Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): Discovery of 5-((3-fluorophenyl)ethynyl)- N -(3-methyloxetan-3-yl)picolinamide (ML254)

Article

pubs.acs.org/jmc

Exploration of Allosteric Agonism Structure−Activity Relationships

within an Acetylene Series of Metabotropic Glutamate Receptor 5

(mGlu₅) Positive Allosteric Modulators (PAMs): Discovery of 5‑((3-

Fluorophenyl)ethynyl)‑N‑(3-methyloxetan-3-yl)picolinamide (ML254)

Mark Turlington,^†,‡,§Meredith J. Noetzel,^†,‡Aspen Chun,^†,‡,§Ya Zhou,^†,‡,§Rocco D. Gogliotti,^†,‡,§

Elizabeth D._†,N_‡guyen,^¶Karen J. Gregory,^†,‡,⊥Paige N. Vinson,^†,‡Jerri M. Rook,^†,‡Kiran K. Gogi,^†,‡

Zixiu Xiang, Thomas M. Bridges,^†,‡,§J. Scott Daniels,^†,‡,§Carrie Jones,^†,‡,§Colleen M. Niswender,^†,‡,§

Jens Meiler,^†,∥,¶,#P. Jeﬀrey Conn,^†,‡,§Craig W. Lindsley,^{†,‡,§,∥}and Shaun R. Stauﬀer*^{,†,‡,§,∥}

^†Department of Pharmacology, ^‡Vanderbilt Center for Neuroscience Drug Discovery, ^¶Center for Structural Biology, and ^#Vanderbilt

Institute for Chemical Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States

^§Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, Tennessee 37232, United States

^∥Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States

^⊥Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia

S

* Supporting Information

ABSTRACT: Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5

(mGlu₅) represent a promising therapeutic strategy for the treatment of schizophrenia. Both

allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic proﬁle

of some mGlu₅PAMs; however, these hypotheses remain to be adequately addressed. To

develop tool compounds to probe these hypotheses, the structure−activity relationship of

allosteric agonism was examined within an acetylenic series of mGlu₅PAMs exhibiting

allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low

glutamate fold-shift allosteric ligand (maximum fold-shift ∼3.0), was selected as a potent PAM

with no agonism in the in vitro system used for compound characterization and in two native

electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to

probe the relative contribution of cooperativity and allosteric agonism to the adverse eﬀect

liability and neurotoxicity associated with this class of mGlu₅PAMs.

plagued by extra-pyramidal side eﬀects (movement disorders),

atypical antipsychotics often oﬀer improved side-eﬀect proﬁles

but are associated with signiﬁcant weight gain.⁸In addition to

the considerable adverse eﬀect proﬁles, neither class of

antipsychotics has a substantial impact on the negative and

cognitive symptoms of the disorder, and 20% of patients are

nonresponsive to treatment.¹These severe limitations highlight

the need to develop new treatments for schizophrenia.

In addition to the dopaminergic pathways, disruptions in

many neuronal circuits including the glutamatergic, GABAergic,

and cholinergic pathways are observed in schizophrenic

patients.³Importantly, abnormalities in glutamatergic circuits

have been linked with all three symptom clusters of

schizophrenia, fueling the development of the glutamate

hypothesis of schizophrenia as a means to address all symptom

clusters. Clinical observations have revealed that phenyl-

cyclidene (PCP) and ketamine, antagonists of the ionotropic

N-methyl-^D-aspartate glutamate receptor (NMDAR), produce

INTRODUCTION

■

A largely unmet medical need aﬀecting approximately 1% of the

world’s population, schizophrenia is a complex mental illness

characterized by three symptom clusters including positive

symptoms (hallucinations, paranoia, disorganized behavior),

negative symptoms (social withdrawal, anhedonia, ﬂat aﬀect),

and cognitive dysfunction (deﬁcits in attention, learning, and

memory).¹⁻⁴Current treatments for schizophrenia were

developed based on the dopaminergic hypothesis of schizo-

phrenia, which points to overactivation of subcortical dopamine

D₂receptors as a causative factor for the positive symptoms of

the disease.^5,6Accordingly, ﬁrst-generation typical antipsy-

chotics (e.g., haloperidol) act as D₂antagonists, and second-

generation atypical antipsychotics (e.g., clozapine, risperidone)

act as mixed D₂/5-HT_2Aantagonists, as well as having activity at

other receptors.^5,6Both classes are routinely used to treat the

positive symptoms of schizophrenia, and several statistical

analyses have revealed that there is little evidence for improved

eﬃcacy of atypical over typical antipsychotics except in severe

cases of schizophrenia.^7,8However, the two classes are diﬀerent

in their side-eﬀect proﬁles. Whereas typical antipsychotics are

Received: July 8, 2013

Published: September 19, 2013

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Figure 1. (a) Representative mGlu₅receptor (protomer of dimer) with orthosteric and allosteric binding sites. (b) Orthosteric mGlu₅ligands. (c)

First generation mGlu₅PAMs.

Figure 2. mGlu₅PAM chemotypes and reported PAM potencies.

schizophrenic-like symptoms.^1−4,9−13Furthermore, administra-

tion of high doses of the NMDA co-agonists glycine, ^D-serine,

and ^D-cycloserine improves positive, negative, and cognitive

symptoms in schizophrenic patients,^1,2,14,15generating excite-

ment that enhancement of glutamatergic neurotransmission

could be a novel strategy for the treatment of schizophrenic

symptoms. While direct activation of NMDAR results in

toxicity associated with ion channel overactivation,^1,2the

metabotropic glutamate receptor 5 (mGlu₅) is closely

associated with NMDAR function and may provide an indirect

means to rescue NMDAR hypofunction with a lower

propensity for toxicity. Numerous studies indicate that mGlu₅

plays a critical role in modulating neurotransmission in

forebrain circuits implicated in NMDAR antagonist mediated

psychotomimetic eﬀects, and the ability to more subtly

modulate NMDAR activity has been proposed to result in a

potentially lower propensity for toxicity.^{1,2,4,13,16,17}

mGlu₅is a member of the Family C G protein-coupled

receptors (GPCRs) that bear a large extracellular “Venus

ﬂytrap-like” domain that serves as the binding site for

glutamate. The high sequence homology of the glutamate

binding sites among the eight known metabotropic glutamate

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Figure 3. Acetylene mGlu₅PAMs used to assess seizure activity in animal models.^37,38

receptor (mGlu) subtypes has made the development of

subtype selective mGlu₅orthosteric agonists diﬃcult.^2,4In

contrast, spatially distinct allosteric binding sites that reside in

the transmembrane domain of the receptor (Figure 1) possess

less sequence conservation across the mGlu subtypes, making

possible the discovery of mGlu₅selective ligands. Allosteric

ligands can activate the receptor directly (allosteric agonists) or

can modulate the activity of the receptor in the presence of the

endogenous ligand glutamate, either enhancing (positive

allosteric modulators, PAMs) or diminishing (negative

allosteric modulators, NAMs) receptor activity.¹⁸⁻²⁰Allosteric

modulators with no agonist activity may provide a more subtle

and physiologically relevant approach to restoring target

function in comparison to orthosteric/allosteric agonists as

receptor response occurs only in the presence of the

endogenous agonist.¹⁸⁻²⁰In addition, allosteric modulators

often oﬀer improvements in chemical tractability and improved

properties for central nervous system (CNS) exposure over

traditional orthosteric glutamate analogues such as quisqualate

and (S)-3,5-DHPG, which have diﬃculty passing the blood−

brain barrier.^16,18−20

physiochemical properties.^16,17Representatives of major

chemotypes are shown in Figure 2, and many of these

compounds have demonstrated eﬃcacy in antipsychotic and

cognition models.²⁵⁻³⁶In addition, Lilly recently revealed

mGlu₅PAMs LSN2814617 (10) and LSN2463359 (14).²⁸

Both 10 and 14 are reported to shift a concentration−response

curve for the group I orthosteric agonist, DHPG, in rat cortical

neurons with relatively weak eﬃcacies compared to historical

mGlu₅PAMs (10, FS = 2.8 at 3 μM; 14, FS = 2.0 at 1 μM). In

vivo, these compounds have been reported to exert signiﬁcant

eﬀects in multiple models including the methylazoxymethanol

(MAM) neurodevelopmental model of schizophrenia, wake-

promoting properties in acute sleep-wake electroencephalo-

gram (EEG), and reversal of NMDAR antagonist SDZ 220,581-

induced disruptions in delayed-matching-to-position

(DMTP).²⁸Interestingly, these low eﬃcacy PAMs do not

substantially impact baseline performance on their own in

DMTP, nor do they have signiﬁcant eﬀects in reversal of

hyperlocomotion in the AHL model, which is in contrast to

previously reported mGlu₅PAMs that maintain higher eﬃcacies

(orthosteric agonist concentration−response curve EC₅₀fold-

shift greater than 3.0).^{17,23−25,30,33,36}

The advantages of allosteric modulators have fueled the

development of highly subtype-selective and CNS-penetrant

mGlu₅PAMs.^16,17,21The earliest mGlu₅PAMs are represented

by the four chemotypes shown in Figure 1. 3,3′-Diﬂuor-

obenzaldazine (DFB), developed by Merck & Co. in 2003,

represented the ﬁrst subtype-selective mGlu₅PAM, and the

ensuing discovery of N-(4-chloro-2-((1,3-dioxoisoindolin-2-

yl)methyl)phenyl)-2-hydroxybenzamide (CPPHA) in 2004

provided a compound with improved potency and properties

suitable for use in native preparations.²¹Shortly thereafter, the

ﬁrst highly useful in vivo tool compound, 3-cyano-N-(1,3-

diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) was discovered

by Merck scientists,^22,23and researchers at Addex Pharmaceut-

icals reported a chemically distinct mGlu₅PAM, ADX-47273

(7), with suitable properties for in vivo studies.²⁴Utilizing these

compounds, studies in animal models have added evidence to

the promise of mGlu₅allosteric activation as a novel therapeutic

strategy for the treatment of schizophrenia. PAM 6 has been

shown to possess eﬃcacy in animal models predictive of

positive symptoms (amphetamine-induced hyperlocomotion

(AHL), prepulse inhibition of acoustic startle reﬂex), cognitive

deﬁcits (behavioral and cognitive ﬂexibility, Morris water maze

(MWM)), and negative symptoms (sucrose prefer-

ence).^16,22,23,36Studies with 7 have revealed similar eﬃcacy in

reversal of positive symptoms (conditioned avoidance respond-

ing, apomorphine-induced climbing, AHL) and improvements

in cognition (novel object recognition, ﬁve-choice serial

reaction time test, MWM).^16,24,36In the wake of these studies,

numerous novel mGlu₅PAM chemotypes have been discovered

and optimized, leading to improvements in potency and

Despite the therapeutic promise of mGlu₅PAMs, recent

ﬁndings have begun to reveal a CNS adverse-eﬀect (AE)

liability for certain classes of mGlu₅PAMs.³⁷⁻³⁹The seizure-

inducing eﬀects of group I mGlu orthosteric agonists is well-

known;^40,41however, the potential for mechanism-related AEs

associated with allosteric mGlu₅receptor activation was not

appreciated until recently. Studies in our laboratories have

demonstrated that allosteric modulator 19 (VU0424465,

ML273, Figure 3) causes seizure activity in rodents.³⁷This

eﬀect can be blocked by treatment with 2-methyl-6-(2-

phenylethynyl) pyridine (MPEP), an allosteric mGlu₅NAM,

pointing to the role of mGlu₅in the observed neurotoxicity.

Our recombinant mGlu₅cell line with low receptor density is

predicted to have a better correlation with the functional

response observed in native systems and thus serves as a more

deﬁnitive assay system to detect allosteric agonism.⁴³Allosteric

modulator 19 displays both allosteric agonism and PAM

activity (ago-PAM activity) in a mGlu₅cell line with low

receptor density, as well as in native systems such as

astrocytes.³⁷In contrast, nicotinamide acetylene allosteric

modulator 20 (VU0361747, Figure 3) was devoid of agonism

in these low-expressing mGlu₅cells and did not display a

seizure liability in vivo.³⁷Thus, allosteric agonist activation of

mGlu₅, similar to orthosteric activation, can contribute to an AE

liability in vivo and epileptogenesis in native systems. Further

evidence in support of this hypothesis surfaced from proﬁling of

21 (VU0403602, Figure 3). Similarly to ago-PAM 19,

picolinamide acetylene 21 demonstrates agonist activity in a

mGlu₅low-expressing cell line and native systems and possesses

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Figure 4. Merck-Addex compounds found to induce neurotoxicity.³⁹

Figure 5. SAR development of Vanderbilt acetylene mGlu₅PAMs. Potency values refer to the low density rat mGlu₅receptor cell line.⁴³

a seizure liability in animal models.³⁸Further complicating the

pharmacological eﬀects of 21, its principle in vivo metabolite 22

(VU0453103) also displays signiﬁcant ago-PAM activity,

suggesting that formation of active metabolites is possible and

may contribute to an adverse eﬀect proﬁle in vivo. These

ﬁndings highlight the need to avoid an ago-PAM pharmaco-

logical proﬁle in both the parent compound and its major

metabolites in the quest to develop allosteric ligands for mGlu₅

receptor activation.

