Discovery of 1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-(1 H)-pyridone (JNJ-40411813): A novel positive allosteric modulator of the metabotropic glutamate 2 receptor

Article

pubs.acs.org/jmc

Discovery of 1‑Butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-

(1H)‑pyridone (JNJ-40411813): A Novel Positive Allosteric Modulator

of the Metabotropic Glutamate 2 Receptor

Jose María Cid,*^,†Gary Tresadern,^†Guillaume Duvey,^⊥,○Robert Lutjens,^⊥,○Terry Finn,^⊥

́

̈

Jean-Philippe Rocher,^⊥Sonia Poli,^⊥Juan Antonio Vega,^†Ana Isabel de Lucas,^†Encarnacion Matesanz,^†

́

María Lourdes Linares,^†Jose Ignacio Andres,^†Jesus Alcazar,^†Jose Manuel Alonso,^†

́

Gregor J. Macdonald,^‡Daniel Oehlrich,^‡Hilde Lavreysen,^§Abdelah Ahnaou,^§Wilhelmus Drinkenburg,^§

Claire Mackie,^∥Stefan Pype,^#David Gallacher,^∇and Andres A. Trabanco^†

́

^†Neuroscience Medicinal Chemistry, Janssen Research & Development, Janssen-Cilag S.A., C/Jarama 75, 45007 Toledo, Spain

^‡Neuroscience Medicinal Chemistry, Neuroscience Biology, Discovery ADME/Tox, Molecular Sciences, Project Management

§

∥

⊥

#

Oﬃce, ^∇Translational Sciences, Janssen Research & Development, Turnhoutseweg 30, B-2340, Beerse, Belgium

^○Addex Therapeutics, 12 Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland

ABSTRACT: We previously reported the discovery of 4-aryl-

substituted pyridones with mGlu2 PAM activity starting from the

HTS hit 5. In this article, we describe a diﬀerent exploration from 5

that led to the discovery of a novel subseries of phenylpiperidine-

substituted pyridones. The optimization strategy involved the

introduction of diﬀerent spacers between the pyridone core and

the phenyl ring of 5. The ﬁne tuning of metabolism and hERG

followed by diﬀerentiation of advanced leads that were identiﬁed on the basis of PK proﬁles and in vivo potency converged on

lead compound 36 (JNJ-40411813). Full in vitro and in vivo proﬁles indicate that 36 displayed an optimal interplay between

potency, selectivity, favorable ADMET/PK and cardiovascular safety proﬁle, and central EEG activity. Compound 36 has been

investigated in the clinic for schizophrenia and anxious depression disorders.

with the NMDA receptor antagonist phencyclidine (PCP) and

mixed mGlu2/3 receptor agonists provided the ﬁrst insights

that revealed that mGlu2 receptor activation may represent a

novel target for the treatment of schizophrenia.^8,9In its ﬁrst

clinical trial, (1R,4S,5S,6S)-2-thiabicyclo[3.1.0]hexane-4,6-di-

carboxylic acid 4-[(2S)-2-amino-4-(methylthio)-1-oxobutyl]-

amino-2,2-dioxide monohydrate 2 (LY2140023), a prodrug of

(−)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-

4,6-dicarboxylic acid 3 (LY404039), demonstrated improve-

ments in positive and negative symptoms compared to placebo

in schizophrenic patients.¹⁰However, these positive data could

not be conﬁrmed in follow-up studies,^11,12and very recently, Eli

Lilly and Co. announced the decision to stop the phase III

development of 2 for the treatment of patients suﬀering from

schizophrenia based on eﬃcacy results.¹³

On the other hand, the anxiolytic potential of 1 was

conﬁrmed in healthy human volunteers, showing activity in

fear-potentiated startle and panic induction after CO₂

challenge.¹⁴

Despite the interesting ﬁndings, there remain hurdles in

developing clinically useful agonists. First, all agonists identiﬁed

so far lack mGlu2 receptor subtype selectivity and also act on

INTRODUCTION

■

Glutamate is the major excitatory neurotransmitter in the

central nervous system (CNS) of vertebrates and elicits and

modulates synaptic responses in the CNS by activating

ionotropic glutamate (iGlu) receptors and metabotropic

glutamate (mGlu) receptors.¹To date, eight mGlu receptor

subtypes and multiple splice variants have been cloned and

classiﬁed into three groups based on sequence homology,

pharmacological proﬁle, and preferential signal transduction

pathways:^2,3group I (mGlu1 and mGlu5), group II (mGlu2

and mGlu3), and group III mGlu receptors (mGlu4, mGlu6,

mGlu7, and mGlu8). Pharmaceutical companies and academic

groups have primarily focused on the pharmacology and

development of ligands acting at group I (mGlu1 and 5) and II

(mGlu2 and 3) receptors, whereas group III (mGlu4, 6, 7, and

8) receptors have started to attract more attention recently.^4,5

Group II mGlu receptors, which are located presynaptically in

the periphery of the synapse, reduce glutamatergic transmission

in brain regions such as the prefrontal cortex and hippo-

campus,⁶where excessive glutamatergic transmission has been

implicated in the pathophysiology of anxiety and schizophrenia.

Mixed mGlu2/3 orthosteric agonists such as (+)-2-

aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1

(LY354740) have shown activity in a wide range of preclinical

animal models of anxiety and schizophrenia.⁷Animal studies

Received: March 28, 2014

Published: July 17, 2014

6495

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Figure 1. Strategies followed for the exploration of HTS hit 5.

a

Scheme 1. Preparation of 4-Bromopyridone Building Blocks 41a−g, 46, and 47a−c

a

Reagents and conditions: (a) K₂CO₃, MeCN, 100 °C, 16 h; (b) NaOH, H₂O, reﬂux, 18 h; (c) POBr₃, DMF, 110 °C, 1−3 h; (d) NIS, AcOH, rt, 1

h; (e) FSO₂CF₂CO₂Me, CuI, DMF, 100 °C, 5 h; (f) H₂, Pd/C, EtOH, rt, 2 h.

the mGlu3 receptor. It has been shown that the mGlu2, and not

the mGlu3 receptor, mediates the actions of the mGlu2/3

receptor agonists (1S,2R,5R,6R)-2-amino-4-oxabicyclo[3.1.0]-

hexane-2,6-dicarboxylic acid (LY379268) and 3 in mouse

models predictive of antipsychotic activity.^15,16Second, the

development of tolerance by group II mGlu receptor agonists

has been observed in several rodent behavioral models.¹⁷

Receptor activation with positive allosteric modulators

(PAMs), which act via alternative allosteric site(s), oﬀers

several advantages.¹⁸Allosteric molecules are likely to be less

polar and to have improved CNS penetration, as they do not

require amino-acid functional groups for activity at the

orthosteric site. Furthermore, the less conserved nature of

allosteric site(s) can allow for improved mGlu receptor subtype

selectivity. Finally, allosteric modulators exert their eﬀects only

in the presence of endogenous glutamate, so they may be less

prone to cause receptor desensitization,^19,20and they respond

to physiological glutamate ﬂuctuations. Given these potential

6496

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

advantages, the number of reported mGlu2 receptor-selective

PAMs has increased in recent years.^21,22Some of these

compounds have been extensively characterized in a number

of animal models, providing preclinical proof of concept that

PAMs can mimic the eﬀects of orthosteric agonists.²³⁻³²

To date, two mGlu2 PAM molecules have advanced into

clinical trials: 4 (AZD8529) from AstraZeneca AB (structure

undisclosed) and 36³³⁻³⁵(also known as ADX71149 in Figure

1) from Janssen Pharmaceuticals and Addex Therapeutics. On

January 2011, the originating company announced the

discontinuation of 4 phase IIa POC study in schizophrenic

patients due to lack of eﬃcacy.^36,37More promisingly, it has

been disclosed that, in an exploratory phase IIa study in

schizophrenia, 36 proved to meet the primary objectives of

safety and tolerability. Moreover, patients treated with

antipsychotics who experience residual negative symptoms

were identiﬁed as the subgroup of patients who may potentially

beneﬁt from add-on treatment with 36, although this is yet to

be established in a formal proof-of-concept study.^38,39

iodopyridone 44, which was subsequently converted into the

corresponding 3-triﬂuoromethylpyridone 45 by treatment with

the nucleophilic triﬂuoromethyl-copper complex generated

from ﬂuorosulfonyldiﬂuoroacetate. Finally, cleavage of the

benzyl ether in intermediates 43a−c and 45 followed by

reaction with POBr₃under thermal conditions yielded the

target intermediates 47a−c and 46, respectively.

The synthesis of the ﬁnal compounds 8−21, 26−29, 31, 32,

and 34 is illustrated in Scheme 2. Treatment of bromopyridone

Scheme 2. Synthesis of the Final Compounds 8−21, 26−29,

a

31, 32, and 34

Additionally, a phase II POC study with 36 as adjunctive

therapy in patients with major depressive disorder (MDD) with

signiﬁcant anxiety symptoms has been completed.⁴⁰Overall, 36

was well-tolerated, and treatment emergent adverse events

reported were similar to those seen in previous clinical studies.

On the basis of a preliminary analysis of the primary eﬃcacy

end point, the 6-Item Hamilton Anxiety Subscale (HAM-A6),

36 did not meet the criterion for eﬃcacy signal detection versus

placebo. Despite a lack of signal on the primary outcome

measure, treatment with 36 showed eﬃcacy signals on several

anxiety measures (HDRS17 anxiety somatization factor, IDS-

C30 anxiety subscale) and on all depression measures

(HDRS17, HAM-D6, and IDS-C30).⁴¹Although eﬃcacy

signals were evident, the data overall does not support the

further development of 36 in anxious depression. Further

exploration of 36 in other indications remains of potential

interest.

a

Reagents and conditions: (a) amine, 2-(2′-di-tert-butylphosphine)-

biphenylpalladium(II) acetate, K₃PO₄, 1,4-dioxane, 85 °C, 2−12 h; (b)

amine, DIPEA, CH₃CN, 140−170 °C, 10−35 min, microwave; (c)

amine, Pd(OAc)₂, BINAP, BuONa, toluene, 100 °C, 16 h.

t

intermediates 41a−g and 46 with an array of commercially

available amines either under palladium-catalyzed cross-

coupling reaction or by nucleophilic substitution under

microwave heating conditions aﬀorded the target compounds

8−21, 26−29, 31, 32, and 34, whose chemical structures are

shown in Tables 1−3.

We have recently reported on a series of pyridones derived

from the optimization of the HTS hit 5 that led to the

identiﬁcation of compound 6.^29,42Compound 6 combined

moderate in vitro activity, good pharmacokinetic proﬁle, and

robust in vivo activity. The optimization strategy involved the

introduction of a variety of substituents in the para position of

the phenyl ring in hit 5 (Figure 1; mapping). Herein, we report

on a new optimization strategy consisting of the exploration of

diﬀerent spacers (Figure 1; linker) between the pyridone core

and the aromatic ring in hit 5.^43,44This strategy resulted in the

identiﬁcation of our clinical lead, 36, whose identiﬁcation path,

SAR, and a general overview of its pharmacological proﬁle are

reported herein.

The 4-substituted-1-cyclopropylmethyl-3-cyano-2-pyridones

22−25 and 30 were prepared following the synthesis sequence

shown in Scheme 3. Nucleophilic substitution of the 4-

bromopyridone 41a with the corresponding amines led to

intermediates 48a−d and 49. Catalytic hydrogenation using

Pd/C as the catalyst of compounds 48a−b and 48d yielded the

target compounds 22, 23, and 30. For intermediate 48c, the

hydrogenation reaction was accomplished by using PtO₂as

catalyst, yielding compound 25. Treatment of intermediate 49

with triethylsilane in TFA led to the ﬁnal compound 24.

The synthesis of the 3-chloropyridone derivatives 33, 35, and

36 was achieved in two steps by Buchwald coupling reaction of

the corresponding amines with intermediates 47a−c followed

by chlorination of the resulting pyridones 50a−c with NCS in

DCM (Scheme 4).

Finally, compounds 6 and 7 were prepared following the

reaction pathway depicted in Scheme 5. Sonogashira coupling

reaction of intermediate 41d with phenylacetylene gave

derivative 51, which was converted into the target compound

6 by catalytic hydrogenation of the triple bond. Alkylation of

40d with benzyl bromide under microwave irradiation aﬀorded

compound 7.

CHEMISTRY

■

The synthesis of the 1,3,4-trisubstituted 2-pyridone derivatives

6−36^32,42is outlined in Schemes 1−5. The key bromopyridone

intermediates, 41a−g, 46, and 47a−c, were prepared as shown

in Scheme 1. Treatment of 2-hydroxypyridine derivative 37

with the required alkyl halides 38a−g aﬀorded the N-alkylated

2-pyridones 39a−g. Subsequent cleavage of the methyl ether

followed by treatment of the resulting hydroxypyridones 40a−g

with POBr₃yielded the intermediates 41a−g. In a similar way,

intermediates 43a−c were obtained by alkylation of the 2-

hydroxy-4-benzyloxypyridine 42 with the corresponding alkyl

halides 38a−c. Iodination of compound 43a (R¹= cyclo-

propylmethyl) with N-iodosuccinimide (NIS) aﬀorded the 3-

RESULTS AND DISCUSSION

■

Guided by our previously reported overlay model for mGlu2

receptor PAM chemotypes,³⁰we expanded the SAR around the

1,4-pyridone series by focusing on introducing a spacer (X)

6497

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

a

Scheme 3. Synthesis of the Final Compounds 22−25 and 30

a

Reagents and conditions: (a) amine, DIPEA, CH₃CN, 140−170 °C, 10−35 min, microwave; (b) H₂, Pd/C, MeOH or AcOEt, rt, 4−16 h; (c) H₂,

PtO₂, AcOH, MeOH, rt, 72 h; (d) Et₃SiH, TFA, rt, 24 h.

a

Scheme 4. Synthesis of the Final Compounds 33, 35, and 36

a

t

Reagents and conditions: (a) amine, Pd(OAc)₂, BINAP, BuONa, toluene, 100 °C, 16 h; (b) NCS, DCM, rt, 10−20 min.

a

Scheme 5. Synthesis of the Final Compounds 6 and 7

a

Reagents and conditions: (a) POBr₃, DMF, 110 °C, 1−3 h; (b) phenylacetylene, PdCl₂(PPh₃)₂, Et₃N, CuI, PPh₃, THF, 90 °C, 10 h; (c) H₂, Pd/C,

MeOH, rt, 3 h; (d) BnBr, Cs₂CO₃, CH₃CN/DMF, 130 °C, 20 min, microwave.

between the pyridone core and the phenyl ring (Figure 1).

Representative compounds illustrating the new SAR generated

along with mGlu2 receptor functional activity and metabolic

stability data are shown in Table 1. Introduction of ethylene

and methoxy linkers (6 and 7) led to a 13- and 3-fold increase

in potency (EC₅₀⁴⁵), respectively, when compared to that of 5.

However, that improvement was not observed in compound 8

that has methylamino as the linker. Conformational restriction

of compound 8 by means of cyclization of the nitrogen spacer

onto the phenyl ring, as in isoquinoline derivative 9, resulted in

a remarkable 12-fold potency increase (9, EC₅₀= 871 nM).

Further modiﬁcation around the isoquinoline group in 9 while

keeping the piperidine fragment led to compounds 10 and 11

bearing a phenylpiperidine substituent. To our delight, the 4-

phenylpiperidine derivative 10 exhibited a further boost in

potency (EC₅₀= 102 nM), whereas the regioisomer 11,

featuring 3-phenyl substitution on the piperidine, was markedly

less active (EC₅₀= 3311 nM).

The distal aromatic group in 10 seemed to be essential for

achieving good activity, as the truncated analogue 12 showed

barely measurable functional PAM activity of the mGlu2

receptor. Both ﬁndings suggest the importance of a lipophilic

group linearly oriented by a spacer from the pyridone core.

