Tetrahydronaphthyridine and dihydronaphthyridinone ethers as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu 5)

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Tetrahydronaphthyridine and Dihydronaphthyridinone Ethers As

Positive Allosteric Modulators of the Metabotropic Glutamate

Receptor 5 (mGlu₅)

Mark Turlington,^†,‡,§Chrysa Malosh,^†,‡,§Jon Jacobs,^†,‡,§Jason T. Manka,^†,‡,§Meredith J. Noetzel,^†,‡

Paige N. Vinson,^†,‡Satyawan Jadhav,^†,‡Elizabeth J. Herman,^†,‡Hilde Lavreysen,^#Claire Mackie,^∇

Jose M. Bartolome-Nebreda,^○Susana Conde-Ceide,^○M. Luz Martín-Martín,^○Han Min Tong,^○

́

Silvia Lopez,^○Gregor J. MacDonald,^#Thomas Steckler,^#J. Scott Daniels,^†,‡,§C. David Weaver,^†,⊥

́

Colleen M. Niswender,^†,‡,§Carrie K. Jones,^†,‡,§P. Jeﬀrey Conn,^†,‡,§Craig W. Lindsley,^{†,‡,§,∥}

and Shaun R. Stauﬀer*^{,†,‡,§,∥}

^†Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States

^‡Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, 1205 Light Hall, Nashville, Tennessee

37232-0697, United States

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

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

^⊥Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States

^#Neuroscience, Janssen Research and Development, Turnhoutseweg 30, B-2340, Beerse, Belgium

^∇Discovery Sciences ADME/Tox, Janssen Research and Development, Turnhoutseweg 30, B-2340, Beerse, Belgium

^○Neuroscience Medicinal Chemistry, Janssen Research and Development, Jarama 75, 45007 Toledo, Spain

S

* Supporting Information

ABSTRACT: Positive allosteric modulators (PAMs) of metabotropic glutamate

receptor 5 (mGlu₅) represent a promising therapeutic strategy for the treatment of

schizophrenia. Starting from an acetylene-based lead from high throughput

screening, an evolved bicyclic dihydronaphthyridinone was identiﬁed. We describe

further reﬁnements leading to both dihydronaphthyridinone and tetrahydronaph-

thyridine mGlu₅PAMs containing an alkoxy-based linkage as an acetylene

replacement. Exploration of several structural features including western pyridine

ring isomers, positional amides, linker connectivity/position, and combinations

thereof, reveal that these bicyclic modulators generally exhibit steep SAR and within

speciﬁc subseries display a propensity for pharmacological mode switching at mGlu₅

as well as antagonist activity at mGlu₃. Structure−activity relationships within a

dihydronaphthyridinone subseries uncovered 12c (VU0405372), a selective mGlu₅

PAM with good in vitro potency, low glutamate fold-shift, acceptable DMPK

properties, and in vivo eﬃcacy in an amphetamine-based model of psychosis.

proﬁles that plague both classes of antipsychotics has been

gained. These include extrapyramidal side eﬀects, sexual

dysfunction, weight gain (metabolic syndrome), agranulocy-

tosis, increased cardiac risk, and poor patient compliance.^3,6,7

The development of positive allosteric modulators (PAMs)

of metabotropic glutamate receptor 5 (mGlu₅)⁸as a novel

approach to test the glutamate or N-methyl-^D-aspartate

(NMDA) receptor hypofunction hypothesis of schizophre-

nia⁹⁻¹⁴has provided preclinical evidence for therapeutic

potential in multiple psychosis and cognitive animal mod-

INTRODUCTION

■

Schizophrenia is a complex mental illness characterized by

positive (hallucinations, paranoia, disorganized behavior) and

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

well as cognitive dysfunction (deﬁcits in attention, learning, and

memory).¹⁻⁴Current treatments, including typical and second-

generation atypical antipsychotics, are based largely upon the

dopaminergic hypothesis of schizophrenia, which targets

overactivation of subcortical dopamine D₂receptors.⁵Both

classes treat the positive symptoms; however, neither class of

antipsychotics has made a substantial impact on the negative

and cognitive symptoms.¹⁻⁴After several decades of clinical

use, a signiﬁcant understanding of the risks and side eﬀect

Received: February 18, 2014

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els,^10,12−14and, for more than a decade, industry and academic

drug discovery groups have been in pursuit of brain penetrant

small molecule mGlu₅potentiators.^15,16A testament to the

success of the ﬁeld has been the identiﬁcation of over eight

reported chemotypes^15,16with in vivo eﬃcacy in preclinical

models, beginning with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-

yl)benzamide (CDPPB, 1),¹⁷piperidinyl 1,2,4-oxadiazoles from

Addex (ADX-47273, 2),^18,19and subsequently acetylenes N-

methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA, 3),²⁰

VU0360172 (4),²¹LSN2463359 (8),^22,23piperazines

VU0364289 (5)²⁴and 1-(4-(2-chloro-4-ﬂuorophenyl)-

piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone (CPPZ, 6),²⁵

ether VU0404251 (7),²⁶and triazole LSN2814617 from Lilly

(9).²³However, recent ﬁndings from Merck-Addex and at the

Vanderbilt Center for Neuroscience Drug Discovery

(VCNDD) have unveiled a target mediated CNS adverse-

eﬀect (AE) liability, driven by excessive glutamate fold

potentation²⁷or allosteric agonism,^28,29respectively; suggesting

that PAMs with lower functional cooperativity with glutamate

(e.g., glutamate fold-shift or potentiation) and devoid of

allosteric agonism may be preferred for improved therapeutic

index.³⁰More recently, we disclosed a phenoxy-based ether

dihydrothiazolopyridone series,³¹with the representative in

vivo tool compound VU0408899 (10) (Figure 1). Ether 10

within the dihydrothiazolopyridones (two examples tested);³¹

however, this trend was not found within monocyclic

nicotinamides of type 7.²⁶Thus, it was unknown if alteration

of the linker moiety within the context of a bicyclic system to

more closely mimic 7 (e.g., dihydronaphthyridinone core

structures 12−17, Figure 2), would be productive for mGlu₅

Figure 2. Evolution of tetrahydronaphthyridine and dihydronaphthyr-

idinone ether based mGlu₅allosteric modulators (12−20) from

nicotinamide 7²⁶and acetylenic modulator scaﬀold 11.³²

receptor potentiation. Prior studies within a dihydronaphthyr-

idinone class utilizing an acetylene-based linker, initially

identiﬁed from an HTS screen, led to PAMs with

submicromolar potency,³²including dihydronaphthyridinones

11, suggested that linkers employing either −CH₂O− or

−OCH₂− might be successful as an acetylene replacement.

Interestingly, evidence of subtle “molecular switches”^15,33

within acetylenic dihydronaphthyridinones 11 via simple

amide congeners led to fundamental changes in the mode of

pharmacology (e.g., from PAMs to negative allosteric

modulators or NAMs).¹⁵The pursuit of ethers 12−20 were

undertaken as part of a larger comprehensive study aimed to

target four key structure−activity relationships: (1) endocyclic

versus exocyclic amide, (2) phenoxy versus benzyloxy linker,

(3) central ring pyridine isomers, and (4) site of the pendant

group attachment to central heterocycle. It was unclear at the

outset if ethers 12−20 would exhibit subtle changes in the

mode of pharmacology similar to 11³²and other chemo-

types^30,34,35or would maintain a higher ﬁdelity for potentiation

similar to CDPPB (1),^17,36,37N-[5-chloro-2-[(−1,3-dioxoisoin-

dolin-2-yl)methyl]phenyl]-2-hydroxybenzamide

(CPPHA),³⁸⁻⁴⁰piperazines (5−6),^24,25,41and glycine sulfona-

mide (ML332)⁴²based mGlu₅PAMs. Herein we describe the

synthesis and pharmacological proﬁle of a series of tetrahy-

dronaphthyridine and dihydronaphthyridinone ethers (12−20)

as positive allosteric modulators of mGlu₅.⁴³⁻⁴⁵Low

cooperativity PAM 12c (VU0405372) was ultimately identiﬁed

and found to demonstrate a suitable balance of in vitro and in

vivo properties for proof-of-concept studies in an amphetamine

hyperlocomotor challenge model. In contrast to previously

reported and related monocyclic ether series,^26,31these bicyclic

modulator scaﬀolds were overall found to exhibit narrow SAR,

low eﬃcacy, and have a higher propensity for pharmacological

“mode switching”¹⁵upon chemical modiﬁcation, demonstrating

an SAR proﬁle reminiscent of related acetylenic PAMs.³²

Figure 1. mGlu₅receptor PAMs with reported eﬃcacy in preclinical

models of schizophrenia and cognition.^17−26,31

represents, in part, eﬀorts to constrain amides of type 7;

however, due to inherent instability in acidic media (20% HP-β-

CD in water, pH = 4) leading to the corresponding 2-hydroxy-

dihydrothiazolopyridone fragment, full exploration of the

analogous system containing a benzyloxy ether linkage was

not possible. Limited in vitro structure−activity relationships

(SAR) suggested a preference for the phenoxy-based linker

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a

Scheme 1. Synthesis of Alkoxy Dihydro-1,6-naphthyridinone Lactams 12

a

Reagents and conditions: (a) 21a, R¹CH₂OH, KOt-Bu, DMF, 100 °C, 4−16 h, 23−63%; (b) lactam, R²CH₂Br or R²CH₂Cl, KOt-Bu, DMF, 60 °C,

2−4 h, 40−75%; (c) lactam, CuI (0.2 equiv), Ar/HetBr, N,N-dimethylethylene diamine, K₂CO₃, toluene, 120 °C, 18 h, 35−59%.

a

Scheme 2. Synthesis of Alkoxymethyl Dihydro-1,6-naphthyridinone Lactams 13

a

Reagents and conditions: (a) 21a, PMBCl, KOt-Bu, DMF, 60 °C, 2 h, 66%; (b) vinyl potassium triﬂuoroborate, Cs₂CO₃, Pd(PPh₃)₄, EtOH, 80 °C,

2 h, 74%; (c) O₃, CH₂Cl₂:MeOH, −78 °C, 40 min, NaBH₄, 0 °C; (d) R¹OH, DIAD, PPh₃, THF, 0 °C, 16 h, 35−40% (over two steps); (e) CAN,

CH₃CN:H₂O, rt, 50 min, 60−65%; (f) 13a−b, R²CH₂Br or R²CH₂Cl, KOt-Bu, DMF, 60 °C, 2−4 h, 44−68%; (g) 13a−b, CuI (0.2 equiv), Ar/

HetBr, N,N-dimethylethylene diamine, K₂CO₃, toluene, 120 °C, 18 h, 25−89%.

a

Scheme 3. Synthesis of Phenoxymethyl Dihydro-1,7-naphthyridinones Lactams 14

a

Reagents and conditions: (a) 24, BH₃THF, 0 °C, 16 h, 68%; (b) Zn(CN)₂, Cs₂CO₃, Pd(PPh₃)₄, DMF, 100 °C, 3 h, 56%; (c) DBAB, phenol, PPh₃,

THF, 0−120 °C, 40 min, 80%; (d) NaOH, 115 °C, 3 h, quant; (e) CH₃I, K₂CO₃, DMF, rt, 16 h, 73%; (f) Bu₃Sn(allyl), Pd(PPh₃)₄, toluene, 125 °C,

16 h, 56%; (g) NaIO₄, OsO₄, THF, 0 °C−rt, 2 h, 74%; (h) RNH₂, THF-MeOH, NaBH(OAc)₃, rt, 16 h, 15−25%.

heteroaryl halides using a copper iodide−diamine ligand

protocol to eﬀect a modiﬁed Ullman lactam cross-coupling to

give the respective alkyl (12i−12n, 12s−12t) and N-aryl

(12o−12p, 12r) lactam products.