Concurrent with these ﬁndings, a collaborative eﬀort

between Merck and Addex Pharmaceuticals revealed that

mGlu₅PAMs within their piperidine and caprolactam chemo-

types induced signiﬁcant convulsions and neuronal cell death in

mice (Figure 4).³⁹In contrast mGlu₅knockout mice that were

administered the compounds did not display these adverse

eﬀects. These compounds did not possess ago-PAM activity in

astrocytes, suggesting allosteric agonism is not necessarily

required to induce adverse eﬀects, although the pharmaco-

logical proﬁle of the major metabolites was not reported. While

the most toxic compound reported, 5PAM523 (23, Figure 4),

had a therapeutic index of just 2-fold (C_maxminimum active

dose amphetamine-induced hyperlocomotion versus C_max

minimum adverse eﬀect dose), higher therapeutic indices

were observed for the additional compounds reported. Of

these, 5PAM916 (24) was found to have the highest

therapeutic index (18-fold) and the lowest glutamate EC₅₀

fold-shift (6-fold at 10 μM). These observations suggest that

the eﬃcacy to shift a glutamate concentration−response curve,

or more precisely, PAM cooperativity, may play an important

role in neurotoxicity, such that improved safety margins may be

realized by designing compounds possessing lower coopera-

tivity.³⁹

preferable for developing compounds with optimal safety

margins. To further investigate the safety implications of ago-

PAM pharmacology, we initiated medicinal chemistry eﬀorts to

explore the structural determinants of ago-PAM activity in the

acetylene scaﬀold 19.³⁷We targeted the development of highly

potent (≤10 nM) tool compounds structurally similar to 19

that did not exhibit allosteric agonism in our in vitro assay

systems. In addition, the glutamate fold-shift proﬁle of

promising pure PAMs was also investigated as an indicator of

cooperativity. Herein we report a detailed structure−activity

relationship (SAR) of allosteric agonism within this class of

acetylene-based mGlu₅PAMs. This study led to the

optimization and characterization of low cooperativity PAM

acetylenes that possess in vitro pharmacological and

pharmacokinetic proﬁles that will enable further studies to

address ongoing questions concerning the therapeutic index of

mGlu₅.

RESULTS AND DISCUSSION

■

Structure−Activity Relationship (SAR) Design. Our

development of acetylenic mGlu₅PAMs originated with a high

throughput screen (HTS) lead VU0092273 (25, Figure 5) that

was quickly optimized to the nicotinamide analogue,

VU0360172 (26, Figure 5).⁴²The introduction of the central

pyridyl ring in 26 enabled salt formation leading to improved

water solubility and the ﬁrst orally active acetylene mGlu₅PAM,

despite a high degree of plasma protein binding (98.9%) and

low CNS exposure (brain to plasma ratio = 0.13). Subsequent

eﬀorts to improve physiochemical properties within this class

led to the discovery that picolinamide analogues, as represented

by 21 (Figure 5),³⁸which show a general improvement in

potency, however still possess a high degree of plasma protein

binding (>99% bound in human and rat). Both 21 and 26 were

found to undergo substantial oxidative metabolism of the

eastern cyclobutane ring (see Figure 3).³⁸With these ﬁndings

These reports, along with the adverse eﬀect liability of

allosteric agonism observed in our laboratories, suggest that

PAMs devoid of agonism in situations where mGlu₅expression

is low and possessing moderate to low eﬃcacy may be

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a

Scheme 1. Synthesis of (R)-3-Amino-2-methylbutan-2-ol

a

Reagents and conditions: (a) SOCl₂, MeOH, 0 °C to rt; (b) Boc₂O, Et₃N, CH₂Cl₂, 0 °C to rt, 88% over 2 steps; (c) MeMgBr (3.0 equiv), THF, 0

°C to rt, 63%; (d) TFA/CH₂Cl₂(1:2), 0 °C, 97%.

a

Scheme 2. Synthesis of Western Aryl Analogues

a

Reagents and conditions: (a) 29, HATU, DIPEA, DMF, rt, 81%; (b) terminal acetylene, CuI, PdCl₂(PPh₃)₂, Et₂NH, DMF, 90 °C, 50−90%; (c)

trimethylsilylacetylene, CuI, PdCl₂(PPh₃)₂, Et₂NH, DMF, 90 °C, 77%; (d) K₂CO₃, MeOH/THF, rt, 95%; (e) aryl bromide, CuI, PdCl₂(PPh₃)₂,

Et₂NH, DMF, 90 °C, 43−62%.

a

Scheme 3. Synthesis of Core Analogues

a

Reagents and conditions: (a) 1-ethynyl-3-ﬂuorobenzene, CuI, PdCl₂(PPh₃)₂, Et₂NH, DMF, 90 °C, 30−97%; (b) 29, HATU, DIPEA, DMF, rt, (c)

LiOH, THF/H₂O, rt.

we turned our eﬀorts toward reducing lipophilicity and

addressing metabolism of the eastern alkyl group through the

introduction of functional groups bearing additional hydrogen

bond donors and/or acceptors in this portion of the molecule.

This eﬀort led to the discovery of 19 bearing a tertiary carbinol,

which contributed to improved solubility, reduced metabolism,

and reduced plasma protein binding (2.8% bound in rat, 2.7%

bound in human).

As 19 represented one of the ﬁrst compounds within the

acetylene class to possess ago-PAM activity in the low-

expressing receptor cell line,³⁷we initiated exploration of

SAR around this analogue to probe the structural determinants

of allosteric agonism. Analysis of 19 yielded three important

structural features to explore: the western aryl ring, the

heteroaryl core, and the eastern amide region. The western

aryl ring was hypothesized to be a structural region that might

impact in vitro PAM to NAM proﬁle mode switching as

observed in related acetylenes.¹⁷The importance of the core

region was suggested by the fact that nicotinamide containing

acetylenes 26 and 20 were active as PAMs but devoid of agonist

activity (Figures 3 and 5). Lastly, the hydroxyl motif in the

eastern amide region of 19 and metabolite 22³⁸was identiﬁed

as a common structural motif among acetylenes possessing

adverse eﬀect proﬁles; therefore, alternate functional groups

designed to maintain good pharmacokinetic and physiochem-

ical properties were targeted.

Chemistry. To explore western aryl and core analogues (R)-

3-amino-2-methylbutan-2-ol, 29, was ﬁrst prepared as shown in

Scheme 1.³⁴Starting from D-alanine, the amino acid was

transformed into the methyl ester upon treatment with SOCl₂

in MeOH. Protection of the amine as the tert-butyl carbamate

yielded intermediate 27 in 88% yield over the 2 steps. Reaction

with MeMgBr (3.0 equiv) yielded tertiary alcohol 28 in 63%

yield, and deprotection in the presence of TFA/CH₂Cl₂

aﬀorded (R)-3-amino-2-methylbutan-2-ol, 29. While the

amino alcohol could be stored as the TFA salt and used over

a period of several weeks, best results were obtained by

removing the tert-butyl carbamate immediately prior to use.

The desired analogues to explore the structural properties of

compounds possessing allosteric agonism were accessed in the

parallel synthetic sequences shown in Schemes 2−4. The

western aryl region was rapidly diversiﬁed utilizing key

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a

Scheme 4. Synthesis of Eastern Amide Analogues

a

Reagents and conditions: (a) 1-ethynyl-3-ﬂuorobenzene, CuI, PdCl₂(PPh₃)₂, Et₂NH, DMF, 90 °C, 65%; (b) amine, HATU, DIPEA, DMF, rt; (c)

NaH, MeI, THF, 0 °C to rt, 58%; (d) DAST, CH₂Cl₂, −78 °C to rt, 30%.

intermediates 30 and 33. 5-Bromo-picolinamide 30 was

accessed via a HATU-mediated amide coupling of 5-

bromopicolinic acid with 29 in 81% yield. Palladium-catalyzed

Sonogoshira coupling of 30 with aryl and cyclic alkyl acetylenes

provided the desired western analogues 31 in 50−90% yield.

When the desired acetylene coupling partners were unavailable,

analogues were synthesized from terminal acetylene 33.

Sonogoshira coupling of 5-bromo-picolinamide 30 with

trimethylsilylacetylene to yield 32, followed by removal of the

silyl protecting group with K₂CO₃in MeOH/THF, provided

terminal acetylene 33 in 73% yield over 2 steps. Sonogoshira

coupling of the resulting terminal acetylene with aryl and

heteoraryl bromides provided the desired analogues in 43−62%

yield.

Synthesis of heteroaryl core analogues was achieved as

displayed in Scheme 3. Sonogoshira coupling of 4-bromo-

heteroaryl carboxylic acids 34a and 1-ethynyl-3-ﬂuorobenzene

yielded biaryl acetylenes 35a in 30−97% yield, which were then

subjected to a HATU-mediated amide bond coupling with 29

to access core analogues 36. Where the desired carboxylic acids

were not available, the corresponding esters 34b were used to

access intermediate 34b via a Sonogoshira coupling with 1-

ethynyl-3-ﬂuorobenzene. Saponiﬁcation of ester 35b with

LiOH in THF/H₂O and amide coupling with 29 yielded the

desired analogues.

The eastern amide region was explored utilizing ethynyl-

picolinic acid 37 as shown in Scheme 4. Following coupling of

5-bromopicolinic acid with 1-ethynyl-3-ﬂuorobenzene to yield

37, a variety of amines were installed through HATU-mediated

amide bond formation. To understand the eﬀect of the chiral

center, (S)-3-amino-2-methylbutan-2-ol was prepared from ^L-

alanine in a sequence analogous to that shown in Scheme 1,

and a variety of commercially available chiral amines bearing

the (R)- and (S)-conﬁguration at the chiral center were

investigated. In addition, to systematically explore the eﬀects of

the eastern hydroxyl on ago-PAM activity, several additional

analogues were prepared directly from 19. Methylation of the

tertiary hydroxyl by treatment with NaH in THF followed by

reaction with MeI aﬀorded methyl ether 38f. The hydroxyl

group was also transformed into the corresponding ﬂuoro-

analogue via reaction of the alcohol with DAST (CH₂Cl₂, −78

°C) to access 38e.

protocol in accord with previously published procedures.^42,43

Activity observed using the low receptor expressing cell line

correlates with the functional response observed in native

systems and allows for detection of allosteric agonism. The

“triple add” protocol allows compounds to be evaluated for

agonism as well as positive and negative allosteric modulation

simulataneously.⁴³

The structure−activity relationship (SAR) are shown in

Tables 1−3. SAR around the western aryl region reveals that

most manipulations to this portion of the molecule do not

eliminate ago-PAM activity. As seen in compounds 19, 31b,

and 31c, positioning of the ﬂuorine atom only modestly

aﬀected potency and eﬃcacy with des-ﬂuoro compound 31a

and 3-ﬂuoro congener 19 preferred for both agonist and PAM

potency. Incorporation of the sterically larger methyl group led

to a concomitant decrease of PAM and agonist activity, except

in the case of meta-substituted analogue 31g. While agonism

was retained for ortho- and meta-substituted derivatives 31f and

31g, methyl substitution at the para-position led to PAM 31h.

Introduction of a western pyridyl also proved to impact agonist

activity, leading to the discovery of PAM 31i, bearing a western

2-pyridine. Structurally, 31i is intriguing as PAM to NAM mode

switching has been observed upon the introduction of the 2-

pyridyl functionality within the tetralone acetylene series

reported by Merz Pharmaceuticals²⁹and in the acetylene

scaﬀold reported by Ritzen

́

and co-workers (Figure 6).^31,44

Thus, it appears that, within the acetylene scaﬀold, the 2-pyridyl

functionality can engender inhibitory eﬀects in some cases.

Moreover, when functionalities are present in other regions of

the molecule that lead to receptor activation (e.g., agonism or

ago-PAM), the 2-pyridyl group appears to moderate these

strong agonist eﬀects. The positioning of the pyridyl nitrogen

also appears to be critical for attenuation of the agonism, as the

3- and 4-substituted pyridines (31j, 31k) maintain ago-PAM

activity. Given that the 2-pyridiyl motif is structurally

reminiscent of mGlu₅antagonist MPEP, we also investigated

the methyl thiazole motif found in the mGlu₅antagonist MTEP

to give 31l, which also elicited a PAM proﬁle with no observed

agonism. Finally, cyclic alkyl groups were explored, with

cyclopropyl derivative 31m found to be a PAM, although with

greatly reduced potency. Ago-PAM cyclopentyl derivative 31n

exhibited a smaller degree of allosteric agonism relative to aryl

analogues.

In Vitro Pharmacology. Compounds were proﬁled in our

rat mGlu₅low receptor expression cell line using a “triple add”

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Table 1. Rat mGlu₅Potency and Percent GluMax Response for Western Aryl Analogues

a

pEC₅₀, EC₅₀, and % GluMax response are the average of at least three independent measurements performed in duplicate or triplicate.

In parallel, explorations of the core region of the molecule

revealed interesting structural features of allosteric agonism.

Deletion of the nitrogen in benzamide 36a yielded a highly

potent ago-PAM (Table 2). Interestingly, although nicotina-

mide analogue 36b displayed reduced potency in comparison

with picolinamide 19, it still displayed signiﬁcant allosteric

agonism. As nicotinamide acetylene analogues exempliﬁed by

20 (Figure 3) and 26 (Figure 5) are generally PAMs devoid of

agonsim, these results suggest that the eastern amide portion of

the molecule is partially responsible for the observed allosteric

agonism. Core modiﬁcations were able to eliminate allosteric

agonism in some instances, as 3- and 4-substituted methyl

analogues 36d and 36e as well as pyrimidine 36f were found to

lack apparent agonist activity. Regioisomeric pyrimidine 36g,

however, displayed ago-PAM activity, as did pyridazine,

pyrazine, and thiazole core analogues (36h−36j).

We next turned our attention to exploring the eastern amide

region within the picolinamide core as this structural motif

appeared to have a signiﬁcant bias toward ago-PAM activity.

Maintaining the propyl backbone found in 19, we prepared a

number of analogues, systematically varying substituents to

explore the SAR proﬁle of allosteric agonism (Table 3). On the

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Figure 6. (a) PAM to NAM mode switching with incorporation of the 2-pyridyl motif.^29,44(b) Acetylene-based allosteric antagonists.