Replacement of the piperidine spacer by piperazine (13) led to

a ∼6-fold reduction in potency compared to that of the

corresponding analogue 10. Lastly, introducing a protonatable

6498

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Table 1. Functional Activity and Metabolic Stability Data of Representative mGlu2 Receptor PAMs 5−15

a

b

c

Values represent the mean for at least two experiments. CI = conﬁdence interval. HLM and RLM data refer to the percent of compound

d

metabolized after incubation with microsomes for 15 min at a 5 μM concentration. CI and SD not determined.

partially basic nitrogen on the 4-phenylpiperazine template (14

and 15) was detrimental for the activity (EC₅₀values of 3090

and 4677 nM respectively).

Alongside the assessment of activity, compounds 6−15 were

assessed for in vitro P450-mediated oxidative stability in human

and rat liver microsomes (HLM and RLM, respectively). The

results shown in Table 1 indicate that most analogues suﬀered

from extensive metabolic degradation in RLM, with somewhat

lower metabolic turnover in HLM. Interestingly analogue 10,

despite having high degradation in RLM (97% metabolized),

was relatively stable in HLM (28% metabolized). Metabolic

proﬁling studies on 10 suggested that the turnover in RLM

could be attributed to oxidation of the N-isopentyl chain.⁴⁶

6499

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Table 2. Functional Activity, Metabolic Stability Data, and Calculated LogP of Representative 4-Phenylpiperidine-Substituted

mGlu2 Receptor PAMs 10 and 16−21

a

b

c

Values represent the mean for at least two experiments. CI = conﬁdence interval. HLM and RLM data refer to the percent of compound

d

metabolized after 15 min at 5 μM concentration. cLogP calculated with Biobyte software.

Given the notable increase in potency observed upon

incorporation of the 4-phenylpiperidine substituent, we turned

our attention to address the high metabolic turnover of 10 in

RLM. The exploration began by focusing on replacing the

isopentyl chain in 10 by alternative aliphatic groups that would

decrease the overall lipophilicity of the molecule (see calculated

LogP in Table 2, compounds 16−21). As seen in Table 2, some

isopentyl replacements (17 and 19−21) led to a slight decrease

in activity. Some others, such as n-butyl (16), showed

comparable potency, and cyclobutylmethyl (18) was the the

only replacement that showed superior potency (EC₅₀= 55

nM) to that of 10. Unfortunately, the metabolic stability in

RLM of 18 did not improve signiﬁcantly. Compound 19,

containing a cyclopropylmethyl chain and having the lowest

lipophilicity (cLogP = 3.16), presented the best balance of

potency and metabolism (EC₅₀= 151 nM and HLM and RLM

25 and 44% metabolized, respectively). Detailed character-

ization of 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-

1-yl)-pyridine-2(1H)-one 19 (JNJ-40068782) has been recently

published.⁴⁷

With the more optimal N-cyclopropylmethyl substituent

identiﬁed, a sequential SAR expansion focusing ﬁrst on the

eﬀect of substitution on the phenyl ring of 19 and second on

alternative groups to the cyano substituent at the 3 position of

the pyridone core was conducted. A representative set of

analogues to exemplify the SAR trends identiﬁed is shown in

Table 3. Overall, both electron-withdrawing and -donating

substituents on the phenyl ring (22−32) were well-tolerated

for potency, and the low metabolic turnover seen for 19 was

retained for most examples. The ortho and para ﬂuoro

substituent in compounds 22 and 24 led to a 2-fold potency

increase, whereas the meta ﬂuoro substituent (23) retained the

activity observed with the nonsubstituted phenyl derivative 19.

When the ﬂuorine atom in compounds 22 and 24 was replaced

with the more lipophilic chlorine (25 and 26), an additional 2-

fold increase in potency was obtained. The triﬂuoromethyl

analogue 27 showed a consistent potency increase, with an

EC₅₀of 46 nM.

In the case of electron-donating substituents, such as

methoxy, ortho substitution was preferred versus para

substitution and thus compound 28 (EC₅₀= 35 nM) was

found to be ∼3-fold more active than 29 (EC₅₀= 180 nM).

Unfortunately, methoxy-substituted compounds were prone to

higher metabolic degradation in both HLM and RLM. Some

additive eﬀects were observed while mapping the phenyl ring

with two ﬂuorine substituents (30−32), with compound 31,

featuring the bis-ortho substitution, showing the better activity

(EC₅₀= 31 nM). Finally, SAR from previously reported series³¹

prompted us to study the eﬀect of replacing the cyano group by

chlorine and triﬂuoromethyl substituents. Relative to 19,

compound 33, bearing a chlorine substituent, displayed slightly

lower potency (EC₅₀= 170 nM) and comparable metabolic

stability. The triﬂuoromethyl-substituted analogue 34 was

found to be ∼2-fold less potent than 19 (EC₅₀= 251 nM)

and presented a pronounced metabolic turnover in RLM. In

contrast to previous SAR trends,³²the increased lipohilicity of

33 and 34 relative to 19 did not result in more potent

compounds.

A set of compounds (19−24, 26−28, and 31−33) was then

investigated for their ability to inhibit the hERG channel in an

6500

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Table 3. Functional Activity, Metabolic Stability Data, Calculated LogP, and hERG Inhibition Data of Representative Set of Aryl

Modiﬁed mGlu2 Receptor PAMs 19 and 22−34

6501

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Table 3. continued

a

b

c

Values represent the mean for at least two experiments. CI = conﬁdence interval. HLM and RLM data refer to the percent of compound

d

e

metabolized after 15 min at 5 μM concentration. cLogP calculated with Biobyte software. Experiments were performed using HEK293 cells stably

transfected with the hERG potassium channel. Whole-cell currents were recorded with an automated patch-clamp system (PatchXpress 7000A).

Three concentrations of compound were tested (0.1, 0.3, and 3 μM; n = 3) and compared to vehicle (0.1% DMSO; n = 3). Data are presented as the

percent inhibition at the highest concentration tested (3 μM).

Table 4. Functional Activity, Metabolic Stability Data, Calculated LogP, and hERG Inhibition Data of Representative Set of

Aryl-Modiﬁed mGlu2 Receptor PAMs 33, 35, and 36

a

b

c

Values represent the mean for at least two experiments. CI = conﬁdence interval. HLM and RLM data refer to the percent of compound

d

e

metabolized after 15 min at 5 μM concentration. cLogP calculated with Biobyte software. Experiments were performed using HEK293 cells stably

transfected with the hERG potassium channel. Whole-cell currents were recorded with an automated patch-clamp system (PatchXpress 7000A).

Three concentrations of compound were tested (0.1, 0.3, and 3 μM; n = 3) and compared to vehicle (0.1% DMSO; n = 3). Data are presented as the

percent inhibition at the highest concentration tested (3 μM).

a

Table 5. PK Data for Compounds 19, 33, 35, and 36 after Oral and i.v. Administration in Male Sprague−Dawley Rats and in

Vivo Activity in the swEEG Model in Rats

b

c

d

compd

Cl (mL, min⁻¹kg⁻¹

)

T_1/2(h)

T_max(h)

AUC_0−inf(ng h/mL)

C_max(ng/mL)

F (%)

B/P

swEEG LAD/LAC

19

33

35

36

18 8.0

24 4.0

13 4.0

23 1.0

2.1 0.1

3.5 0.3

2.6 0.5

2.3 0.5

0.5 0.0

0.7 0.3

0.5 0.0

4350 895

2312 516

3455 734

2250 417

1407 391

986 154

1357 214

938 300

51

34

25

31

0.3

0.9

0.8

1

3/420

>10/>986

3/407

3/281

a

b

Study in male Sprague−Dawley rats dosed at 10 mg/kg p.o. and 2.5 mg/kg iv, formulated in 20% HP-β-CD + HCl solution at pH 4. Values are the

c

mean of three animals SEM. Ratios calculated after 1 h of a single dose at 10 mg/kg sc in 1 mg/mL in 20% HP-β-CD + HCl solution at pH 4 in

the Swiss mouse. Compound given orally. LAD stands for lowest active dose expressed in mg/kg. LAC stands for lowest active concentration in

d

plasma, at active doses estimated on the basis of C_maxexpressed in ng/mL.

electrophysiology patch-clamp study. The inhibition of the

hERG channel is an indicator for potential QTc prolongation

and potential for serious cardiac arrhythmias. Unfortunately,

most of the compounds inhibited the hERG channel in >55%

at the highest concentration tested (3 μM). Thus, in the case of

3-cyanopyridones, the more lipohilic molecules (26, 27, and

32) showed higher hERG inhibitory activity. Interestingly,

despite its high lipophilicity (cLogP = 4.25), the 3-

chloropyridone derivative 33 exhibited the lowest hERG

inhibition value (51%). However, more detailed in vivo

cardiovascular safety studies are required to put the hERG

inhibition data into a meaningful context. On the basis of the

described proﬁle, compounds 18 and 33 were progressed to in

vivo pharmacokinetics studies to further understand the

potential of the series.

Thus, 19 and 33 were assessed for their ability to cross the

blood−brain barrier (BBB) after a subcutaneous dose of 10

mg/kg in mice. As shown in Table 5, compound 33 displayed a

higher brain-to-plasma (B/P) ratio of 0.9 vs 0.3 for 19. Given

the requirement for adequate BBB penetration, further

exploration of 3-cyanopyridones was halted while we instead

sought to ﬁne tune the hERG interaction of the 3-

chloropyridone lead 33. Two compounds with diﬀerent alkyl

chains attached to the pyridone nitrogen were synthesized (35

and 36), and their data are shown in Table 4. Thus, compounds

35 and 36, having an iso-butyl or butyl chain, respectively,

showed comparable potency (35: EC₅₀= 195 nM and 36: EC₅₀

= 147 nM) and metabolic stability to that of 33. More

interestingly, both compounds had reduced hERG inhibition

(35: 24%; 36: 43% at 3 μM).

6502

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Figure 2. Eﬀect of 36 or vehicle (20% CD + 1HCl) on sleep−wake organization in rats during 20 consecutive hours and after a single oral dose.

Mean percentage of occurrence per 30 min period is indicated for each sleep−wake state. Shaded area indicates dark period. Small bar charts indicate

amounts of vigilance states in minutes ( SEM, n = 8 for each group) during the ﬁrst 4 h postadministration. Dots on the lines of the graph indicate p

< 0.05 versus vehicle (Wilcoxon−Mann−Whitney rank sum); * indicates p < 0.05: Wilcoxon−Mann−Whitney rank sum tests compared to vehicle

values.

Because of their overall favorable attributes, compounds 35

and 36 along with 19 and 33 were investigated further to

ascertain their drug-like properties as well as in vivo activity.

Overall, the four compounds exhibited relatively comparable rat

PK data (Table 5). After intravenous (i.v.) administration, all

compounds presented low-to-moderate plasma clearance (Cl),

with compounds 33 and 36 having higher values. When dosed

orally (p.o.), plasma concentrations declined in comparable

magnitude for all of the compounds with relatively short half-

lives (T_1/2between 2.1 and 3.5 h). The mean maximum plasma

concentrations (C_max) were reached at 0.5−0.7 h postdose and

were in the range of ∼900 to ∼1400 ng h/mL, indicating a fast

absorption rate. Plasma exposure (AUC_0−inf) was relatively

good for all compounds, ranging between ∼2000 and ∼4350 ng

h/mL, and this resulted in moderate absolute oral bioavail-

abilities (F %) between 25 and 51%. As expected, B/P ratios

observed of compounds 35 and 36 were comparable to that of

compound 33, previously discussed.

As in previously reported series, the in vivo pharmacological

activity of the compounds was investigated in the sleep−wake

EEG (swEEG) paradigm.^29,31,32It has been previously shown

that selective activation or positive allosteric modulation of the

mGlu2 receptor in vivo aﬀects the sleep−wake architecture in

rats and mice.^48,49After acute p.o. administration of a 3 mg/kg

dose, compounds 19, 35, and 36, but not 33, signiﬁcantly

decreased the amount of REM sleep in rats during the ﬁrst 4 h

without clear eﬀects on the other sleep−wake stages (see 36 in

Figure 2, bottom right panel, as a representative example). The

active dose of 3 mg/kg p.o. corresponded to plasma

concentrations of ∼420, 407, and 281 ng/mL for compounds

19, 35, and 36, respectively.

Compound 36 showed the best overall in vitro and in vivo

proﬁle among all of the analogues prepared, which prompted us

to select it for an extensive characterization. A general proﬁle of

36 is presented in Table 6. Compound 36 did not have

signiﬁcant activity against other mGlu receptor subtypes⁵⁰with

≥30-fold selectivity, exhibiting also ≥100-fold selectivity versus

a panel of diverse targets⁵¹with only mild antagonist eﬀect for

the 5HT_2Areceptor (K_b= 1.1 μM). Further characterization of

the 5HT_2Ainteraction will be described in more detail in a

separate publication.

Radioligand binding studies were performed using [³H]19 as

an allosteric mGlu2 receptor radioligand⁴⁷and conﬁrmed that

36 binds to an allosteric site with high aﬃnity (IC₅₀= 68 nM)

Compound 36 showed low aqueous solubility (<0.001 mg/

mL), which was not inﬂuenced by the pH; nevertheless, the

solubility of 36 increased in complexation with cyclodextrins

and thus with 20% HP-β-CD it reached 2.20 mg/mL at pH

4.16. Caco-2 data showed high permeability with no indication

for an eﬄux mechanism such as P-gp. Elimination half-lives

were moderate across diﬀerent species. Volume of distribution

across species was equal to or a little higher than total body

water (0.8−1.8 L/kg), indicative of compound distribution

outside of the plasma. Oral bioavailability was low to moderate

across mouse, rat, dog and monkey (6−31%). The predicted in

vitro hepatic clearance for the rat, dog, monkey, and human

suggested that phase 1 metabolism via the CYP450 enzymes

contributes largely to the in vivo plasma clearance of the

6503

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

of eﬀects but rather used as a signal generation study, 36 met

the primary objectives of safety and tolerability and also

demonstrated an eﬀect in patients with residual negative

symptoms. Further evaluation of this signal in a formal proof-

of-concept study is warranted in patients with residual negative

symptoms. In contrast, in patients with MDD with signiﬁcant

anxiety symptoms, 36 failed to meet the criterion for eﬃcacy

signal. Despite this lack of signal on the primary outcome

measure, treatment with 36 showed mixed and modest eﬃcacy

signals on several anxiety measures and on all depression

measures.

The data collected from the clinical trials with 36

undoubtedly provides valuable information and should help

to better assess the potential of the mGlu2 receptor as a viable

target for the treatment of CNS disorders. To that end, ﬁnding

the right patient population that may be beneﬁt from treatment

with an mGlu2 modulator is still a challenge that must be

addressed.

Table 6. Overview of 36

physicochemical

properties

MW = 344

Experimental pK_a< 2

Experimental logP = 4.6 at pH 6.8

mGlu2 PAM EC₅₀= 147 42 nM; E_max= 273 32%

in vitro

pharmacology

mGlu2 PAM binding IC₅₀∼ 68 29 nM

Selectivity (mGlu 1.3−8; CEREP) ≥ 30-fold vs

mGlu2 PAM but 5HT_2A(∼10-fold)

ADME proﬁle

Solubility <0.001 mg/mL in water and 2.2 mg/mL

20% HP-β-CD at pH 4

Plasma Protein Binding (%) = 99.5 (h), 99.6 (r),

99.2 (ms), 99.4 (dog)

Permeability (Caco-2 monolayers): High

EXPERIMENTAL SECTION

(AB/BA P_app× 10 cm s⁻¹= 12/6)

■

Chemistry. Unless otherwise noted, all reagents and solvents were

obtained from commercial suppliers and used without further

puriﬁcation. Synthesis of intermediates 39a,c,d,e, 40a,c,d,e, and

41a,c,d,e and ﬁnal compound 5 were previously described.²⁸Thin-

layer chromatography (TLC) was carried out on silica gel 60 F254

plates (Merck). Flash column chromatography was performed on silica

gel (particle size 60 Å, mesh = 230−400 (Merck)) using standard

techniques. Microwave-assisted reactions were performed in a single-

mode reactor, Biotage Initiator Sixty microwave reactor (Biotage), or

in a multimode reactor, MicroSYNTH Labstation (Milestone, Inc.).