Evaluation of the alternate ether linkage based upon an

aryloxy methyl dihydronaphthyridinone, series 13, began

synthetically from 21a as shown in Scheme 2. Protection

with para-methoxybenzyl chloride (PMBCl) aﬀorded 21b in

good yield. Suzuki−Miyaura coupling under Molander’s

conditions using vinyl potassium triﬂuoroborate salt gave

RESULTS AND DISCUSSION

■

Chemistry. To prepare bicyclic analogues that most closely

mimic the structural features found in 11, we initially prepared

a series of dihydro-1,6-naphthyridin-5(6H)-ones (12a−12t,

Scheme 1). Starting from known compound 2-chloro-7,8-

dihydro-1,6-naphthyridin-5(6H)-one (21a, Supporting Infor-

mation), substitution under basic conditions in the presence of

various alcohols with heating aﬀorded 12a−12h, 12q, and 12s

in low to moderate yield. A subset was treated with alkylating

agents under basic conditions, or with various aryl and

C

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Scheme 4. Synthesis of Phenoxymethyl-dihydro-2,7-naphthyridinones 15

a

Reagents and conditions: (a) 29, POCl₃, 16 h, 95 °C, 68%; (b) cat. CH₃ReO₃, H₂O₂, CH₂Cl₂, 16 h, rt, quant; (c) Ac₂O, 120 °C, 45 min, 11%; (d)

K₂CO₃, MeOH, rt, 15 min, 92%; (e) DBAB, phenol, PPh₃, THF, 60 °C, 16 h, 73%; (f) Bu₃(vinyl)Sn, Pd(PPh₃)₄, toluene−dioxane, 90 °C, 16 h,

76%; (g) amine/ammonia in THF or MeOH, sealed tube, 100 °C, 16 h, 39−93%.

vinyl dihydronaphthyridinone intermediate 22. Subsequent

ozonolysis and reductive work-up gave intermediate alcohol

23. Mitsunobu alkylation in the presence of either phenol or 3-

ﬂuorophenol provided 23a−23b in 35−40% yield over two

steps from 22. Removal of the PMB protecting group using

cerium ammonium nitrate proceeded smoothly to give 13a−

13b. Remaining analogues 13c−13i were prepared similarly as

demonstrated for series 12, using alkylating conditions or

copper-based arylation conditions to give alkyl and N-aryl

lactam products 13c−13e and 13f−13i, respectively.

Scheme 5. Synthesis of 3-Benzyloxy-tetrahydro-1,6-

naphthyridine Amides 20d−20g

a

Reagents and conditions: (a) 34, HATU, DIPEA, DMF, rt, 16 h, 60−

90%; (b) 35, ArCH₂OH, Cs₂CO₃, 1,10-Phen, CuI, toluene, 110 °C, 4

h, 20−35%.

Within a third class of lactams 14 (Scheme 3), a benzyloxy

linkage was retained and an alternate dihydro-1,7-naphthyr-

idinone ring system was explored, placing the pyridine nitrogen

at the position ortho to the lactam amide. Synthesis of series 14

began with borane reduction of 5,6-dichloronicotinic acid 24

and subsequent cyanation using Pd(0) catalysis to give 25 in

moderate yield. Treatment of 25 under Mitsunobu conditions

using phenol and di-tert-butyl azodicarboxylate (DBAB),

followed by hydrolysis and ester formation, aﬀorded chloroester

27. Allylation of 27 using Stille conditions with allyl tributyltin,

followed by osmium tetroxide−sodium periodate mediated

cleavage, provided key aldehyde intermediate 28 in good yield

for the two steps. At this stage, installation of the lactam could

be accomplished using a variety of amines in a one-pot two-step

reductive alkylation, ring-cyclization reaction using sodium

triacetoxyborohydride in THF−MeOH to provide examples

14a−14d in low to moderate yield.

Synthesis of phenoxymethyl-dihydro-2,7-naphthyridinones

15 was accomplished using a seven-step sequence from

methyl-6-methylnicotinate (29) as shown in Scheme 4 and

represented the fourth class of dihydronaphthyridinones

investigated. Chlorination followed by pyridine N-oxide

formation using catalytic methyltrioxorhenium(VII) in the

presence of hydrogen peroxide gave intermediate 30.

Polonovski rearrangement to give acetate 31 followed in low

yield. Hydrolysis and subsequent Mitsunobu alkylation

provided ether 32 in good yield. Installation of the vinyl

moiety using Stille conditions provided key pyridine

intermediate 33 in good yield. Vinylpyridine 33 smoothly

underwent one-pot Michael addition and lactam cyclization

using ammonia or several alkyl and aryl amines to give 15a−

15d in moderate to excellent yield.

give amides 35a−35c. Ullman-based etheriﬁcation using CuI

and 1,10-phenanthroline in toluene with heat aﬀorded targets

20d−20g. The related aryloxymethyl targets retaining a similar

substitution pattern and acyl-tetrahydronaphthyridine were

prepared according to Scheme 6. Initial introduction of amide

provided intermediate 35c in 96% yield. Introduction of 4-

ﬂuoro-benzamide provided intermediate 35c, followed by Stille

cross-coupling to give vinyl 36. Subsequent ozonolysis and

reductive work-up gave alcohol 37, which was elaborated via

Mitsunobu alkylation to provide 20a−20c in low to moderate

yields.

In Vitro Pharmacology. As reported previously,³⁰

compounds were proﬁled in a rat mGlu₅low receptor

expression cell line using a “triple add” protocol allowing for

detection of agonism as well as positive and negative allosteric

modulation simultaneously.^21,46Importantly, we have shown

that detection of allosteric agonism observed using the low

receptor expressing cell line correlates with functional response

observed in native systems and modulators with an allosteric

agonist proﬁle have been shown to be associated with

epileptiform activity and behavioral convulsions in ro-

dents.²⁸⁻³⁰In contrast, an allosteric modulator devoid of

agonism in these low-expressing mGlu₅cells was found not to

display a seizure liability in vivo.²⁸Thus, this recombinant

mGlu₅cell line in conjunction with the “triple add” protocol

provided an important initial in vitro assessment of both

agonist and potentiation activity at mGlu₅, which could be

utilized to prioritize analogues for further progression.

Dihydronaphthyridinones. SAR are shown in Tables 1−4

(12−15, 20) and Figures 4−6 (16−19) and summarize in vitro

potency optimization eﬀorts from 10 distinct subseries. Initial

eﬀorts within Tables 1−2 were focused on further expanding

our previously utilized pharmacophore noted within several

chemotypes, including acetylene 4,²¹ethers 7 and 10,^26,31

which retain a pyridyl nitrogen and an amide oxygen as

Alternative tetrahydronaphthyridines wherein the ether

linkage is appended at the 3-position as a benzyloxy with an

exocyclic amide were achieved according to Scheme 5. Starting

bromide 34 was treated with HATU coupling conditions to

D

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a

Scheme 6. Synthesis of 3-Aryloxymethyl-tetrahydro-1,6-naphthyridine Amides 20a−20c

a

Reagents and conditions: (a) 34, 4-ﬂuorobenzoic acid, HATU, DIPEA, DMF, rt, 16 h, 96%; (b) Bu₃(vinyl)Sn, Pd(PPh₃)₄, toluene, 120 °C, 1 h,

82%; (c) O₃, CH₂Cl₂:MeOH, −78 °C, 30 min, NaBH₄, 0 °C; (d) R¹OH, DIAD, polystyrene−PPh₃, THF, rt, 16 h, 25−40% (over two steps).

potential hydrogen bond accepting groups in an optimal spatial

arrangement as illustrated in Figure 3. As can be seen in Table

congeners 12o and 12p demonstrated comparable potency

below 250 nM, with 12p slightly more eﬃcacious (Glu max

50% vs 38%). Both the western ﬂuoro and the eastern amide N-

aryl SAR parallel the recently reported thiazolyl core structure

system;³¹however, interestingly, N-alkyl derivatives were

signiﬁcantly better tolerated in the thiazolyl ring system.

We next turned to the alternate 2-phenoxymethyl derivatives

shown in Table 2. Studies by AstraZeneca Pharmaceuticals⁴⁸

and within the dihydrothiazolopyridone series 10³¹have

demonstrated successful utilization of the both benzyloxy-

and phenoxymethyl-based spacers as replacements for the

acetylene moiety, with varied potency depending on the nature

of the heterocycle scaﬀold.^{26,31,48−50}Interestingly, in contrast to

the unsubstituted benzyloxy lactams 12a−12b, alkoxymethyl-

dihydronaphthyridinones 13a−13b were weak PAMs (a weak

PAM results in a response that is greater than a 10% increase

compared to the submaximal glutamate addition (EC₂₀) but

does not potentiate the overall glutamate EC₂₀response by at

least 2-fold). Similarly, N-alkyl derivatives were not productive

as PAMs, leading to compounds categorized as weak or inactive

(13c−13e, 13i). Direct N-aryl derivatives were more beneﬁcial,

with the western 3-ﬂuorophenyl congener 13g bearing an N-4-

ﬂuoro-phenyl proving to be potent (EC₅₀= 97 nM) and

eﬃcacious (Glu max = 72%); the 2-pyridyl derivative 13h was

5-fold less active (EC₅₀= 595 nM) and less eﬃcacious.

