.

3-(4-(3-Hydroxy-4-isobutyryl-2-methylphenoxy)butoxy)-4-

methoxybenzoic Acid (63). Colorless solid (0.279 g, 67%); 82−

84 °C. ¹H NMR (DMSO): δ 7.84 (d, J = 9.2 Hz, 1H), 7.50 (d, J = 10.5

Hz, 1H), 7.42 (s, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 9.2 Hz,

1H), 4.14 (brs, 2H), 4.07 (brs, 2H), 3.77 (s, 3H), 3.74−3.63 (m, 1H),

1.95 (s, 3H), 1.88 (brs, 4H), 1.08 (d, J = 6.4 Hz, 6H). ¹³C NMR

(DMSO): δ 210.2, 167.6, 163.0, 162.3, 153.3, 148.1, 130.4, 123.7,

113.7, 112.7, 112.2, 111.7, 103.9, 68.4, 56.2, 26.0, 19.9, 8.1. LC−MS

(ESI) calcd for C₂₃H₂₈O₇[M + H]⁺: 417.18. Found: 417.05. HRMS

(ESI) calcd for C₂₃H₂₈O₇[M + H]⁺: 417.1908. Found: 417.1895.

3-(4-(3-Hydroxy-4-isobutyryl-2-methylphenoxy)butoxy)-2-

methylbenzoic Acid (64). Colorless solid (0.264 g, 66%); mp 115−

117 °C. ¹H NMR (DMSO): δ 7.85 (d, J = 9.2 Hz, 1H), 7.28 (d, J = 6.8

Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 7.3 Hz, 1H), 6.64 (d, J =

9.2 Hz, 1H), 4.16−4.05 (m, 4H), 3.66−3.54 (m, 1H), 2.03 (s, 3H),

1.93 (s, 3H), 1.91−1.88 (m, 4H), 1.10 (d, J = 6.8 Hz, 6H). ¹³C NMR

(DMSO): δ 210.2, 169.7, 162.9, 157.4, 133.2, 130.4, 127.1, 126.8,

121.9, 114.8, 112.7, 112.2, 103.9, 68.3, 68.1, 34.3, 26.0, 19.9, 13.1, 8.0.

LC−MS (ESI) calcd for C₂₃H₂₈O₆[M + H]⁺: 401.19. Found: 401.05.

HRMS (ESI) calcd for C₂₃H₂₈O₆[M + H]⁺: 401.1959. Found:

401.1948.

2-Fluoro-5-(4-(3-hydroxy-4-isobutyryl-2-methylphenoxy)-

butoxy)benzoic Acid (65). Colorless solid (0.234 g, 58%); mp 122−

124 °C. ¹H NMR (DMSO): δ 7.82 (d, J = 9.2 Hz, 1H), 7.27−7.13 (m,

3H), 6.61 (d, J = 8.7 Hz, 1H), 4.12−4.02 (m, 4H), 3.64−3.46 (m,

1H), 1.94 (s, 3H), 1.90−1.86 (m, 4H), 1.08 (d, J = 6.9 Hz, 6H). ¹³C

NMR (DMSO): δ 210.2, 165.5, 162.9, 162.3, 154.8, 154.6, 130.5,

118.2, 116.4, 112.7, 112.2, 103.9, 68.3, 33.6, 25.8, 19.9, 8.0. LC−MS

(ESI) calcd for C₂₂H₂₅FO₆[M + H]⁺: 405.16. Found: 405.00. HRMS

(ESI) calcd for C₂₂H₂₅FO₆[M + H]⁺: 405.1708. Found: 405.1705.

4-(4-(3-Hydroxy-4-isobutyryl-2-methylphenoxy)butoxy)-3-

methylbenzoic Acid (66). Colorless solid (0.224 g, 56%); mp 160−

162 °C. ¹H NMR (DMSO): δ 7.84 (d, J = 9.2 Hz, 1H), 7.75−7.71 (m,

1H), 7.76 (s, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.61 (d, J = 9.2 Hz, 1H),

4.14−4.09 (m, 4H), 3.71−3.61 (m, 1H), 2.12 (s, 3H), 1.94 (s, 3H),

1.94−1.90 (m, 4H), 1.08 (d, J = 5.5 Hz, 6H). ¹³C NMR (DMSO): δ

210.2, 167.7, 162.9, 162.3, 160.8, 132.0, 130.5, 129.7, 126.2, 122.8,

1127, 112.3, 111.2, 103.9, 68.3, 67.9, 34.6, 25.9, 19.9, 16.4, 8.0.

LC−MS (ESI) calcd for C₂₃H₂₈O₆[M + H]⁺: 401.19. Found: 401.05.

HRMS (ESI) calcd for C₂₃H₂₈O₆[M + Na]⁺: 423.1778. Found:

423.1778.