Nuclear magnetic resonance (NMR) spectra were recorded with either

a Bruker DPX-400 or a Bruker AV-500 spectrometer (broaduker AG)

with standard pulse sequences, operating at 400 and 500 MHz,

respectively, using CDCl₃and DMSO-d₆as solvents. Chemical shifts

(δ) are reported in parts per million (ppm) downﬁeld from

tetramethylsilane (δ = 0). Coupling constants are reported in Hertz.

Splitting patterns are deﬁned by s (singlet), d (doublet), dd (double

doublet), t (triplet), q (quartet), quin (quintet), sex (sextet), sep

(septet), or m (multiplet). Liquid chromatography combined with

mass spectrometry (LC−MS) was performed on a HP 1100 HPLC

system (Agilent Technologies) comprising a quaternary or binary

pump with degasser, an autosampler, a column oven, a diode-array

detector (DAD), and a column. Flow from the column was split to a

MS spectrometer. The MS detector was conﬁgured either with an

electrospray ionization source or an ESCI dual ionization source

(electrospray combined with atmospheric pressure chemical ioniza-

tion). Nitrogen was used as the nebulizer gas. Data acquisition was

performed with MassLynx-Openlynx software or with Chemsation-

Agilent Data Browser software. Gas chromatography combined with

mass spectrometry (GC−MS) was performed using a 6890 series gas

chromatograph (Agilent Technologies) system comprising a 7683

series injector and autosampler, a column oven, and a J&W HP-5MS

coupled to a 5973N MSD mass selective detector (single quadrupole,

Agilent Technologies). The MS detector was conﬁgured with an

electronic impact ionization source/chemical ionization source (EI/

CI). EI low-resolution mass spectra were acquired by scanning from 50

to 550 at a rate of 14.29 scans/s. The source temperature was

maintained at 230 °C. Helium was used as the nebulizer gas. Data

acquisition was performed with Chemstation-Open Action software.

Melting point (mp) values are peak values obtained with experimental

uncertainties that are commonly associated with this analytical method

and were determined in open capillary tubes on a Mettler FP62

apparatus with a temperature gradient of 10 °C/min. Maximum

temperature was 300 °C.

CYP450 inh. = 1A2, 2D6, 3A4 (>18 μM),

2C9 (6 μM), 2C19 (7 μM)

rat PK: 2.5 mg/kg i.v. Cl = 23 mL/min/kg; V_dss= 2.3 L/kg; T_max= 0.5 h;

; 10 mg/kg p.o.

T_1/2= 2.3 h

AUC_0−inf= 2250 ng h/mL; C_max= 938 ng/mL;

F (%) = 31; B/P ratio = 1

dog PK: 1 mg/kg i.v.; Cl = 23 mL/min/kg; V_dss= 1.8 L/kg; T_max= 0.5 h;

2.5 mg/kg p.o.

T_1/2= 3.7 h

AUC_0−inf= 743 ng.h/mL; C_max= 373 ng/mL;

F (%) = 20

swEEG rat p.o.

CV safety

LAD = 3 mg/kg (free plasma conc. ∼ 0.9 ng/mL)

a

hERG PC ; rabbit Purkinje ﬁber; anesthetized guinea-

b

pig, and anesthetized dog; all margin ≥38

c

genotoxicity

Ames TA98 : clean up to 125 mg/mL

d

In vitro Micronucleus : clean up to 150 μg/mL

a

Experiments were performed using HEK293 cells stably transfected

with the hERG potassium channel. Whole-cell currents were recorded

with an automated patch-clamp system (PatchXpress7000A). Three

concentrations of compound were tested (0.1, 0.3, and 3 μM; n = 3)

and compared with vehicle (0.1% DMSO; n = 3). Data are presented

as the percent inhibition at the highest concentration tested (3 μM).

b

Based on eﬃcacy free concentration in sw-EEG model (∼0.9 ng/

c

mL). In vitro bacterial reverse mutation assay in Salmonella

typhimurium TA98. Study conducted in human lymphoblastoid

d

TK6 cell line in the presence or absence of metabolic activation (rat

liver S9).

compound. The calculated hepatic clearance corresponded to

∼45−55% of the hepatic blood ﬂow across the species.

Compound 36 does not inhibit CYP450 enzymes signiﬁcantly

and is predominantly metabolized by CYP 3A4. Plasma protein

binding was consistently high across the species. Compound 36

showed good safety margins in various cardiovascular studies

and no indication of genotoxicity or cytotoxicity.

CONCLUSIONS

■

Further optimization of the 1,4-disubstituted cyanopyridone

series of mGlu2 receptor PAMs was performed. Strategic

exploration of the SAR led to a novel generation of pyridones

with a more optimal balance between potency, PK, and CV

safety. Our lead optimization eﬀorts have led to the discovery of

36 as a potent, selective mGlu2 receptor PAM, which is orally

bioavailable and safe. In an exploratory phase 2a study in

schizophrenia, not powered to determine statistical signiﬁcance

Purities of all new compounds were determined by analytical

reverse-phase RP-HPLC using the area percentage method on the UV

6504

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

trace recorded at a wavelength of 254 nm and were found to have

≥95% purity, unless otherwise speciﬁed.

phenylpiperidine (42.5 mg, 0.26 mmol) and following the procedure

described for 8, compound 11 was obtained as a white gummy solid

1

1-Isopentyl-2-oxo-4-phenethyl-1,2-dihydropyridine-3-car-

bonitrile (6). A mixture of 1-isopentyl-2-oxo-4-(2-phenylethynyl)-1,2-

dihydropyridine-3-carbonitrile 39d (45 mg, 0.2 mmol) and 10% Pd/C

(0.01 g, catalyst) in MeOH (5 mL) was hydrogenated for 3 h at room

temperature. The catalyst was ﬁltered oﬀ under nitrogen atmosphere,

and the ﬁltrate solvent was evaporated. The crude was puriﬁed by ﬂash

column chromatography (silica gel, MeOH in CH₂Cl₂, 0:100 to 4:96).

Desired fractions were collected, and the solvent was evaporated. The

residue was further puriﬁed by preparative reverse phase HPLC (C18

XSelect 19 × 100 mm 5 μm, 20−100% CH₃CN/0.1% NH₄CO₃H/

(13 mg, 30%). H NMR (400 MHz, CDCl₃) δ 0.95 (d, J = 6.4 Hz,

6H), 1.53−1.67 (m, 3H), 1.76−1.88 (m, 2H), 1.90−2.01 (m, 1H),

2.06−2.17 (m, 1H), 2.91 (tt, J = 11.4, 3.7 Hz, 1H), 3.03−3.18 (m,

2H), 3.80−3.87 (m, 2H), 4.12−4.21 (m, 1H), 4.24−4.33 (m, 1H),

5.85 (d, J = 7.9 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 7.22−7.37 (m, 5H).

LC−MS m/z 350 [M + H]⁺.

Isopentyl-2-oxo-4-(1-piperidyl)pyridine-3-carbonitrile (12).

Starting from 41d (110 mg, 0.60 mmol) and piperidine hydrobromide

(119.5 mg, 0.72 mmol) and following the procedure described for 8,

compound 12 was obtained as a white solid (125 mg, 82%). mp 75.9

1

NH₄OH pH 9 solution in water) to yield 6 (1.6 mg, 4%). H NMR

°C. H NMR (500 MHz, CDCl₃) δ 0.94 (d, J = 6.4 Hz, 6H), 1.53−

(500 MHz, CDCl₃) δ 0.96 (d, J = 6.4 Hz, 6H), 1.58−1.65 (m, 3H),

2.92−3.04 (m, 4H), 3.92−3.97 (m, 2H), 6.04 (d, J = 6.9 Hz, 1H),

7.16−7.24 (m, 3H), 7.27−7.32 (m, 2H), 7.35 (d, J = 6.9 Hz, 1H).

LC−MS m/z 295 [M + H]⁺.

1.66 (m, 3H), 1.71 (br s, 6H), 3.58−3.67 (m, 4H), 3.81−3.87 (m,

2H), 5.92 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H). LC−MS m/z

274 [M + H]⁺.

Isopentyl-2-oxo-4-(4-phenylpiperazinn-1-yl)pyridine-3-car-

bonitrile (13). Starting from 41d (120 mg, 0.40 mmol) and 1-

phenylpiperazine (77.87 mg, 0.48 mmol) and following the procedure

described for 8, compound 13 was obtained as a white solid (155 mg,

4-Benzyloxy-1-isopentyl-2-oxo-1,2-dihydropyridine-3-car-

bonitrile (7). Benzyl bromide (88.2 μL, 0.7 mmol) was added to a

mixture of 40d (102 mg, 0.5 mmol) and CsCO₃(319 mg, 1 mmol) in

CH₃CN (2 mL) and DMF (1 mL). The reaction mixture was heated

in a microwave oven at 130 °C for 20 min. The solid was ﬁltered oﬀ

through a Celite pad and washed with EtOAc (10 mL). The combined

organic ﬁltrates were evaporated in vacuo, and the residue was puriﬁed

by ﬂash column chromatography (silica gel, silica gel, MeOH in

CH₂Cl₂, 0:100 to 4:96). Desired fractions were collected, and the

solvent was evaporated to yield compound 7 as a white solid (115 mg,

1

99%). mp > 300 °C. H NMR (400 MHz, CDCl₃) δ 0.95 (d, J = 6.0

Hz, 6H), 1.54−1.67 (m, 3H), 3.29−3.37 (m, 4H), 3.79−3.90 (m, 6H),

5.92 (d, J = 7.9 Hz, 1H), 6.87−6.96 (m, 3H), 7.23−7.32 (m, 3H).

LC−MS m/z 351 [M + H]⁺.

Isopentyl-2-oxo-4-[4-(2-pyridyl)piperazin-1-yl]pyridine-3-

carbonitrile (14). Starting from 41d (50 mg, 0.20 mmol) and 1-(2-

pyridyl)piperazine (40.8 μL, 0.24 mmol) and following the procedure

described for 8, compound 14 was obtained as a white solid (60 mg,

95%). ¹H NMR (400 MHz, CDCl₃) δ 0.95 (d, J = 6.2 Hz, 6H), 1.54−

1.69 (m, 3H), 3.71−3.77 (m, 4H), 3.81−3.89 (m, 6H), 5.91 (d, J = 7.9

Hz, 1H), 6.64 (d, J = 8.5 Hz, 1H), 6.68 (ddd, J = 7.2, 4.9, 0.7 Hz, 1H),

7.25 (d, J = 7.9 Hz, 1H), 7.53 (ddd, J = 8.7, 7.0, 2.1 Hz, 1H), 8.20

(ddd, J = 5.0, 1.9, 0.8 Hz, 1H). LC−MS m/z 352 [M + H]⁺.

Isopentyl-2-oxo-4-(4-benzylpiperazin-1-yl)pyridine-3-car-

bonitrile (15). Starting from 41d (120 mg, 0.40 mmol) and 1-

benzylpiperazine (84.60 mg, 0.48 mmol) and following the procedure

described for 8, compound 15 was obtained as a white solid (150 mg,

1

79%). mp 173.8 °C. H NMR (400 MHz, CDCl₃) δ 0.95 (d, J = 5.8

Hz, 6H), 1.54−1.67 (m, 3H), 3.88−3.94 (m, 2H), 5.28 (s, 2H), 6.06

(d, J = 7.7 Hz, 1H), 7.31−7.45 (m, 6H). LC−MS m/z 297 [M + H]⁺.

4-[Benzyl(methyl)amino]-1-isopentyl-2-oxo-1,2-dihydropyr-

idine-3-carbonitrile (8). A mixture of 41d (50 mg, 0.2 mmol), N-

methylbenzylamine (28.8 mg, 0.2 mmol), 2-(2′-di-tert-

butylphosphine)biphenylpalladium(II) acetate (4.6 mg, 0.01 mmol),

K₃PO₄(90 mg, 0.4 mmol), and 1,4-dioxane (6 mL) was heated at 85

°C for 6 h. The mixture was cooled to room temperature and ﬁltered

through a Celite pad. The solid was washed with more 1,4-dioxane (10

mL). The organic ﬁltrate was evaporated, and the residue was puriﬁed

by ﬂash column chromatography (silica gel, silica gel, MeOH−NH₃in

CH₂Cl₂, 0:100 to 4:96). Desired fractions were collected, and the

solvent was evaporated to yield compound 8 (39 mg, 66%). ¹H NMR

(400 MHz, CDCl₃) δ 0.94 (d, J = 6.2 Hz, 6H), 1.53−1.67 (m, 3H),

3.35 (s, 3H), 3.79−3.86 (m, 2H), 4.73 (s, 2H), 5.77 (d, J = 8.1 Hz,

1H), 7.10 (d, J = 8.1 Hz, 1H), 7.15−7.20 (m, 2H), 7.28−7.34 (m,

1H), 7.34−7.41 (m, 2H). LC−MS m/z 310 [M + H]⁺.

4-(3,4-Dihydro-1H-isoquinolin-2-yl)-1-isopentyl-2-oxo-1,2-

dihydropyridine-3-carbonitrile (9). Starting from 41d (120 mg, 0.4

mmol) and 1,2,3,4-tetrahydroisoquinoline following the procedure

described for 8, compound 9 was obtained as a white solid (78 mg,

54%). mp > 300. ¹H NMR (400 MHz, CDCl₃) δ 0.95 (d, J = 6.4 Hz,

6H), 1.53−1.66 (m, 3H), 3.05 (t, J = 5.9 Hz, 2H), 3.82−3.88 (m, 2H),

3.91 (t, J = 5.9 Hz, 2H), 4.78 (s, 2H), 5.92 (d, J = 8.1 Hz, 1H), 7.12−

7.26 (m, 5H). LC−MS m/z 322 [M + H]⁺.

1

93%). mp 131.6 °C. H NMR (400 MHz, CDCl₃) δ 0.94 (d, J = 6.2

Hz, 6H), 1.52−1.66 (m, 3H), 2.53−2.60 (m, 4H), 3.54 (s, 2H), 3.63−

3.69 (m, 4H), 3.80−3.86 (m, 2H), 5.85 (d, J = 7.9 Hz, 1H), 7.21 (d, J

= 7.9 Hz, 1H), 7.23−7.40 (m, 5H). LC−MS m/z 365 [M + H]⁺.

Butyl-2-oxo-4-(4-phenyl-1-piperidyl)pyridine-3-carbonitrile

(16). Starting from 41c (1000 mg, 3.92 mmol) and 4-phenylpiperidine

(821.6 mg, 5.09 mmol) and following the procedure described for 10,

compound 16 was obtained as a cream solid (1.24 g, 94%). mp 138.2

°C. ¹H NMR (500 MHz, CDCl₃) δ 0.95 (t, J = 7.4 Hz, 3H), 1.36 (sxt,

J = 7.5 Hz, 2H), 1.69 (quin, J = 7.5 Hz, 2H), 1.85 (qd, J = 12.7, 3.3 Hz,

2H), 1.97−2.05 (m, 2H), 2.82 (tt, J = 12.1, 3.5 Hz, 1H), 3.23 (br t, J =

12.7 Hz, 2H), 3.84 (t, J = 7.4 Hz, 2H), 4.28−4.35 (m, 2H), 5.87 (d, J =

8.1 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.19−7.25 (m, 3H), 7.29−7.35

(m, 2H). LC−MS m/z 336 [M + H]⁺.

Isobutyl-2-oxo-4-(4-phenyl-1-piperidyl)pyridine-3-carboni-

trile (17). Starting from 41b (510 mg, 2.0 mmol) and 4-

phenylpiperidine (386.9 mg, 2.4 mmol) and following the procedure

described for 10, compound 17 was obtained as a white solid (606 mg,

1-Isopentyl-2-oxo-4-(4-phenyl-1-piperidyl)-1,2-dihydropyri-

dine-3-carbonitrile (10). DIPEA (0.233 mL, 1.34 mmol) was added

to a mixture of 41d (300 mg, 1.12 mmol) and 4-phenylpiperidine (198

mg, 1.23 mmol) in CH₃CN (3 mL). The reaction mixture was heated

at 150 °C for 10 min in a microwave oven. The solvent was

evaporated, and the residue was puriﬁed by ﬂash column

chromatography (silica gel, silica gel, EtOAc in CH₂Cl₂, 0:100 to

20:80). Desired fractions were collected, and the solvent was

evaporated to yield compound 10 (296 mg, 63%) as a colorless oil

that solidiﬁed upon standing. mp > 300. ¹H NMR (400 MHz, CDCl₃)

δ 0.96 (d, J = 6.2 Hz, 6H), 1.54−1.68 (m, 3H), 1.84 (qd, J = 12.5, 3.9

Hz, 2H), 1.97−2.05 (m, 2H), 2.82 (tt, J = 12.1, 3.8 Hz, 1H), 3.18−

3.27 (m, 2H), 3.82−3.89 (m, 2H), 4.27−4.35 (m, 2H), 5.89 (d, J = 7.9

Hz, 1H), 7.18 (d, J = 7.9 Hz, 1H), 7.20−7.26 (m, 3H), 7.29−7.36 (m,

2H). LC−MS m/z 350 [M + H]⁺.