Fortunately, introduction of a 3-ﬂuoro moiety on the distal

phenyl consistently boosted potency (∼2−3-fold) and proved

transferrable among the subseries (e.g., 13f vs 13g). The

potency for 13g−13h was overall similar to the benzyloxy

direct comparators 12o−12p; however, phenoxymethyl-based

PAMs 13g−13h have a 20−30% elevated Glu max response.

Despite modiﬁcation of the linker, PAM activity was retained,

with no evidence of allosteric agonism.

Figure 3. Heterocycle, amide, linker pharmacophore relationships, and

relative proﬁle for “endocyclic” series 12−15 dihydronaphthyridinones

and hypothetical benzyloxy lactam core regioisomers of 14−15.

1, in the case of 2-benzyloxy derivatives, half of the analogues

were active as mGlu₅PAMs. Among actives, the parent lactams

(R = H, 12a−12h, 12q) showed a range of potency from 62 to

1171 nM with low to moderate maximal eﬃcacy (glutamate

max 43−68%). Notably, no evidence for allosteric agonism was

observed for actives in the series. SAR on the R¹group

demonstrated toleration for meta-substituted ﬂuorine and

chlorine congeners 12c and 12e, respectively, representing

two of the more interesting actives from this subseries on the

basis of overall potency and eﬃcacy (EC₅₀< 250 nM, Glu max

>40%). n-Butyloxy derivatives 12q and 12r, reminiscent of a

previously disclosed non-MPEP ether PAM, were not

successful.⁴⁷The 2 and 3-pyridyl congeners 12g and 12h

were also not tolerated at R¹. Additionally, eastern alkyl

derivatives 12i−12n containing linear, branched, cyclic, phenyl,

and heterocyclic systems were either inactive (a <10% increase

in the baseline EC₂₀glutamate ﬂuorescence response in calcium

assays at the highest concentration tested of 30 μM is deﬁned

as “inactive”) or had moderate potency and low eﬃcacy (EC₅₀

> 300 nM, PAM Glu max <40%, 12l−12n, 12s−12t). N-Aryl

In parallel, we evaluated isomeric dihydronaphthyridinones

14−15 (Table 3), which retain the linkage site for the distal

phenyl para to the lactam amide; however, the pyridine

nitrogen is adjacent to the lactam amide in a position

reminiscent of a hydrogen bond accepting (HBA) pharmaco-

phore proposed in a related ether series by Varnes and co-

workers.⁴⁸Within both series 14 (Y = N) and series 15 (X =

N) NH lactams (14a, 15a), N-methyl analogues (14b, 15b),

and chiral (R)-3,3-dimethylbutan-2-yl substitute analogues,

inspired by monocyclic ether 7, all proved to be inactive. The

lack of activity in the calcium assay for parent lactams 14a and

15a versus the benzyloxy comparator 12a, in addition to

western 3-ﬂuoro congeners 13b (inactive) versus 12c (EC₅₀

=

225 nM), suggests that for series 12−15 wherein the distal

E

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Table 1. SAR of 2-Alkoxy-dihydronaphthyridinones 12

Table 2. SAR of 2-Alkoxymethyl-dihydronaphthyridinones

13

a

Calcium mobilization assay using HEK293 cells stably expressing rat

mGlu₅receptors; values are the average of three or more independent

determinations; Inactive, less than 10% change in calcium ﬂuorescence

b

compared to the EC₂₀glutamate response. Expressed as amplitude of

response using 30 μM test compound (percentage of maximal

response versus 100 μM glutamate).

4-Fluorophenyl PAMs 14d and 15d, which diﬀer in the location

of the central pyridine nitrogen, have comparable potency with

dissimilar eﬃcacy, with pyridine isomer 14d being one of the

most eﬃcacious PAMs reported across the tetrahydronaph-

thyridine subseries described. The related congener 13f was

∼2.5-fold more potent (EC₅₀= 198 nM) with 68% glutamate

max, and 13g was 2-fold more potent than 13f. Relative to

series 12, 12o is equipotent to 13g; however, 13g revealed a

∼2-fold higher maximal glutamate response (72% vs 38%).

Although a more comprehensive survey was undertaken for

series 12−13 relative to 14−15, on the basis of direct

comparisons, general trends were noted between the series. A

summary of the potency and eﬃcacy proﬁles are found within

Figure 3. Neither molecular switches nor allosteric agonism

proved to be issues for 12−15 and SAR was generally steep.

Active compounds within 12−13 have capacity for high

potency (EC₅₀≤ 200 nM) and moderate to high eﬃcacy (%

Glu max ≥50%), whereas actives within series 14−15, although

more limited, exhibited moderate potency and variable eﬃcacy

(%Glu max 30% and 81%). Subtle changes in structure−

eﬃcacy/cooperativity relationships are best addressed via

glutamate fold-shift experiments; however, glutamate max

trends were consistent within series 12−15, and series 12

shows variable eﬃcacies that encompass the full range reported

for all subseries (Figure 3). In general, phenoxy linkages show

improved eﬃcacy over benzyloxy linkages for N-arylated

analogues. The emerging pharmacophore for 12−13 suggested

these dihydronaphthyridinone scaﬀolds interact at the allosteric

a

Calcium mobilization assay using HEK293 cells stably expressing rat

mGlu₅receptors; values are the average of three or more independent

determinations; Inactive, less than 10% change in calcium ﬂuorescence

compared to the EC₂₀glutamate response. Expressed as amplitude of

response using 30 μM test compound (percentage of maximal

response versus 100 μM glutamate).

b

phenyl is maintained para to the lactam amide, the heteroatom

arrangement between the linker oxygen and the pyridine

nitrogen within series 12 is more preferred. However, N-aryl

analogues have a unique SAR, suggesting that in this context

the central tetrahydronaphthyridine scaﬀold is more critical for

mGlu₅potentiation than the nature of the linker, as 12 and 13

are equally tolerated in the context of an N-aryl substituent. N-

F

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which were either inactive or weak PAMs (EC₅₀> 10 μM).

Interestingly, a related more rigid, acetylene tetralone scaﬀold

recently reported by Merz Pharmaceuticals GmbH⁵¹displays a

similar array of HBA moieties and was reported to have

excellent PAM activity (EC₅₀< 100 nM) in a recombinant

CHO cell line expressing human mGlu₅.

Table 3. SAR of Isomeric Phenoxymethyl-

dihydronaphthyridinones 14−15

Acyl-tetrahydronaphthyridines. Inspired in part by the

Addex piperidine chemotype 2,^15,52we postulated that perhaps

a more extended pharmacophore placing the amide carbonyl

moiety exocyclic of the tetrahydronaphthyridine scaﬀold as

found within the Addex chemotype might prove viable (Figure

5). PAM 12c served as a precursor (Scheme 1) and allowed for

a

Calcium mobilization assay using HEK293 cells stably expressing rat

mGlu₅receptors; values are the average of three or more independent

determinations; Inactive, less than 10% change in calcium ﬂuorescence

b

compared to the EC₂₀glutamate response. Expressed as amplitude of

response using 30 μM test compound (percentage of maximal

response versus 100 μM glutamate).

Figure 5. Relationship between 2 and 18−20 and resulting ﬁrst-

generation exocyclic amides 18 utilizing 2-benzyloxy linkage.

site in a manner distinct than that proposed by Varnes and co-

workers⁴⁸and likely more similar to a monocyclic series of

nicotinamides previously described.²⁶Although formally

benzyloxy variants of 14 and 15 (Figure 3) remained as

potential mGlu₅PAMs, the template was not pursued. This

decision was made in part on the basis of the overall eﬃcacy

and potency observed within subseries 13, thus phenoxy-based

systems remained higher priority for the remaining survey.

We next pursued alternative sites for linker attachment of the

pendant aryl examining 3-substituted dihydronaphthyridinones

based on either a benzyloxy (16) or phenoxy-based (17)

system (Figure 4). Brief surveys of both N-alkyl and N-aryl and

heteroaryl substituents, including several congeners related to

those previously described (Table 1−3), were unsuccessful in

identifying favorable starting points and led to modulators

facile access to representative benzyloxy targets 18a−d in two

steps via borane reduction and subsequent amide coupling.

Intriguingly, all proved to be weak to moderate antagonists of

mGlu₅(Figure 5). At this juncture, in light of the unexpected

switch in the mode of pharmacology for subseries 18, as well as

the general challenges of multidimensional combinations of

linker, core, attachment site, and lactam versus exocyclic amide

(minimum of 24 formal combinations/scaﬀolds), we employed

a ligand-based computational tool previously reported⁴²to

prioritize and potentially streamline medicinal chemistry eﬀorts

and ligand design. Using this approach, we assessed ligand-

based conformational ensembles of known highly eﬃcacious

PAMs, including Addex ligand 2⁵²and other lead mole-

cules,^53,54with PAM activity below 150 nM and robust Glu

max values above 70%. These low energy ensembles of PAMs

served as ﬂexible “hypotheses” or reference PAMs for a mutual

ﬂexible low energy shape-based alignment of proposed

dihydronaphthyridinone and tetrahydronaphthyridines using

the Surﬂex-Sim algorithm as implemented in Sybyl.^55,56The

molecular similarity and alignment optimization algorithms

used in Surﬂex-Sim docking utilize a functional term to

minimize the volume of molecular superpositions to construct

an objective function for scoring superpositions of multiple

molecules. The objective function can then be used as targets

for virtual screening, or in this case, for scaﬀold hopping

prioritization. The results from these studies using 2 are

summarized in Figure 6 with a depicted overlay of the lowest

energy structures for 2 versus 2-phenoxy methyl model amide

19 and 3-phenoxy methyl model amide 20. A Surﬂex-Sim score

Figure 4. SAR summary of 3-benzyloxy and 3-phenoxymethyl-

dihydronaphthyridinones 16−17.