(4-(4-Butyryl-3-hydroxy-2-methylphenoxy)butoxy)-3-

methoxybenzoic Acid (67). Colorless solid (0.270 g, 65%); mp

148−150 °C. ¹H NMR (DMSO): δ 7.80 (d, J = 9.2 Hz, 1H), 7.48 (d,

J = 10.5 Hz, 1H), 7.39 (s, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.60 (d, J =

8.7 Hz, 1H), 4.13−4.07 (m, 4H), 3.75 (s, 3H), 2.93 (t, J = 7.5 Hz,

2H), 1.94 (s, 3H), 1.91−1.88 (m, 4H), 1.61−1.57 (m, 2H), 0.90 (t, J =

7.3 Hz, 3H). ¹³C NMR (DMSO): δ 206.4, 167.4, 162.9, 161.7, 152.4,

148.9, 130.7, 123.6, 123.5, 112.5, 112.4, 103.8, 68.5, 68.3, 55.9, 26.0,

25.7, 18.4, 14.1, 8.0. LC−MS (ESI) calcd for C₂₃H₂₈O₇[M + H]⁺:

417.18. Found: 417.00. HRMS (ESI) calcd for C₂₃H₂₈O₇[M + H]⁺:

417.1908. Found: 417.1903.

3-(4-(4-Butyryl-3-hydroxy-2-methylphenoxy)butoxy)-4-

methoxybenzoic Acid (68). Colorless solid (0.279 g, 67%); mp

129−131 °C. ¹H NMR (DMSO): δ 7.79 (d, J = 9.1 Hz, 1H), 7.43 (d,

J = 10.0 Hz, 1H), 7.43 (s, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.63 (d, J =

9.2 Hz, 1H), 4.13−4.07 (m, 4H), 3.79 (s, 3H), 2.99 (t, J = 7.3 Hz,

2H), 1.96 (s, 3H), 1.89−1.86 (m, 4H), 1.63−1.61 (m, 2H), 0.92 (d,

J = 7.6 Hz, 3H). ¹³C NMR (DMSO): δ 206.4, 167.6, 162.9, 161.7,

153.3, 148.0, 130.6, 123.7, 123.4, 113.6, 112.4, 111.6, 103.8, 68.44,

68.41, 56.2, 26.0, 25.6, 18.4, 14.1, 8.0. LC−MS (ESI) calcd for

C₂₃H₂₈O₇[M + H]⁺: 417.18. Found: 417.00. HRMS (ESI) calcd for

C₂₃H₂₈O₇[M + H]⁺: 417.1908. Found: 417.1896.

4-(4-(3-Hydroxy-2-methyl-4-propionylphenoxy)butoxy)-3-

methoxybenzoic Acid (57). Colorless solid (0.253 g, 63%);

1

mp 128−130 °C. H NMR (DMSO): δ 7.80 (d, J = 9.2 Hz, 1H),

7.49 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.01 (d, J = 8.7 Hz, 1H),

6.60 (d, J = 9.2 Hz, 1H), 4.13−4.03 (m, 4H), 3.74 (s, 3H), 2.99 (q, J =

7.5 Hz, 2H), 1.96 (s, 3H), 1.90−1.88 (m, 4H), 1.07 (t, J = 7.3 Hz,

3H). ¹³C NMR (DMSO): δ 206.8, 162.9, 161.5, 152.3, 148.9, 130.5,

123.6, 113.5, 112.6, 112.4, 103.9, 98.5, 68.5, 66.9, 66.2, 60.6, 60.0, 31.1,

26.0, 25.7, 8.9, 8.0. LC−MS (ESI) calcd for C₂₂H₂₆O₇[M + H]⁺:

403.17. Found: 403.00. HRMS (ESI) calcd for C₂₂H₂₆O₇[M + Na]⁺:

425.1571. Found: 425.1568.

3-(4-(3-Hydroxy-2-methyl-4-propionylphenoxy)butoxy)-4-

methoxybenzoic Acid (58). Colorless solid (0.293 g, 73%); mp

120−122 °C. ¹H NMR (DMSO): δ 7.77 (d, J = 8.6 Hz, 1H), 7.50 (d,

J = 10.5 Hz, 1H), 7.42 (s, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.60 (d, J =

9.1 Hz, 1H), 4.13−4.03 (m, 4H), 3.77 (s, 3H), 2.98 (q, J = 7.5 Hz,

2H), 1.95 (s, 3H), 1.90−1.88 (m, 4H), 1.05 (t, J = 7.3 Hz, 3H). ¹³C

NMR (DMSO): δ 206.8, 167.7, 162.9, 161.5, 153.3, 148.0, 130.4,

123.7, 113.7, 113.4, 112.4, 111.6, 103.8, 68.5, 68.4, 56.2, 30.6, 25.5,

25.4, 8.9, 8.0. LC−MS (ESI) calcd for C₂₂H₂₆O₇[M + H]⁺: 403.17.

Found: 403.35. HRMS (ESI) calcd for C₂₂H₂₆O₇[M + Na]⁺:

425.1571. Found: 425.1569.

3-(4-(3-Hydroxy-2-methyl-4-propionylphenoxy)butoxy)-2-

methylbenzoic Acid (59). Colorless solid (0.228 g, 59%); 103−

1

105 °C. H NMR (DMSO): δ 7.77 (d, J = 9.2 Hz, 1H), 7.24 (d, J =

9.2 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.59 (d,

J = 9.2 Hz, 1H), 4.12−4.02 (m, 4H), 3.00 (q, J = 7.3 Hz, 2H), 2.28 (s,

3H), 1.94 (s, 3H), 1.90−1.89 (m, 4H), 1.05 (t, J = 7.4 Hz, 3H). ¹³C

NMR (DMSO): δ 206.8, 169.7, 162.8, 161.5, 157.4, 133.2, 130.4,

127.1, 126.8, 121.9, 114.8, 113.5, 112.4, 103.8, 63.3, 31.8, 24.6, 13.1,

8.9, 8.0. LC−MS (ESI) calcd for C₂₂H₂₆O₆[M + H]⁺: 387.17. Found:

387.05. HRMS (ESI) calcd for C₂₂H₂₆O₆[M + H]⁺: 387.1802. Found:

387.1802.