1

94%). H NMR (500 MHz, CDCl₃) δ 0.94 (d, J = 6.6 Hz, 6H), 1.86

(qd, J = 12.7, 3.6 Hz, 2H), 1.98−2.06 (m, 2H), 2.15 (dquin, J = 13.7,

6.8 Hz, 1H), 2.82 (tt, J = 12.0, 3.6 Hz, 1H), 3.19−3.28 (m, 2H), 3.65

(d, J = 7.5 Hz, 2H), 4.29−4.36 (m, 2H), 5.87 (d, J = 8.1 Hz, 1H), 7.13

(d, J = 7.8 Hz, 1H), 7.20−7.26 (m, 3H), 7.29−7.36 (m, 2H). LC−MS

m/z 336 [M + H]⁺.

1-(Cyclobutylmethyl)-2-oxo-4-(4-phenyl-1-piperidyl)-

pyridine-3-carbonitrile (18). Starting from 41e (600 mg, 2.24

mmol) and 4-phenylpiperidine (435 mg, 2.69 mmol) and following

the procedure described for 10, compound 18 was obtained as a cream

1

solid (749.4 m g, 96%). mp 158.1 °C. H NMR (400 MHz, DMSO-

d₆) δ 1.62−1.98 (m, 10H), 2.63 (quin, J = 7.7 Hz, 1H), 2.86 (tt, J =

12.1, 3.5 Hz, 1H), 3.17−3.27 (m, 2H), 3.81 (d, J = 7.4 Hz, 2H), 4.21−

4.29 (m, 2H), 6.16 (d, J = 8.1 Hz, 1H), 7.18−7.23 (m, 1H), 7.24−7.34

(m, 5H). LC−MS m/z 348 [M + H]⁺.

Isopentyl-2-oxo-4-(3-phenyl-1-piperidyl)pyridine-3-carboni-

trile (11). Starting from 41d (60 mg, 0.22 mmol) and 3-

6505

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

1-(Cyclopropylmethyl)-2-oxo-4-(4-phenyl-1-piperidyl)-

pyridine-3-carbonitrile (19). Starting from 41a (0.3 g, 1.18 mmol)

and following the procedure described for 10, compound 19 was

room temperature for 72 h. The mixture was ﬁltered through a Celite

pad, and the residue was washed with CH₂Cl₂. The ﬁltrate was

evaporated in vacuo, and the crude was puriﬁed by ﬂash column

chromatography (silica gel, silica gel, MeOH in CH₂Cl₂, 0:100 to

1

obtained as a solid (285 mg, 73%). mp 128.4. H NMR (500 MHz,

1

CDCl₃) δ 0.31−0.42 (m, 2H), 0.55−0.66 (m, 2H), 1.15−1.24 (m,

1H), 1.85 (qd, J = 12.7, 3.8 Hz, 2H), 1.97−2.05 (m, 2H), 2.82 (tt, J =

12.1, 3.8 Hz, 1H), 3.19−3.28 (m, 2H), 3.71 (d, J = 7.2 Hz, 2H), 4.28−

4.37 (m, 2H), 5.90 (d, J = 7.8 Hz, 1H), 7.20−7.26 (m, 3H), 7.28−7.36

(m, 3H). LC−MS m/z 334 [M + H]⁺.

5:95) to give 25 as a white solid (289.6 mg, 42%). H NMR (500

MHz, DMSO-d₆) δ 0.29−0.39 (m, 2H), 0.41−0.51 (m, 2H), 1.11−

1.20 (m, 1H), 1.72 (qd, J = 12.6, 3.6 Hz, 2H), 1.89 (br d, J = 12.4 Hz,

2H), 3.24−3.32 (m, 3H), 3.63 (d, J = 6.9 Hz, 2H), 4.29 (br d, J = 13.6

Hz, 2H), 6.19 (d, J = 8.1 Hz, 1H), 7.25 (td, J = 7.8, 1.7 Hz, 1H), 7.33

(td, J = 7.5, 1.2 Hz, 1H), 7.40 (dd, J = 7.8, 1.7 Hz, 1H), 7.44 (dd, J =

7.9, 1.3 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H). LC−MS m/z 368 [M +

H]⁺.

4-[4-(4-Chlorophenyl)-1-piperidyl]-1-(cyclopropylmethyl)-2-

oxo-pyridine-3-carbonitrile (26). Starting from 41a (0.35 g, 1.38

mmol) and following the procedure described for 10, compound 26

was obtained as a solid (177.42 mg, 35%). ¹H NMR (400 MHz,

CDCl₃) δ 0.15−0.27 (m, 2H), 0.38−0.51 (m, 2H), 0.98−1.10 (m,

1H), 1.65 (qd, J = 12.7, 3.9 Hz, 2H), 1.79−1.87 (m, 2H), 2.64 (tt, J =

12.1, 3.8 Hz, 1H), 3.01−3.11 (m, 2H), 3.56 (d, J = 7.3 Hz, 2H), 4.12−

4.19 (m, 2H), 5.73 (d, J = 8.1 Hz, 1H), 6.97−7.03 (m, 2H), 7.11−7.18

(m, 3H). LC−MS m/z 368 [M + H]⁺.

1-(2-Cyclopropylethyl)-2-oxo-4-(4-phenyl-1-piperidyl)-

pyridine-3-carbonitrile (20). Starting from 41f (0.2 g, 0.748 mmol)

and following the procedure described for 10, compound 20 was

1

obtained as a solid (200 mg, 77%). mp > 300. H NMR (500 MHz,

CDCl₃) δ 0.03−0.10 (m, 2H), 0.40−0.52 (m, 2H), 0.59−0.69 (m,

1H), 1.57−1.66 (m, 2H), 1.79−1.90 (m, 2H), 1.97−2.05 (m, 2H),

2.77−2.86 (m, 1H), 3.23 (br t, J = 12.9 Hz, 2H), 3.92 (br t, J = 6.8 Hz,

2H), 4.32 (br d, J = 13.3 Hz, 2H), 5.86 (d, J = 8.1 Hz, 1H), 7.18−7.26

(m, 4H), 7.29−7.36 (m, 2H). LC−MS m/z 348 [M + H]⁺.

2-Oxo-4-(4-phenyl-1-piperidyl)-1-(4,4,4-triﬂuorobutyl)-

pyridine-3-carbonitrile (21). Starting from 41g (0.25 g, 0.81 mmol)

and following the procedure described for 10, compound 21 was

obtained as a solid (300 mg, 95%). mp 113.2 °C. ¹H NMR (500 MHz,

CDCl₃) δ 1.78 (qd, J = 12.7, 3.8 Hz, 2H), 1.89−2.02 (m, 4H), 2.04−

2.16 (m, 2H), 2.77 (tt, J = 12.1, 3.6 Hz, 1H), 3.14−3.23 (m, 2H), 3.85

(br t, J = 7.1 Hz, 2H), 4.27 (br d, J = 13.3 Hz, 2H), 5.85 (d, J = 8.1 Hz,

1H), 7.07 (d, J = 7.8 Hz, 1H), 7.12−7.21 (m, 3H), 7.22−7.29 (m,

2H). LC−MS m/z 390 [M + H]⁺.

1-(Cyclopropylmethyl)-2-oxo-4-[4-[3-(triﬂuoromethyl)-

phenyl]-1-piperidyl]pyridine-3-carbonitrile (27). DIPEA (0.72

mL, 4.14 mmol) was added to a mixture of 41a (0.35 g, 1.38 mmol)

and 4-(3-triﬂuoromethyl-phenyl)-piperidine (0.41 g, 1.79 mmol) in

CH₃CN (8 mL). The reaction mixture was heated at 170 °C for 35

min in a microwave oven. The mixture was washed with a saturated

solution of NH₄Cl and extracted with CH₂Cl₂(2 × 5 mL).The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica gel, silica gel, MeOH−NH₃in CH₂Cl₂, 0:100

to 1:99). Desired fractions were collected, and the solvent was

evaporated to yield compound 27 (0.414 g, 75%) as a white solid. mp

1-(Cyclopropylmethyl)-4-[4-(2-ﬂuorophenyl)-1-piperidyl]-2-

oxo-pyridine-3-carbonitrile (22). A solution of 48a (0.185 g, 0.52

mmol) in EtOAc (15 mL) was hydrogenated (atmospheric pressure)

with Pd/C (37 mg) as a catalyst at room temperature for 4 h. The

mixture was ﬁltered through a Celite pad, and the ﬁltrate was

evaporated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica gel, MeOH−NH₃in CH₂Cl₂, 0:100 to 2:98)

1

111.9 °C. H NMR (400 MHz, CDCl₃) δ 0.31−0.44 (m, 2H), 0.55−

1

to give 22 as a pale yellow solid (148.3 mg, 80%). H NMR (400

0.68 (m, 2H), 1.15−1.27 (m, 1H), 1.87 (qd, J = 12.8, 3.7 Hz, 2H),

2.00−2.09 (m, 2H), 2.90 (tt, J = 12.1, 3.7 Hz, 1H), 3.19−3.30 (m,

2H), 3.73 (d, J = 7.3 Hz, 2H), 4.29−4.39 (m, 2H), 5.91 (d, J = 7.9 Hz,

1H), 7.32 (d, J = 8.1 Hz, 1H), 7.39−7.56 (m, 4H). LC−MS m/z 402

[M + H]⁺.

MHz, CDCl₃) δ 0.31−0.44 (m, 2H), 0.55−0.68 (m, 2H), 1.16−1.25

(m, 1H), 1.88 (qd, J = 12.4, 3.7 Hz, 2H), 1.97−2.05 (m, 2H), 3.18 (tt,

J = 12.0, 3.9 Hz, 1H), 3.26 (td, J = 12.9, 2.4 Hz, 2H), 3.72 (d, J = 7.3

Hz, 2H), 4.29−4.38 (m, 2H), 5.90 (d, J = 8.1 Hz, 1H), 7.01−7.08 (m,

1H), 7.09−7.15 (m, 1H), 7.18−7.26 (m, 2H), 7.30 (d, J = 7.9 Hz,

1H). LC−MS m/z 352 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(2-methoxyphenyl)-1-piperid-

yl]-2-oxo-pyridine-3-carbonitrile (28). Starting from 41a (0.35 g,

1.38 mmol) and 4-(2-methoxy-phenyl)-piperidine (0.342 g, 1.79

mmol) following the procedure described for 27, compound 28 was

obtained as a white solid (0.319 g, 64%). mp 148.1 °C. ¹H NMR (400

MHz, CDCl₃) δ 0.30−0.43 (m, 2H), 0.54−0.68 (m, 2H), 1.15−1.25

(m, 1H), 1.81 (qd, J = 12.6, 3.7 Hz, 2H), 1.95−2.03 (m, 2H), 3.21−

3.32 (m, 3H), 3.72 (d, J = 7.3 Hz, 2H), 3.85 (s, 3H), 4.29−4.37 (m,

2H), 5.90 (d, J = 8.1 Hz, 1H), 6.88 (dd, J = 8.2, 1.1 Hz, 1H), 6.94 (td,

J = 7.5, 1.0 Hz, 1H), 7.17 (dd, J = 7.6, 1.8 Hz, 1H), 7.19−7.25 (m,

1H), 7.29 (d, J = 7.9 Hz, 1H). LC−MS m/z 364 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(4-methoxyphenyl)-1-piperid-

yl]-2-oxo-pyridine-3-carbonitrile (29). Starting from 41a (0.35 g,

1.38 mmol) and 4-(4-methoxy-phenyl)-piperidine (0.376 g, 1.66

mmol) following the procedure described for 27, compound 29 was

obtained as a white solid (0.284 g, 48%). mp 247.6 °C. ¹H NMR (500

MHz, CDCl₃) δ 0.32−0.43 (m, 2H), 0.56−0.66 (m, 2H), 1.16−1.25

(m, 1H), 1.81 (qd, J = 12.7, 3.6 Hz, 2H), 1.99 (br d, J = 12.1 Hz, 2H),

2.78 (tt, J = 12.1, 3.5 Hz, 1H), 3.22 (br t, J = 12.9 Hz, 2H), 3.72 (d, J =

7.2 Hz, 2H), 3.80 (s, 3H), 4.32 (br d, J = 13.3 Hz, 2H), 5.90 (d, J = 7.8

Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.30 (d, J

= 7.8 Hz, 1H). LC−MS m/z 364 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(3-ﬂuorophenyl)-1-piperidyl]-2-

oxo-pyridine-3-carbonitrile (23). Starting from 48b (0.215 g, 0.61

mmol) and following the procedure described for 22, compound 23

1

was obtained as a pale yellow solid (156 mg, 72%). H NMR (400

MHz, CDCl₃) δ 0.33−0.46 (m, 2H), 0.56−0.70 (m, 2H), 1.16−1.28

(m, 1H), 1.85 (qd, J = 12.8, 3.8 Hz, 2H), 1.99−2.08 (m, 2H), 2.85 (tt,

J = 12.1, 3.7 Hz, 1H), 3.19−3.30 (m, 2H), 3.74 (d, J = 7.0 Hz, 2H),

4.30−4.38 (m, 2H), 5.91 (d, J = 7.9 Hz, 1H), 6.91−6.98 (m, 2H), 7.02

(br d, J = 7.9 Hz, 1H), 7.27−7.35 (m, 1H), 7.32 (d, J = 7.9 Hz, 1H).

LC−MS m/z 352 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(4-ﬂuorophenyl)-1-piperidyl]-2-

oxo-pyridine-3-carbonitrile (24). Triethylsilane (1.11 mL, 9.55

mmol) was added to a solution of 49 (0.351 g, 0.955 mmol) in

CF₃CO₂H (0.5 mL, 6.49 mmol). The mixture was stirred at room

temperature for 18 h and then diluted with CH₂Cl₂(15 mL) and

washed with 1 N NaOH (2 × 30 mL). The organic layer was

separated, dried (Na₂SO₄), ﬁltered, and concentrated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica gel,

MeOH−NH₃in CH₂Cl₂, 0:100 to 2:98) to give 24 as a white solid

1

(225 mg, 67%). mp 92.3 °C. H NMR (400 MHz, CDCl₃) δ 0.26−

0.39 (m, 2H), 0.48−0.61 (m, 2H), 1.11−1.21 (m, 1H), 1.75 (qd, J =

12.7, 3.7 Hz, 2H), 1.90−1.98 (m, 2H), 2.78 (tt, J = 12.1, 3.8 Hz, 1H),

3.13−3.23 (m, 2H), 3.67 (d, J = 7.0 Hz, 2H), 4.24−4.32 (m, 2H), 5.94

(d, J = 8.1 Hz, 1H), 6.92−6.99 (m, 2H), 7.10−7.18 (m, 2H), 7.36 (d, J

= 7.9 Hz, 1H). LC−MS m/z 352 [M + H]⁺.