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Table 4. “Exocyclic” Tetrahydronaphthyridine Amides 20

a

Calcium mobilization assay using HEK293 cells stably expressing rat

mGlu₅receptors; values are the average of three or more independent

Figure 6. Example of ﬂexible alignment of model 2 and 3-

phenoxymethyl-tetrahydronaphthyridines 19 and 20 (green surface)

vs 2 (purple surface).

determinations; Inactive, less than 10% change in calcium ﬂuorescence

b

compared to the EC₂₀glutamate response. Expressed as amplitude of

response using 30 μM test compound (percentage of maximal

c

response versus 100 μM glutamate). Weak antagonist, data represents

(SS) that approaches the reference hypothesis, arbitrarily set to

10, is indicative of a template that more closely mimics the

reference conformer ensemble. Using 2 as a template, series 20,

containing a 3-phenoxymethyl, was predicted to have greater

molecular similarity (SS = 9.43) than the 2-phenoxymethyl

prototype 19 (SS = 8.90). Unlike 2-benzyloxy derivatives

within series 18, compounds within series 19 were not

antagonists and proved inactive in the primary calcium assay

(nine analogues were prepared and tested from a 3-ﬂuoro 1-(2-

((3-ﬂuorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-

6(5H)-yl)ethanone scaﬀold, see Supporting Information);

however, at this time, it is unclear if compounds within series

19 have neutral cooperativity and thus retained aﬃnity for

mGlu₅(a silent allosteric modulator or SAM).^44,57With this

caveat in mind, it is also recognized that the PAM EC₅₀is better

described as a composite of four parameters encompassing

aﬃnity of the modulator for the receptor, allosteric eﬀects on

binding and signaling of the orthosteric ligand and potential

intrinsic agonist eﬃcacy of the modulator. Thus, although

within closely related series, the Surﬂex-Sim approach appears

to facilitate scaﬀold prioritization based on PAM EC₅₀activity

at of Glu₅, the power of the Surﬂex-Sim methodology warrants

a broader more rigorous retrospective analysis with these

additional facets of modulator activity taken into consideration.

With some insight that perhaps 3-substituted tetrahyrdo-

naphthyridines 20 were preferred from ligand-based modeling,

additional analogues were prioritized. Selected derivatives 20a−

20c, which retains a 3-phenoxy methyl linker, and 3-benzyloxy

variants 20d−20g, are shown in Table 4. Within benzyloxy

derivatives 20d−20g, compounds covered a spectrum of

proﬁles ranging from inactive (20d, 20f), to weak PAMs

(20e), to weak partial antagonists (20g); the latter eﬀect driven

by subtle changes in ﬂuorine substitution patterns, a

phenomenon not uncommon among certain mGlu₅modulator

chemotypes.³³Within the phenoxy subseries analogues 20a−

pLC₅₀/IC₅₀from EC₈₀window and %Glu represents E_minnot E_max

.

20c, success was found with 4-ﬂuorobenzamide derivatives; in

particular, the western phenyl and 3-ﬂuoro phenyl derivatives

20a and 20b have statistically comparable potency and eﬃcacy,

with EC₅₀s of 640 and 1090 nM, respectively, and a Glu max of

69%. In contrast, the 4-ﬂuoro congener 20c was 2−3-fold less

active. Three signiﬁcant ﬁndings are evident among these

lactam and exocyclic amide templates examined: (1) parent

dihydronaphthyridinone 12c remains a confounding singleton

relative to regioisomeric and positional congeners 13b, 14a,

15a, 16, and 17, (2) relative to earlier acetylenic dihyronaph-

thyridinones 11,³²no molecular switches were noted within

dihydronaphthyridinone systems, however, steep SAR was

evident, and (3) in contrast to dihydronaphthyridinones,

exocyclic acyl-tetrahydronaphthyridines were highly sensitive

to the linker attachment site, as 2-substituted based ethers 18

and 19, proved to be either weak antagonists or functionally

inactive in the calcium assay. In addition, within the exocyclic

acyl-tetrahydronaphthyridines, phenoxymethyl-based ethers

were better tolerated and less susceptible to changes in the

mode of pharmacology (e.g., 20a versus 20g); however, overall

potency was generally diminished relative to series 12−13.

In light of the overall mGlu₅PAM potency (EC₅₀< 250 nM)

and eﬃcacy (Glu max >50%) observed for compounds 12c,

12p, 13f, and 13g, these PAMs were selected for further

characterization (Table 5). In addition, the moderately potent

PAM 14d (EC₅₀= 538 nM) was considered for further

evaluation based upon its enhanced eﬃcacy (Glu max = 81%).

Among the PAMs further examined, 12p, 13f, 13g, and 14d

have similar cLogP and molecular weight (MW) by virtue of

their common lactam N-aryl substituent, while the simple NH

lactam 12c stands out as a low MW modulator (MW = 272)

with excellent calculated ligand eﬃciency metrics (LipE and

LELP, see Table 5).^58,59PAM 12c maintains the highest

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Table 5. Rat mGlu₅Potency Values and Data for Selected Key Compounds

a

b

c

d

e

compd

rmGlu₅EC₅₀(nM)

cLogP

LipE

LELP

CL_int(h, r)

CL_HEP(h, r)

PPB f_u(h, r)

12c

12p

13f

225

212

198

97

2.62

3.96

3.72

3.88

3.60

4.03

2.71

2.98

3.13

2.67

5.76

11.71

10.55

10.92

25/175

18/138

18/108

8.8/121

19/33

11/50

9.6/47

9.6/43

6.2/44

10/22

0.047/0.034

0.010/0.025

0.021/0/036

0.014/0.029

0.096/0.110

13g

14d

538

a

b

c

For the mGlu₅pEC₅₀assay see Tables 1−4 and Experimental Section. cLogP was calculated using Adriana’s XLogP code. LipE = pEC50−cLogP;

LELP = ligand eﬃciency/cLogP.^58,59CL_int, intrinsic clearance; CL_HEP, predicted hepatic clearance, human and rat (h, r), mL/min/kg. PPB f_u,

d

e

plasma protein binding fraction unbound.

calculated LipE and lowest LELP among the set, suggesting a

highly favorable enthalpy driven eﬀect on cooperativity. Despite

the less favorable physicochemical and LE properties for 12p,

13f, 13g, and 14d relative to 12c, intrinsic clearance in human

and rat microsomes suggested moderate predicted hepatic

clearance and 1−10% fraction unbound. Subsequent selectivity

proﬁling against mGlu_1−4,6−8using 12c, 13g, and 14d revealed

that N-aryl congeners 13g and 14d were also active as mGlu₃

negative allosteric modulators (NAMs) with negative cooper-

ativity fold-shift values between 0.2 and 0.3, while 12c was fully

selective for mGlu₅(mGlu_1−4,6−8EC₅₀> 10 μM, see Supporting

Information).

Additional Pharmacological Characterization of 12c. In

light of the unexpected lack of selectivity for 13g and 14d

which maintain a similar N-aryl endocyclic amide pharmaco-

phore found within 12p and 13f, we elected to continue to

proﬁle 12c in more depth based on its overall potency,

selectivity, and excellent physicochemical and ligand eﬃciency

parameters (see Figure 7 for overall proﬁle). Positive allosteric

mGlu₅receptor revealed a small leftward shift in the glutamate

concentration response curve (CRC) with increasing concen-

tration of 12c (55 nM to 30 μM), with no signiﬁcant eﬀect on

the maximal response. A 2.2-fold leftward shift in the glutamate

EC₅₀was observed in the presence of 30 μM 12c, and a

predicted allosteric modulator aﬃnity of 2.17 μM and an

eﬃcacy cooperativity factor (log β) between glutamate and

indicated allosteric modulator of 0.23 (cooperativity ∼1.71)

was deﬁned as calculated using the operational model of

allosterism.⁶⁰These results support a positive allosteric

interaction between glutamate and 12c and were conﬁrmed

in a cell line expressing the human mGlu₅receptor,⁴⁶where 10

μM 12c produced a 3.3-fold leftward shift in the glutamate

CRC and a PAM CRC potency of 370 nM (71% Glu max). In

comparison to recently reported PAMs from our lab that have

been proﬁled in glutamate progressive fold-shift studies up to

30 μM, including acetylenic picolinamides (e.g., ML254,

maximum rat mGlu₅fold-shift 3.0)³⁰and dihydrothiazolopyr-

idone 10 (maximum rat mGlu₅fold-shift 5.4),³¹12c represents

one of the weakest mGlu₅allosteric modulators reported in

terms of measurable positive cooperativity.

Extended DMPK Characterization and Ancillary Pharma-

cology of 12c. Rat brain homogenate binding to assess fraction

unbound in brain revealed a reasonable brain f_uof 2.0% for 12c.

Inhibition of the major human cytochrome P450 (CYP)

enzymes (2C9, 2D6, 3A4, 1A2) was measured in a cocktail

assay using human liver microsomes and known substrates.

PAM 12c was found to display weak CYP inhibitory activity at

1A2 (IC₅₀= 25.7 μM), while no activity was observed against

the other CYPs tested (IC₅₀> 30 μM). In kinetic aqueous

solubility assays, 12c displayed moderate to good solubility (pH

2, 4, 7.4 buﬀer: 38, 29, and 28 μM) and moderate solubility in

fasted simulated intestinal ﬂuid (Fassif, pH = 7.2) of 31 μg/mL

(114 μM). In a nontoxic vehicle screen using 20% HP β-

cyclodextrin, 12c was a solution at 1 mg/mL and a

microsuspension at 3−5 mg/mL (pH 4.0). Rat pharmacoki-

netic studies (1 mg/kg IV, 10 mg/kg PO) revealed a high

plasma clearance (CL_p) of 75 mL/min/kg and a short half-life

(T_1/2) of 0.3 h. After oral administration (10 mg/kg, 0.5%

methyl cellulose, 1 mg/mL, noncrossover) to male Sprague−

Dawley rats, 12c reached an average maximal concentration

(C_max) of 3.36 μM with a corresponding time to reach C_max

(T_max) of 0.5 h and an AUC_0−24hof 6.2 μM-h, thus aﬀording an

estimated %F of 75. We performed a single dose in vivo screen

to study the ability of 12c to reverse amphetamine-induced

hyperlocomotion in rats (PO, 56.6 mg/kg 20% HP β-

cyclodextrin). Robust reversal of the hyperlocomotion response

was noted at this dose (>30%, data not shown) and high brain

exposure was observed at 1.5 h (C_brain= 16.6 μM, 332 nM

unbound), producing an average brain-to-plasma (B:P) ratio of

1.67 and unbound brain level above the rat in vitro EC₅₀.