2-Fluoro-5-(4-(3-hydroxy-2-methyl-4-propionylphenoxy)-

butoxy)benzoic Acid (60). Colorless solid (0.254 g, 65%); mp 123−

125 °C. ¹H NMR (DMSO): δ 7.76 (d, J = 8.7 Hz, 1H), 7.26−7.13 (m,

3H), 6.59 (d, J = 9.2 Hz, 1H), 4.11−4.02 (m, 4H), 2.98 (q, J = 7.3 Hz,

2H), 1.93 (s, 3H), 1.90−1.86 (m, 4H), 1.05 (t, J = 7.3 Hz, 3H). ¹³C

NMR (DMSO): δ 206.8, 165.5, 162.8, 161.5, 154.8, 154.6, 130.4,

118.4, 116.5, 113.5, 112.4, 103.8, 68.3, 31.1, 25.8, 8.9, 8.0. LC−MS

(ESI) calcd for C₂₁H₂₃FO₆[M + H]⁺: 391.15. Found: 391.00. HRMS

(ESI) calcd for C₂₁H₂₃FO₆[M + Na]⁺: 413.1371. Found: 413.1366.

4-(4-(3-Hydroxy-2-methyl-4-propionylphenoxy)butoxy)-3-

methylbenzoic Acid (61). Colorless solid (0.205 g, 53%); mp 178−

180 °C. ¹H NMR (DMSO): δ 7.80−7.78 (m, 2H), 7.70 (s, 1H), 6.99

(d, J = 8.7 Hz, 1H), 6.63 (d, J = 9.2 Hz, 1H), 4.15−4.10 (m, 4H), 3.02

(q, J = 7.3 Hz, 2H), 2.24 (s, 3H), 1.96 (s, 3H), 1.95−1.92 (m, 4H),

1.08 (t, J = 7.3 Hz, 3H). ¹³C NMR (DMSO): δ 206.8, 167.7, 162.8,

161.5, 160.8, 132.0, 130.5, 129.7, 126.2, 122.8, 113.5, 112.4, 111.2,

103.8, 68.3, 67.9, 31.1, 25.9, 25.8, 16.4, 8.9, 8.0. LC−MS (ESI) calcd

for C₂₂H₂₆O₆[M + H]⁺: 387.17. Found: 387.05. HRMS (ESI) calcd

for C₂₂H₂₆O₆[M + Na]⁺: 409.1622. Found: 409.1625.

4-(4-(3-Hydroxy-4-isobutyryl-2-methylphenoxy)butoxy)-3-

methoxybenzoic Acid (62). Colorless solid (0.283 g, 68%); mp

118−120 °C. ¹H NMR (DMSO): δ 7.84 (d, J = 8.7 Hz, 1H), 7.49 (d,

J = 10.5 Hz, 1H), 7.39 (s, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.61 (d, J =

9.2 Hz, 1H), 4.14−4.07 (m, 4H), 3.74 (s, 3H), 3.74−3.63 (m, 1H),

1.94 (s, 3H), 1.90−1.88 (m, 4H), 1.09 (d, J = 6.9 Hz, 6H). ¹³C NMR

(DMSO): δ 210.3, 167.6, 163.0, 162.3, 152.4, 148.9, 130.5, 123.6,

123.5, 112.7, 112.6, 112.4, 112.2, 103.9, 68.5, 56.0, 34.8, 26.0, 25.7,

3-(4-(4-Butyryl-3-hydroxy-2-methylphenoxy)butoxy)-2-

methylbenzoic Acid (69). Colorless solid (0.244 g, 61%); mp 97−

99 °C. ¹H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz, 1H), 7.27 (d, J = 7.3

Hz, 1H), 7.17 (t, J = 8.2 Hz, 1H), 7.08 (d, J = 7.8 8 Hz, 1H), 6.59 (d,

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J = 9.2 Hz, 1H), 4.13−4.02 (m, 4H), 2.92 (t, J = 7.3 Hz, 2H), 2.28 (s,

3H), 1.94 (s, 3H), 1.89−1.86 (m, 4H), 1.61−1.57 (m, 2H), 0.89 (t, J =

7.8 Hz, 3H). ¹³C NMR (DMSO): δ 206.4, 169.6, 162.9, 161.7, 157.4,

133.1, 130.6, 127.1, 126.8, 121.8, 114.8, 113.7, 112.5, 103.8, 68.3,

68.2, 26.0, 18.4, 14.1, 13.1, 8.0. LC−MS (ESI) calcd for C₂₃H₂₈O₆

[M + H]⁺: 401.19. Found: 401.05. HRMS (ESI) calcd for C₂₃H₂₈O₆

[M + Na]⁺: 439.1727. Found: 439.1727.

5-(4-(4-Butyryl-3-hydroxy-2-methylphenoxy)butoxy)-2-ﬂuo-

robenzoic Acid (70). Colorless solid (0.255 g, 63%); mp 88−90 °C.