Table 2. Rat mGlu₅Potency and Percent GluMax Response for Core Analogues

a

pEC₅₀, EC₅₀, and % GluMax response are the average of at least three independent measurements performed in duplicate or triplicate.

basis of our initial hypothesis that the tertiary carbinol

contributes to allosteric agonism and adverse eﬀects, our ﬁrst

round of SAR involved deletion and modiﬁcation of the tertiary

hydroxyl. Surprisingly, tert-butyl analogues 38a and 38b and

isopropyl analogues 38c and 38d displayed potent ago-PAM

activity, and replacement of the hydroxyl with ﬂuorine (38e)

resulted in a potent ago-PAM.

For optically active compounds 38a−38d a slight preference

was observed for the (R)-enantiomer. Interestingly, capping the

tertiary carbinol as the methyl ether (38f) resulted in an

inactive compound. As changes to the tertiary carbinol were not

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Table 3. Rat mGlu₅Potency and Percent GluMax Response for Eastern Amide Analogues Lacking Polar Functionalities

a

pEC₅₀, EC₅₀, and % GluMax response are the average of at least three independent measurements performed in duplicate or triplicate.

successful in preventing ago-PAM activity, we deleted the

methyl group adjacent to the amide while maintaining the

eastern alkyl chain carbon backbone length found in 19. This

exploration proved fruitful with isobutyl analogue 38h and

methyl-cyclopropyl analogue 38j found to be potent PAMs;

however, the eﬀect was subtle and the trend was not entirely

clear as the propyl analogue 38g, tert-butyl analogue 38i, and

methyl-cyclobutyl analogue 38k displayed weak agonism.

Since deletion of the tertiary carbinol did not yield a general

strategy to prevent allosteric agonism, we next explored

analogues bearing the eastern alcohol in order to gain an

understanding of the SAR of allosteric agonism when a

hydrogen bond donor was maintained (Table 4). The eﬀect of

the conﬁguration of the stereogenic center was ﬁrst explored,

and surprisingly, the opposite enantiomer of 19, (S)-38l, was

inactive, suggesting that the position of the tertiary hydroxyl

group is critical in receptor activation at the allosteric binding

site. In contrast, both enantiomers of optically active derivatives

38a−38d were active ago-PAMs, with only a slight preference

for the (R)-enantiomer. Removal of steric bulk surrounding the

alcohol (38m) and adjacent to the amide (38n) did not remove

allosteric agonism, while alcohol 38o possessing no steric bulk

was inactive. Interestingly, extension of the carbon backbone in

analogue 38p resulted in a loss of agonist activity, suggesting

that allosteric agonism within this series is sensitive to the

location of the tertiary carbinol.

desirable physiochemical and DMPK properties, which inten-

tionally deter formation of hydroxylated metabolites speciﬁcally

within the eastern amide moiety that may engender undesirable

pharmacology on their own.³⁸Fluorinated alkyl amides were

explored, and triﬂuoroethyl derivative 38q was found to be an

ago-PAM with moderate potency. Triﬂuoroalkyl derivatives

possessing a chiral center aﬀorded increased activity (38r−

38s). Interestingly, these analogues yielded enantiospeciﬁc

activity, with (R)-38r representing a highly potent PAM and

(S)-38s displaying ago-PAM activity. We also explored oxetanes

since this motif has been utilized as a surrogate for geminal

dimethyl groups and represent a polar alternative for the

introduction of steric bulk as well as possessing hydrogen bond

accepting properties.⁴⁵In particular, the oxetane was appealing

as a means to address the high lipophilicity and plasma protein

binding associated with our recently reported PAM

VU0405386 (43);⁴⁶thus, we prepared the 3-methyl-substituted

oxetane as a tert-butyl replacement (38t, Figure 7). The oxetane

can also be envisioned as an isosteric replacement for the

cyclobutyl moiety found in 21 (Figure 3), which was found to

generate the 3-hydroxy metabolite 22 (Figure 3) that

contributed in part to an adverse eﬀect proﬁle. Incorporation

of the oxetane moiety proved successful and 38t represents the

most potent PAM (EC₅₀= 9.3 nM) discovered thus far within

this series. Maintaining the oxetane and extending the carbon

chain length one carbon led to equipotent PAM 38u. Further

extension to the two-carbon variant 39v maintained potentia-

tion activity; however, weak allosteric agonism returned (EC₅₀

= 2.8 μM, GluMax 16.3%). Collectively, the eastern amide SAR

We next pursued alternatives to the tertiary carbinol,

investigating functional groups that conceptually could serve

as a “molecular lock”, such that the functionality possesses

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Table 4. Rat mGlu₅Potency and Percent GluMax Response for Eastern Amide Analogues Bearing Polar Functionalities

a

pEC₅₀, EC₅₀, and % GluMax response are the average of at least three independent measurements performed in duplicate or triplicate.

Figure 7. Oxetane surrogate for steric bulk with decreased lipophilicity.

strongly suggests that the general structural elements of the 2-

methylbutan-2-ol motif (29) introduced to increase solubility

are strongly biased toward allosteric agonism and that other

eastern amide groups can be identiﬁed with favorable

physiochemical properties that result in PAMs lacking agonist

activity.

In light of these results, a ﬁnal round of SAR was pursued to

probe the eﬀects of the basicity of the pyridyl nitrogen in pure

PAM 31i and to investigate combination of the western pyridyl

with a small subset of eastern alkyl groups such as the oxetane

motif. We ﬁrst studied the eﬀects of the basicity of the pyridyl

nitrogen through the introduction of electron-withdrawing

ﬂuorine atoms around the pyridine ring. If a basic nitrogen at

the 2-position of the pyridine ring helps to stabilize a

conformation of the receptor not disposed toward allosteric

agonism, we hypothesized that decreased basicity of the pyridyl

nitrogen at this position might weaken this interaction and

restore allosteric agonism. As shown in Table 5, introduction of

ﬂuorine reveals that the basicity of the pyridyl nitrogen is in fact

important, as ﬂuoro-substituted pyridyls 31o−31r display

allosteric agonism. We subsequently designed several analogues

to explore whether the 2-pyridyl functionality could serve as a

“molecular lock” to prevent allosteric agonism in compounds

bearing eastern amide alkyl groups found to possess agonism

when combined with the 3-ﬂuorophenyl western aryl ring. This

study revealed that the 2-pyridyl motif prevented allosteric

agonism in some but not all cases. Modulator 44b and 44c

exhibited PAM activity; however, tert-butyl analogue 44a

displayed ago-PAM activity. We then pursued a hybrid

picolinamide acetylene containing the western 2-pyridyl and

eastern oxetane amide motifs since these substructures were

discovered to be two of the most preferred structural elements

to maintain PAM activity without apparent agonism (44d).

Unexpectedly, this combination led to mode switching, yielding

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Table 5. Rat mGlu₅Potency and Percent GluMax Response for Pyridyl Analogues

a

b

pEC₅₀, EC₅₀, and % GluMax response are the average of at least three independent measurements performed in duplicate or triplicate. pIC₅₀, IC₅₀,

and GluMin are the average of three independent measurements performed in triplicate.

an antagonist. Moving the nitrogen to the 4-position and

maintaining the oxetane (45) restored some potentiation,

resulting in a weak PAM.

In an eﬀort to gain further insights into the interaction of

ago-PAM versus PAM preferring functional groups within the

mGlu₅binding pocket, PAMs 31i and 38t along with ago-PAM

19 were docked into our recently generated comparative model

of the transmembrane region of mGlu₅(Figure 8).⁴⁷

Interestingly, in comparison with previously docked picolina-

mide PAMs (e.g., 43 and 21),⁴⁷all three compounds sit higher

in the binding pocket; this may be attributable to the inclusion

Figure 8. mGlu₅binding pose for pure-PAMs 31i (cyan color) and 38t

(green color) and ago-PAM 19 (magenta).

of the additional hydrogen bond acceptor on the eastern amide.

Similar to what we observed for other acetylenic PAMs,

computationally it was diﬃcult to diﬀerentiate whether the

eastern amide is buried deep within the pocket, or points

toward the extracellular space, likely a reﬂection of the highly

linear structure for the class. However, models wherein the

eastern amide points toward the extracellular space placed the

compounds in proximity of residues previously found to be

critical for the function of acetylene PAMs and MPEP (Figure

8).⁴⁷The hydroxyl and amide carbonyl of the modulators were

within 3 Å of S808, and the 2-pyridyl western aryl ring was

within 3−5 Å from Y658, T780, and W784. This pose was

chosen as the most likely binding conformation due to its

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Table 6. Rat mGlu₅Cooperativity (Fold-Shift) of Selected PAMs Lacking Apparent Allosteric Agonist Activity

a

PAM concentration tested was 10 μM. Values represent the average of three independent measurements performed in triplicate.

consistency with existing data, and the second possible pose is

shown in the Supporting Information (see section VI).

a gain in allosteric agonist activity at W784A, a mutation known

to reduce the negative cooperativity of MPEP and increase

positive cooperativity of other PAMs.⁴⁷On the basis of the

pose depicted in Figure 8 and the 2-pyridyl “molecular switch”

trends discussed previously (vide supra, see Figure 6), it may be

hypothesized that the 2-pyridyl western aryl within acetylenic

mGlu₅modulators has a key interaction with W784, favoring

less active receptor states. In the W784A mutant where this

interaction is absent, modulator 31i more readily facilitates

active receptor conformations.

Having gained insights into the allosteric agonism SAR and

potential models for receptor/residue-ligand interaction, we

turned our attention to PAM glutamate cooperativity due to its

impact on therapeutic index. We selected PAMs lacking agonist

activity from this investigation that display potency values ≤200

nM and evaluated their ability to left shift the glutamate

concentration−response curve at a concentration of 10 μM

(Table 6). This analysis revealed a range of glutamate fold-shift

values from 1.3 to 5.3. This distribution of fold-shift values

provides useful tool compounds to probe the impact of

cooperativity on therapeutic index in PAMs within the biaryl

acetylene chemotype. As a result of its excellent potency and

In an attempt to probe these putative poses and elucidate the

molecular determinants of agonism further, we examined the

impact of four key point mutations (Y658V, T780A, W784A

and S808A) on modulator aﬃnity, cooperativity, and agonism

by applying an operational model of allosterism to glutamate

concentration−response curves in the absence and presence of

varying concentrations of each PAM, i.e., a progressive fold-

shift experiment (Supporting Information, see sections II−V).

All three compounds were sensitive to alanine substitution of

T780A; however, the observed reductions in aﬃnity (10−30-

fold) were not as substantial as those reported for previous

picolinamides (e.g., 43 and 21).⁴⁷The overall proﬁle of 38t

across all four point mutations was comparable to the prior

picolinamides,⁴⁷and in particular both Y658V and S808A

engendered a NAM switch identical to previously reported (5-

((3-ﬂuorophenyl)ethynyl)pyridin-2-yl)(3-hydroxyazetidin-1-

yl)methanone (VU0405398).⁴⁷The most striking diﬀerences

are observed for 31i, as Y658V had no eﬀect on aﬃnity or

cooperativity, despite causing marked reductions in aﬃnity,

including abolishment of PAM activity, for all other

picolinamide PAMs tested to date. Furthermore, 31i showed

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low cooperativity, PAM 38t was selected for further character-

ization and declared an MLPCN probe.

To validate that 38t interacts with mGlu₅at the MPEP

binding site, radioligand binding studies were performed with

[³H]methoxyPEPy. Increasing concentrations of 38t resulted in

complete inhibition of [³H]methoxyPEPy binding, supporting a

competitive interaction between the two ligands (Figure 9). 38t

metabolism with a predicted hepatic clearance (CL_HEP) of 1.6

mL/min/kg in rat and 0.2 mL/min/kg in human, a signiﬁcant

improvement in comparison to similar compounds within this

series (19, predicted CL_HEPof 34.5 mL/min/kg in rat and 3.5

mL/min/kg in human; 21, predicted CL_HEPof 55.6 mL/min/

kg in rat³⁸). PAM 38t also possesses improved fraction

unbound (f_u) as measured by plasma protein binding assay

using equilibrium dialysis, with oxetane 38t 3.5% unbound in

human plasma and 3.6% unbound in rat plasma. In comparison,

more lipophilic 21 displays human and rat f_uplasma values

<1%.³⁸Rat brain homogenate binding was used to determine

fraction unbound in brain for 38t; these studies revealed f_u

brain values of 1.6%. To assess drug−drug interactions,

inhibition of the major human cytochrome P450 (CYP)

enzymes (2C9, 2D6, 3A4, 1A2) was measured in human liver

microsomes, and 38t was found to display inhibitory activity at

1A2 (IC₅₀= 5.30 μM), while no activity was observed against

the other CYPs tested (IC₅₀>30 μM). Solubility of 38t was

found to be modest with a Fassif (fasted simulated intestinal

ﬂuid) solubility of 10−23 μg/mL.

Figure 9. Compound 38t fully displaces [³H]methoxyPEPy binding.