Figure 7. Proﬁle summary of 12c.

modulator activity was investigated by fold-shift experiments to

determine the degree of cooperativity with glutamate. As shown

in Figure 8, progressive fold-shift experiments utilizing the rat

Figure 8. Glutamate CRC in the presence of increasing concentrations

of 12c.

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Furthermore, in vivo B:P and in vitro f_u,plasma/f_u,brain(1.7)

measures were identical, with a calculated K_p,uuof 1.1,

suggesting passive CNS penetration for 12c in rat.⁶¹Thus,

12c continued to demonstrate promising properties to warrant

further evaluation in vivo. With respect to ancillary

pharmacology, in a broad panel selectivity screen against 68

GPCRs, ion channels and transporters using 10 μM 12c, no

signiﬁcant oﬀ-target activity was noted (Euroﬁns Inc.).

In Vivo Pharmacological Characterization. Encouraged by

its overall proﬁle as a low fold-shift PAM, 12c was evaluated

across a range of doses for its ability to reverse amphetamine-

induced hyperlocomotion (AHL), an established model of

antipsychotic activity (Figure 8).^17,36As seen in Figure 9, 12c

CONCLUSIONS

■

Diverse mGlu₅modulator pharmacological proﬁles were

attained within a series of tetrahydronaphthyridine and

dihydronaphthyridinone scaﬀolds through modiﬁcations of

heteroatoms within the linker, core structure, and location of

the pendant linkage moiety. Up to 10 diverse subseries were

examined, and ﬁve of these series, 12−15 and 20, were

described in detail, leading to preferred endocyclic series 12

and 13 which, although free from allosteric agonism and

molecular switches, tended to have steep SAR. N-Aryl

dihydronaphthyridinone congeners were nonselective and

displayed negative cooperativity at mGlu₃and thus were not

further progressed. Parent benzyloxy lactam 12c emerged as a

suitable tool compound with good potency, excellent

selectivity, and pharmacokinetic properties suitable for acute

studies. Despite behaving in vitro as an ultralow cooperativity

PAM in recombinant systems, 12c showed robust, dose-

dependent eﬀects in the reversal of amphetamine-induced

hyperlocomotion, with a lowest active dose of 30 mg/kg PO,

that produced an estimated brain unbound level 3-fold above its

in vitro potency. Maximal eﬃcacy of 12c was observed at a dose

of 100 mg/kg with no signiﬁcant motor impairment or overt

neurological side eﬀects. 12c provides another tool compound

to complement the available mGlu₅PAMs with well

characterized cooperativity proﬁles^30,31to better enable

comparative studies with other chemotypes in native systems

and in preclinical animal models.

EXPERIMENTAL SECTION

■

Figure 9. Dose-dependent eﬀect and calculated terminal unbound

brain of 12c on the reversal of amphetamine-induced hyperlocomotion

in rats.

General. All reagents purchased from commercial suppliers were

used without puriﬁcation. Unless noted, all solvents used were

anhydrous and all reactions were carried out under argon atmosphere.

Microwave assisted reactions were performed in a single-mode reactor:

Emrys Optimizer microwave reactor (Biotage). Analytical thin layer

chromatography was performed on Analtech silica gel GF 250 μm

plates and on silica gel 60 F254 plates (Merck) under standard

techniques. Unless otherwise speciﬁed, preparative reverse-phase high

performance liquid chromatography (RP-HPLC) puriﬁcation was

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

Phenomenex Luna C18 column (50 mm × 30 mm I.D., 5 μm), with

an acetonitrile (unmodiﬁed)−0.1% triﬂuoroacetic acid in water

gradient. Normal-phase silica gel preparative puriﬁcation was

performed using an automated Combi-ﬂash Rf from ISCO using

either ISCO or Merck ready-to-connect cartridges. Analytical LC/MS

was performed using the following instruments: (A) Agilent 1200

series with UV detection at 215 and 254 nm and ELSD detection

(Polymer Laboratories PL-ELS 2100), utilizing an Accucore C18 2.6 μ,

2.1 mm × 30 mm column, a 1.1 min gradient, 7% [CH₃CN/0.1%

TFA]−95% [CH₃CN/0.1%TFA] and a G6130 single quadrupole mass

spectrometer or (B) ultra performance liquid chromatography

(UPLC) measurement performed using an Acquity UPLC (Waters)

system comprising a sampler organizer, a binary pump with degasser, a

four column’s oven, a diode array detector (DAD), and a BEH-C18

column (1.7 μm, 2.1 mm × 50 mm) from Waters, with a ﬂow rate of

1.0 mL/min at 50 °C without split to the MS detector. The gradient

conditions consisted of 95% A (0.5 g/L ammonium acetate solution +

5% acetonitrile), 5% B (acetonitrile), to 40% A, 60% B in 3.8 min, to

5% A, 95% B in 4.6 min, kept until 5.0 min without split to the MS

detector. The MS detector was conﬁgured with an ESCI dual

ionization source (electrospray combined with atmospheric pressure

chemical ionization). Nitrogen was used as the nebulizer gas. The

source temperature was maintained at 140 °C. Low-resolution mass

spectra (single quadrupole, SQD detector) were acquired by scanning

from 100 to 1000 in 0.1 s using an interchannel delay of 0.08 s. The

capillary needle voltage was 3 kV. The cone voltage was 25 V for

positive ionization mode and 30 V for negative ionization mode. Data

dosed orally showed robust dose-dependent eﬀects in reversal

of hyperlocomotion, with a maximal eﬀect (55%) observed at

the highest dose tested of 100 mg/kg and an average terminal

unbound brain concentration of 1.5 μM. The lowest active dose

of 30 mg/kg aﬀorded an estimated average terminal unbound

brain concentration of ∼712 nM, which represents a 3-fold

multiple of the rat in vitro potency and according to the

progressive fold-shift studies (Figure 8), represents a left-ward

shift in the glutamate CRC in the ∼1.2−1.4 fold range. Despite

the overall low positive cooperativity, 12c retains eﬃcacy in

AHL at unbound brain exposures that are a multiple of the in

vitro EC₅₀and thus illustrates that high cooperativity with

glutamate is not fully required for an mGlu₅PAM to maintain

activity in the AHL model. This observation appears to be in

contrast to data reported recently for low fold-shift PAMs 8−9

reported by Lilly, wherein little eﬀect was noted in a similar

amphetamine-induced hyperlocomotor activity assay using

male Lister hooded rats.²³Rotarod studies were performed

using 12c at a doses of 30, 56.6, and 100 mg/kg PO (20% HP-

β-CD in water) to assess balance and motor coordination (see

Supporting Information). PAM 12c showed no statistically

signiﬁcant eﬀect on general motor output (120 s cutoﬀ). In a

modiﬁed Irwin neurological test battery in rats, 12c, when

dosed at 100 mg/kg PO (20% HP-β-CD in water), had no

performance deﬁcits on any autonomic or somatomotor

nervous system functions out to 4 h. Collectively, these results

highlight that PAM 12c is free from overt neurological side

eﬀects and motor behavior deﬁcits at doses that produce

maximal eﬃcacy in reversal of amphetamine-induced hyper-

locomotion (100 mg/kg PO).

J

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acquisition was performed with MassLynx-Openlynx software. GC/

MS measurement was performed using a 6890 series gas chromato-

graph (Agilent Technologies) system comprising a 7683 series injector

and autosampler, 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. GC/MS was carried out on a J&W HP-5MS column

(20 m × 0.18 mm, 0.18 μm) from Agilent Technologies, with a ﬂow

rate of 0.7 mL/min. The temperature gradient applied was: initial

temperature 50 °C, hold for 2.0 min, then a 50 °C/min ramp applied

for 5.0 min until 300 °C and hold for 3.0 min in a 10 min run. Front

inlet temperature was 250 °C. Split injection mode was used, 0.2 μL

injection volume, with a 50/1 ratio into the GC/MS system. HRMS

were obtained using a Micromass (Waters) Q-Tof API-US calibrated

and veriﬁed with sodium iodide. The samples were diluted with a

50:50 0.1% formic acid (in Milli-Q):acetonitrile solution, directly

infused using leucine−enkephalin (M + H = 556.2771) as a lockmass.

Scan range was from 100 to 1000 Da, using a scan time of one second.

The [M + H] or [M + Na] ion was observed. Purity of all ﬁnal

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

extraction, washing, and chromatography were HPLC grade. NMR

spectra were recorded on either a Bruker DPX-400 or a Bruker AV-

adsorbed onto silica and puriﬁed (0 to 80% EtOAc/hexanes) to give a

semipure solid. This material was further puriﬁed on RP-HPLC

(eluting with 40−90% MeCN/H₂O with 0.1% TFA modiﬁer) to give

840 mg of the title compound (35%). ¹H NMR (400 MHz, CDCl₃) δ

8.44 (1H, d, J = 7.6 Hz), 7.38 (1H, dd, J = 14.6, 7.6 Hz), 7.24 (1H, d, J

= 7.5 Hz), 7.22 (1H, d, J = 9.6 Hz), 7.04 (1H, t, J = 8 Hz), 6.79 (1H, d,

J = 8.4 Hz), 5.79 (1H, br s), 5.45 (2H, s), 3.64 (2H, t, J = 6.4 Hz), 3.10

(2H, t, J = 6.4 Hz). LCMS t_R= 0.736 min, > 98% at 215 and 254 nm,

m/z = 273.1 [M + H]. HRMS (ES+, M + H) calcd for C₁₅H₁₄FN₂O₂,

273.0961; found, 273.0963.