¹H NMR (DMSO): δ 7.78 (d, J = 8.7 Hz, 1H), 7.27−7.13 (m, 3H),

6.59 (d, J = 9.2 Hz, 1H), 4.11−4.02 (m, 4H), 2.92 (t, J = 7.3 Hz, 2H),

1.94 (s, 3H), 1.89−1.87 (m, 4H), 1.61−1.59 (m, 2H), 0.89 (t, J = 7.3

Hz, 3H). ¹³C NMR (DMSO): δ 206.4, 165.4, 162.9, 161.7, 154.8,

154.6, 130.6, 118.2, 116.4, 113.6, 112.4, 103.8, 68.3, 25.8, 18.3, 14.1,

8.0. LC−MS (ESI) calcd for C₂₂H₂₅FO₆[M + H]⁺: 405.43. Found:

405.05. HRMS (ESI) calcd for C₂₂H₂₅FO₆[M + Na]⁺: 427.1527.

Found: 427.1523.

HRMS (ESI) calcd for C₂₄H₂₉FO₆[M + Na]⁺: 455.1840. Found:

455.1840.

mGlu receptor in Vitro Assays. Human embryonic kidney

(HEK-293) cell lines coexpressing rat mGlu receptors 2, 3, 4, 6, 7, or

8 and G protein-coupled inwardly rectifying potassium (GIRK)

channels⁵⁵were grown in growth medium containing 45% DMEM,

45% F-12, 10% FBS, 20 mM HEPES, 2 mM ^L-glutamine, antibiotic/

antimycotic, nonessential amino acids, 700 μg/mL G418, and

0.6 μg/mL puromycin at 37 °C in the presence of 5% CO₂. Cells

expressing rat mGlu₁and mGlu₅receptor were cultured as described

in Hemstapat et al.⁵⁶All cell culture reagents were purchased from

Invitrogen Corp. (Carlsbad, CA) unless otherwise noted. Calcium

assays were used to assess activity of compounds at mGlu₁and mGlu₅,

as previously described by Engers et al.⁵⁷Calcium assays at mGlu₃

were performed as described for mGlu₅with the exception that

TREx293 mGlu₃G_α15cells were treated with tetracycline at 20 ng/mL

for 20 h prior to assay.

Compound activity at group II (mGlu₂and mGlu₃) and group III

(mGlu₄, mGlu₆, mGlu₇, and mGlu₈) was assessed using thallium ﬂux

through GIRK channels, a method that has been described in detail.⁵⁵

Brieﬂy, cells were plated into 384-well, black-walled, clear-bottomed

poly-^D-lysine-coated plates at a density of (15 000 cells/20 μL)/well

in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and

100 units/mL penicillin/streptomycin (assay medium). Plated cells

were incubated overnight at 37 °C in the presence of 5% CO₂. The

following day, the medium was exchanged from the cells to assay

buﬀer [Hanks’ balanced salt solution (Invitrogen) containing 20 mM

HEPES, pH 7.3] using an ELX405 microplate washer (BioTek),

leaving 20 μL/well, followed by the addition of 20 μL/well FluoZin2-

AM (330 nM ﬁnal concentration) indicator dye (Invitrogen, prepared

as a stock in DMSO and mixed in a 1:1 ratio with Pluronic acid F-127)

in assay buﬀer. Cells were incubated for 1 h at room temperature, and

the dye was exchanged to assay buﬀer using an ELX405, leaving

20 μL/well. Test compounds were diluted to 2 times their ﬁnal desired

concentration in assay buﬀer (0.3% DMSO ﬁnal concentration).

Agonists were diluted in thallium buﬀer [125 mM sodium bicarbonate

(added fresh on the morning of the experiment), 1 mM magnesium

sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium

sulfate, and 10 mM HEPES, pH 7.3] at 5 times the ﬁnal concentration

to be assayed. Cell plates and compound plates were loaded onto

a kinetic imaging plate reader (FDSS 6000 or 7000; Hamamatsu

Corporation, Bridgewater, NJ). Appropriate baseline readings were

taken (10 images at 1 Hz; excitation, 470 20 nm; emission, 540

30 nm), and test compounds were added in a 20 μL volume and

incubated for approximately 2.5 min before the addition of 10 μL of

thallium buﬀer with or without agonist. After the addition of agonist,

data were collected for approximately an additional 2.5 min. Data were

analyzed using Excel (Microsoft Corp, Redmond, WA). The slope of

the ﬂuorescence increase beginning 5 s after thallium/agonist addition and

ending 15 s after thallium/agonist addition was calculated, corrected to

vehicle and maximal agonist control slope values, and plotted using either

XLﬁt (ID Business Solutions Ltd.) or Prism software (GraphPad Software,

San Diego, CA) to generate concentration−response curves. Potencies

were calculated from ﬁts using a four-point parameter logistic equation.

For concentration−response curve experiments, compounds were serially

diluted 1:3 into 10 point concentration−response curves and were

transferred to daughter plates using an Echo acoustic plate reformatter

(Labcyte, Sunnyvale, CA). Test compounds were applied and followed

by EC₂₀concentrations of glutamate. For selectivity experiments, full

concentration−response curves of glutamate or L-AP4 (for mGlu₇) were

obtained in the presence of a 10 μM concentration of compound, and

compounds that aﬀected the concentration−response by less than

2-fold in terms of potency or eﬃcacy were designated as inactive.