4-[4-(2-Chlorophenyl)-1-piperidyl]-1-(cyclopropylmethyl)-2-

oxo-pyridine-3-carbonitrile (25). A solution of 48c (0.68 g, 1.86

mmol) in MeOH (5 mL) and CH₃CO₂H (1 mL) was hydrogenated

(atmospheric pressure) with PtO₂(4 mg, 0.018 mmol) as a catalyst at

1-(Cyclopropylmethyl)-4-[4-(2,4-diﬂuorophenyl)-1-piperid-

yl]-2-oxo-pyridine-3-carbonitrile (30). 48c (0.9 g, 2.67 mmol) and

10% Pd/C (0.0028 g, 0.026 mmol) in MeOH (15 mL) were

hydrogenated for 16 h. The crude was ﬁltered over Celite and washed

with MeOH, and the solution was dried over Na₂SO₄, ﬁltered, and

concentrated. The crude was puriﬁed by ﬂash column chromatography

(silica gel, silica gel, MeOH−NH₃in CH₂Cl₂, 0:100 to 1:99). Desired

fractions were collected, and the solvent was evaporated to yield

compound 30 (0.614 g, 62%) as a white solid. mp 111.9 °C. ¹H NMR

6506

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

1

(500 MHz, DMSO-d₆) δ 0.31−0.37 (m, 2H), 0.43−0.48 (m, 2H),

1.11−1.20 (m, 1H), 1.74 (qd, J = 12.4, 3.2 Hz, 2H), 1.81−1.88 (m,

2H), 3.14 (tt, J = 12.1, 3.8 Hz, 1H), 3.22−3.29 (m, 2H), 3.63 (d, J =

7.2 Hz, 2H), 4.26 (br d, J = 13.6 Hz, 2H), 6.18 (d, J = 7.8 Hz, 1H),

7.04 (td, J = 8.5, 2.3 Hz, 1H), 7.17−7.23 (m, 1H), 7.40 (td, J = 8.5, 6.9

Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H). LC−MS m/z 370 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(2,6-diﬂuorophenyl)-1-piperid-

yl]-2-oxo-pyridine-3-carbonitrile (31). Starting from 41a (0.35 g,

1.38 mmol) and 4-(2,6-diﬂuoro-phenyl)-piperidine acetate (0.426 g,

1.65 mmol) following the procedure described for 27, compound 31

was obtained as a white solid (0.39 g, 82%). mp 149.4 °C. H NMR

(400 MHz, CDCl₃) δ 0.95 (t, J = 7.3 Hz, 3H), 1.31−1.42 (m, 2H),

1.68−1.78 (m, 2H), 1.85−1.98 (m, 4H), 2.64−2.73 (m, 1H), 2.87−

2.98 (m, 2H), 3.82 (brd, J = 12.1 Hz, 2H), 3.93 (t, J = 7.3, 2H), 6.03

(d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.19−7.28 (m, 3H),

7.29−7.37 (m, 2H). LC−MS m/z 345 [M + H]⁺.

1-Isobutyl-4-methoxy-2-oxo-1,2-dihydropyridine-3-carboni-

trile (39b). Starting from isobutyl bromide 38b (11.5 mL g, 103

mmol) and 37 (8 g, 53.3 mmol) and following the procedure

described for synthesis of 39a,c,d,e,²⁹compound 39b was obtained as

1

a white solid (11.0 g, quantitative). H NMR (400 MHz, CDCl₃) δ

was obtained as a white solid (0.184 g, 31%). mp 144.2 °C. H NMR

0.93 (d, J = 6.7 Hz, 6H), 2.10−2.22 (m, 1H), 3.74 (d, J = 7.4 Hz, 2H),

4.00 (s, 3H), 6.05 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H). LC−

MS m/z 207 [M + H]⁺.

(400 MHz, CDCl₃) δ 0.30−0.44 (m, 2H), 0.54−0.69 (m, 2H), 1.13−

1.27 (m, 1H), 1.87 (br d, J = 12.5 Hz, 2H), 2.16−2.30 (m, 2H), 3.16−

3.37 (m, 3H), 3.72 (d, J = 7.2 Hz, 2H), 4.34 (br d, J = 13.6 Hz, 2H),

5.90 (d, J = 7.9 Hz, 1H), 6.81−6.90 (m, 2H), 7.10−7.24 (m, 1H), 7.30

d, J = 7.9 Hz, 1H). LC−MS m/z 370 [M + H]⁺.

1-(2-Cyclopropylethyl)-4-methoxy-2-oxo-1,2-dihydropyri-

dine-3-carbonitrile (39f). Starting from 2-cyclopropylethyl bromide

38g (2.59 g, 17.3 mmol) and 37 (7.1 g, 19.0 mmol) and following the

procedure as described for synthesis of 39a,c,d,e,²⁹compound 39f was

obtained as a white solid (2.63 g, 70%). ¹H NMR (500 MHz, CDCl₃)

δ 0.00−0.06 (m, 2H), 0.42−0.53 (m, 2H), 0.57−0.66 (m, 1H), 1.64

(quin, J = 6.72 Hz, 2H), 3.99 (s, 3H), 4.01 (t, J = 6.90 Hz, 2H), 6.07

(d, J = 7.80 Hz, 1H), 7.56 (d, J = 7.80 Hz, 1H). LC−MS m/z 219 [M

+ H]⁺.

1-(Cyclopropylmethyl)-4-[4-(3,5-diﬂuorophenyl)-1-piperid-

yl]-2-oxo-pyridine-3-carbonitrile (32). Starting from 41a (0.35 g,

1.38 mmol) and 4-(3,5-diﬂuoro-phenyl)-piperidine (0.3 g, 1.52 mmol)

following the procedure described for 27, compound 32 was obtained

1

as a white solid (0.28 g, 50%). H NMR (500 MHz, DMSO-d₆) δ

0.29−0.39 (m, 2H), 0.41−0.51 (m, 2H), 1.10−1.19 (m, 1H), 1.70 (qd,

J = 12.6, 3.6 Hz, 2H), 1.86−1.94 (m, 2H), 2.93 (tt, J = 12.0, 3.5 Hz,

1H), 3.17−3.25 (m, 2H), 3.63 (d, J = 7.2 Hz, 2H), 4.27 (br d, J = 13.3

Hz, 2H), 6.18 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 9.2 Hz, 3H), 7.70 (d, J

= 7.8 Hz, 1H). LC−MS m/z 370 [M + H]⁺.

1-(4,4,4-Triﬂuorobutyl)-4-methoxy--2-oxo-1,2-dihydropyri-

dine-3-carbonitrile (39g). Starting from 38g (0.107 mL, 0.867

mmol) and following the procedure as described for synthesis of

39a,c,d,e,²⁹compound 39g was obtained as a white solid (0.142 g,

82%). ¹H NMR (400 MHz, CDCl₃) δ 1.98−2.08 (m, 2H), 2.10−2.24

(m, 2H), 3.99−4.04 (m, 2H), 4.02 (s, 3H), 6.13 (d, J = 7.9 Hz, 1H),

7.52 (d, J = 7.6 Hz, 1H). LC−MS m/z 261 [M + H]⁺.

3-Chloro-1-(cyclopropylmethyl)-4-(4-phenyl-1-piperidyl)-

pyridin-2-one (33). NCS (0.81 g, 6.09 mmol) was added to a

mixture of 50a (1.89 g, 6.15 mmol) in CH₂Cl₂(300 mL). The reaction

mixture was stirred for 10 min at room temperature. The mixture was

washed with a saturated solution of NaHCO₃and extracted with

CH₂Cl₂(2 × 10 mL). The combined organic layers were dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude was puriﬁed by

ﬂash column chromatography (silica gel, silica gel, MeOH−NH₃in

CH₂Cl₂, 0:100 to 3:97). Desired fractions were collected, and the

solvent was evaporated to yield compound 33 (1.88 g, 85%) as a white

1-Isobuthyl-4-hydroxy-2-oxo-1,2-dihydropyridine-3-car-

bonitrile (40b). Starting from 39b (10.93 g, 53 mmol) and following

the procedure as described for synthesis of 40a,c,d,e,²⁹compound 40b

1

was obtained as a white solid (4.83 g, 47%). H NMR (400 MHz,

DMSO-d₆) δ 0.83 (d, J = 6.9 Hz, 6H), 1.99 (s, 1H), 3.65 (d, J = 7.4

Hz, 2H), 6.04 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 12.69 (br

s, 1H). LC−MS m/z 193 [M + H]⁺.

1

solid. H NMR (400 MHz, CDCl₃) δ 0.31−0.45 (m, 2H), 0.53−0.67

1-(2-Cyclopropylethyl)-4-hydroxy-2-oxo-1,2-dihydropyri-

dine-3-carbonitrile (40f). Starting from 39f (2.63 g, 12 mmol) and

following the procedure described as described for synthesis of

39a,c,d,e,²⁹compound 40f was obtained as a white solid (1.15 g,

47%). ¹H NMR (500 MHz, DMSO-d₆) δ −0.09−0.02 (m, 2H) 0.31−

0.42 (m, 2H) 0.56−0.65 (m, 1H) 1.48 (q, J = 7.13 Hz, 2H) 3.89 (t, J =

6.94 Hz, 2H) 6.03 (d, J = 7.80 Hz, 1H) 7.84 (d, J = 7.51 Hz, 1H)

12.61 (br s, 1H). LC−MS m/z 205 [M + H]⁺.

(m, 2H), 1.20−1.31 (m, 1H), 1.85−1.99 (m, 4H), 2.64−2.76 (m, 1H),

2.86−2.99 (m, 2H), 3.77−3.87 (m, 2H), 3.81 (d, J = 7.2 Hz, 2H), 6.05

(d, J = 7.6 Hz, 1H), 7.19−7.29 (m, 4H), 7.29−7.37 (m, 2H). LC−MS

m/z 343 [M + H]⁺.

3-Triﬂuoromethyl-1-(cyclopropylmethyl)-4-(4-phenyl-1-

piperidyl)pyridin-2-one (34). To a solution of 46 (0.3 g, 1.01

mmol) in toluene (7 mL) were added 4-phenylpiperidine (0.33 g, 2.02

mmol), Pd(OAc)₂(0.012 g, 0.005 mmol), NaO^tBu (0.24 g, 2.52

mmol), and BINAP (0.05 g, 0.08 mmol). The mixture was heated at

100 °C for 16 h in a sealed tube. The cooled mixture was partitioned

between water and EtOAc. The crude was extracted with EtOAc (3 ×

5 mL), and the combined organic layers were dried (Na₂SO₄) and

concentrated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica gel, silica gel, MeOH in CH₂Cl₂, 0:100 to

4:96). Compound 34 was obtained as white solid (0.11 g, 31%). mp

1-(4,4,4-Triﬂuorobutyl)-4-hydroxy-2-oxo-1,2-dihydropyri-

dine-3-carbonitrile (40g). Starting from 39g (6.8 g, 26.1 mmol) and

following the procedure as described for synthesis of 39a,c,d,e,²⁹

1

compound 40g was obtained as a white solid (5.9 g, 92%). H NMR

(400 MHz, DMSO-d₆) δ 1.77−1.87 (m, 2H), 2.19−2.36 (m, 2H), 3.89

(t, J = 7.3 Hz, 2H), 6.06 (d, J = 7.6 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H),

12.69 (br s, 1H). LC−MS m/z 247 [M + H]⁺.

4-Bromo-1-isobutyl-2-oxo-1,2-dihydropyridine-3-carboni-

trile (41b). Starting from 40b (4.83 g, 25.1 mmol) and following the

procedure as described for synthesis of 41a,c,d,e,²⁹compound 41b was

obtained as a white solid (4.21 g, 66%). ¹H NMR (400 MHz, CDCl₃)

δ 0.95 (d, J = 6.7 Hz, 6H), 2.11−2.23 (m, 1H), 3.76 (d, J = 7.4 Hz,

2H), 6.49 (d, J = 7.2 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H). LC−MS m/z

256 [M + H]⁺.

1

177.2 °C. H NMR (500 MHz, CDCl₃) δ 0.33−0.38 (m, 2H), 0.45−

0.50 (m, 2H), 1.13−1.22 (m, 1H), 1.64−1.75 (m, 2H), 1.64−1.75 (m,

2H), 1.84 (br d, J = 11.0 Hz, 2H), 2.73−2.80 (m, 1H), 3.14 (br t, J =

12.1 Hz, 2H), 3.59 (br d, J = 13.0 Hz, 2H), 3.65 (d, J = 7.2 Hz, 2H),

6.21 (d, J = 7.8 Hz, 1H), 7.19−7.23 (m, 1H), 7.24−7.29 (m, 2H), 7.73

(d, J = 7.8 Hz, 1H). LC−MS m/z 377 [M + H]⁺.

3-Chloro-1-isobutyl-4-(4-phenyl-1-piperidyl)pyridin-2-one

(35). Starting from 50b (0.35 g, 1.38 mmol) and N-chlorosuccinimide

(0.62 g, 4.65 mmol) following the procedure described for 33,

4-Bromo-1-(2-cyclopropylethyl)-2-oxo-1,2-dihydropyridine-

3-carbonitrile (41f). Starting from 40f (1.15 g, 5.63 mmol) and

following the procedure as described for synthesis of 41a,c,d,e,²⁹

1

compound 35 was obtained as a white solid (1.42 g, 83%). H NMR

compound 41f was obtained as a white solid (1.04 g, 69%). H NMR

(400 MHz, CDCl₃) δ 0.94 (d, J = 6.7 Hz, 6H), 1.86−1.99 (m, 4H),

2.21 (dquin, J = 13.7, 6.9 Hz, 1H), 2.65−2.76 (m, 1H), 2.88−2.97 (m,

2H), 3.74 (d, J = 7.4 Hz, 2H), 3.80−3.87 (m, 2H), 6.02 (d, J = 7.6 Hz,

1H), 7.07 (d, J = 7.6 Hz, 1H), 7.20−7.29 (m, 3H), 7.31−7.38 (m,

2H). LC−MS m/z 345 [M + H]⁺.

3-Chloro-1-butyl-4-(4-phenyl-1-piperidyl)pyridin-2-one (36).

Starting from 50c (0.43 g, 1.40 mmol) and N-chlorosuccinimide (0.19

g, 1.4 mmol) following the procedure described for 33, compound 36

(400 MHz, CDCl₃) δ 0.00−0.12 (m, 2H), 0.42−0.55 (m, 2H), 0.57−

0.69 (m, 1H), 1.67 (q, J = 7.1 Hz, 2H), 4.04 (t, J = 6.9 Hz, 2H), 6.50

(d, J = 7.2 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H). LC−MS m/z 268 [M +

H]⁺.

4-Bromo-1-(4,4,4-triﬂuorobutyl)-2-oxo-1,2-dihydropyridine-

3-carbonitrile (41g). Starting from 40g (5.1 g, 20.7 mmol) and

following the procedure as described for synthesis of 41a,c,d,e,²⁹

1

compound 41g was obtained as a white solid (5.2 g, 82%). H NMR

6507

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

(400 MHz, CDCl₃) δ 2.00−2.11 (m, 2H), 2.12−2.27 (m, 2H), 4.05 (t,

J = 7.3 Hz, 2H), 6.58 (d, J = 7.4 Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H).

LC−MS m/z 310 [M + H]⁺.

(m, 1H), 3.76 (d, J = 7.2 Hz, 2H), 6.34 (dd, J = 7.2, 2.3 Hz, 1H), 6.82

(d, J = 2.1 Hz, 1H), 7.26 (d, J = 7.2 Hz, 1H). LC−MS m/z 228 [M +

H]⁺.

4-Benzyloxy-1-cyclopropylmethyl-1H-pyridin-2-one (43a).

Cyclopropylmethyl bromide 38a (2.64 mL, 27.33 mmol) and

K₂CO₃(10.3 g, 74.52 mmol) was added to a solution of 42 (5 g,

24.84 mmol) in CH₃CN (200 mL). The mixture was stirred at reﬂux

for 16 h. The cooled mixture was ﬁltered through a Celite pad and

concentrated in vacuo. The residue thus obtained was precipitated by

treatment with diethyl ether. The resulting solid was ﬁltered oﬀ and

4-Bromo-1-isobutyl-pyridin-2-one (47b). Starting from 43b

(6.78 g, 26.35 mmol) and following the procedure described for 46,

compound 47b was obtained (3.5 g, 58%). ¹H NMR (500 MHz,

CDCl₃) δ 0.93 (d, J = 6.6 Hz, 6H), 2.10−2.20 (m, 1H), 3.70 (d, J = 7.5

Hz, 2H), 6.33 (dd, J = 7.2, 2.3 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 7.08

(d, J = 7.2 Hz, 1H). LC−MS m/z 230 [M + H]⁺.