To verify its PAM pharmacological proﬁle in native systems,

38t was examined for induction of long-term depression (LTD)

at the Schaﬀer collateral-CA1 (SC-CA1) synapse in the

hippocampal formation. LTD at this synapse is known to be

modulated by mGlu₅activation, and orthosteric mGlu₅agonists

such as (S)-3,5-DHPG have been shown to elicit LTD.⁴⁸

Similarly ago-PAM 19 induces LTD;³⁸however, 38t does not

induce LTD on its own (Figure 11; 100.3 3.7% baseline 55

exhibited a K_iof 90 nM, representing a ∼10-fold higher

functional activity (EC₅₀= 9.3 nM) compared to binding. To

utilize compounds in in vivo assays it is important to determine

if they are selective for mGlu₅compared with other mGlu

subtypes. A 10 μM concentration of PAM 38t did not shift the

glutamate (or L-AP4) concentration−response curve when

evaluated using cells expressing any of the other mGlu subtypes

(mGlu_1−4,6−8, see Supporting Information section I) demon-

strating high selectivity for mGlu₅. In addition, screening of 10

μM 38t against a panel of 68 GPCRs, ion channels, and

transporters revealed no signiﬁcant oﬀ-target activity (Euroﬁns

Inc.). Finally, oxetane 38t was evaluated in progressive fold-

shift experiments (50 nM to 30 μM). The shift in the glutamate

concentration−response curve in the presence of increasing

concentrations of modulator is shown in Figure 10. Increasing

Figure 11. Compound 38t has no eﬀect on long-term depression at

the SC-CA1 synapse in rat hippocampus.

min after compound washout). This provides further evidence

that 38t does not elicit a response on its own in native systems.

In addition, prior studies involving 19 showed the induction of

epileptiform activity in CA3 pyramidal neurons in hippocampal

preparations. We performed similar studies with 38t to assess

agonist activity in this native CNS preparation. PAM 38t had

no signiﬁcant eﬀect on either the interevent interval (127.9

7.7% of baseline) or amplitude (101.2 5.0% of baseline) of

spontaneous ﬁring supporting an agonism-free proﬁle for 38t

(data not shown). These data demonstrate that 38t acts as a

pure PAM in two hippocampal native systems.

Figure 10. Compound 38t shifts the glutamate concentration−

response curve leftward with increasing concentrations of modulator:

1.7 at 50 nM; 1.7 at 100 nM; 2.2 at 500 nM; 2.7 at 1 μM; 3.0 at 5 μM;

2.8 at 10 μM and 3.2 at 30 μM.

concentration of modulator resulted in a fold-shift that reached

a maximum of approximately 3.0-fold at 5 μM with a predicted

aﬃnity of −6.81 (154 nM) and an eﬃcacy cooperativity factor

(log β) between glutamate and indicated allosteric modulator

of 0.34 (cooperativity ∼2.2).

The in vitro drug metabolism and pharmacokinetic (DMPK)

proﬁle of 38t was next determined, with the hope that the

oxetane motif would help to mitigate previously observed

metabolism and improve physiochemical properties within this

chemotype. Gratifyingly, oxetane 38t displayed low in vitro

CONCLUSION

■

Although mGlu₅PAMs represent a promising therapeutic

strategy for the treatment of schizophrenia, recent reports have

raised concerns over a seizure liability and neurotoxicity

associated with some chemotypes. Allosteric agonism within

the 19 acetylene chemotype and a high cooperativity

(glutamate fold-shift) within the Merck-Addex piperidine and

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spectra were recorded on a Bruker 400 MHz spectrometer. ¹H

chemical shifts are reported as δ values in CDCl₃or CDOD₃. Data are

reported as follows: chemical shift, integration, multiplicity (s = singlet,

bs = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet, hex

= hextet, sep = septet, dd = doublet of doublets, dq = doublet of

quartets, m = multiplet), coupling constant reported in Hz. ¹³C

chemical shifts are reported in δ values in CDCl₃or CDOD₃as

follows: chemical shift, C−F coupling constants (J_C−F) reported in Hz.

Low resolution mass spectra were obtained on an Agilent 1200 series

6130 mass spectrometer. HRMS were obtained using a Micromass

(Waters) Q-Tof API-US calibrated and veriﬁed with sodium iodide.

The samples were diluted with a 50:50 0.1% formic acid (in Milli-Q)/

acetonitrile solution, directly infused using leucine-enkephalin (M + H

= 556.2771) as a lockmass. Scan range was from 100 to 1000 Da, using

a scan time of 1 s. The [M + H] or [M + Na] ion was observed.

Optical rotation values were obtained on a JASCO P-2000

polarimeter.

Chemistry. (R)-tert-Butyl (3-Hydroxy-3-methylbutan-2-yl)-

carbamate (28). ^D-Alanine (15 g, 0.168 mol, 1 equiv) was dissolved

in MeOH (75 mL, 2.2 M) and cooled to 0 °C. SOCl₂(20.8 mL, 0.286

mol, 1.7 equiv) was slowly added taking care to control the exotherm.

The reaction mixture was stirred overnight warming to room

temperature. After the reaction was determined to be complete by

TLC, the MeOH and excess SOCl₂were carefully removed by vacuum

distillation. The resulting oil was redissolved in MeOH (50 mL), and

the solvent was removed by rotary evaporation. The resulting methyl

ester was dissolved in CH₂Cl₂(150 mL, 1.1 M) and cooled to 0 °C.

Et₃N (70.2 mL, 0.504 mol, 3 equiv) and Boc₂O (44 g, 0.202 mol, 1.2

equiv) were added, and the reaction mixture was allowed to warm to

room temperature and stirred overnight. After the reaction was

determined to be complete by TLC, the precipitates formed during the

reaction were removed via ﬁltration through Celite rinsing with

CH₂Cl₂. The organic layer was washed with citric acid (satd aq, 1 × 50

mL), dried with Na₂SO₄, and concentrated via rotary evaporation

caprolactam series are pharmacological proﬁles that have been

associated with an adverse eﬀect liability. We have extensively

explored the SAR of allosteric agonism within the acetylene

amide chemotype, providing insight into the structural

elements contributing to allosteric agonism. In general, the

structural elements of the eastern amide were found to have the

greatest impact on the presence or absence of allosteric

agonism. Replacement of the western 3-ﬂuorophenyl with the

2-pyridyl motif was found to eliminate allosteric agonism in

many but not all cases. The computational docking and

mutagenesis data highlight the subtleties of interactions within

the common allosteric site, wherein key residues, in particular

W784 and S808, are hypothesized to be engaged in key

interactions important for receptor−modulator interaction and

function. Future studies to understand if the functional

consequences of these mutants are indicative of a direct

residue side chain−modulator interaction or an indirect

allosteric interaction will be important to pursue.

Because of the potential impact of cooperativity on adverse

eﬀects, the glutamate fold-shift proﬁle of potent PAMs was

examined, revealing PAMs possessing low to moderate eﬃcacy

as assessed by glutamate fold-shift. This distribution of

cooperativity proﬁles will enable studies to test the impact of

glutamate fold-shift on neurotoxicity within this chemotype. On

the basis of its eﬃcacy proﬁle and lack of apparent agonism in

vitro, highly potent oxetane 38t was further characterized and

found to possess signiﬁcantly improved DMPK properties

compared with other acetylenes within this series. 38t was also

proﬁled in native systems and exhibited no allosteric agonism

for the induction of LTD at the Schaﬀer collateral-CA1 (SC-

CA1) synapse or epileptiform activity in CA3 pyramidal

neurons. Preliminary experiments using a high dose of 38t in an

in vivo model of psychosis demonstrate robust reversal of

amphetamine-induced hyperlocomotion (data not shown) with

no overt behavioral disturbances; however, deﬁnitive PK-PD

studies in this and other models, including ﬂuorojade

neurotoxicity studies, are needed in order to fully ascertain

the anticipated in vivo properties of PAM 38t. PAM 38t

represents a highly potent tool compound with acceptable

pharmacokinetic properties that will enable further studies to

probe the therapeutic index of low fold-shift PAMs. Such

studies involving 38t and other structurally diverse PAMs are

underway and will be reported in due course. mGlu₅PAM 38t

(ML254) is an MLPCN probe and is freely available upon

request.⁶⁰

yielding (R)-methyl 2-((tert-butoxycarbonyl)amino)propanoate as a

1

yellow oil (30 g, 88% yield). [α]²⁰= 3.6 (c 1.3, CHCl₃); H NMR

D

(400 MHz, CDCl₃) δ 5.07 (1H, bs), 4.28 (1H, m), 3.71 (3H, s), 1.41

(9H, s), 1.35 (3H, d, J = 7.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

173.8, 155.0, 79.7, 52.2, 49.1, 28.2, 18.5; HRMS (ES+, M + H) calcd

for C₉H₁₈NO₄204.1236, found 204.1236.

(R)-Methyl 2-((tert-butoxycarbonyl)amino)propanoate (5 g, 24.6

mmol, 1 equiv) was dissolved in THF (125 mL, 0.2 M) and cooled to

0 °C. MeMgBr (32.8 mL, 98.4 mmol, 5 equiv, 3.0 M soln in Et₂O) was

added slowly, and the reaction was allowed to warm to room

temperature and stirred overnight. The reaction was then quenched

carefully with NH₄Cl (satd aq) and extracted with EtOAc (3 × 75

mL). The organic layer was dried with Na₂SO₄and concentrated. The

crude oil was puriﬁed by silica gel chromatography eluting with Hex/

EtOAc (0−75% EtOAc) with the desired product eluting between

30% and 50% EtOAc. (R)-tert-Butyl (3-hydroxy-3-methylbutan-2-

yl)carbamate was isolated as a clear oil (3.15 g, 63% yield). [α]²⁰

=

D

1

1.9 (c 1.4, CHCl₃); H NMR (400 MHz, CDCl₃) δ 4.82 (1H, d, J =

7.2 Hz), 3.54 (1H, bs), 2.58 (1H, bs), 1.41 (9H, s), 1.19 (3H, s), 1.14

(3H, s), 1.09 (3H, d, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

156.3, 79.3, 72.8, 54.5, 28.3, 27.3, 25.6, 16.1; HRMS (ES+, M + H)

calcd for C₁₀H₂₂NO₃204.1600, found 204.1598.

EXPERIMENTAL SECTION

■

General. All reagents purchased from commercial suppliers were

used without puriﬁcation. Unless noted all solvents used were

anhydrous and all reactions were carried out under argon atmosphere.

Analytical thin layer chromatography was performed on Analtech silica

gel GF 250 μm plates. Preparative RP-HPLC puriﬁcation was

performed on a Gilson Inc. preparative UV-based system using a

Phenomenex Luna C18 column (50 mm × 30 mm i.d., 5 μm) with an

acetonitrile (unmodiﬁed)/0.1% triﬂuoroacetic acid in water gradient.

Normal-phase silica gel preparative puriﬁcation was performed using

an automated Combi-ﬂash Rf from ISCO. Analytical LC−MS was

performed on an Agilent 1200 Series with UV detection at 215 and

254 nm and ELSD detection (Polymer Laboratories PL-ELS 2100),

utilizing an Accucore C18 2.6 μm, 2.1 mm × 30 mm column, a 1.1 min

gradient, 7% [CH₃CN/0.1% TFA] to 95% [CH₃CN/0.1% TFA], and

a G6130 single quadrupole mass spectrometer. Purity of all ﬁnal

compounds was determined to be >98% by analytical HPLC. Solvents

for extraction, washing, and chromatography were HPLC grade. NMR

(R)-3-Amino-2-methylbutan-2-ol (29). In a scintillation vial,

(R)-tert-butyl (3-hydroxy-3-methylbutan-2-yl)carbamate (1 equiv) was

dissolved in CH₂Cl₂(0.1 M) and cooled to 0 °C. Triﬂuoroacetic acid

(0.2M) was added, and the reaction mixture was stirred for 1 h. The

starting material was determined to be consumed by TLC, and the

reaction mixture was concentrated, resulting in a brown oil. The

resulting (R)-3-amino-2-methylbutan-2-ol TFA salt was dissolved in

DMF (0.2 M) and used without puriﬁcation.

(R)-5-Bromo-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide

(30). In a scintillation vial, (R)-tert-butyl (3-hydroxy-3-methylbutan-2-

yl)carbamate (1.5 g, 7.4 mmol, 1 equiv) was dissolved in CH₂Cl₂(6

mL, 0.12 M) and cooled to 0 °C. Triﬂuoroacetic acid (3 mL, 0.25 M)

was added, and the reaction mixture was stirred for 1 h. The starting

material was determined to be consumed by TLC, and the reaction

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mixture was concentrated via rotary evaporation resulting in a brown

oil. The resulting (R)-3-amino-2-methylbutan-2-ol TFA salt was

dissolved in DMF (5 mL) and used without puriﬁcation. In a 100

mL round-bottom ﬂask 5-bromopicolinic acid (1.49 g, 7.4 mmol, 1.0

equiv) and HATU (3.1 g, 8.14 mmol, 1.1 equiv) were combined in

DMF (25 mL, 0.3 M). N,N-Diisopropylethylamine (3.2 mL, 18.25

mmol, 5 equiv) was then added, and the reaction was stirred for 15

min. The (R)-3-amino-2-methylbutan-2-ol TFA salt was added as a

solution in DMF, and the reaction was stirred 18 h after which the

reaction was determined to be complete by LC−MS. The reaction

mixture was partitioned between EtOAc (75 mL) and H₂O (25 mL).

After separating the aqueous layer the organic layer was washed again

with H₂O (2 × 15 mL). The organic layer was then dried with

Na₂SO₄, concentrated, and puriﬁed via silica gel chromatography

eluting with Hex/EtOAc (0−75% EtOAc) with the aryl bromide

bs), 1.29 (3H, d, J = 6.9 Hz), 1.28 (6H, d, J = 5.6 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 163.6, 150.6, 149.8, 149.0, 140.1, 130.3, 125.6, 121.8,

121.7, 91.5, 89.8, 72.9, 53.7, 27.6, 25.8, 15.9; HRMS (ES+, M + H)

calcd for C₁₈H₂₀N₃O₂310.1556, found 310.1554.