TREx293 mGlu₃G_α15Cell Line Creation. In order to generate a

tetracycline (Tet) inducible rat mGlu₃stable cell line to be used for a

calcium mobilizaion assay, TREx293 cells (Invitrogen) were transfected

with mouse G_α15-pCMV6 plasmid (Origene) using Fugene6 (Promega).

The cells were selected for G_α15expression with 1 mg/mL G418 in the

presence of 10 μg/mL blasticidins to maintain Tet repressor expression.

Two weeks after the selection, polyclonal TREx293 G_α15cells were

4-(4-(4-Butyryl-3-hydroxy- 2-methylphenoxy)butoxy)-3-

methylbenzoic Acid (71). Colorless solid (0.232 g, 58%); mp

161−163 °C. ¹H NMR (DMSO): δ 7.79 (d, J = 9.2 Hz, 1H), 7.71 (d,

J = 8.7 Hz, 1H), 7.68 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.59 (d, J = 8.7

Hz, 1H), 4.13−4.08 (m, 4H), 2.92 (t, J = 7.3 Hz, 2H), 2.12 (s, 3H),

1.94 (s, 3H), 1.90−1.88 (m, 4H), 1.63−1.61 (m, 2H), 0.89 (t, J = 7.3

Hz, 3H). ¹³C NMR (DMSO): δ 206.4, 167.7, 162.9, 161.7, 160.8,

132.0, 130.7, 129.7, 126.3, 122.8, 113.7, 112.5, 111.2, 103.9, 68.3, 67.9,

26.1, 25.9, 18.4, 16.3, 14.1, 8.0. LC−MS (ESI) calcd for C₂₃H₂₈O₆

[M + H]⁺: 401.19. Found: 401.05. HRMS (ESI) calcd for C₂₃H₂₈O₆

[M + Na]⁺: 423.1778. Found: 423.1778.

3-(4-(4-(3,3-Dimethylbutanoyl)-3-hydroxy-2-methylphenoxy)-

butoxy)-4-methoxybenzoic Acid (72). Colorless solid (0.270 g,

1

61%); mp 140−142 °C. H NMR (DMSO): δ 7.84 (d, J = 9.2 Hz,

1H), 7.50 (d, J = 10.0 Hz, 1H), 7.49 (s, 1H), 7.00 (d, J = 8.7 Hz, 1H),

6.58 (d, J = 9.2 Hz, 1H), 4.13−4.03 (m, 4H), 3.79 (s, 3H), 2.80

(s, 2H), 1.94 (s, 3H), 1.89−1.87 (m, 4H), 0.96 (s, 9H). ¹³C NMR

(DMSO): δ 206.4, 167.6, 163.1, 162.1, 153.3, 148.1, 131.8, 123.7,

123.5, 114.9, 113.6, 111.6, 103.6, 68.4, 56.2, 49.1, 32.1, 30.5, 26.0, 25.8,

8.0. LC−MS (ESI) calcd for C₂₅H₃₂O₇[M + H]⁺: 445.21. Found:

445.00. HRMS (ESI) calcd for C₂₅H₃₂O₇[M + Na]⁺: 467.2040.

Found: 467.2041.

4-(4-(4-(3,3-Dimethylbutanoyl)-3-hydroxy-2-methylphenoxy)-

butoxy)-5-methoxycyclohexa-1,3-dienecarboxylic Acid (73).

Colorless solid (0.258 g, 58%); mp 133−135 °C. ¹H NMR

(DMSO): δ 7.86 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 10.1 Hz, 1H),

7.39 (s, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.58 (d, J = 9.2 Hz, 1H), 4.13−

4.07 (m, 4H), 3.77 (s, 3H), 2.80 (s, 2H), 1.94 (s, 3H), 1.89−1.87 (m,

4H), 0.96 (s, 9H). ¹³C NMR (DMSO): δ 206.4, 167.6, 163.1, 162.1,

152.4, 148.9, 131.8, 123.6, 114.9, 112.6, 112.4, 103.6, 68.4, 55.9, 49.1,

32.1, 30.5, 26.0, 25.8, 8.0. LC−MS (ESI) calcd for C₂₅H₃₂O₇

[M + H]⁺: 445.21. Found: 445.00. HRMS (ESI) calcd for C₂₅H₃₂O₇

[M + Na]⁺: 467.2040. Found: 467.2045.

3-(4-(4-(3,3-Dimethylbutanoyl)-3-hydroxy-2-methylphenoxy)-

butoxy)-2-methylbenzoic Acid (74). Colorless solid (0.278 g, 65%);

mp 123−125 °C. ¹H NMR (DMSO): δ 7.84 (d, J = 8.7 Hz, 1H), 7.26

(d, J = 7.8 Hz, 1H), 7.16 (t, J = 8.2 Hz, 1H), 7.08 (d, J = 7.3 Hz, 1H),

6.58 (d, J = 9.2 Hz, 1H), 4.13−4.02 (m, 4H), 2.80 (s, 2H), 2.28

(s, 3H), 1.94 (s, 3H), 1.90−1.87 (m, 4H), 0.96 (s, 9H). ¹³C NMR

(DMSO): δ 206.4, 169.7, 163.0, 162.1, 157.4, 131.1, 131.8, 127.1,

126.8, 121.9, 114.9, 114.8, 112.4, 103.6, 68.3, 66.8, 49.1, 32.1, 30.4,

13.1, 8.0. LC−MS (ESI) calcd for C₂₅H₃₂O₇[M + H]⁺: 429.22.