2-Butoxy-7,8-dihydro-1,6-naphthyridin-5(6H)-one (12q). Starting

from 2-chloro-7,8-dihydro-1,6-naphthyridin-5(6H)-one (100 mg, 0.55

mmol) and following the general procedure outlined in 12a and 12c,

RP-HPLC aﬀorded the title compound as a powder (72 mg, 60%). ¹H

NMR (400 MHz, CDCl₃) δ 8.17 (1H, d, J = 8.6 Hz), 6.67 (1H, d, J =

8.6 Hz), 5.84 (1H, bs), 4.33 (2H, t, J = 6.6 Hz), 3.61 (2H, t, J = 6.8

Hz), 3.06 (2H, t, J = 6.8 Hz), 1.77 (2H, p, J = 7.9 Hz), 1.48 (2H, hex, J

= 7.5 Hz), 0.98 (3H, t, J = 7.4 Hz). ¹³C NMR (100 MHz, CDCl₃) δ

166.6, 165.7, 158.1, 138.1, 117.9, 109.4, 66.3, 39.2, 30.9, 30.8, 19.1,

13.7. HRMS (ES+, M + H) calcd for C₁₂H₁₇N₂O₂, 221.1290; found,

221.1290.

2-Butoxy-6-(2,4-diﬂuorophenyl)-7,8-dihydro-1,6-naphthyridin-

5(6H)-one (12r). 2-Butoxy-7,8-dihydro-1,6-naphthyridin-5(6H)-one

(12q, 50 mg, 0.23 mmol), 1-bromo-2,4-diﬂuorobenzene (52 μL,

0.46 mmol), CuI (9.1 mg, 0.047 mmol), N¹,N²-dimethylethane-1,2-

diamine (14 μL, 0.129 mmol), and potassium carbonate (64 mg, 0.46

mmol) were combined in a 2.0 mL microwave vial and placed under

an argon atmosphere. Degassed toluene was added and the mixture

heated at 110 °C for 6 h. The mixture was cooled to rt, ﬁltered over

Celite, and the ﬁltrate concentrated to dryness. RP-HPLC puriﬁcation

aﬀorded the title compound as a powder (45 mg, 59%). ¹H NMR (400

MHz, CDCl₃) δ 8.22 (1H, d, J = 8.6 Hz), 7.32 (1H, m), 6.92 (2H, m),

6.68 (1H, d, J = 8.6 Hz), 4.35 (2H, t, J = 6.6 Hz), 3.91 (2H, t, J = 6.7

Hz), 3.19 (2H, t, J = 6.6 Hz), 1.77 (2H, p, J = 7.9 Hz), 1.48 (2H, hex, J

= 7.6 Hz), 0.98 (3H, t, J = 7.4 Hz). ¹³C NMR (100 MHz, CDCl₃) δ

1

500 MHz spectrometer with standard pulse sequences. H chemical

shifts are reported as δ values in CDCl₃or DMSO-d₆. Data are

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

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

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

triplets, dq = doublet of quartets, m = multiplet), coupling constant J

reported in Hz. ¹³C chemical shifts are reported in δ values in CDCl₃

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

Hz. For a number of compounds, melting points (mp) were

determined in open capillary tubes on a FP62 or on a FP81HTFP90

apparatus (Mettler). Melting points were measured with a temperature

gradient of 10 °C/min (maximum temperature 300 °C), and the

melting point was read from a digital display. Melting point values are

peak values and were obtained with experimental uncertainties that are

commonly associated with this analytical method. Optical rotation

values were obtained on a JASCO P-2000 polarimeter.

165.9, 163.9, 161.4(dd, J_C−F= 247.6, 11.3 Hz), 157.8 (dd, J_C−F

=

250.8, 12.3 Hz), 157.66, 139.00, 129.61 (dd, J_C−F= 9.8, 2.6 Hz),

126.42, (dd, J_C−F= 12.8, 3.9 Hz), 118.0, 111.55 (dd, J_C−F= 22.2, 3.6

Hz), 109.7, 104.9 (dd, J_C−F= 26.0, 24.0 Hz), 66.4, 48.5, 31.2, 31.0,

19.2, 13.8. HRMS (ES+, M + H) calcd for C₁₈H₁₉F₂N₂O₂, 333.1415;

found, 333.1413.

6-Benzyl-2-(benzyloxy)-7,8-dihydro-1,6-naphthyridin-5(6H)-one

(12s). 2-(Benzyloxy)-7,8-dihydro-1,6-naphthyridin-5(6H)-one (12a,

30 mg, 0.117 mmol) and benzyl bromide (19 μL, 0.158 mmol)

were dissolved into DMF (0.5 mL) and KOt-Bu added (16 mg, 0.143

mmol). The mixture was stirred for 3 h at 60 °C and then cooled to rt.

Water (1.0 mL) was added to the mixture and extracted with EtOAc

(3×). The combined organic layers were evaporated to dryness and

the crude product mixture puriﬁed by RP-HPLC to aﬀord 16 mg of

the desired product (40%). ¹H NMR (400 MHz, CDCl₃) δ 8.28 (1H,

d, J = 8.6 Hz), 7.45 (2H, m), 7.33 (8H, m), 6.76 (1H, d, J = 8.6 Hz),

5.41 (2H, s), 4.77 (2H, s), 3.53 (2H, t, J = 6.9 Hz), 3.02 (2H, t, J = 6.9

Hz). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 164.1, 156.9, 139.0, 137.3,

136.8, 128.7, 128.5, 128.1, 128.0, 127.9, 127.5, 118.7, 109.9, 68.1, 50.2,

44.5, 30.7. HRMS (ES+, M + H) calcd for C₂₂H₂₁N₂O₂, 345.1603;

found, 345.1600.

2-(Benzyloxy)-6-(pyridin-2-ylmethyl)-7,8-dihydro-1,6-naphthyri-

din-5(6H)-one (12t). 2-(Benzyloxy)-7,8-dihydro-1,6-naphthyridin-

5(6H)-one (12a, 30 mg, 0.117 mmol) and 2-bromomethylpyridine-

hydrogenbromide (40 mg, 0.158 mmol) were dissolved into DMF (0.5

mL) and KOt-Bu added (36 mg, 1.35 mmol). The mixture was stirred

for 3 h at 60 °C and then cooled to rt. Water (1.0 mL) was added to

the mixture and extracted with EtOAc (3×). The combined organic

layers were evaporated to dryness and the crude product mixture

puriﬁed by RP-HPLC to aﬀord 16.2 mg of 12t (40%). ¹H NMR (400

MHz, CDCl₃) δ 0.8.55 (1H, d, J = 4.6 Hz), 8.25 (1H, d, J = 8.6 Hz),

7.66 (1H, dt, J = 7.6, 1.7 Hz), 7.45 (2H, m), 7.35 (4H, m), 7.2 (1H,

m), 6.74 (1H, d, J = 8.6 Hz), 5.41 (2H, s), 4.88 (2H, s), 3.70 (2H, t, J

= 6.9 Hz), 3.07 (2H, t, J = 6.9 Hz). ¹³C NMR (100 MHz, CDCl₃) δ

165.1, 164.2, 157.3, 157.2, 149.2, 140.0, 136.9, 136.8, 128.5, 128.1,

Chemistry. Synthesis of presented compounds was performed

according to Schemes 1−6. Experimental procedures, analytical data,

HRMS, and NMR spectra can be found within Supporting

Information. Synthesis and analytical data for representative members

within series 12−19 are provided below.

2-(Benzyloxy)-7,8-dihydro-1,6-naphthyridin-5(6H)-one (12a).

Benzyl alcohol (85 μL, 0.82 mmol) was dissolved into DMF (1.6

mL) and treated with KOt-Bu (184 mg, 1.64 mmol) and stirred for 30

min. The mixture was treated with 2-chloro-7,8-dihydro-1,6-naphthyr-

idin-5(6H)-one (20, 100 mg, 0.55 mmol, see Supporting Information)

and heated to 100 °C for 4 h. The mixture was cooled to rt, acidiﬁed

with 6 N HCl, and extracted with EtOAc (3×). The combined organic

layers were washed with brine, dried over Na₂SO₄, and evaporated to

dryness. The crude product mixture was puriﬁed by RP-HPLC

(eluting with 40−90% MeCN/H₂O with 0.1% TFA modiﬁer) to

aﬀord the title compound (73 mg, 53%). ¹H NMR (400 MHz, CDCl₃)

δ 8.20 (1H, d, J = 8.6 Hz), 7.46 (2H, m), 7.36 (3H, m), 6.75 (1H, d, J

= 8.6 Hz), 5.85 (1H, bs), 5.43 (2H, s), 3.62 (2H, t, J = 6.8 Hz), 3.09

(2H, t, J = 6.8 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 165.2,

158.0, 138.3, 136.7, 128.4, 128.0, 127.9, 118.3, 109.8, 68.1, 39.2, 30.7.

HRMS (ES+, M + H) calcd for C₁₅H₁₅N₂O₂, 255.1134, found,

255.1130.

2-((3-Fluorobenzyl)oxy)-7,8-dihydro-1,6-naphthyridin-5(6H)-one

(12c). 3-Fluorobenzyl alcohol (1.66 g, 13.14 mmol) was dissolved into

DMF (25 mL) and treated with KOt-Bu (3.2 g, 26.29 mmol) and

stirred for 30 min. The mixture was treated with 2-chloro-7,8-dihydro-

1,6-naphthyridin-5(6H)-one (20, 1.49 g, 8.82 mmol, see Supporting

Information) and heated to 100 °C overnight. The mixture was

K

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128.0, 122.4, 122.3, 118.6, 109.8, 68.1, 52.4, 45.6, 30.7. HRMS (ES+,

M + H) calcd for C₂₁H₂₀N₃O₂, 346.1556; found, 346.1553.

NMR (400 MHz, CDCl₃) δ 8.36 (1H, d, J = 8.0 Hz), 7.50 (1H, d, J =

8.0 Hz), 7.23 (1H, m), 6.75 (1H, m), 6.68 (2H, m), 5.18 (2H, s), 3.75

(2H, t, J = 6.7 Hz), 3.47 (2H, d, J = 7.0 Hz), 3.21 (2H, t, J = 6.7 Hz),

1.07 (1H, m), 0.55 (2H, m), 0.31 (2H, m). ¹³C NMR (100 MHz,

CDCl₃) δ 163.6 (d, J_C−F= 244.2. Hz), 163.1, 159.5, (d, J_C−F= 10.8

Hz), 159.1, 157.5, 137.0, 130.3 (d, J_C−F= 9.9 Hz), 124,4, 119.8, 110.5

(d, J_C−F= 2.8 Hz), 108.2 (d, J_C−F= 21.2 Hz), 102.7 (d, J_C−F= 24.8

Hz), 70.5, 51.4, 45.2, 30.8, 9.5, 3.5. HRMS (ES+, M + H) calcd for

C₁₉H₂₀FN₂O₂, 327.1509; found, 327.1508.