(R)-5-Ethynyl-N-(3-hydroxy-3-methylbutan-2-yl)-

picolinamide (33). In a 100 mL round-bottom ﬂask, (R)-5-bromo-N-

(3-hydroxy-3-methylbutan-2-yl)picolinamide (1.5 g, 5.2 mmol, 1

equiv), PdCl₂(PPh₃)₂(187 mg, 026 mmol, 0.05 equiv), and CuI (99

mg, 0.52 mmol, 0.1 equiv) were combined, placed under argon

atmosphere, and dissolved in DMF (15 mL, 0.35M). Trimethylsily-

lacetylene (1.1 mL, 7.8 mmol, 1.5 equiv) was added, followed by

Et₂NH (3.2 mL, 31.2 mmol, 6 equiv). The reaction mixture heated to

90 °C for 45 min after which the reaction was determined to be

complete by LC−MS. The reaction mixture was diluted with EtOAc

(45 mL) and washed with H₂O (3 × 15 mL). The organic layer was

dried with Na₂SO₄, concenterated, and puriﬁed via silica gel

chromatography eluting with Hex/EtOAc (0 to 75% EtOAc) with

the trimethylsilyl-protected acetylene eluting at 40% EtOAc as a pale

eluting at 40% EtOAc. The product was isolated as a brown oil (1.72 g,

1

81% yield). [α]²⁰= −13.7 (c 0.81, CHCl₃); H NMR (400 MHz,

D

CDCl₃) δ 8.51 (1H, bs), 8.09 (1H, d, J = 9.2 Hz), 8.01 (1H, m), 7.90

(1H, m), 4.04 (1H, m), 3.03 (1H, bs), 1.22 (9 H,bs); ¹³C NMR (100

MHz, CDCl₃) δ 163.4, 149.1, 148.2, 139.8, 123.7, 123.6,72.6, 53.5,

27.4, 25.8, 15.7; HRMS (ES+, M + Na) calcd for C₁₁H₁₅N₂O₂BrNa

309.0215, found 309.0212.

1

yellow oil (1.22 g, 77% yield). H NMR (400 MHz, CDCl₃) δ8.55

(1H, bs), 8.10 (1H, d, J = 9.1 Hz), 8.14 (1H, dd, J = 8.1, 1.8 Hz), 7.85

(1H, dd, J = 8.1, 2.0 Hz), 4.10 (1H, m), 2.65 (1H, bs), 1.27 (3H, d, J =

7.7 Hz), 1.26 (6H, d, J = 5.8 Hz), 0.26 (9H, s).

General Methods for Series 31. Method A, 31a−n. In a

scintillation vial, (R)-5-bromo-N-(3-hydroxy-3-methylbutan-2-yl)-

picolinamide, 31, (1 equiv) was placed under argon atmosphere and

dissolved in DMF (0.25M). PdCl₂(PPh₃)₂(0.05 equiv) and CuI (0.1

equiv) were added, followed by an alkyne (1.25 equiv) and Et₂NH (6

equiv). The reaction mixture was heated to 90 °C for 45 min after

which the reaction was determined to be complete by LC−MS. The

reaction mixture was ﬁltered through a Fisherbrand Nylon 0.45 μm

syringe ﬁlter and puriﬁed directly by preparative RP-HPLC eluting

with 0.1% TFA in H₂O/MeCN (10−90% MeCN).

The trimethylsilyl-protected acetylene (1.22 g, 4.0 mmol, 1 equiv)

was dissolved in MeOH/THF (1:1, 16 mL, 0.25 M), and K₂CO₃(1.1

g, 8.0 mmol, 2 equiv) was added. The reaction was stirred at room

temperature for 1 h after which it was determined to be complete by

LC−MS. The reaction mixture was diluted with H₂O (5 mL) and

extracted with EtOAc (3 × 20 mL). The organic layer was dried with

Na₂SO₄, cocentrated, and puriﬁed via silica gel chromatography eluting

with Hex/EtOAc (0 to 100% EtOAc) with the acetylene eluting at

60−80% EtOAc as a pale yellow oil (883 mg, 95% yield). [α]²⁰

=

D

−4.9 (c 0.73, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.61 (1H, d, J =

1.2 Hz), 8.17 (1H, bs), 8.14 (1H, dd, J = 8.0, 0.3 Hz), 7.90 (1H, dd, J

= 8.1, 2.0 Hz), 4.10 (1H, dq, J = 9.1, 6.9 Hz), 3.34 (1H, s), 2.58 (1H,

s), 1.27 (3H, d, J = 6.9 Hz), 1.26 (6H, d, J = 5.8 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 163.7, 151.0, 148.8, 140.4, 121.8, 121.7, 82.7, 79.9,

73.0, 53.7, 27.6, 25.7, 15.9; HRMS (ES+, M + Na) calcd for

C₁₃H₁₆N₂O₂Na 255.1109, found 255.1107.

Method B, 31o−r. In a scintillation vial, (R)-5-ethynyl-N-(3-

hydroxy-3-methylbutan-2-yl)picolinamide, 33, (1 equiv) was placed

under argon atmosphere and dissolved in DMF (0.25M). An aryl

halide (1 equiv), Pd(PPh₃)₄(0.05 equiv) and CuI (0.1 equiv) were

added, followed by Et₃N (17 equiv). The reaction mixture heated to

60 °C until the reaction was determined to be complete by LC−MS

(1−2 h). The reaction mixture was ﬁltered through a Fisherbrand

Nylon 0.45 μm syringe ﬁlter and puriﬁed directly by preparative RP-

HPLC eluting with 0.1% TFA in H₂O/MeCN (10−90% MeCN).

(R)-5-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

tan-2-yl)-3-methylpicolinamide (31d). LC−MS: t_R= 0.798 min,

General Methods for Series 36. Method A, 36a,b,d,e,h−j. In a

scintillation vial, an aryl halide carboxylic acid, 34a (1 equiv),

PdCl₂(PPh₃)₂(0.05 equiv), and CuI (0.1 equiv) were combined,

placed under argon atmosphere, and dissolved in DMF (0.25M). 3-

Fluorophenylacetylene (1.25 equiv) was added, followed by Et₂NH (6

equiv). The reaction mixture was heated to 90 °C for 45 min after

which the reaction was determined to be complete by LC−MS. The

reaction mixture was ﬁltered through a Fisherbrand Nylon 0.45 μm

syringe ﬁlter and puriﬁed directly by preparative RP-HPLC eluting

with 0.1% TFA in H₂O/MeCN (10−90% MeCN) to yield the desired

acetylene carboxylic acid 35a.

In a scintillation vial, acetylene carboxylic acid 35a (1.0 equiv) and

HATU (1.1 equiv) were combined in DMF (0.25 M). N,N-

Diisopropylethylamine (DIPEA, 5 equiv) was then added. After the

reaction mixture was stirred for 10 min, a solution of freshly prepared

(R)-3-amino-2-methylbutan-2-ol TFA salt in DMF (1.1 equiv) was

added, and the reaction was stirred until determined to be complete by

LC−MS (2−20 h). The reaction mixture was ﬁltered through a

Fisherbrand Nylon 0.45 μm syringe ﬁlter and puriﬁed directly by

preparative RP-HPLC eluting with 0.1% TFA in H₂O/MeCN (10−

90% MeCN).

Method B, 36c,f,g. In a scintillation vial an aryl halide carboxylic

ester, 34b (1 equiv), Pd(PPh₃)₄(0.05 equiv), and CuI (0.1 equiv)

were combined, placed under an argon atmosphere, and dissolved in

DMF (0.25 M). 3-Fluorophenylacetylene (1.25 equiv) was added,

followed by Et₃N (17 equiv). The reaction mixture heated to 60 °C

until the reaction was determined to be complete by LC−MS (1−2 h).

The reaction mixture was ﬁltered through a Fisherbrand Nylon 0.45

μm syringe ﬁlter and puriﬁed directly by preparative RP-HPLC eluting

with 0.1% TFA in H₂O/MeCN (10−90% MeCN) to yield the desired

acetylene carboxylic ester 35b.

>98% at 215 and 254 nm, m/z = 341.2 [M + H]⁺. [α]²⁰= −14.2 (c

D

0.74, CHCl₃); ¹H NMR (400 MH, CDCl₃) δ 8.48 (1H, d, J = 1.6 Hz),

8.24 (1H, d, J = 8.8 Hz), 7.70 (1H, d, J = 1.2 Hz), 7.32 (2H, m), 7.24

(1H, m), 7.10 (1H, m), 4.10 (1H, dq, J = 8.9, 6.9 Hz), 2.73 (3H, s),

2.68 (1H, bs), 1.27 (9H, m); ¹³C NMR (100 MHz, CDCl₃) δ 165.6,

162.3 (d, J_C−F= 245.6 Hz), 147.6, 146.0, 143.0, 135.0, 130.1 (d, J_C−F

=

8.6 Hz), 127.6 (d, J_C−F= 3.0 Hz), 124.0 (d, J_C−F= 9.4 Hz), 121.8,

118.5 (d, J_C−F= 22.8 Hz), 116.4 (d, J_C−F= 21.1 Hz), 92.8, 86.2, 73.1,

53.4, 27.6, 25.5, 20.3, 16.0; HRMS (ES+, M + H) calcd for

C₂₀H₂₂FN₂O₂341.1665, found 341.1663.

(R)-N-(3-Hydroxy-3-methylbutan-2-yl)-5-(pyridin-2-

ylethynyl)picolinamide (31i). LC−MS: t_R= 0.533 min, >98% at

215 and 254 nm, m/z = 310.2 [M + H]⁺. [α]²⁰= −21.1 (c 0.53,

D

CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.71 (1H, bs), 8.64 (1H, d, J

= 4.6 Hz), 8.17 (2H, d, J = 8.1 Hz), 7.99 (1H, dd, J = 8.1, 1.7 Hz), 7.71

(1H, dt, J = 7.8, 1.5 Hz), 7.55 (1H, d, J = 7.9 Hz), 7.28 (1H, m), 4.11

(1H, dq, J = 8.9, 6.7 Hz), 2.70 (1H, bs), 1.27 (3H, d, J = 6.8 Hz), 1.26

(6H, d, J = 8.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.7, 150.7,

150.2, 148.8, 142.4, 140.1, 136.3, 127.4, 123.4, 121.9, 121.7, 93.4, 85.1,

72.9, 53.7, 27.6, 25.8, 15.9; HRMS (ES+, M + H) calcd for

C₁₈H₂₀N₃O₂310.1556, found 310.1554.

(R)-N-(3-Hydroxy-3-methylbutan-2-yl)-5-(pyridin-4-

ylethynyl)picolinamide (31k). LC−MS: t_R= 0.461 min, >98% at

215 and 254 nm, m/z = 310.2 [M + H]⁺. [α]²⁰= −27.8 (c 0.48,

D

1

CHCl₃); H NMR (400 MHz, CDCl₃) δ 8.67 (1H, dd, J = 1.6 Hz),

8.65 (1H, bs), 8.20 (1H, m), 8.17 (1H, bs), 7.97 (1H, dd, J = 8.2, 2.0

Hz), 7.41 (2H, d, J = 4.9 Hz), 4.12 (1H, dq, J = 9.5, 7.0 Hz), 2.59 (1H,

7990

dx.doi.org/10.1021/jm401028t | J. Med. Chem. 2013, 56, 7976−7996

Journal of Medicinal Chemistry

Article

Methyl or ethyl ester 35b (1 equiv) was dissolved in THF/H₂O

(4:1, 0.1 M), and LiOH (3 equiv) was added. The reaction was

vigorously stirred until the starting material was observed to be

consumed by LC−MS (30 min to 16 h). The reaction was quenched

with 2 M HCl and extracted with ethyl acetate (3 × 10 mL). The

organic layers were combined, dried with Na₂SO₄, concentrated, and

used without further puriﬁcation. In a scintillation vial, the resulting

acetylene carboxylic acid (1.0 equiv) and HATU (1.1 equiv) were

combined in DMF (0.25 M). N,N-Diisopropylethylamine (DIPEA, 5

equiv) was then added. After the reaction mixture was stirred for 10

min, a solution of freshly prepared (R)-3-amino-2-methylbutan-2-ol

TFA salt in DMF (1.1 equiv) was added, and the reaction was stirred

until determined to be complete by LC−MS (2−20 h). The reaction

mixture was ﬁltered through a Fisherbrand Nylon 0.45 μm syringe

ﬁlter and puriﬁed directly by preparative RP-HPLC eluting with 0.1%

TFA in H₂O/MeCN (10−90% MeCN).

Hz), 150.5, 147.4, 139.9, 130.2 (d, J_C−F= 8.6 Hz), 127.7 (d, J_C−F= 3.1

Hz), 125.3 (q, J_C−F= 279.3 Hz), 123.8 (d, J_C−F= 9.4 Hz), 123.2,

122.0, 118.6 (d, J_C−F= 22.9 Hz), 116.6 (d, J_C−F= 21.0 Hz), 93.7, 86.1,

46.5 (q, J_C−F= 31.6 Hz), 14.4 (d, J_C−F= 1.5 Hz); HRMS (ES+, M +

H) calcd for C₁₇H₁₃F₄N₂O 337.0964, found 337.0966.

(S)-5-((3-Fluorophenyl)ethynyl)-N-(1,1,1-triﬂuoropropan-2-

yl)picolinamide (38s). LC−MS: t_R= 1.030 min, >98% at 215 and

254 nm, m/z = 336.9 [M + H]⁺. [α]²⁰_D= −10.0 (c 0.82, CHCl₃); ¹H

NMR (400 MHz, CDCl₃) δ 8.68 (1H, d, J = 1.4 Hz), 8.20 (1H, d, J =

8.1 Hz), 8.11 (1H, d, J = 9.8 Hz), 7.97 (1H, dd, J = 7.8, 1.8 Hz), 7.35

(2H, m), 7.27 (1H, m), 7.10 (1H, m), 4.88 (1H, m), 1.46 (3H, d, J =

7.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 162.4 (d, J_C−F= 245.7

Hz), 150.5, 147.4, 139.9, 130.2 (d, J_C−F= 8.6 Hz), 127.7 (d, J_C−F= 3.1

Hz), 125.3 (q, J_C−F= 279.3 Hz), 123.8 (d, J_C−F= 9.4 Hz), 123.2,

122.0, 118.6 (d, J_C−F= 22.9 Hz), 116.6 (d, J_C−F= 21.0 Hz), 93.7, 86.1,

46.5 (q, J_C−F= 31.6 Hz), 14.4 (d, J_C−F= 1.5 Hz); HRMS (ES+, M +

H) calcd for C₁₇H₁₃F₄N₂O 337.0964, found 337.0963.