Found: 429.00. HRMS (ESI) calcd for C₂₅H₃₂O₆[M + Na]⁺:

451.2091. Found: 451.2093.

5-(4-(4-(3,3-Dimethylbutanoyl)-3-hydroxy-2-methylphenoxy)-

butoxy)-2-ﬂuorobenzoic Acid (75). Colorless solid (0.258 g, 60%);

1

mp 130−132 °C. H NMR (DMSO): δ 7.83 (d, J = 9.2 Hz, 1H),

7.27−7.13 (m, 3H), 6.57 (d, J = 9.2 Hz, 1H), 4.11−4.02 (m, 4H), 2.79

(s, 2H), 1.94 (s, 3H), 1.86−1.85 (m, 4H), 0.96 (s, 9H). ¹³C NMR

(DMSO): δ 206.4, 165.5, 163.0, 162.1, 154.8, 154.6, 131.7, 121.2,

118.5, 116.4, 114.9, 112.4, 103.6, 68.4, 66.8, 49.1, 32.2, 30.4, 8.0.

LC−MS (ESI) calcd for C₂₄H₂₉FO₆[M + H]⁺: 433.19. Found: 433.00.

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obtained. The entire coding sequence of rat mGlu₃was ampliﬁed by

polymerase chain reaction (PCR) and cloned into the Tet-inducible

expression plasmid pcDNA5/TO (Invitrogen). Rat mGlu₃-pcDNA5/

TO was transfected into TREx293 G_α15cells and selected for mGlu₃

expression with 200 μg/mL hygromycin in the presence of G418 and

blasticidins. The resulting polyclonal TREx293 mGlu₃G_α15cells were

plated for monoclonal selection, and positive monoclones were

identiﬁed in the calcium mobilization assay. Cells were maintained in

growth medium containing DMEM, 10% Tet-tested FBS (Atlanta

Biogicals), 20 mM HEPES, 2 mM ^L-glutamine, antibiotic/antimycotic,

nonessential amino acids, 500 μg/mL G418, 100 μg/mL hygromycin,

and 5 μg/mL blasticidin S at 37 °C in the presence of 5% CO₂.

HEK293A mGlu₂G_α15Cell Line Creation. In order to generate a

rat mGlu₂stable cell line to be used for a calcium mobilization assay,

HEK293A cells (ATCC) were transfected with mouse G_α15-pCMV6

plasmid (Origene) using Fugene6 (Promega). The cells were sel-

ected for G_α15expression with 1 mg/mL G418. Two weeks after the

selection, polyclonal HEK293A G_α15cells were obtained. The entire

coding sequence of rat mGlu₂was ampliﬁed by PCR and cloned into

the expression plasmid pIRESpuro3 (Invitrogen). Rat mGlu₂-

pIRESpuro3 was transfected into HEK293A G_α15cells and selected

for mGlu₂expression with 0.6 μg/mL puromycin in the presence

of G418. The resulting polyclonal HEK293A mGlu₂G_α15cells were

then utilized for calcium mobilization assays. Cells were maintained

in growth medium containing DMEM, 10% FBS, 20 mM HEPES,

2 mM ^L-glutamine, antibiotic/antimycotic, nonessential amino acids,

700 μg/mL G418, and 0.6 μg/mL puromycin at 37 °C in the presence

of 5% CO₂.

Western Blotting. Western blotting was performed as detailed

previously⁵⁸utilizing 10% polyacrylamide gels and a rabbit polyclonal

mGlu_2/3antibody (Millipore, catalog no. 06-676) for detection of mGlu₃.

Microsomal Stability in Vitro Assay. Pooled rat liver micro-

somes (BD Biosciences, no. 452701) were preincubated with test

compounds at 37.5 °C for 5 min in the absence of NADPH. The

reaction was initiated by addition of NADPH, and the mixture was

incubated under the same conditions. The ﬁnal incubation concen-

trations were 4 μM test compound, 2 mM NADPH, and 1 mg/mL

(total protein) liver microsomes in phosphate buﬀered saline (PBS) at

pH 7.4. One aliquot (100 μL) of the incubation mixture was withdrawn

at 15 min time points and combined immediately with 100 μL of

ACN/MeOH. After mixing, the sample was centrifuged at approx-

imately 13 000 rpm for 12 min. The supernatant was ﬁltered and

transferred into an autosampler vial, and the amount of test compound

was quantiﬁed using a Shimadzu LCMS 2010EV mass spectrometer.

The change of the AUC (area under the curve) of the parent

compound as a function of time was used as a measure of microsomal

stability. Test compounds were run in duplicate with a positive control.

Plasma Stability in Vitro Assay. A 20 μL aliquot of a 10 mM

solution in DMSO of the test compound was added to 2.0 mL of

heparinized rat plasma (Lampire, P1-150N) to obtain a 100 μM ﬁnal

solution. The mixture was incubated for 1 h at 37.5 °C. Aliquots of

100 μL were taken at 15 min intervals and diluted with 100 μL

of MeOH/ACN. After mixing, the sample was centrifuged at

approximately 13 000 rpm for 12 min. The supernatant was ﬁltered

and transferred into an autosampler vial, and the amount of test

compound was quantiﬁed using the Shimadzu LCMS-2010EV system.