4-Bromo-1-butyl-pyridin-2-one (47c). Starting from 43c (2.19

g, 8.5 mmol) and following the procedure described for 46, compound

1

dried in the vacuum oven (50 °C) to yield 43a (6.32 g, 98%). H

1

NMR (500 MHz, CDCl₃) δ 0.31−0.41 (m, 2H), 0.53−0.64 (m, 2H),

1.21 (m, J = 7.80 Hz, 1H), 3.74 (d, J = 6.94 Hz, 2H), 4.98 (s, 2H),

5.97 (dd, J = 7.51, 2.60 Hz, 1H), 5.99 (d, J = 2.89 Hz, 1H), 7.25 (d, J =

7.51 Hz, 1H), 7.31−7.42 (m, 5H). LC−MS m/z 258 [M + H]⁺.

4-Benzyloxy-1-isobutyl-1H-pyridin-2-one (43b). Starting from

ⁱbutyl bromide 38b (5.89 mL, 54.66 mmol) and 42 (10 g, 49.69

mmol) and following the procedure described for 43a, compound 43b

47c was obtained (1.82 g, 93%). H NMR (400 MHz, CDCl₃) δ 0.95

(t, J = 7.3 Hz, 3H), 1.36 (dq, J = 15.1, 7.4 Hz, 2H), 1.66−1.75 (m,

2H), 3.89 (t, J = 7.4 Hz, 2H), 6.34 (dd, J = 7.2, 2.1 Hz, 1H), 6.84 (d, J

= 2.3 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H). LC−MS m/z 230 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(2-ﬂuorophenyl)-3,6-dihydro-

2H-pyridin-1-yl]-2-oxo-pyridine-3-carbonitrile (48a). Starting

from 41a (0.23 g, 0.91 mmol) and 4-(2-ﬂuorophenyl)-1,2,3,6-

tetrahydropyridine (0.2 g, 1.13 mmol) and following the procedure

described for 10, compound 48a was obtained as a white solid (263

1

was obtained as a white solid (11.25 g, 88%). H NMR (400 MHz,

CDCl₃) δ 0.93 (d, J = 6.7 Hz, 6H), 2.09−2.21 (m, 1H), 3.67 (d, J = 7.4

Hz, 2H), 4.98 (s, 2H), 5.94 (dd, J = 7.4, 2.8 Hz, 1H), 5.99 (d, J = 2.8

Hz, 1H), 7.08 (d, J = 7.4 Hz, 1H), 7.32−7.43 (m, 5H). LC−MS m/z

258 [M + H]⁺.

1

mg, 83%). H NMR (400 MHz,CDCl₃) δ 0.30−0.43 (m, 2H), 0.53−

0.66 (m, 2H), 1.14−1.26 (m, 1H), 2.71−2.78 (m, 2H), 3.72 (d, J = 7.2

Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 4.32 (br q, J = 2.6 Hz, 2H), 5.95 (d,

J = 8.1 Hz, 1H), 5.98−6.02 (m, 1H), 7.02−7.08 (m, 1H), 7.10−7.15

(m, 1H), 7.22−7.28 (m, 2H), 7.35 (d, J = 7.9 Hz, 1H). LC−MS m/z

350 [M + H]⁺.

4-Benzyloxy-1-butyl-1H-pyridin-2-one (43c). Starting from

ⁿbutyl bromide 38c (8.80 mL, 81.99 mmol) and 42 (15 g, 74.54

mmol) and following the procedure described for 43a, compound 43c

1

1-(Cyclopropylmethyl)-4-[4-(3-ﬂuorophenyl)-3,6-dihydro-

2H-pyridin-1-yl]-2-oxo-pyridine-3-carbonitrile (48b). Starting

from 41a (0.23 g, 0.91 mmol) and 4-(3-ﬂuorophenyl)-1,2,3,6-

tetrahydropyridine (0.2 g, 1.13 mmol) and following the procedure

described for 6, compound 48b was obtained as a white solid (246 mg,

77%). ¹H NMR (400 MHz, CDCl₃) δ 0.30−0.43 (m, 2H), 0.53−0.67

(m, 2H), 1.14−1.25 (m, 1H), 2.71−2.78 (m, 2H), 3.72 (d, J = 7.2 Hz,

2H), 3.93 (t, J = 5.5 Hz, 2H), 4.30 (q, J = 2.7 Hz, 2H), 5.93 (d, J = 8.1

Hz, 1H), 6.09−6.13 (m, 1H), 6.98 (tdd, J = 8.3, 2.5, 0.9 Hz, 1H), 7.08

(dt, J = 10.5, 2.2 Hz, 1H), 7.15−7.19 (m, 1H), 7.31 (td, J = 8.0, 6.0 Hz,

1H), 7.34 (d, J = 8.1 Hz, 1H). LC−MS m/z 350 [M + H]⁺.

4-[4-(2-Chlorophenyl)-3,6-dihydro-2H-pyridin-1-yl]-1-(cyclo-

propylmethyl)-2-oxo-pyridine-3-carbonitrile (48c). Starting

from 41a (0.47 g, 1.88 mmol) and 4-(2-chlorophenyl)-1,2,3,6-

tetrahydropyridine (0.4 g, 2.07 mmol) and following the procedure

described for 10, compound 48c was obtained as a white solid (680

mg, 100%). ¹H NMR (500 MHz, CDCl₃) δ 0.32−0.42 (m, 2H), 0.54−

0.65 (m, 2H), 1.16−1.24 (m, 1H), 2.65−2.71 (m, 2H), 3.72 (d, J = 7.2

Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 4.31 (br q, J = 2.9 Hz, 2H), 5.72−

5.75 (m, 1H), 5.94 (d, J = 7.8 Hz, 1H), 7.16−7.20 (m, 1H), 7.21−7.26

(m, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.36−7.39 (m, 1H). LC−MS m/z

366 [M + H]⁺.

1-(Cyclopropylmethyl)-4-[4-(2,4-diﬂuorophenyl)-3,6-dihy-

dro-2H-pyridin-1-yl]-2-oxo-pyridine-3-carbonitrile (48d). Start-

ing from 41a (0.67 g, 2.67 mmol) and 4-(2,4-diﬂuorophenyl)-1,2,3,6-

tetrahydropyridine (0.57 g, 2.94 mmol) and following the procedure

described for 10, compound 48d was obtained as a white solid (900

mg, 92%). ¹H NMR (500 MHz, CDCl₃) δ 0.32−0.42 (m, 2H), 0.54−

0.65 (m, 2H), 1.15−1.24 (m, 1H), 2.71 (br s, 2H), 3.72 (d, J = 7.2 Hz,

2H), 3.90 (t, J = 5.5 Hz, 2H), 4.30 (br q, J = 2.6 Hz, 2H), 5.93 (d, J =

8.1 Hz, 1H), 5.95−5.98 (m, 1H), 6.81 (ddd, J = 11.2, 8.7, 2.6 Hz, 1H),

6.84−6.89 (m, 1H), 7.23 (td, J = 8.7, 6.4 Hz, 1H), 7.34 (d, J = 7.8 Hz,

1H). LC−MS m/z 368 [M + H]⁺.

was obtained as a white solid (13.5 g, 70%). H NMR (360 MHz,

CDCl₃) δ 0.93 (t, J = 7.32 Hz, 3H), 1.28−1.40 (m, 2H), 1.62−1.73

(m, 2H), 3.84 (t, J = 7.32 Hz, 2H), 4.95 (s, 2H), 5.93 (dd, J = 7.32,

2.90 Hz, 1H), 5.97 (d, J = 2.93 Hz, 1H), 7.11 (d, J = 7.32 Hz, 1H),

7.27−7.42 (m, 5H). LC−MS m/z 258 [M + H]⁺.

4-Benzyloxy-1-cyclopropylmethyl-3-iodo-1H-pyridin-2-one

(44). NIS (2.64 g, 11.74 mmol) was added to a solution of 43a (3 g,

11.74 mmol) in acetic acid (40 mL). The mixture was stirred at room

temperature for 1 h. The mixture was concentrated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica gel, silica

gel, MeOH in CH₂Cl₂, 0:100 to 3:97) to give the desired product 44

1

(4.12 g, 92%). H NMR (500 MHz, CDCl₃) δ 0.32−0.42 (m, 2H),

0.54−0.65 (m, 2H), 1.19−1.29 (m, 1H), 3.82 (d, J = 7.2 Hz, 2H), 5.24

(s, 2H), 6.00 (d, J = 7.5 Hz, 1H), 7.31−7.37 (m, 2H), 7.38−7.47 (m,

4H). LC−MS m/z 382 [M + H]⁺.

4-Benzylox-1-cyclopropylmethyl-3-triﬂuoromethyl-1H-pyri-

din-2-one (45). Methyl 2,2-diﬂuoro-2-(ﬂuorosulfonyl)acetate (0.67

mL, 5.24 mmol) and intermediate 44 were added to a solution of CuI

(0.99 g, 5.24 mmol) in DMF (30 mL). The mixture was heated at 100

°C for 5 h. The cooled mixture was ﬁltered through a Celite pad and

concentrated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica gel, DCM) to give the desired product 45

1

(0.76 g, 89%). H NMR (500 MHz, CDCl₃) δ 0.30−0.41 (m, 2H),

0.56−0.67 (m, 2H), 1.16−1.27 (m, 1H), 3.75 (d, J = 7.2 Hz, 2H), 5.22

(s, 2H), 6.08 (d, J = 7.8 Hz, 1H), 7.31−7.43 (m, 5H), 7.52 (d, J = 7.8

Hz, 1H). LC−MS m/z 324 [M + H]⁺.

4-Bromo-1-(cyclopropylmethyl)-3-(triﬂuoromethyl)pyridin-

2-one (46). A solution of 45 (2 g, 6.18 mmol) in ethanol (60 mL) was

hydrogenated (atmospheric pressure) with Pd/C (400 mg) as a

catalyst at room temperature for 2 h. The mixture was ﬁltered through

Celite pad, and the ﬁltrate was evaporated in vacuo. The residue was

dissolved in DMF (28 mL), and phosphorus oxybromide (3.54 g,

12.36 mmol) was added. The mixture was heated at 100 °C for 1 h.

The cooled mixture was partitioned between water and EtOAc. After

three extractions with EtOAc, the combined organic layers were dried

(Na₂SO₄) and concentrated in vacuo. The crude was puriﬁed by ﬂash

column chromatography (silica gel, DCM) to yield 46 (0.769 g, 42%).

¹H NMR (400 MHz, CDCl₃) δ 0.33−0.49 (m, 2H), 0.57−0.73 (m,

1-(Cyclopropylmethyl)-4-[4-(4-ﬂuorophenyl)-4-hydroxy-1-

piperidyl]-2-oxo-pyridine-3-carbonitrile (49). To a solution of 46

(0.77 g, 1.88 mmol) in CH₃CN (10 mL) were added 4-(4-ﬂuoro-

phenyl)-piperidin-4-ol (0.369 g, 1.89 mmol) and DIPEA (0.697 mL, 4

mmol). The mixture was heated under microwave irradiation at 140

°C for 30 min. The cooled mixture was evaporated in vacuo. The

residue was taken up in CH₂Cl₂and washed with saturated solution of

NaHCO₃. The combined organic layers were dried over Na₂SO₄and

concentrated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica gel, silica gel, MeOH−NH₃in CH₂Cl₂, 0:100

to 2:98); compound 49 was obtained as a white solid (0.53 g, 73%).

2H), 1.18−1.31 (m, 1H), 3.79 (d, J = 7.40 Hz, 2H), 6.51 (d, J = 7.17

Hz, 1H), 7.47 (d, J = 7.17 Hz, 1H). LC−MS m/z 296 [M + H]⁺.

4-Bromo-1-(cyclopropylmethyl)pyridin-2-one (47a). Starting

from 43a (2.196 g, 8.6 mmol) and following the procedure described

1

for 46, compound 47a was obtained (1.82 g, 93%). H NMR (400

MHz, CDCl₃) δ 0.31−0.44 (m, 2H), 0.55−0.69 (m, 2H), 1.16−1.28

6508

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

¹H NMR (500 MHz, CDCl₃) δ 0.32−0.42 (m, 2H), 0.56−0.67 (m,

6.2 Hz, 6H), 1.56−1.71 (m, 3H), 3.94−4.01 (m, 2H), 6.36 (d, J = 6.8

Hz, 1H), 7.36−7.47 (m, 3H), 7.53 (d, J = 7.0 Hz, 1H), 7.59−7.65 (m,

2H). LC−MS m/z 291 [M + H]⁺.

2H), 1.15−1.24 (m, 1H), 1.60 (br s, 1H), 1.90−1.97 (m, 2H), 2.20

(td, J = 13.2, 4.2 Hz, 2H), 3.66 (td, J = 12.9, 2.2 Hz, 2H), 3.72 (d, J =

6.9 Hz, 2H), 4.09−4.16 (m, 2H), 5.91 (d, J = 8.1 Hz, 1H), 7.07 (br t, J

= 8.7 Hz, 2H), 7.30 (d, J = 7.8 Hz, 1H), 7.44−7.51 (m, 2H). LC−MS

m/z 368 [M + H]⁺.

Biology. Membrane Preparation. CHO cells expressing the

human mGlu2 receptor were grown until they were 80% conﬂuent,

washed in ice-cold phosphate buﬀered saline, and stored at −20 °C

until membrane preparation. After thawing, cells were suspended in 50

mM Tris-HCl, pH 7.4, and collected through centrifugation for 10 min

at 23 500g at 4 °C. Cells were lysed in 5 mM hypotonic Tris-HCl, pH

7.4, and after recentrifugation for 20 min at 30 000g at 4 °C, the pellet

was homogenized with an Ultra Turrax homogenizer in 50 mM Tris-

HCl, pH 7.4. Protein concentrations were measured by the Bio-Rad

protein assay using bovine serum albumin as standard.

1-(Cyclopropylmethyl)-4-(4-phenyl-1-piperidyl)pyridin-2-

one (50a). To a solution of 47a (0.4 g, 1.58 mmol) in toluene (5 mL)

were added 4-phenylpiperidine (0.509 g, 3.16 mmol), Pd(OAc)₂

(0.017 g, 0.079 mmol), NaO^tBu (0.38 g, 3.95 mmol), and BINAP

(0.073 g, 0.11 mmol). The mixture was heated at 100 °C for 16 h in a

sealed tube. The cooled mixture was partitioned between water and

EtOAc. The crude was extracted with EtOAc (3 × 5 mL), and the

combined organic layers were dried (Na₂SO₄) and concentrated in

vacuo. The crude was puriﬁed by ﬂash column chromatography (silica

gel, silica gel, MeOH−NH₃in CH₂Cl₂, 0:100 to 1:99); compound 50a

was obtained as brown oil (0.48 g, 98%). ¹H NMR (500 MHz, CDCl₃)

δ 0.31−0.42 (m, 2H), 0.53−0.64 (m, 2H), 1.19−1.28 (m, 1H), 1.77

(qd, J = 12.7, 3.9 Hz, 2H), 1.89−1.96 (m, 2H), 2.76 (tt, J = 12.1, 3.6

Hz, 1H), 2.96 (td, J = 12.8, 2.2 Hz, 2H), 3.73 (d, J = 7.2 Hz, 2H),

3.90−3.97 (m, 2H), 5.82 (d, J = 2.9 Hz, 1H), 5.98 (dd, J = 7.8, 2.9 Hz,

1H), 7.17−7.26 (m, 4H), 7.30−7.35 (m, 2H). LC−MS m/z 309 [M +

H]⁺.

1-Isobutyl-4-(4-phenyl-1-piperidyl)pyridin-2-one (50b). To a

solution of 47b (2 g, 8.69 mmol) in toluene (35 mL) were added 4-

phenylpiperidine (1.962 g, 12.16 mmol), Pd(OAc)₂(0.098 g, 0.43

mmol), NaO^tBu (2.088 g, 21.72 mmol), and 2,2′-bis (diphenylphos-

phino)-1,1′-binaphthyl (0.406 g, 0.652 mmol). The mixture was

heated at 100 °C for 16 h in a sealed tube. The cooled mixture was

partitioned between water and EtOAc. The crude was extracted with

EtOAc (3 × 5 mL), and the combined organic layers were dried

(Na₂SO₄) and concentrated in vacuo. The crude was puriﬁed by ﬂash

column chromatography (silica gel, silica gel, MeOH in CH₂Cl₂, 0:100

to 4:96); compound 50b was obtained as a white solid (1.9 g, 65%).