6-(4-Fluorophenyl)-2-(phenoxymethyl)-7,8-dihydro-1,6-naph-

thyridin-5(6H)-one (13f). 2-(Phenoxymethyl)-7,8-dihydro-1,6-naph-

thyridin-5(6H)-one (13a, 50 mg, 0.19 mmol), 1-bromo-2,4-diﬂuor-

obenzene (42 μL, 0.38 mmol), CuI (7.2 mg, 0.038 mmol), and

potassium carbonate (52 mg, 0.38 mmol) were combined in a 2.0 mL

microwave vial and placed under an argon atmosphere. Degassed

toluene was added followed by N¹,N²-dimethylethane-1,2-diamine (12

μL, 0.110 mmol) and the mixture heated at 120 °C for 16 h. The

mixture was cooled to rt, ﬁltered over Celite, and the ﬁltrate

concentrated to dryness. RP-HPLC puriﬁcation aﬀorded the title

compound as a powder (16 mg, 31%). ¹H NMR (400 MHz, CDCl₃) δ

8.43 (1H, d, J = 8.0 Hz), 7.60 (1H, d, J = 8.0 Hz), 7.33 (4H, m), 7.12

(2H, m), 6.99 (3H, m), 5.25 (2H, m), 4.06 (2H, t, J = 6.7 Hz), 3.36

(2H, t, J = 6.6 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 160.9 (d,

J_C−F= 245.0 Hz), 160.7, 158.1, 157.5, 138.4 (d, J_C−F= 3.2 Hz), 137.5,

2-(Phenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one

(13a). [2-(Phenoxy)methyl)-6-(4-methoxybenzyl)-7,8-dihydro-1,6-

naphthyridin-5(6H)-one (14 mg, 0.0.037 mmol) was dissolved in

CH₃CN (0.25 mL) and water (0.1 mL) added. Cerium ammonium

nitrate (24.3 mg, 0.0.04 mmol) was added, and after 15 min, the

reaction was partitioned between EtOAc (5 mL) and brine (1 mL).

The mixture was extracted with EtOAc (2×), and the organic layers

combined, concentrated, and puriﬁed by RP-HPLC to give title

1

compound (2.6 mg, 28%). H NMR (400 MHz, CDCl₃) δ 8.32 (1H,

d, J = 8.0 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.25 (2H, m), 6.76 (1H, m),

6.69 (2H, m), 6.49 (1H, bs), 5.10 (2H, s), 3.65 (2H, dt, J = 6.9, 2.5

Hz), 3.20 (2H, t, J = 6.7 Hz). LCMS t_R= 0.732 min, > 98% at 215 and

254 nm, m/z = 255.1 [M + H]. HRMS (ES+, M + H) calcd for

C₁₅H₁₄N₂O₂, 255.1055; found, 255.1057.

2-((3-Fluorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-

5(6H)-one (13b). [2-((3-Fluorophenoxy)methyl)-6-(4-methoxyben-

zyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one (23b, 300 mg, 0.77

mmol) was dissolved in CH₃CN (5 mL) and water (2 mL) added.

Cerium ammonium nitrate (419 mg, 0.77 mmol) was added, and after

15 min, the reaction was partitioned between EtOAc (10 mL) and

brine (10 mL). The organic layers were concentrated and puriﬁed by

1

RP-HPLC to give title compound as a solid (74 mg, 76%). H NMR

(400 MHz, CDCl₃) δ 8.36 (1H, d, J = 8.0 Hz), 7.53 (1H, d, J = 8.0

Hz), 7.25 (1H, m), 6.76 (1H, m), 6.69 (2H, m), 6.49 (1H, bs), 5.20

(2H, s), 3.68 (2H, dt, J = 6.7, 2.5 Hz), 3.21 (2H, t, J = 6.7 Hz). ¹³C

NMR (100 MHz, CDCl₃) δ 165.3, 163.6 (d, J_C−F= 244.2 Hz), 159.9,

159.4 (d, J_C−F= 10.9 Hz), 158.3, 136.8, 130.4 (d, J_C−F= 9.8 Hz),

123.9, 119.9, 110.5 (d, J_C−F= 2.9 Hz), 108.3 (d, J_C−F= 21.1 Hz), 102.7

(d, J_C−F= 24.9 Hz), 70.5, 39.4, 30.9. HRMS (ES+, M + H) calcd for

C₁₅H₁₄FN₂O₂, 273.1039; found, 273.1037.

129.6, 127.0 (d, J_C−F= 8.5 Hz), 124.3, 121.4, 120.1, 115.9 (d, J_C−F

22.5 Hz), 114.8, 70.1, 48.7, 31.2. HRMS (ES+, M + H) calcd for

C₂₁H₁₈FN₂O₂, 349.1352; found, 349.1351.

=

2-((3-Fluorophenoxy)methyl)-6-(5-ﬂuoropyridin-2-yl)-7,8-dihy-

dro-1,6-naphthyridin-5(6H)-one (13h). 2-((3-Fluorophenoxy)-

methyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one (13b, 26 mg, 0.095

mmol), 2-bromo-5-ﬂuoropyridine (33 mg, 0.19 mmol), CuI (3.8 mg,

0.02 mmol), and potassium carbonate (26 mg, 0.19 mmol) were

combined in a 2.0 mL microwave vial and placed under an argon

atmosphere. Degassed toluene (1.0 mL) was added followed by N¹,N²-

dimethylethane-1,2-diamine (5.8 μL, 0.053 mmol) and the mixture

heated at 120 °C for 16 h. The mixture was cooled to rt and ﬁltered

over Celite. The ﬁltrate was diluted with H₂O and extracted with

EtOAc (3×). The organic layers were combined and concentrated to

dryness. RP-HPLC puriﬁcation aﬀorded the title compound as a

2-((3-Fluorophenoxy)methyl)-6-methyl-7,8-dihydro-1,6-naph-

thyridin-5(6H)-one (13c). 2-((3-Fluorophenoxy)methyl)-7,8-dihydro-

1,6-naphthyridin-5(6H)-one (13b) (10.0 mg, 0.04 mmol) was

dissolved in DMF (0.5 mL). NaH (2.0 mg, 0.08 mmol) was added

into the solution and stirred at rt. After 30 min, MeI (5.0 μL) was

added into the mixture and allowed to stir for 1 h. The mixture was

then quenched with H₂O (2.0 mL) and extracted with EtOAc (3×).

The organic layers were combined and concentrated to aﬀord product

as a white solid (9.2 mg, 87%). LCMS t_R= 0.81 min, > 98% at 215 and

1

powder (31 mg, 89%). H NMR (400 MHz, CDCl₃) δ 8.46 (1H, d, J

= 8.0 Hz), 8.29 (1H, d, J = 3.0 Hz), 8.01 (1H, dd, J = 9.1, 4.0 Hz), 7.57

(1H, d, J = 8.1 Hz), 7.47 (1H, m), 7.23 (1H, m), 6.77 (1H, m), 6.71

(2H, m), 5.23 (2H, s), 4.38 (2H, t, J = 6.7 Hz), 3.32 (2H, t, J = 6.6

Hz). ¹³C NMR (100 MHz, CDCl₃) δ 163.6 (d, J_C−F= 244.4 Hz),

163.6, 160.4, 159.4 (d, J_C−F= 10.8 Hz), 158.4, 156.8 (d, J_C−F= 252.0

Hz), 149.5 (d, J_C−F= 7.8 Hz), 137.6, 135.1 (d, J_C−F= 25.2 Hz), 130.4

1

254 nm, m/z = 287.2 [M + H]. H NMR (400 MHz, CDCl₃) δ 8.30

(1H, d, J = 8.0 Hz), 7.45 (1H, d, J = 8.0 Hz), 7.25 (2H, m), 6.76 (1H,

m), 6.62 (2H, m), 5.10 (2H, s), 3.65 (2H, dt, J = 6.9, 2.5 Hz), 3.20

(2H, t, J = 6.7 Hz), 3.16 (s, 3 H).

2-((3-Fluorophenoxy)methyl)-6-isobutyl-7,8-dihydro-1,6-naph-

thyridin-5(6H)-one (13d). 2-((3-Fluorophenoxy)methyl)-7,8-dihydro-

1,6-naphthyridin-5(6H)-one (13b) (100 mg, 0.41 mmol) was

dissolved in DMF (2.0 mL). KOt-Bu (55 mg, 0.5 mmol) was added

followed by 1-bromo-2-methylpropane (70 μL, 0.5 mmol). The

mixture was heated to 60 °C for 4 h. The mixture was then quenched

with H₂O (2.0 mL) and extracted with EtOAc (3×). The organic

layers were combined, washed with brine, and concentrated. The

resulting crude mixture was puriﬁed by RP-HPLC to give title

compound as a powder (43 mg, 44%). ¹H NMR (400 MHz, CDCl₃) δ

8.37 (1H, d, J = 8.0 Hz), 7.51, (1H, d, J = 8.0 Hz), 7.22 (1H, m), 6.76

(1H, m), 6.69 (2H, m), 5.19 (2H, s), 3.65 (2H, t, J = 6.8 Hz), 3.40

(2H, d, J = 7.5 Hz), 3.19 (2H, t, J = 6.7 Hz), 2.05 (1H, sep, J = 6.8

Hz), 0.97 (6H, d, J = 6.7 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 163.6

(d, J_C−F= 244.2 Hz), 163.4, 159.5 (d, J_C−F= 10.7 Hz), 159.1, 157.4,

137.1, 130.4 (d, J_C−F= 10.0 Hz), 124.5, 119.9, 110.5 (d, J_C−F= 2.9

Hz), 108.2 (d, J_C−F= 21.2 Hz), 102.7 (d, J_C−F= 24.9 Hz), 70.5, 54.7,

45.8, 30.9, 27.2, 20.1. HRMS (ES+, M + H) calcd for C₁₉H₂₂FN₂O₂,

329.1665; found, 329.1662.