5-((3-Fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)-

picolinamide (38t). LC−MS: t_R= 0.879 min, >98% at 215 and 254

nm, m/z = 310.9 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.64 (1H,

d, J = 1.4 Hz), 8.29 (1H, bs), 8.13 (1H, d, J = 7.8 Hz), 7.93 (1H, dd, J

= 8.1, 2.0 Hz), 7.33 (2H, m), 7.24 (1H, m), 7.09 (1H, m), 4.94 (2H, d,

J = 6.4 Hz), 4.57 (2H, d, J = 6.5 Hz), 1.77 (3H, s); ¹³C NMR (100

MHz, CDCl₃) δ 162.9, 162.3 (d, J_C−F= 245.9 Hz), 150.4, 148.2, 139.8,

130.1 (d, J_C−F= 8.6 Hz), 127.6 (d, J_C−F= 3.0 Hz), 123.8 (d, J_C−F= 9.3

Hz), 122.7, 121.3, 118.5 (d, J_C−F= 22.9 Hz), 116.5 (d, J_C−F= 21.0

Hz), 93.5 (d, J_C−F= 3.5 Hz), 86.2, 81.8, 53.6, 23.6; HRMS (ES+, M +

H) calcd for C₁₈H₁₆FN₂O₂311.1196, found 311.1197.

(R)-6-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

tan-2-yl)nicotinamide (36b). LC−MS: t_R= 0.765 min, >98% at 215

and 254 nm, m/z = 326.9 [M + H]⁺. [α]²⁰= −7.5 (c 0.57, CHCl₃);

D

¹H NMR (400 MHz, CDCl₃) δ 8.98 (1H, d, J = 1.8 Hz), 8.10 (1H, dd,

J = 8.1, 2.3 Hz), 7.55 (1H, d, J = Hz), 7.32 (3H, m), 7.08 (1H, m),

6.76 (1H, d, J = 8.8 Hz), 4.13 (1H, dq, J = 8.8, 6.8 Hz), 2.62 (1H, bs),

1.29 (6H, d, J = 4.4 Hz), 1.27 (3H, d, J = 6.8 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 164.8, 162.3 (d, J_C−F= 245 Hz), 148.3, 145.3, 135.3,

130.1 (d, J_C−F= 8.5 Hz), 129.1, 128.0 (d, J_C−F= 7.0 Hz), 126.9, 123.5

(d, J_C−F= 9.4 Hz), 118.8 (d, J_C−F= 23.0), 116.8 (d, J_C−F= 21.0 Hz),

90.0 (d, J_C−F= 3.4 Hz), 88.7, 72.6, 53.7, 28.0, 26.3, 15.7 ; HRMS (ES+,

M + H) calcd for C₁₉H₂₀FN₂O₂: 327.1509, found 327.1510.

5-((3-Fluorophenyl)ethynyl)-N-((3-methyloxetan-3-yl)-

(R)-6-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

methyl)picolinamide (38u). LC−MS: t_R= 0.797 min, >98% at 215

tan-2-yl)-5-methylnicotinamide (36e). LC−MS: t_R= 0.816 min,

1

and 254 nm, m/z = 325.1 [M + H]⁺. H NMR (400 MHz, CDCl₃) δ

>98% at 215 and 254 nm, m/z = 341.2 [M + H]⁺. [α]²⁰= −17.9 (c

D

0.43, CHCl₃); ¹H NMR (400 MH, CDCl₃) δ 8.57 (1H, s), 8.17 (1H,

d, J = 7.2 Hz), 8.07 (1H, s), 7.36 (2H, s), 7.24 (1H, m), 7.09 (1H, m),

4.12 (1H, dq, J = 8.9, 6.9 Hz), 2.55 (3H, s), 1.71 (1H, bs), 1.29 (3H, d,

J = 6.8 Hz), 1.28 (6H, d J = 9.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

8.67 (1H, d, J = 1.3 Hz), 8.29 (1H, bs), 8.20 (1H, d, J = 7.6 Hz), 7.97

(1H, dd, J = 6.0, 2.0 Hz), 7.35 (2H, m), 7.26 (1H, m), 7.10 (1H, m),

4.59 (2H, d, J = 6.0 Hz), 4.44 (2H, d, J = 6.0 Hz), 3.68 (2H, d, J = 6.5

Hz), 1.40 (3H, s); ¹³C NMR (100 MHz, CDCl₃) δ 164.2, 162.4 (d,

J_C−F= 245.9 Hz), 150.5, 148.2, 139.9, 130.2 (d, J_C−F= 8.7 Hz), 127.7

(d, J_C−F= 3.0 Hz), 123.9 (d, J_C−F= 9.4 Hz), 122.7, 121.8, 118.6 (d,

J_C−F= 22.8 Hz), 116.6 (d, J_C−F= 21.1 Hz), 93.4 (d, J_C−F= 3.7 Hz),

86.3, 80.3, 45.9, 40.2, 22.0; HRMS (ES+, M + H) calcd for

C₁₉H₁₈FN₂O₂325.1352, found 325.1353.

164.2, 162.4 (d, J_C−F=245.7 Hz), 150.6, 150.5, 148.2, 130.1 (d, J_C−F

=

8.6 Hz), 127.6 (d, J_C−F= 3.0 Hz), 124.2 (d, J_C−F= 9.2 Hz), 122.9,

122.6, 118.6 (d, J_C−F= 22.7 Hz), 116.4 (d, J_C−F= 21.1 Hz), 96.8, 85.4,

73.1, 53.8, 27.6, 25.6, 20.4, 15.9; HRMS (ES+, M + H) calcd for

C₂₀H₂₂FN₂O₂341.1665, found 341.1664.

5-((3-Fluorophenyl)ethynyl)picolinic Acid (37). In a 100 mL

round-bottom ﬂask, 5-bromopicolinic acid (3g, 14.8 mmol, 1 equiv),

PdCl₂(PPh₃)₂(519 mg, 0.74 mmol, 0.05 equiv), and CuI (282 mg,

1.48 mmol, 0.1 equiv) were combined, place under an argon

atmosphere, and dissolved in DMF (50 mL, 0.3 M). 3-

Fluorophenylacetylene (2.05 mL, 17.76 mmol, 1.2 equiv) was added,

followed by Et₂NH (9.2 mL, 88.8 mmol, 6 equiv). The reaction

mixture heated to 90 °C for 45 min after which the reaction was

determined to be complete by LC−MS. The crude reaction mixture

was diluted with EtOAc (50 mL) and H₂O (50 mL). After separating

the organic layer, the aqueous layer was washed with EtOAc (2 × 25

mL). The aqueous layer was then acidiﬁed to ∼pH = 2 with 2 M HCl,

and extracted with EtOAc (3 × 50 mL). The organic layer was dried

with Na₂SO₄, concentrated, and used without further puriﬁcation. The

(R)-2-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

tan-2-yl)pyrimidine-5-carboxamide (36f). LC−MS: t_R= 0.708

min, >98% at 215 and 254 nm, m/z = 327.9 [M + H]⁺. [α]²⁰_D= −8.9

1

(c 0.67, CHCl₃); H NMR (400 MHz, CDCl₃) δ 9.12 (2H, s), 7.43

(1H, d, J = 7.8 Hz), 7.34 (2H, m), 7.14 (1H, td, J = 9.2, 2.4 Hz), 6.75

(1H, d, J = 8.8 Hz), 4.14 (1H, dq, J = 8.8, 6.9 Hz), 2.31 (1H, bs), 1.30

(6H, s), 1.28 (3H, d, J = 6.9 Hz); ¹³C NMR (100 MHz, CDCl₃) δ

162.8, 162.2 (d, J_C−F= 246.1 Hz), 156.1, 154.3, 130.2 (d, J_C−F= 8.4

Hz), 128.6 (d, J_C−F= 3.1 Hz), 125.9, 122.6 (d, J_C−F= 9.4 Hz), 119.4

(d, J_C−F= 23.1 Hz), 117.5 (d, J_C−F= 21.2 Hz), 88.6 (d, J_C−F= 3.5 Hz),

88.2, 72.5, 53.7, 28.0, 26.4, 15.7; HRMS (ES+, M + H) calcd for

C₁₈H₁₉FN₃O₂328.1461, found 328.1459.

5-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutyl)-

picolinamide (38p). LC−MS: t_R= 0.870 min, >98% at 215 and 254

nm, m/z = 326.9 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.61 (1H,

bs), 8.42 (1H, m), 8.15 (1H, d, J = 8.1 Hz), 7.90 (1H, dd, J = 8.1, 2.0

Hz), 7.31 (2H, m), 7.22 (1H, m), 7.07 (1H, m), 3.63 (2H, q, J = 6.1

Hz), 2.44 (1H, bs), 1.82 (2H, t, J = 7.0 Hz), 1.30 (6H, s); ¹³C NMR

(100 MHz, CDCl₃) δ 163.8, 162.3 (d, J_C−F= 245.7 Hz), 150.4, 148.7,

139.7, 130.1 (d, J_C−F= 8.6 Hz), 127.6 (d, J_C−F= 3.1 Hz), 123.9 (d,

1

product was isolated as a white solid (2.32 g, 65%). H NMR (400

MHz, CD₃OD) δ 8.81 (1H, bs), 8.16 (2H, m), 7.44 (2H, m), 7.34

(1H, d, J = 9.7 Hz), 7.21 (1H, m); ¹³C NMR (100 MHz, CD₃OD) δ

167.0, 163.9 (d, J_C−F= 244.4 Hz), 152.5, 148.0, 141.3, 131.7 (d, J_C−F

8.6 Hz), 129.0, 125.8, 125.2 (d, J_C−F= 9.3 Hz), 124.8, 119.4 (d, J_C−F

=

23.4 Hz), 117.7 (d, J_C−F= 21.4 Hz), 94.8, 86.8; HRMS (ES+, M + H)

calcd for C₁₄H₉FNO₂242.0617, found 242.0618.

J_C−F= 9.4 Hz),122.7, 121.5, 118.5 (d, J_C−F= 22.8 Hz), 116.4 (d, J_C−F

=

Amide Analogues 19, 38a−d,g−v. In a scintillation vial, 5-((3-

ﬂuorophenyl)ethynyl)picolinic acid, 37, (1.0 equiv) and HATU (1.1

equiv) were combined in DMF (0.25 M). N,N-Diisopropylethylamine

(DIPEA, 3 equiv) was then added. The reaction mixture was stirred for

10 min after which an amine (1.1 equiv) was added. The reaction was

stirred until determined to be complete by LC−MS (2−20 h). The

reaction mixture was ﬁltered through a Fisherbrand Nylon 0.45 μm

syringe ﬁlter and puriﬁed directly by preparative RP-HPLC eluting

with 0.1% TFA in H₂O/MeCN (10−90% MeCN).

21.1 Hz), 93.1 (d, J_C−F= 3.5 Hz), 86.3, 70.5, 42.0, 35.8, 29.6; HRMS

(ES+, M + H) calcd for C₁₉H₂₀FN₂O₂327.1509, found 327.1508.

(R)-5-((3-Fluorophenyl)ethynyl)-N-(1,1,1-triﬂuoropropan-2-

yl)picolinamide (38r). LC−MS: t_R= 1.030 min, >98% at 215 and

1

254 nm, m/z = 336.9 [M + H]⁺. [α]²⁰= 10.5 (c 0.59, CHCl₃); H

D

NMR (400 MHz, CDCl₃) δ 8.68 (1H, d, J = 1.4 Hz), 8.20 (1H, d, J =

8.1 Hz), 8.11 (1H, d, J = 9.8 Hz), 7.97 (1H, dd, J = 7.8, 1.8 Hz), 7.35

(2H, m), 7.27 (1H, m), 7.10 (1H, m), 4.88 (1H, m), 1.46 (3H, d, J =

7.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 162.4 (d, J_C−F= 245.7

7991

dx.doi.org/10.1021/jm401028t | J. Med. Chem. 2013, 56, 7976−7996

Journal of Medicinal Chemistry

Article

(R)-5-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

tan-2-yl)picolinamide (19). LC−MS: t_R= 0.731 min, >98% at 215

and 254 nm, m/z = 327.1 [M + H]⁺. [α]²⁰_D= −22.2 (c 0.43, CHCl₃);

¹H NMR (400 MHz, CDCl₃) δ 8.65 (1H, dd, J = 1.3, 0.7 Hz), 8.19

(2H, dd, J = 8.1, 0.7 Hz), 7.95 (1H, dd, J = 8.1, 2.0 Hz), 7.34 (2H, m),

7.24 (1H, m), 7.09 (1H, m), 4.13 (1H, dq, J = 9.1, 6.8 Hz), 2.50 (1H,

bs), 1.29 (3H, d, J = 6.8 Hz), 1.28 (6H, d, J = 8.0 Hz); ¹³C NMR (100

MHz, CDCl₃) δ 163.8 162.3 (d, J_C−F= 245.7 Hz), 150.4, 148.4, 139.8,

130.1 (d, J_C−F= 8.6 Hz), 127.7 (d, J_C−F= 3.0 Hz), 123.9 (d, J_C−F= 9.3

Hz), 122.5, 121.8, 118.5 (d, J_C−F= 22.9 Hz), 116.5 (d, J_C−F= 21.0

Hz), 93.3, 86.3, 73.1, 53.8, 27.6, 25.7, 15.9; HRMS (ES+, M + H)

calcd for C₁₉H₂₀FN₂O₂327.1509, found 327.1507.