¹H NMR (500 MHz, CDCl₃) δ 0.93 (d, J = 6.6 Hz, 6H), 1.76 (qd, J =

[³⁵S]GTPγS Binding Assay. For [³⁵S]GTPγS measurements,

compound and glutamate were diluted in buﬀer containing 10 mM

HEPES acid, 10 mM HEPES salt, pH 7.4, containing 100 mM NaCl, 3

mM MgCl₂, and 10 μM GDP. Membranes were thawed on ice and

diluted in the same buﬀer, supplemented with 14 μg/mL saponin

(ﬁnal assay concentration of 2 μg/mL saponin). Final assay mixtures

contained 7 μg of membrane protein and were preincubated with

compound alone (determination of agonist eﬀects) or together with an

EC₂₀concentration (4 μM) of glutamate (determination of PAM

eﬀects) for 30 min at 30 °C. [³⁵S]GTPγS was added at a ﬁnal

concentration of 0.1 nM and incubated for another 30 min at 30 °C.

Reactions were terminated by rapid ﬁltration through Uniﬁlter-96 GF/

B ﬁlter plates (Packard) using a 96-well Packard ﬁltermate harvester.

Filters were washed six times with ice-cold 10 mM NaH₂PO₄/10 mM

Na₂HPO₄, pH 7.4, and ﬁlter-bound radioactivity was counted in a

microplate scintillation and luminescence counter from Packard.

Data Analysis. Data were processed using an internal software

interface and were calculated as the percent of the control agonist

challenge. Sigmoid concentration−response curves plotting these

percentages versus the log concentration of the test compound were

analyzed using nonlinear regression analysis. The EC₅₀is the

concentration of a compound that causes a half-maximal potentiation

of the glutamate response.The pEC₅₀values are calculated as the −log

EC₅₀(wherein EC₅₀is expressed in mol L⁻¹).

Patch-Clamp Assay. Experiments were performed using HEK293

cells stably expressing the hERG potassium channel. Cells were grown

at 37 °C and 5% CO₂in culture ﬂasks in MEM medium supplemented

with 10% heat-inactivated fetal bovine serum, 1% ^L-glutamine−

penicillin−streptomycin solution, 1% nonessential amino acids

(100×), 1% sodium pyruvate (100 mM), and 0.8% Geneticin (50

mg/mL). Before use, the cells were subcultured in MEM medium in

the absence of 5 mL of ^L-glutamine−penicillin−streptomycin. For use

in the automated patch-clamp system PatchXpress 7000A (Axon

Instruments), cells were harvested to obtain cell suspension of single

cells. The extracellular solution contained the following (mM): 150

NaCl, 4 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, 5 glucose (pH 7.4 with

NaOH). The pipette solution contained the following (mM): 120

KCl, 10 HEPES, 5 EGTA, 4 ATP-Mg2, 2 MgCl₂, 0.5 CaCl₂(pH 7.2

with KOH). Patch-clamp experiments were performed in the voltage-

clamp mode, and whole-cell currents were recorded with an automated

patch-clamp assay utilizing the PatchXpress 7000A system (Axon

Instruments). Current signals were ampliﬁed and digitized by a

multiclamp ampliﬁer, stored, and analyzed by using the PatchXpress

and DataXpress software and Igor 5.0 (Wavemetrics). The holding

potential was −80 mV. The hERG current (K⁺-selective outward

current) was determined as the maximal tail current at −40 mV after a

2 s depolarization to +60 mV. The pulse cycling rate was 15 s. Before

each test pulse, a short pulse (0.5 s) from the holding potential to −60

mV was given to determine (linear) leak current. After establishment

of whole-cell conﬁguration and a stability period, the vehicle was

applied for 5 min followed by the test substance at increasing

concentrations of 10⁻⁷, 3 × 10⁻⁷, and 3 × 10⁻⁶M. Each concentration

of the test substance was applied twice. The eﬀect of each

concentration was determined after 5 min as an average current of

three sequential voltage pulses. To determine the extent of block, the

residual current was compared with vehicle pretreatment.

12.8, 3.5 Hz, 2H), 1.87−1.94 (m, 2H), 2.11−2.21 (m, 1H), 2.74 (tt, J

= 12.1, 3.5 Hz, 1H), 2.94 (td, J = 12.8, 2.5 Hz, 2H), 3.64 (d, J = 7.5

Hz, 2H), 3.88−3.95 (m, 2H), 5.78 (d, J = 2.9 Hz, 1H), 5.93 (dd, J =

7.8, 2.6 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 7.17−7.25 (m, 3H), 7.28−

7.34 (m, 2H). LC−MS m/z 311 [M + H]⁺.

1-Butyl-4-(4-phenyl-1-piperidyl)pyridin-2-one (50c). To a

solution of 47c (2 g, 8.69 mmol) in toluene (75 mL) were added 4-

phenylpiperidine (2.1 g, 13.038 mmol), Pd(OAc)₂(0.98 g,0.435

mmol), NaO^tBu (2.09 g, 21.73 mmol), and BINAP (0.406 g,0.652

mmol). The mixture was heated at 100 °C for 16 h in a sealed tube.

The cooled mixture was partitioned between water and EtOAc. The

crude was extracted with EtOAc (3 × 5 mL), and the combined

organic layers were dried over Na₂SO₄and concentrated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica gel, silica

gel, MeOH−NH₃in CH₂Cl₂, 0:100 to 4:96); compound 50c was

obtained (1.8 g, 66%). ¹H NMR (400 MHz, CDCl₃) δ 0.95 (t, J = 7.4

Hz, 3H), 1.31−1.43 (m, 2H), 1.65−1.82 (m, 4H), 1.86−1.95 (m, 2H),

2.74 (tt, J = 12.1, 3.7 Hz, 1H), 2.94 (td, J = 12.8, 2.5 Hz, 2H), 3.84 (t, J

= 7.4 Hz, 2H), 3.88−3.95 (m, 2H), 5.79 (d, J = 2.8 Hz, 1H), 5.95 (dd,

J = 7.7, 2.9 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 7.17−7.25 (m, 3H),

7.28−7.35 (m, 2H). LC−MS m/z 311 [M + H]⁺.

1-Isopentyl-2-oxo-4-(2-phenylethynyl)pyridine-3-carboni-

trile (51). To a solution of 41d (0.15 g, 0.6 mmol) in THF (6 mL)

were added phenylacetylene (0.064 mL, 0.6 mmol), PdCl₂(PPh₃)₂

(0.02 g, 0.028 mmol), Et₃N (0.0787 mL, 2.2 mmol), and PPh₃(0.0037

g, 0.014 mmol). The mixture was degassed for 5 min, and then CuI

(0.0013 g, 0.007 mmol) was added. The mixture was heated at 90 °C

for 10 h in a sealed tube. The mixture was cooled to room temperature

and treated with aqueous Na₂S₂O₄and CH₂Cl₂. The layers were

separated, the organic layer was washed with a saturated aqueous

solution of NaHCO₃, and the combined organic layers were dried

(Na₂SO₄) and concentrated in vacuo. The crude was puriﬁed by

automated ﬂash column chromatography over silica gel (silica gel,

silica gel, MeOH−NH₃in CH₂Cl₂, 0:100 to 2:98); compound 51 was

1

obtained (0.097 g, 60%). H NMR (400 MHz, CDCl₃) δ 0.97 (d, J =

6509

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

Sleep−Wake EEG: Animals, Drug Treatment, and Exper-

imental Procedure. All in vivo experimental procedures were

performed according to the applicable European Communities

Council Directive of November 24, 1986 (86/609/EEC) and

approved by the Animal Care and Use Committee of Janssen

Pharmaceutical Companies of Johnson & Johnson and by the local

ethical committee. Male Sprague−Dawley rats (Charles River, France)

weighing 250−300 g were used. Animals were chronically implanted

with electrodes for recording the cortical electroencephalogram

(EEG), electrical neck muscle activity (EMG), and ocular movements

(EOG). All animals were housed in individually ventilated cages under

environmentally controlled conditions (ambient temperature, 22 °C;

humidity, 60%) on a 12 h light/dark cycle (lights on from 12:00 a.m.

to 12:00 p.m., illumination intensity of ∼100 lx). The animals had free

access to food and tap water. The eﬀects of the tested molecule and

vehicle on sleep−wake distribution during the lights-on period were

investigated in 16 rats (n = 8 each group). Two EEG recording

sessions were performed: the ﬁrst recording session started at 13:30 h

and lasted 20 h following oral administration of saline. The second

recording session was performed during the same consecutive

circadian time and for the same duration following administration of

either vehicle (20% CD + 2H2T) or tested compound. Sleep

polysomnographic variables were determined oﬄine as described

elsewhere using a sleep stages analyzer, scoring each 2 s epoch before

averaging stages over 30 min periods. Sleep−wake state classiﬁcations

were assigned on the basis of the combination of dynamics of ﬁve EEG

frequency domains, integrated EMG, EOG, and body activity level:

active wake (AW); passive wake (PW); intermediate stage (pre-REM

transients); rapid eye movement sleep (REM); light non-REM sleep

(lSWS); deep non-REM sleep (dSWS). Diﬀerent sleep−wake

parameters were investigated over 20 h postadministration, and time

spent in each vigilance state, sleep parameters, latencies for ﬁrst REM

sleep period and the number of transitions between states were

determined.

GCMS, gas chromatography mass spectrometry; GPCR, G-

protein-coupled receptor; h, hours; HLM, human liver

microsome; HP-β-CD, 2-hydroxylpropyl-β-cyclodextrin; HTS,

high-throughput screening; iGlu, ionotropic glutamate; iv,

intravenous; LAD, lowest active dose; LC−MS, liquid

chromatography combined with mass spectrometry; mGlu2,

metabotropic glutamate 2; min, minutes; lSWS, light non-REM

sleep; MDD, major depressive disorder; MeOH, methanol; mp,

melting point; MW, molecular weight; NCS, N-chlorosuccini-

mide; ND, not determined; NM, not measurable; NIS, N-

iodosuccinimide; NMDA, N-methyl-^D-aspartate; NMR, nuclear

magnetic resonance; NOEL, no eﬀect level; NT, not tested;

PAM, positive allosteric modulator; PCP, phencyclidine; PK,

pharmacokinetics; po, per oral; PW, passive wake; SAR,

structure−activity relationship; SEM, standard error of mean;

po, oral; REM, rapid eye movement; RLM, rat liver microsome;

ROL, REM sleep onset latency; rt, room temperature; RP-

HPLC, reverse-phase high-performance liquid chromatography;

sc, subcutaneous; SD, standard deviation; sw-EEG, sleep−wake

electroencephalogram; TFA, triﬂuoroacetic acid; THF, tetrahy-

drofuran; TLC, thin-layer chromatography; TPSA, total polar

surface area; V_d, volume of distribution

REFERENCES

■

(1) Kew, J. N.; Kemp, J. A. Ionotropic and metabotropic glutamate

receptor, structure and pharmacology. Psychoharmacology 2005, 182,

320.

(2) Pin, J. P.; Galvez, T.; Prezeau, L. Evolution, structure, and

activation mechanism of family 3/C G-protein-coupled receptors.

Pharmacol. Ther. 2003, 98, 325−354.

(3) Brauner-Osborne, H.; Wellendorph, P.; Jensen, A. A. Structure,

pharmacology and therapeutic prospects of family C G-protein

coupled receptors. Curr. Drug Targets 2007, 8, 169−184.

(4) Lavreysen, H.; Dautzenberg, F. Therapeutic potential of group III

metabotropic glutamate receptors. Curr. Med. Chem. 2008, 15, 671−

684.

(5) Niswender, C. M.; Conn, P. J. Metabotropic glutamate receptors:

physiology, pharmacology, and disease. Annu. Rev. Pharmacol. Toxicol.

2010, 50, 295−322.

(6) Wright, R. A.; Johnson, B. G.; Zhang, C.; Salhoff, C.; Kingston, A.

E.; Calligaro, D. O.; Monn, J. A.; Schoepp, D. D.; Marek, G. J. CNS

distribution of metabotropic glutamate 2 and 3 receptors: transgenic

mice and [3H]LY459477 autoradiography. Neuropharmacology 2013,

66, 89−98.

(7) Schoepp, D. D.; Marek, G. J. Preclinical pharmacology of

mGluR2/3 receptor agonists: novel agents for schizophrenia? Curr.

Drug Targets: CNS Neurol. Disord. 2002, 1, 215−225.

(8) Moghaddam, B.; Adams, B. W. Reversal of phencyclidine effects

by a group II metabotropic glutamate receptor agonist in rats. Science

1998, 281, 477−484.

(9) Cartmell, J.; Monn, J. A.; Schoepp, D. D. The metabotropic

glutamate 2/3 receptor agonists LY354740 and LY379268 selectively

attenuate phencyclidine versus d-amphetamine motor behaviors in

rats. J. Pharmacol. Exp. Ther. 1999, 291, 161−170.

(10) Patil, S. T.; Zhang, L.; Martenyi, F.; Lowe, S. L.; Jackson, K. A.;

Andreev, B. V.; Avedisova, A. S.; Bardenstein, L. M.; Gurovich, I. Y.;

Morozova, M. A.; Mosolov, S. N.; Neznanov, N. G.; Reznik, A. M.;

Smulevich, A. B.; Tochilov, V. A.; Johnson, B. G.; Monn, J. A.;

Schoepp, D. D. Activation of mGlu2/3 receptors as a new approach to

treat schizophrenia: a randomized Phase 2 clinical trial. Nat. Med.

2007, 13, 1102−1107.

Statistical Analysis. Time spent in each vigilance state (AW, PW,

lSWS, dSWS, IS, and REMS) was expressed as a percentage of the

recording period. A statistical analysis of the obtained data was carried

out by a nonparametric analysis of variance of each 30 min period,

followed by a Wilcoxon−Mann−Whitney rank sum test of

comparisons with the control group.

AUTHOR INFORMATION

Corresponding Author

*Tel: +34 925 245767. Fax: +34 925 245771. E-mail: jcid@its.

jnj.com.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank Addex Therapeutics for its collaboration on

the mGlu2PAM discovery program, members of the

puriﬁcation and analysis team from Toledo, the biology and

ADMET team from Janssen R & D, and Lieve Heylen, Heidi

Szel, and Ria Wouters for technical assistance.

ABBREVIATIONS USED

■

AcOH, acetic acid; ADME, absorption, distribution, metabo-

lism, excretion; AUC, area under the curve; AW, active wake;

BBB, blood−brain barrier; BINAP, 2, 2′-bis-

(diphenylphosphino)-1,1′-binaphthyl; B/P, brain plasma ratio;

Cl, clearance; C_max, maximum plasma concentrations; CNS,

central nervous system; CV, cardiovascular; dSWS, deep non-

REM sleep; DCM, dichloromethane; DIPEA, diisopropylethyl-

amine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;

EEG, electroencephalogram; EMG, electrical neck muscle

activity; EOG, ocular movement; EtOAc, ethyl acetate;

(11) Kinon, B. J.; Zhang, L.; Millen, B. A.; Osuntokun, O. O.;

Williams, J. E.; Kollack-Walker, S.; Kimberley, J.; Ludmila, K.; Natalia,

J. A multicenter, inpatient, phase 2, double-blind, placebo-controled

dose-ranging study of LY2140023 monohydrate in patients with DSM-

IV schizophrenia. J. Clin. Psychopharmacol. 2011, 31, 349−355.

6510

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

(12) Kinon, B. J.; Go

pomaglumetad methionil: a non-dopaminergic treatment for schizo-

phrenia. Neuropharmacology 2013, 66, 82−86.

(13) Lilly Stops Phase III development of pomaglumetad methionil for

the treatment of schizophrenia based on eﬃcacy results; Eli Lilly and Co.:

Indianapolis, IN, 2012; https://investor.lilly.com/releasedetail.

cfm?ReleaseID=703018.

(14) Schoepp, D. D.; Wright, R. A.; Levine, L. R.; Gaydos, B.; Potter,

W. Z. LY354740, an mGlu2/3 receptor agonist as a novel approach to

treat anxiety/stress. Stress 2003, 6, 189−197.