The change of the AUC of the parent compound as a function of time

was used as a measure of plasma stability.

and speciﬁcations but lacking the membrane. The concentrations of the

acceptor and equilibrium solutions were determined using the Shimadzu

LCMS-2010EV and AUC methods. The acceptor plate and equilibrium

plate concentrations were used to calculate the permeability rate (log P_e)

of the compounds. The log P_evalues were calculated using the following

equations:

⎡

⎢

⎛

⎜

⎞⎤⎫

⎧

⎩

[drug]_acceptor

⎟⎥

⎟

⎨

⎬

log P = log C −ln 1 −

e

⎜

⎪

⎢

⎣

[drug]_equilibrium

⎥

⎦

⎝

⎠ ⎭

V_DV_A

C =

(V_D+ V_A)(area)(time)

In this equation, V_D(cm³) is the donor volume (0.150 cm³), V_A(cm³) is

the acceptor volume (0.300 cm³), area (cm²) is the accessible ﬁlter area

(0.168 cm²), and time (s) is the incubation time. Variables [drug]_acceptor

and [drug]_equilibruimare concentrations of the test drug for the sample

(acceptor) and reference (equilibrium) solutions in the acceptor

compartment.

Behavioral Assessments. Subjects. Male Wistar rats (Charles

River Laboratories, Raleigh, NC) weighing 300−350 g at the

beginning of each experiment were housed in pairs in standard rat

Plexiglas cages with food and water available ad libitum except during

food training and the food self-administration experiment (see below).

Rats were maintained in a climate-controlled room at 21 °C on a 12 h

reverse light/dark cycle, and all experiments were conducted during

the dark (i.e., active) phase (7:00 to 19:00 h) of the cycle under

dim red lighting. All procedures were conducted in accordance

with the guidelines from the National Institutes of Health and the

Association for the Assessment and Accreditation of Laboratory

Animal Care and were approved by the Institutional Animal Care and

Use Committee.

Drugs. Cocaine hydrochloride (National Institute on Drug Abuse,

Bethesda, MD) was dissolved in sterile physiological saline and ﬁltered

through a 0.22 μm syringe ﬁlter (Fisher Scientiﬁc, Pittsburgh, PA) for

sterilization purposes. 74 was mixed into a 10% EtOH, 1% Tween 80

solution.

Food Training. Details regarding the experimental procedures have

been described previously.³⁷All rats were placed under food restriction

(20 g of food/day) and trained during daily 1 h sessions to lever-press

for 45 mg food pellets (Research Diets, New Brunswick, NJ) under a

ﬁxed ratio 1 reinforcement schedule with a 1 s time-out period (FR1

TO1s). Successful responses were followed by illumination of a cue

light for the duration of the time-out period, when lever presses had

no consequence. Successful acquisition of food responding, deﬁned as

earning 100 pellets during each session, resulted in progression of the

training program to FR1 TO10s and FR1 TO20s. Training lasted

approximately 5 days.

Cocaine Self-Administration Experiment. After successful acquis-

ition of food training, rats (n = 11) were fed ad libitum, surgically

prepared with intravenous catheters inserted into the right jugular

vein under isoﬂurane anesthesia (1−1.5% isoﬂurane/oxygen mixture),

and allowed 7 days to recover (see Jin et al. (2010) for details).

Rats were then trained to self-administer cocaine under a FR1 TO20s

reinforcement schedule during daily 1 h sessions. Each response at the

active lever resulted in an intravenous infusion of cocaine (0.5 mg/kg/

infusion) over a 2 s period in a volume of 0.05 μL. Rats were trained

for approximately 10 days until responding for cocaine stabilized (i.e.,

>10 infusions/session; <20% variability in number of infusions over

three consecutive sessions). After stabilization of responding, rats were

administered 74 (0, 10, 20, 40 mg/kg, ip; 3 mL/kg volume; 60 min

pretreatment time) according to a within-subjects Latin-square design.

At least 4 days elapsed between drug/vehicle injections to re-establish

stable self-administration behavior (<20% variability over three

consecutive sessions).

Parallel Artiﬁcial Membrane Permeation Assay (PAMPA). A

96-well microtiter plate (Millipore, MSSACCEPTOR) was ﬁlled with

300 μL of aqueous buﬀer solution (in general, phosphate pH 7.2

buﬀer was used) and covered with a microtiter ﬁlterplate (Millipore,

MAIPNTR10) to create a sort of sandwich construction. The hydro-

phobic ﬁlter material was impregnated with a 10% solution of polar

brain lipid extract in chloroform (Avanti) as the artiﬁcial membrane,

and the organic solvent was allowed to completely evaporate.