123.9 (d, J_C−F= 9.3 Hz), 122.5, 121.8, 118.5 (d, J_C−F= 22.9 Hz), 116.5

(d, J_C−F= 21.0 Hz), 93.3, 86.3, 73.1, 53.7, 27.6, 25.7, 15.9; HRMS (ES

+, M + H) calcd for C₁₉H₂₀FN₂O₂327.1509, found 327.1507.

General Amide Coupling and Sonogashira Two-Step

Procedure (44a,b,d, 45). In a scintillation vial, 5-bromopicolinic

acid (1.0 equiv) and HATU (1.1 equiv) were combined in DMF (0.25

M). N,N-Diisopropylethylamine (DIPEA, 5 equiv) was then added.

After the reaction mixture was stirred for 10 min, the desired amine

was added, and the reaction was stirred until determined to be

complete by LC−MS (2−20 h). The reaction mixture was ﬁltered

through a Fisherbrand Nylon 0.45 μm syringe ﬁlter and puriﬁed

directly by preparative RP-HPLC eluting with 0.1% TFA in H₂O/

MeCN (10−90% MeCN).

In a scintillation vial, the aryl bromide (1 equiv), PdCl₂(PPh₃)₂

(0.05 equiv), and CuI (0.1 equiv) were combined, placed under argon

atmosphere, and dissolved in DMF (0.25M). The desired ethynylpyr-

idine (2- or 4-ethynylpyridine) (1.25 equiv) was added, followed by

Et₂NH (6 equiv). The reaction mixture heated to 90 °C for 45 min

after which the reaction was determined to be complete by LC−MS.

The reaction mixture was ﬁltered through a Fisherbrand Nylon 0.45

μm syringe ﬁlter and puriﬁed directly by preparative RP-HPLC eluting

with 0.1% TFA in H₂O/MeCN (10−90% MeCN) to yield products

44a−d and 45.

(R)-N-(3-Methylbutan-2-yl)-5-(pyridin-2-ylethynyl)-

picolinamide (44b). LC−MS: t_R= 0.727 min, >98% at 215 and 254

nm, m/z = 294.2 [M + H]⁺. [α]²⁰_D= −50.0 (c 0.78, CHCl₃); ¹H NMR

(400 MHz, CDCl₃) δ 8.72 (1H, d, J = 1.7 Hz), 8.65 (1H, d, J = 4.7

Hz), 8.20 (1H, d, J = 8.1 Hz), 8.00 (1H, dd, J = 6.1, 2.0 Hz), 7.89 (1H,

d, J = 9.0 Hz), 7.73 (1H, dt, J = 7.7, 1.6 Hz),), 7.57 (1H, d, J = 7.8 Hz),

7.30 (1H, m), 4.05 (1H, m), 1.84 (1H, sep, J = 6.5 Hz), 1.21 (3H, d, J

= 6.7 Hz), 0.97 (6H, dd, J = 6.8, 4.0 Hz); ¹³C NMR (100 MHz,

CDCl₃) δ 162.8, 150.7, 150.2, 149.1, 142.4, 140.1, 136.3, 127.4, 123.4,

121.7, 121.6, 93.3, 85.1, 50.2, 33.1, 18.6, 18.5, 17.6; HRMS (ES+, M +

H) calcd for C₁₈H₂₀N₃O 294.1606, found 294.1607.

(R)-N-(3-Fluoro-3-methylbutan-2-yl)-5-((3-ﬂuorophenyl)-

ethynyl)picolinamide (38e). (R)-5-((3-Fluorophenyl)ethynyl)-N-

(3-hydroxy-3-methylbutan-2-yl)picolinamide, 19, (40 mg, 0.12 mmol,

1.0 equiv) was dissolved in CH₂Cl₂(1.2 mL, 0.1 M) and cooled to

−78 °C. DAST (19 μL, 0.12 mmol, 1.0 equiv) was added dropwise,

and the reaction was allowed to slowly warm to rt over 4 h. The

reaction was determined to be complete by LC−MS and was

quenched carefully by the addition of H₂O (0.5 mL). The aqueous

layer was extracted with EtOAc (3 × 5 mL), dried Na₂SO₄,

concentrated, and puriﬁed by preparative RP-HPLC eluting with

0.1% TFA in H₂O/MeCN (10−90% MeCN) to aﬀord the title

compound in 30% yield. LC−MS: t_R= 0.926 min, >98% at 215 and

1

254 nm, m/z = 329.1 [M + H]⁺. [α]²⁰= −5.9 (c 1.16, CHCl₃); H

D

NMR (400 MHz, CDCl₃) δ 8.68 (1H, d, J = 1.3 Hz), 8.19 (1H, dd, J =

12.0, 0.4 Hz), 8.14 (1H, d, J = 9.8 Hz), 7.96 (1H, dd, J = 8.1, 2.0 Hz),

7.35 (2H, m), 7.27 (1H, m), 7.10 (1H, m), 4.29 (1H, m), 1.57 (1H,

bs), 1.46 (3H, d, J = 16.7 Hz), 1.40 (3H, d, J = 16.7 Hz), 1.33 (3H, d, J

= 6.9 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.2, 162.4 (d, J_C−F

=

22.9 Hz), 150.5, 148.4, 139.8, 130.1 (d, J_C−F= 8.7 Hz), 127.7 (d, J_C−F

= 3.2 Hz), 124.0 (d, J_C−F= 9.4 Hz), 122.6, 121.8, 118.6 (d, J_C−F= 21.8

Hz), 116.5, (d, J_C−F= 21.0 Hz), 96.6 (d, J_C−F= 170.7 Hz), 93.3 (d,

J_C−F= 3.4 Hz), 86.4, 51.8 (d, J_C−F= 22.2 Hz), 24.6 (d, J_C−F= 7.8 Hz),

24.3 (d, J_C−F= 7.4 Hz), 15.6 (d, J_C−F= 3.7 Hz) ; HRMS (ES+, M + H)

calcd for C₁₉H₁₈F₂N₂ONa 351.1285, found 351.1282.

(R)-N-(3-Fluoro-3-methylbutan-2-yl)-5-(pyridin-2-ylethynyl)-

picolinamide (44c). (R)-N-(3-Hydroxy-3-methylbutan-2-yl)-5-(pyr-

idin-2-ylethynyl)picolinamide, 31i, (39 mg, 0.12 mmol, 1.0 equiv) was

dissolved in CH₂Cl₂(1.2 mL, 0.1 M) and cooled to −78 °C. DAST

(19 μL, 0.12 mmol, 1.0 equiv) was added dropwise, and the reaction

was allowed to slowly warm to rt over 4 h. The reaction was

determined to be complete by LC−MS and was quenched carefully by

the addition of H₂O (0.5 mL). The aqueous layer was extracted with

EtOAc (3 × 5 mL), dried Na₂SO₄, concentrated, and puriﬁed by

preparative RP-HPLC eluting with 0.1% TFA in H₂O/MeCN (10−

90% MeCN) to aﬀord the title compound in 81% yield. LC−MS: t_R=

(R)-5-((3-Fluorophenyl)ethynyl)-N-(3-methoxy-3-methylbu-

tan-2-yl)picolinamide (38f). (R)-5-((3-Fluorophenyl)ethynyl)-N-

(3-hydroxy-3-methylbutan-2-yl)picolinamide, 19, (68 mg, 0.21

mmol, 1.0 equiv) was dissolved in THF (2 mL, 0.1 M) and cooled

to 0 °C. NaH (11.1 mg, 0.46 mmol, 2.2 equiv) was added. The

reaction mixture was warmed to rt and stirred for 10 min. Methyl

iodide (15 μL, 0.23 mmol, 1.1 equiv) was added, and the reaction

mixture was stirred at room temperature overnight. Analysis of the

reaction mixture by LC−MS revealed ∼70% of the desired product,

∼20% starting material, and ∼10% dimethylated product. The reaction

was quenched with NH₄Cl (satd, aq) and extracted with EtOAc (3 × 5

mL). The organic layers were dried with Na₂SO₄, concentrated, and

puriﬁed by preparative RP-HPLC eluting with 0.1% TFA in H₂O/

MeCN (10−90% MeCN) to aﬀord the product in 58% yield. LC−MS:

t_R= 0.824 min, >98% at 215 and 254 nm, m/z = 341.2 [M + H]⁺.

[α]²⁰_D= −31.7 (c 0.65, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.66

(1H, d, J = 1.6 Hz), 8.21 (1H, d, J = 9.6 Hz), 8.17 (1H, d, J = 8.1 Hz),

7.92 (1H, dd, J = 8.1, 1.9 Hz), 7.32 (2H, m), 7.23, (1H, m), 7.07 (1H,

m), 4.18 (dq, 1H, J = 9.5, 6.7 Hz), 3.25 (3H, s), 1.23 (3H, d, J = 6.9

Hz), 1.20 (6H, d, J = 11.6 Hz), ; ¹³C NMR (100 MHz, CDCl₃) δ

0.671 min, >98% at 215 and 254 nm, m/z = 312.2 [M + H]⁺. [α]²⁰

=

D

−14.0 (c 0.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.75 (1H, dd,

J = 1.9, 0.6 Hz), 8.67 (1H, d, J = 4.0 Hz), 8.20 (1H, dd, J = 8.1, 0.6

Hz), 8.15 (1H, d, J = 9.8 Hz), 8.02 (1H, dd, J = 8.1, 2.0 Hz), 7.74 (1H,

dt, J = 7.8, 1.8 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.31 (1H, m), 4.28 (1H,

m), 1.45 (3H, d, J = 16.2 Hz), 1.45 (3H, d, J = 16.4 Hz), 1.45 (3H, d, J

= 6.9 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.0, 150.8, 150.3, 148.7,

142.4, 140.1, 136.3, 127.4, 123.5, 122.0, 121.7, 96.5 (d, J_C−F= 170.8

Hz), 93.5, 84.8, 51.4 (d, J_C−F= 22.3 Hz), 24.5 (d, J_C−F= 5.8 Hz), 24.2

(d, J_C−F= 5.3 Hz), 15.5 (d, J_C−F= 3.8 Hz); HRMS (ES+, M + H)

calcd for C₁₈H₁₉FN₃O 312.1512, found 312.1511.

163.0, 162.3 (d, J_C−F= 245.6 Hz), 150.4, 148.8, 139.6, 130.1 (d, J_C−F

=

N-(3-Methyloxetan-3-yl)-5-(pyridin-2-ylethynyl)-

picolinamide (44d). LC−MS: t_R= 0.453 min, >98% at 215 and 254

nm, m/z = 294.2 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.72 (1H,

d, J = 1.2 Hz), 8.66 (1H, d, J = 4.4 Hz), 8.28 (1H, bs), 8.15 (1H, d, J =

8.1 Hz), 8.02 (1H, dd, J = 9.2, 3.0 Hz), 7.73 (1H, td, J = 7.8, 1.6 Hz),

7.58 (1H, d, J = 7.8 Hz), 7.31 (1H, m), 4.94 (2H, d, J = 6.3 Hz), 4.58

(2H, d, J = 6.5 Hz), 1.77 (3H, s); ¹³C NMR (100 MHz, CDCl₃) δ

162.9, 150.8, 150.3, 148.6, 142.4, 140.3, 136.4, 127.4, 123.6, 122.4,

121.4, 93.7, 84.9, 81.9, 53.7, 23.7; HRMS (ES+, M + H) calcd for

C₁₇H₁₆N₃O₂294.1243, found 294.1240.

8.6 Hz), 127.6 (d, J_C−F= 3.0 Hz), 124.0 (d, J_C−F= 9.3 Hz), 122.2,

121.6, 118.5 (d, J_C−F= 22.9 Hz), 116.4 (d, J_C−F= 21.1 Hz), 93.0, 86.4,

75.9, 52.3, 49.4, 22.0, 21.9, 15.5; HRMS (ES+, M + H) calcd for

C₂₀H₂₂FN₂O₂341.1665, found 341.1664.

(S)-5-((3-Fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbu-

tan-2-yl)picolinamide (38l). LC−MS: t_R= 0.738 min, >98% at 215

and 254 nm, m/z = 327.1 [M + H]⁺. [α]²⁰= 24.3 (c 0.48, CHCl₃);

D

¹H NMR (400 MHz, CDCl₃) δ 8.64 (1H, dd, J = 1.3, 0.7 Hz), 8.19

(1H, bs), 8.18 (1H, dd, J = 8.1, 0.7 Hz), 7.94 (1H, dd, J = 8.1, 2.0 Hz),

7.34 (2H, m), 7.24 (1H, m), 7.09 (1H, m), 4.13 (1H, dq, J = 9.1, 6.8

Hz), 2.66 (1H, bs), 1.29 (3H, d, J = 6.8 Hz), 1.28 (6H, d, J = 8.0 Hz);

¹³C NMR (100 MHz, CDCl₃) δ 163.8 162.3 (d, J_C−F= 245.7 Hz),

150.4, 148.4, 139.8, 130.1 (d, J_C−F= 8.6 Hz), 127.6 (d, J_C−F= 3.0 Hz),

N-(3-Methyloxetan-3-yl)-5-(pyridin-4-ylethynyl)-

picolinamide (45). LC−MS: t_R= 0.354 min, >98% at 215 and 254

nm, m/z = 294.2 [M + H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (1H,

d, J = 1.2 Hz), 8.65 (2H, m), 8.29 (1H, bs), 8.16 (1H, d, J = 8.1 Hz),

7992

dx.doi.org/10.1021/jm401028t | J. Med. Chem. 2013, 56, 7976−7996

Journal of Medicinal Chemistry

Article

7.98 (1H, dd, J = 8.1, 1.9 Hz), 7.41 (2H, d, J = 5.6 Hz), 4.93 (2H, d, J

= 6.4 Hz), 4.58 (2H, d, J = 6.4 Hz), 1.77 (3H, s); ¹³C NMR (100

MHz, CDCl₃) δ 162.9, 150.5, 149.9, 148.8, 140.1, 130.2, 125.5, 122.0,

121.4, 91.7, 89.5, 81.8, 53.7, 23.6; HRMS (ES+, M + H) calcd for

C₁₇H₁₆N₃O₂294.1243, found 294.1242.