(15) Woolley, M. L.; Pemberton, D. J.; Bate, S.; Corti, C.; Jones, D.

N. C. The mGlu2 but not the mGlu3 receptor mediates the actions of

the mGluR2/3 agonist, LY379268, in mouse models predictive of

antipsychotic activity. Psychopharmacology 2008, 196, 431−440.

(16) Fell, M. J.; Svensson, K. A.; Johnson, B. G.; Schoepp, D. D.

Evidence for the role of mGlu2 not mGlu3 receptors in the preclinical

antipsychotic pharmacology of the mGlu2/3 receptor agonist

LY404039. J. Pharmacol. Exp. Ther. 2008, 326, 209−217.

(17) Galici, R.; Echemendia, N. G.; Rodriguez, A. L.; Conn, P. J. A

selective allosteric potentiator of metabotropic glutamate (mGlu) 2

receptors has effects similar to an orthosteric mGlu2/3 receptor

agonist in mouse models predictive of antipsychotic activity. J.

Pharmacol. Exp. Ther. 2005, 315, 1181−1187.

(18) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Allosteric

modulators of GPCRs: a novel approach for the treatment of CNS

disorders. Nat. Rev. Drug. Discovery 2009, 8, 41−54.

(19) Pin, J. P.; Parmentier, M.-L.; Prezeau, L. Positive allosteric

modulators for g-aminobutyric acidB receptors open new routes for

the development of drugs targeting family 3 G-protein-coupled

receptors. Mol. Pharmacol. 2001, 60, 881−884.

(20) Gjoni, T.; Urwyler, S. Receptor activation involving positive

allosteric modulation, unlike full agonism, does not result in GABAB

receptor desensitization. Neuropharmacology 2008, 55, 1293−1299.

(21) Trabanco, A. A.; Cid, J. M.; Lavreysen, H.; Macdonald, G. J.;

Tresadern, G. Progress in the development of positive allosteric

modulators of the metabotropic glutamate receptor 2. Curr. Med.

Chem. 2011, 18, 47−68.

(22) Trabanco, A. A.; Cid, J. M. mGluR2 positive allosteric

modulators (PAMs): a patent review (2009−present). Expert Opin.

Ther. Patents 2013, 23, 629−647.

(23) Lorrain, D. S.; Schaffhauser, H.; Campbell, U. C.; Baccei, C. S.;

Correa, L. D.; Rowe, B.; Rodriguez, D. E.; Anderson, J. J.; Varney, M.

A.; Pinkerton, A. B.; Vernier, J.-M.; Bristow, L. J. Group II mGlu

receptor activation suppresses norepinephrine release in the ventral

hippocampus and locomotor responses to acute ketamine challenge.

Neuropsychopharmacology 2003, 28, 1622−1632.

(24) Johnson, M. P.; Baez, M.; Jagdmann, G. E.; Britton, T. C.; Large,

T. H.; Callagaro, D. O.; Tizzano, J. P.; Monn, J. A.; Schoepp, D. D.

Discovery of allosteric potentiators for the metabotropic glutamate 2

receptor: synthesis and subtype selectivity of N-(4-(2-

methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-yl-

methylamine. J. Med. Chem. 2003, 46, 3189−3192.

(25) Pinkerton, A. B.; Vernier, J.-M.; Schaffhauser, H.; Rowe, B. A.;

Campbell, U. C.; Rodriguez, D. E.; Lorrain, D. S.; Baccei, C. S.;

Daggett, L. P.; Bristow, L. J. Phenyl-tetrazolyl acetophenones:

discovery of positive allosteric potentiators for the metabotropic

glutamate 2 receptor. J. Med. Chem. 2004, 47, 4595−4599.

(26) Fell, M. J.; Witkin, J. M.; Falcone, J. F.; Katner, J. S.; Perry, K.

W.; Hart, J.; Rorick-Kehn, L.; Overshiner, C. D.; Rasmussen, K.;

Chaney, S. F.; Benvenga, M. J.; Li, X.; Marlow, D. L.; Thompson, L.

K.; Lueck, S. K.; Wafford, K. A.; Seidel, W. F.; Edgar, D. M.; Quests, A.

T.; Felder, C. C.; Wang, X.; Heinz, B. A.; Nikolayev, A.; Kuo, M. S.;

Mayhugh, D.; Khilevich, A.; Zhang, D.; Ebert, P. J.; Eckstein, J. A.;

Ackermann, B. L.; Swanson, S. P.; Catlow, J. T.; Dean, R. A.; Jackson,

K.; Tauscher-Wisniewski, S.; Marek, G. J.; Schkeryantz, J. M.;

Svensson, K. A. N-(4-((2-(Trifluoromethyl)-3-hydroxy-4-(isobutyryl)-

phenoxy)methyl) benzyl)-1-methyl-1H-imidazole-4-carboxamide

(THIIC), a novel metabotropic glutamate 2 potentiator with potential

anxiolytic/antidepressant properties: in vivo profiling suggests a link

́

mez, J. C. Clinical development of

between behavioral and central nervous system neurochemical change.

J. Pharmacol. Exp. Ther. 2011, 336, 165−177.

(27) Johnson, P. L.; Fitz, S. D.; Engleman, E. A.; Svensson, K. A.;

Schkeryantz, J. M.; Shekhar, A. Group II metabotropic glutamate

receptor type 2 allosteric potentiators prevent sodium lactate-induced

panic-like response in panic-vulnerable rats. J. Psychopharmacol. 2013,

27, 152−61.

(28) Cid, J. M.; Duvey, G.; Cluzeau, P.; Nhem, V.; Macary, K.; Raux,

A.; Poirier, N.; Muller, J.; Bolea, C.; Finn, T.; Urios, I.; Epping-Jordan,

M.; Chamelot, E.; Derouet, F.; Girard, F.; MacDonald, G. J.; Vega, J.

A.; de Lucas, A. I.; Matesanz, E.; Lavreysen, H.; Linares, M. L.;

Oehlrich, D.; Oyarzabal, J.; Tresadern, G.; Trabanco, A. A.; Andres, J.

I.; Le Poul, E.; Imogai, H.; Lutjens, R.; Rocher, J.-P. Discovery of 1,5-

disubstituted pyridones: a new class of positive allosteric modulators of

the metabotropic glutamate 2 receptor. ACS Chem. Neurosci. 2010, 1,

788−795.

(29) Cid, J. M.; Duvey, G.; Tresadern, G.; Nhem, V.; Funary, R.;

Cluzeau, P.; Vega, J. A.; de Lucas, A. I.; Matesanz, E.; Alonso, J. M.;

Linares, M. L.; Andres, J. I.; Poli, S.; Lutjens, R.; Imogai, H.; Rocher, J.-

P.; MacDonald, G. J.; Oehlrich, D.; Lavreysen, H.; Ahnaou, A.;

Drinkenburg, W.; Mackie, C.; Trabanco, A. A. Discovery of 1,4-

disubstituted 3-cyano-2-pyridones: a new class of positive allosteric

modulators of the metabotropic glutamate 2 receptor. J. Med. Chem.

2012, 55, 2388−2405.

(30) Tresadern, G.; Cid, J. M.; MacDonald, G. J.; Vega, J. A.; de

Lucas, A. I.; Garcia, A.; Matesanz, E.; Linares, M. L.; Oehlrich, D.;

Lavreysen, H.; Biesmans, Ilse; Trabanco, A. A. Scaffold hopping from

pyridones to imidazo[1,2-a]pyridines. new positive allosteric modu-

lators of metabotropic glutamate 2 receptor. Bioorg. Med. Chem. Lett.

2010, 20, 175−179.

(31) Trabanco, A. A.; Tresadern, G.; MacDonald, G. J.; Vega, J. A.;

de Lucas, A. I.; Matesanz, E.; Garcia, A.; Linares, M. L.; Alonso de

Diego, S. A.; Alonso, J. M.; Oehlrich, D.; Ahnaou, A.; Drinkenburg,

W.; Mackie, C.; Andres, J. I.; Lavreysen, H.; Cid, J. M. Imidazo[1,2-

a]pyridines: orally active positive allosteric modulators of the

metabotropic glutamate 2 receptor. J. Med. Chem. 2012, 55, 2688−

2701.

(32) Cid, J. M.; Tresadern, G.; Vega, J. A.; de Lucas, A. I.; Matesanz,

E.; Iturrino, L.; Linares, M. L.; Garcia, A.; Andres, J. I.; MacDonald, G.

J.; Ohelrich, G.; Lavreysen, H.; Megens, A.; Ahnaou, A.; Drinkenburg,

W.; Mackie, C.; Pype, S.; Gallacher, D.; Trabanco, A. A. Discovery of

3-cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl-

1,2,4-triazolo[4,3-a]pyridine (JNJ-42153605): a positive allosteric

modulator of the metabotropic glutamate 2 receptor. J. Med. Chem.

2012, 55, 8770−8789.

(33) Kent, J.; Anghelescu, I.-G.; Kezic, I.; Daly, E.; Ceusters, M.; De

Smedt, H.; Van Nueten, L.; De Boer, P. Safety, tolerability and potential

therapeutic eﬃcacy of a novel glutamate modulator as adjunctive treatment

in patients with schizophrenia, 166th Annual Meeting of the American

Psychiatric Association, San Francisco, CA, May 18−22, 2013;

Abstract, 3160-P.

(34) Investigation of the safety, tolerability and potential therapeutic

eﬀects of JNJ-40411813 in patients with schizophrenia. http://

clinicaltrials.gov/show/NCT01323205.

(35) Hopkins, C. R. Is there a path forward for mGlu2 positive

allosteric modulators for the treatment of schizophrenia? ACS Chem.

Neurosci. 2013, 4, 211−213.

(36) AstraZeneca 2010 ﬁnancial results. http://www.astrazeneca.

com/Investors/ﬁ nancial-information/Financial-results/2010-

Financial-results.

(37) Litman, R. E.; Smith, M. A.; Doherty, J.; Cross, A.; Raines, S.;

Zukin, S. AZD8524, a positive allosteric modulator at the mGluR2

receptor, does not improve symptoms in schizophrenia: a proof of principle

study, 53rd NCDEU: an annual meeting sponsored by ASCP,

Hollywood, Florida, May 28−31, 2013; Poster 83.

(38) Addex reports top-line data from a successful phase 2a clinical

study with ADX71149 in schizophrenia patients. http://www.

addextherapeutics.com/investors/press-releases/news-details/article/

6511

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Journal of Medicinal Chemistry

Article

addex-reports-top-line-data-from-a-successful-phase-2a-clinical-study-

with-adx71149-in-schizophrenia/.

IL8B, CXCR2, M1, M2, M3, MC4, NET, NK2, NK3, NPY1, NPY2,

NT1, OP1, OP3, ORL1, V1A, VIP, SST, 5HT1B, α1, α2, BZD, CaCH,

CICH, GABA, KCH, NaCH, and SKCaCH.

(39) Lavreysen, H. Discovery and early clinical development of novel

mGlu2 receptor PAMs, American College of Neuropsychopharmacol-

ogy 52nd Annual Meeting, Hollywood, Florida, Dec 8−12, 2013.

(40) A study of JNJ-40411813 as supplementary treatment to an

antidepressant in adults with depression and anxiety symptoms.

http://clinicaltrials.gov/show/NCT01582815.

(41) Addex Reports top-line data from ADX71149 phase 2a study in

patients with major depressive disorder (MDD) with signiﬁcant

anxiety symptoms. http://www.addextherapeutics.com/investors/

press-releases/news-details/article/addex-reports-top-line-data-from-

adx71149-phase-2a-study-in-patients-with-major-depressive-disorder/.

(42) Cid-Nunez, J. M.; Trabanco-Suarez, A. A.; Macdonald, G. J.;

̃

Duvey, G. A. J.; Lutjens, R. J. Preparation of 1,4-disubstituted 3-

̈

cyanopyridone derivatives as positive mGluR2 receptor modulators. WO/

2008/107480, 2008.

(43) Cid-Nunez, J. M.; Trabanco-Suar

́

ez, A. A.; Macdonald, G. J.;

̃

Duvey, G. A. J.; Lutjens, R. J. Preparation of 3-cyano-4-(4-phenyl-

̈

piperidin-1-yl)-pyridin-2-one derivatives for treating and preventing neurol.

and psychiatric diseases associated with glutamate dysfunction. WO/

2008/107479, 2008.

(44) Cid-Nunez, J. M.; Trabanco-Suar

́

ez, A. A.; Macdonald, G. J.;

̃

Duvey, G. A. J.; Lutjens, R. J.; Finn, T. P. Preparation of bipyridinones as

̈

positive allosteric modulators of mGluR2 for treating neurological and

psychiatric disorders. WO/2009/033704, 2009.

(45) The in vitro [³⁵S]GTPγS assay provides two measures of

compound activity: pEC₅₀, which is deﬁned as the negative logarithm

of the concentration at which half-maximal potentiation of glutamate is

reached, and the maximal increase in observed glutamate response, the

% E_MAX

.

(46) Compound 6 was incubated with human and rat liver

microsomes at 0.4% ﬁnal solvent concentration (0.16% DMSO and

0.24% CH₃CN). The study was conducted to identify the main

metabolites formed after 0 min (control) and 60 min of incubation

time in the presence of an NADPH generating system at 37 °C. Test

concentration of 6 was 5 μM. Samples were compared to the control

incubations in which compound was added at termination. Data were

acquired on a Waters UPLC/QToF Premier mass spectrometer using

a 10 min generic UPLC method and a generic MSe method.

Complementary MS/MS experiments were performed when neces-

sary. An Acquity UPLC C18 (2.1 mm × 100 mm, 1.8 μm) column was

used. Interpretation of data was performed with Waters Metabolynx

software.

(47) Lavreysen, H.; Langlois, X.; Ahnaou, A.; Drinkenburg, W.; te

Riele, P.; Biesmans, I.; Van der Linden, I.; Peeters, L.; Megens, A.;

Wintmolders, C.; Cid, J.; Trabanco, A. A.; Andres, I.; Dautzenberg, F.

M.; Lutjens, R.; Macdonald, G.; Atack, J. R. Pharmacological

characterization of JNJ-40068782, a new potent, selective and

systemically active positive allosteric modulator of the mGlu2 receptor

and its radioligand [3H]JNJ-40068782. J. Pharmacol. Exp. Ther. 2013,

346, 514−527.

(48) Feinberg, I.; Campbell, I. G.; Schoepp, D. D.; Anderson, K. The

selective group mGlu2/3 receptor agonist LY379268 suppresses REM

sleep and fast EEG in the rat. Pharmacol., Biochem. Behav. 2002, 73,

467−74.

(49) Ahnaou, A.; Dautzenberg, F. M.; Geys, H.; Imogai, H.; Gibelin,

A.; Moechars, D.; Steckler, T.; Drinkenburg, W. H. Modulation of

group II metabotropic glutamate receptor (mGlu2) elicits common

changes in rat and mice sleep−wake architecture. Eur. J. Pharmacol.

2009, 603, 62−72.

(50) Compound 36 was tested for agonist or antagonist activity on

mGlu receptors in ﬂuorescent Ca²⁺assays using HEK293 cells

expressing human mGlu1, mGlu3, and mGlu5 receptors or [³⁵S]

GTPgS assays using L929 cells expressing human mGlu4 or CHO cells

expressing rat mGlu6 receptors.

(51) The CEREP selectivity screen was performed on the following

targets: 5HT1A, 5HT2A, 5HT3, 5HT5A, 5HT6, 5HT7, A1, A2A, A3,

AT1, Beta1, BK2, CCKA, CCR1, D1, D2, DAT, ETA, GAL2, H1, H2,

6512

dx.doi.org/10.1021/jm500496m | J. Med. Chem. 2014, 57, 6495−6512

Article Doi

Discovery of 1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-(1 H)-pyridone (JNJ-40411813): A novel positive allosteric modulator of the metabotropic glutamate 2 receptor

DOI: 10.1021/jm500496m

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm500496m

Full text of DOI:10.1021/jm500496m

Products guided by the article

R&D Labs maybe for 1127499-32-4

Relevant to this article

Hot Product