Permeation studies were started by the transfer of 150 μL of a 100 μM

test compound solution on top of the ﬁlter plate. The maximum DMSO

content of the stock solutions was <1.5%. In parallel, an equilibrium

solution lacking a membrane was prepared using the exact concentrations

Food Self-Administration Experiment. To assess nonspeciﬁc

actions of 74, after successful acquisition of food training and stabiliza-

tion of responding (<20% variability over three consecutive sessions),

rats (n = 8) were administered 74 (0, 10, 20, 40 mg/kg, ip; 3 mL/kg

volume; 60 min pretreatment time) according to a within-subjects

4169

dx.doi.org/10.1021/jm5000563 | J. Med. Chem. 2014, 57, 4154−4172

Journal of Medicinal Chemistry

Article

Latin-square design. All test parameters, including the FR1 TO20s

reinforcement schedule, were identical to the parameters under which

cocaine was self-administered.

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Statistical Analyses. The number of cocaine infusions/food pellets

earned during test sessions with 74 was calculated as a percentage

of the average number of infusions/pellets earned during the prior

three baseline sessions. Data were then analyzed with a mixed design

analysis of variance (ANOVA) with 74 dose (within-subjects) and self-

administration (i.e., cocaine vs food; between-subjects) as factors.

Signiﬁcant eﬀects were further analyzed with Tukey post hoc tests.

The level of signiﬁcance was set at α = 0.05.

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ASSOCIATED CONTENT

* Supporting Information

■

S

Computationally determined plasma−protein binding for 11

compounds and oﬀ-target proﬁling data against a panel of CNS

receptors for compound 72. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: 858-646-3100. Fax: 858-795-5225. E-mail: ncosford@

sanfordburnham.org.

■

Author Contributions

^#R.-P.D. and D.J.S. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

(13) Chaki, S.; Ago, Y.; Palucha-Paniewiera, A.; Matrisciano, F.; Pilc,

A. mGlu2/3 and mGlu5 receptors: potential targets for novel

antidepressants. Neuropharmacology 2013, 66, 40−52.

(14) Conn, P. J.; Lindsley, C. W.; Jones, C. K. Activation of

metabotropic glutamate receptors as a novel approach for the

treatment of schizophrenia. Trends Pharmacol. Sci. 2009, 30, 25−31.

(15) Sheﬄer, D. J.; Conn, P. J. Activation of Group II Metabotropic

Glutamate Receptors (mGluR2 and mGluR3) as a Novel Approach for

Treatment of Schizophrenia. In Glutamate-Based Therapies for

ACKNOWLEDGMENTS

■

This work was supported by National Institutes of Health

Grant R01 DA023926 to N.D.P.C. and NIDA grant DA011946

to A.M. D.J.S. is a recipient of a National Alliance for Research

on Schizophrenia and Depression (NARSAD), Dylan Tauber

Young Investigator Award. The binding proﬁle for compound

72 was generously provided by the National Institute of Mental

Health’s Psychoactive Drug Screening Program, Contract

HHSN-271-2008-00025-C (NIMH PDSP).

Psychiatric Disorders; Skolnick, P., Ed.; Birkhauser: Basel, Switzerland,

̈

2010; pp 101−116.

(16) Kenny, P. J.; Markou, A. The ups and downs of addiction: role

of metabotropic glutamate receptors. Trends Pharmacol. Sci. 2004, 25,

265−272.

ABBREVIATIONS USED

■

mGlu₂, metabotropic glutamate receptor subtype 2; PAM,

positive allosteric modulator; BINA, 3′-((2-cyclopentyl-6,7-

dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yloxy)methyl)-

biphenyl-4-carboxylic acid; HTS, high-throughput screening;

PK, pharmacokinetic; POC, proof-of-concept; ADME, absorp-

tion, distribution, metabolism, excretion; AUC, area under

curve; LOQ, limit of quantitation; HEK, human embryonic

kidney; GIRK, G protein-coupled inwardly rectifying potas-

sium; PBS, phosphate buﬀered saline; DMEM, Dulbecco’s

modiﬁed Eagle medium; HEPES, 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid; FBS, fetal bovine serum

(17) Xi, Z. X.; Ramamoorthy, S.; Baker, D. A.; Shen, H.; Samuvel, D.

J.; Kalivas, P. W. Modulation of group II metabotropic glutamate

receptor signaling by chronic cocaine. J. Pharmacol. Exp. Ther. 2002,

303, 608−615.

(18) Adewale, A. S.; Platt, D. M.; Spealman, R. D. Pharmacological

stimulation of group II metabotropic glutamate receptors reduces

cocaine self-administration and cocaine-induced reinstatement of drug

seeking in squirrel monkeys. J. Pharmacol. Exp. Ther. 2006, 318, 922−

931.

(19) Aujla, H.; Martin-Fardon, R.; Weiss, F. Rats with extended

access to cocaine exhibit increased stress reactivity and sensitivity to

the anxiolytic-like effects of the mGluR 2/3 agonist LY379268 during

abstinence. Neuropsychopharmacology 2008, 33, 1818−1826.

(20) Baptista, M. A.; Martin-Fardon, R.; Weiss, F. Preferential effects

of the metabotropic glutamate 2/3 receptor agonist LY379268 on

conditioned reinstatement versus primary reinforcement: comparison

between cocaine and a potent conventional reinforcer. J. Neurosci.

2004, 24, 4723−4727.

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Article Doi

Design and synthesis of systemically active metabotropic glutamate subtype-2 and -3 (mGlu2/3) receptor positive allosteric modulators (PAMs): Pharmacological characterization and assessment in a rat model of cocaine dependence

DOI: 10.1021/jm5000563

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