Electrophysiology (LTD and Epileptiform Studies). All

animals used in these studies were cared for in accordance with the

NIH Guide for the Care and Use of Laboratory Animals. Either 30−40

(LTD experiments) or 24−30 (epileptiform experiments) day old

male Sprague−Dawley rats were used. The brains were quickly

removed and submerged into ice-cold cutting solution (in mM: 110

sucrose, 60 NaCl, 3 KCl, 1.25 NaH₂PO₄, 28 NaHCO₃, 5 glucose, 0.6

(+)-sodium-^L-ascorbate, 0.5 CaCl₂, 7 MgCl₂). All solutions were

continuously bubbled with 95% O₂/5% CO₂. Transverse slices (400

μm) were made using a vibratome (Leica VT100S). For LTD

experiments, individual hippocampi were microdissected out, trans-

ferred to a room temperature mixture containing equal volumes of

cutting solution and artiﬁcial cerebrospinal ﬂuid (ACSF; in mM: 125

NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 25 glucose, 2 CaCl₂, 1

MgCl₂), and equilibrated for 30 min, followed by room temperature

ACSF for 1 h. For epileptiform experiments, individual hippocampi

were transferred directly into room temperature ACSF (in mM: 124

NaCl, 5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 glucose, 2 CaCl₂, 1.2

MgSO₄) and equilibrated for 1 h. Slices were transferred to a

submersion recording chamber and equilibrated for 5−10 min at 30−

32 °C. A bipolar-stimulating electrode was placed in the stratum

radiatum near the CA3-CA1 border in order to stimulate the Schaﬀer

collaterals. Recording electrodes were ﬁlled with ACSF and placed in

the stratum radiatum of area CA1 (LTD experiments) or in the

pyramidal cell body layer of CA3 (epileptiform experiments). Field

potential recordings were acquired using a Multiclamp 700B (Warner

Instruments) ampliﬁer and pClamp 9.2 software. For stimulation-

based experiments an intensity that produced 50−60% of the

maximum was used as the baseline stimulation. mGlu₅compounds

were diluted to the appropriate concentrations in DMSO and applied

to the bath using a perfusion system. Sampled data was analyzed by

averaging three sequential ﬁeld excitatory postsynaptic potentials

(fEPSPs) slopes, followed by normalizing to the average slope

calculated during the predrug period (percent of baseline). For

epileptiform experiments, spontaneous events were measured using

MiniAnalysis (Synaptosoft Inc., NJ), and inter-event interval (IEI) was

normalized to the baseline response.

Comparative Modeling of receptor. The comparative model of

mGlu₅was constructed as described previously.⁴⁷In brief, the X-ray

crystal structure for human β2-adrenergic receptor (PDB ID: 2RH1)⁴⁹

was chosen as a template on the basis of its high sequence similarity to

mGlu₅. A proﬁle to proﬁle sequence alignment of TM regions between

Class C hepta-helical transmembrane regions and Class A crystal

structure templates was directly adopted from Muhlemann et al.,⁵⁰

with the exception of TM2, TM4, and TM7, which were based on the

alignment of CaSR with Class A hepta-helical regions.⁵¹The sequence

alignment was used to thread the amino acid sequence of the mGlu₅

transmembrane helical region onto the backbone coordinates of the

β2-adrenergic receptor. The protein structure prediction software

package Rosetta 3.4⁵²was used to rebuild the loop regions between

the helices using Monte Carlo Metropolis (MCM) fragment

replacement combined with cyclic coordinate descent loop closure

(CCD).^53,54The resulting full sequence models were then subjected to

eight iterative cycles of side chain repacking and gradient minimization

of ϕ, ψ, and χ angles in Rosetta Membrane.⁵⁵Over 5,000 comparative

models of mGlu₅were generated and clustered for structural similarity

using bcl::Cluster.⁵⁶The lowest energy model from the largest cluster

was used for further ligand docking studies.

Fluorescence-Based Calcium Flux Assay (Concentration−

Response Curve (Potency) and Glutamate Fold Shift (Eﬃcacy).

For measurement of compound-evoked increases in intracellular

calcium, HEK293 cells stably expressing rat mGlu₅were plated in 384-

well,⁴⁴poly-D-lysine coated, black-walled, clear-bottomed plates in 20

μL of assay medium (DMEM supplemented with 10% dialyzed fetal

bovine serum, 20 mM HEPES, and 1 mM sodium pyruvate) at a

density of 15,000 cells/well. Cells were grown overnight at 37 °C/5%

CO₂. The next day, medium was removed from the cells, and they

were incubated with 20 μL/well of 1 μM Fluo-4AM (Invitrogen,

Carlsbad, California) prepared as a 2.3 mM stock in dimethyl sulfoxide

(DMSO) and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127

and diluted in calcium assay buﬀer (Hank’s Balanced Salt Solution

(HBSS; Invitrogen, Carlsbad, CA) supplemented with 20 mM HEPES

and 2.5 mM probenecid, pH 7.4) for 50 min at 37 °C. Dye loading

solution was removed and replaced with 20 μL/well of assay buﬀer.

For PAM potency curves, mGlu₅compounds were diluted in calcium

assay buﬀer and added to the cells followed by the addition of an EC₂₀

concentration of glutamate 140 s later and then an EC₈₀concentration

of glutamate 60 s later. For fold-shift experiments either a single

concentration (10 μM) or multiple ﬁxed concentrations (50 nM to 30

μM) of mGlu₅compound or vehicle were added followed by the

addition of a concentration−response curve (CRC) of glutamate 140 s

later. Calcium ﬂux was measured over time as an increase in

ﬂuorescence using a Functional Drug Screening System 6000 (FDSS

6000, Hamamatsu, Japan). The change in relative ﬂuorescence over

basal was calculated before normalization to the maximal response to

glutamate.

Selectivity Screening. mGlu₁. To assess the eﬀect of test

compounds at mGlu₁, Ca²⁺mobilization assays were performed as

described previously.^35,43Brieﬂy HEK293 cells stably expressing rat

mGlu₁were plated in black-walled, clear-bottomed, poly-D-lysine-

coated 384-well plates (Greiner Bio-One, Monroe, NC) in assay

medium at a density of 20,000 cells/well. Calcium ﬂux was measured

over time as an increase in ﬂuorescence of the Ca²⁺indicator dye Fluo-

4AM using a FDSS 6000. Either vehicle or a ﬁxed concentration of test

compound (10 μM, ﬁnal concentration) was added followed 140 s

later by a CRC of glutamate. Data were analyzed as described above.

Group II and Group III mGlus. The functional activity of the

compounds of interest was assessed at the rat group II and III mGlu

receptors by measuring thallium ﬂux through GIRK channels as

previously described.⁶¹Brieﬂy, HEK293-GIRK cells expressing mGlu

subtypes 2, 3, 4, 6, 7, or 8 were plated into 384-well, black-walled,

clear-bottom poly-^D-lysine coated plates at a density of 15,000 cells/

well in assay medium. A single concentration of test compound (10

μM) or vehicle was added followed 140 s later by a CRC of glutamate

(or L-AP4 for mGlu₇) diluted in thallium buﬀer (125 mM NaHCO₃, 1

mM MgSO₄, 1.8 mM CaSO₄, 5 mM glucose, 12 mM thallium sulfate,

10 mM HEPES), and ﬂuorescence was measured using a FDSS 6000.

Data were analyzed as described previously.⁶¹

Radioligand Binding. Membranes were prepared from HEK293A

cells expressing rat mGlu₅. Cells were harvested and pelleted by

centrifugation, resuspended in ice-cold homogenization buﬀer (50 mM

Tris-HCl, 10 mM EDTA, 0.9% NaCl, pH7.4), and homogenized by 3

× 10 s bursts. Cell fractions were separated by centrifugation, and the

resulting pellet resuspended in ice-cold assay buﬀer (50 mM Tris-HCl,

0.9% NaCl, pH7.4). For inhibition binding experiments, membranes

(50 μg/well) were incubated with 7 nM [³H]methoxyPEPy and a

range of concentrations of test ligand for 1 h at room temperature with

shaking in assay buﬀer. Ten micromolar MPEP was used to determine

nonspeciﬁc binding. Assays were terminated by rapid ﬁltration using a

Brandel 96-well plate Harvester and washed three times with ice-cold

assay buﬀer. The next day MicroScint20 was added, and radioactivity

was counted.

Computational Docking of Ligands. Ligands 19, 38t, and 31i

were computationally docked into the comparative model of mGlu₅

using Rosetta Ligand.⁵⁷⁻⁵⁹Each modulator was allowed to sample

docking poses in a 5 Å radius centered at the putative binding site for

allosteric modulation, determined by the residues known to aﬀect

MPEP aﬃnity.⁴⁷Once a binding mode had been determined by the

docking procedure, 10 low energy conformations of the ligand created

by MOE (Molecular Operating Environment, Chemical Computing

Group, Ontario, Canada) were tested within the site. Side-chain

rotamers around the ligand were optimized simultaneously in a Monte

Carlo minimization algorithm. The energy function used during the

docking procedure contains terms for van der Waals attractive and

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Journal of Medicinal Chemistry

Article

repulsive forces, hydrogen bonding, electrostatic interactions between

pairs of amino acids, solvation, and a statistical term derived from the

probability of observing a side-chain conformation from the Protein

Data Bank. For each modulator, over 5,000 docked complexes were

generated and clustered for structural similarity using bcl::Cluster.⁵⁷

The lowest energy binding mode from the two largest clusters for each

modulator, encompassing ligand positions for which the eastern amide

was pointing either toward or away from the extracellular surface, were

used for further analysis.

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ASSOCIATED CONTENT

* Supporting Information

■

S

mGlu selectivity for 38t, progressive glutamate fold-shift

analysis, pK_Band log β calculations, DMPK procedures,

compound characterization, and NMR data for compounds.

This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Tel: 615-936-8407. E-mail: shaun.stau ﬀ er@vanderbilt.edu.

■

Notes

The authors declare no competing ﬁnancial interest.

(12) Marek, G. J.; Behl, B.; Bespalov, A. Y.; Gross, G.; Lee, Y.;

Schoemaker, H. Glutamatergic (N-methyl-^D-aspartate receptor)

hypofrontality in schizophrenia: too little juice or a miswired brain?

Mol. Pharmacol. 2010, 77, 317−326.

ACKNOWLEDGMENTS

■

Funding for E.D.N. is provided by Public Health Service award

T32 GM07347 from the National Institute of General Medical

Studies for the Vanderbilt Medical-Scientist Training Program

(MSTP) and the Paul Calabresi Medical Student Research

Fellowship from the Pharmaceutical Research and Manufac-

turers of America (PhRMA) Foundation. This work was

supported in part by grants from the NIH (NS031373 and

MH062646). Vanderbilt is a member of the MLPCN and

houses the Vanderbilt Specialized Chemistry Center for

Accelerated Probe Development (U54MH084659).

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receptors: Physiology, pharmacology, and disease. Annu. Rev.

Pharmacol. Toxicol. 2010, 50, 295−322.

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G.; Javitt, D. C. Placebo-controlled trial of D-cycloserine added to

conventional neuroleptics, olanzapine, or risperidone in schizophrenia.

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ABBREVIATIONS USED

■

(16) Lindsley, C. W.; Stauffer, S. R. Metabotropic glutamate receptor

5-positive allosteric modulators for the treatment of schizophrenia

(2004−2012). Pharm. Pat. Anal. 2013, 2, 93−108.

mGlu, metabotropic glutamate receptor; PAM, positive

allosteric modulator; NAM, negative allosteric modulator;

SAM, silent allosteric modulator; PCP, phenylcyclidene;

NMDAR, ionotropic N-methyl-^D-aspartate glutamate receptor;

DHPG, dihydroxyphenylglycine; LTD, long-term depression;

MTEP, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine; MPEP, 2-

methyl-6-(phenylethynyl)pyridine; DFB, 3,3′-diﬂuorobenzalda-

zine; CPPHA, N-(4-chloro-2-((1,3-dioxoisoindolin-2-yl)-

methyl)phenyl)-2-hydroxybenzamide; CDPPB, 3-cyano-N-

(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; AHL, amphet-

amine-induced hyperlocomotion; MWM, Morris water maze;

MAM, methylazoxymethanol; EEG, electroencephalogram;

DMTP, delayed-matching-to-position; MLPCN, molecular

libraries probe production centers network

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the metabotropic glutatamate receptor subtype 5 (mGlu₅). ACS Chem.

Neurosci. 2011, 2, 450−470.

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from classical modes of modulation to allosteric mechanisms. ACS

Chem. Biol. 2008, 3, 530−541.

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Allosteric modulation of seven transmembrane spanning receptors:

theory, practice, and opportunities for central nervous system drug

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Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): Discovery of 5-((3-fluorophenyl)ethynyl)- N -(3-methyloxetan-3-yl)picolinamide (ML254)

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