(d, J_C−F= 9.9 Hz), 124.4, 124.3 (d, J_C−F= 19.7 Hz), 120.9 (d, J_C−F

=

4.5 Hz), 119.9, 110.4 (d, J_C−F= 2.9 Hz), 108.3 (d, J_C−F= 21.1 Hz),

102.7 (d, J_C−F= 24.9 Hz), 70.5, 45.0, 31.2. HRMS (ES+, M + H) calcd

for C₂₀H₁₆F₂N₃O₂, 368.1211; found, 368.1210.

6-Benzyl-2-((3-ﬂuorophenoxy)methyl)-7,8-dihydro-1,6-naphthyr-

idin-5(6H)-one (13i). Starting from 2-((3-ﬂuorophenoxy)methyl)-7,8-

dihydro-1,6-naphthyridin-5(6H)-one (13b) (100 mg, 0.41 mmol) and

(bromomethyl)benzene (67 μL, 0.55 mmol), the general procedure

outlined in 13d was performed. RP-HPLC aﬀorded the title

compound as a powder (74 mg, 66%). ¹H NMR (400 MHz,

CDCl₃) δ 8.44 (1H, d, J = 8.0 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.32 (5H,

m), 7.24 (1H, m), 6.76 (1H, m), 6.69 (2H, m), 5.19 (2H, s), 4.80 (2H,

s), 3.59 (2H, t, J = 6.8 Hz), 3.15 (2H, t, J = 6.8 Hz). ¹³C NMR (100

MHz, CDCl₃) δ 163.6 (d, J_C−F= 244.3 Hz), 163.4, 159.4 (d, J_C−F

10.6 Hz), 159.3, 157.5, 137.2, 136.9, 130.4 (d, J_C−F= 10.0 Hz), 128.8,

128.1, 127.7, 124.2, 119.9, 110.5 (d, J_C−F= 2.9 Hz), 108.2 (d, J_C−F

=

21.4 Hz), 102.7 (d, J_C−F= 24.9 Hz), 70.5, 50.4, 44.5, 30.7. HRMS (ES

+, M + H) calcd for C₂₂H₂₀FN₂O₂, 363.1509; found, 363.1507.

3-Phenoxymethyl-6,7-dihydro-5H-[1,7]naphthyridin-8-one (14a).

Ring-closure step: Starting from 3-(2-oxo-ethyl)-5-phenoxymethyl-

pyridine-2-carboxylic acid methyl ester (128 mg, 0.45 mmol) and

benzylamine (0.059 mL, 0.54 mmol) in CH₂Cl₂(8 mL), the mixture

was stirred at rt for 1 h and sodium triacetoxyborohydride (95 mg,

0.45 mmol) was added and the mixture was stirred at rt for 16 h. The

6-(Cyclopropylmethyl)-2-((3-ﬂuorophenoxy)methyl)-7,8-dihydro-

1,6-naphthyridin-5(6H)-one (13e). Starting from 2-((3-

ﬂuorophenoxy)methyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one

(13b) (100 mg, 0.41 mmol) and (bromomethyl)cyclopropane (50 μL,

0.50 mmol), the general procedure outlined in 13d was performed.

RP-HPLC aﬀorded the title compound as a powder (64 mg, 66%). ¹H

L

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mixture was diluted with CH₂Cl₂and washed with a saturated solution

of NaHCO₃and brine. The organic layer was separated, dried

(Na₂SO₄), ﬁltered, and the solvents evaporated in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (silica; 7N

solution of ammonia in methanol in CH₂Cl₂0/100 to 4/96). The

desired fractions were collected and concentrated to yield, 7-benzyl-3-

phenoxymethyl-6,7-dihydro-5H-[1,7]naphthyridin-8-one as an oil (41

mg, 23%). LCMS t_R= 2.83 min, > 90% at 215 and 254 nm, m/z =

255.1 [M + H]. Debenzylation step: Triﬂuoromethanesulfonic acid

0.020 mL, 0.23 mmol) was added to a solution of 7-benzyl-3-

phenoxymethyl-6,7-dihydro-5H-[1,7]naphthyridin-8-one (20 mg,

0.058 mmol) in toluene (0.5 mL) in a sealed tube. The mixture was

stirred at 150 °C for 15 min under microwave irradiation. DOWEX 1

× 2−100 (strongly basic anion exchanger) (300 mg) was added,

followed by MeOH (1 mL). The mixture was shaken 4 h at rt, and the

resin was ﬁltered and washed with CH₂Cl₂(1 mL), MeOH (1 mL),

CH₂Cl₂(1 mL), and MeOH (1 mL). The ﬁltrate was evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica, 7 M ammonia in methanol in CH₂Cl₂0/100 to 5/95).

The desired fractions were collected and the solvents evaporated in

vacuo to yield an impure fraction which was puriﬁed by RP-HPLC on

(C18 XBridge 19 mm × 100 mm 5 μm). Mobile phase (gradient from

80% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 20% CH₃CN

to 0% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 100%

CH₃CN) to yield 14a as a white solid (6.26 mg, 42% yield). LCMS t_R

diluted with CH₂Cl₂and washed with a saturated solution of NaHCO₃

and brine. The organic layer was separated, dried (Na₂SO₄), ﬁltered,

and the solvents evaporated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (silica; 7N solution of ammonia in

methanol in CH₂Cl₂0/100 to 4/96). The desired fractions were

collected and the solvents evaporated in vacuo to yield an impure

fraction which was repuriﬁed by RP-HPLC on (C18 XBridge 19 mm

× 100 mm, 5 μm). Mobile phase (gradient from 80% 0.1%

NH₄CO₃H/NH₄OH pH 9 solution in water, 20% CH₃CN to 0%

0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 100% CH₃CN), to

yield 14d as a white solid (15 mg, 15%). LCMS t_R= 2.00 min, > 98%

1

at 215 and 254 nm, m/z = 349.1 [M + H]. H NMR (500 MHz,

CDCl₃) δ 8.78 (d, J = 1.7 Hz, 1 H), 7.76 (br. s, 1 H), 7.42−7.36 (m, 2

H), 7.35−7.29 (m, 2 H), 7.16−7.07 (m, 2 H), 7.01 (t, J = 7.5 Hz, 1

H), 6.98 (d, J = 7.8 Hz, 2 H), 5.17 (s, 2 H), 4.01 (t, J = 6.4 Hz, 2 H),

3.22 (t, J = 6.4 Hz, 2 H).

6-Phenoxymethyl-3,4-dihydro-2H-[2,7]naphthyridin-1-one (15a).

A 7N solution of ammonia in methanol (5.5 mL) was added to 6-

phenoxymethyl-4-vinyl-nicotinic acid methyl ester (33, 55 mg, 0.2

mmol). The mixture was stirred at 100 °C for 16 h. The solvents were

evaporated in vacuo. The crude product was puriﬁed by ﬂash

chromatography (silica; AcOEt in CH₂Cl₂0/100 to 100/0). The

desired fractions were collected and the solvents evaporated in vacuo

to yield an impure fraction which was triturated with Et₂O and

repuriﬁed by RP-HPLC on (C18 XBridge 30 mm × 100 mm, 5 μm).

Mobile phase (gradient from 80% 0.1% NH₄CO₃H/NH₄OH pH 9

solution in water, 20% CH₃CN to 0% 0.1% NH₄CO₃H/NH₄OH pH 9

solution in water, 100% CH₃CN) to yield 15a as a white solid (20 mg,

39%); mp 193 °C. LCMS t_R= 1.47 min, > 98% at 215 and 254 nm, m/

z = 255.1 [M + H]. ¹H NMR (400 MHz, CDCl₃) δ 9.18 (s, 1 H), 7.43

(d, J = 0.5 Hz, 1 H), 7.36−7.28 (m, 2 H), 7.06−6.94 (m, 3 H), 6.26

(br s, 1 H), 5.24 (s, 2 H), 3.61 (td, J = 6.6, 2.9 Hz, 2 H), 3.03 (t, J =

6.7 Hz, 2 H)..

2-Methyl-6-phenoxymethyl-3,4-dihydro-2H-[2,7]naphthyridin-1-

one (15b). Starting from 6-phenoxymethyl-4-vinyl-nicotinic acid

methyl ester (33, 55 mg, 0.2 mmol) and 2 M methylamine in THF

(5.5 mL) following the procedure described for compound 15a,

compound 15b was obtained as a white solid (51 mg, 93%); mp 134

°C. LCMS t_R= 1.72 min, > 98% at 215 and 254 nm, m/z = 269.1 [M

+ H]. ¹H NMR (500 MHz, CDCl₃) δ 9.18 (s, 1 H), 7.38 (s, 1 H), 7.31

(s, 2 H), 7.04−6.95 (m, 3 H), 5.23 (s, 2 H), 3.59 (t, J = 6.6 Hz, 2 H),

3.16 (s, 3 H), 3.03 (t, J = 6.6 Hz, 2 H)..

6-Phenoxymethyl-2(R)-(1,2,2-trimethyl-propyl)-3,4-dihydro-2H-

[2,7]naphthyridin-1-one (15c). Acetic acid (0.006 mL, 0.11 mmol)

was added to a solution of 6-phenoxymethyl-4-vinyl-nicotinic acid

methyl ester (33, 29 mg, 0.11 mmol) and (R)-(+)-3,3-dimethyl 2-

aminobutane (0.030 mL, 0.225 mmol) in MeOH (1 mL). The mixture

was stirred at 100 °C for 16 h. Then (R)-(+)-3,3-dimethyl 2-

aminobutane (0.030 mL, 0.225 mmol) and acetic acid (0.006 mL, 0.11

mmol) were added, and the mixture was heated at 100 °C for 2 days.

The solvents were evaporated in vacuo and the residue diluted with

CH₂Cl₂and extracted with a saturated solution of NaHCO₃. The

organic layer was separated, dried (Na₂SO₄), ﬁltered, and the solvents

evaporated in vacuo. The crude product was puriﬁed by ﬂash

chromatography (silica; AcOEt in CH₂Cl₂0/100 to 30/70). The

desired fractions were collected and the solvents evaporated in vacuo

to yield compound 15c as a colorless oil which solidiﬁed upon

standing at room temperature (29 mg, 79%). LCMS t_R= 3.10 min, >

98% at 215 and 254 nm, m/z = 339.1 [M + H]. ¹H NMR (500 MHz,

CDCl₃) δ 9.19 (s, 1 H), 7.38 (s, 1 H), 7.31 (t, J = 8.1 Hz, 2 H), 7.03−

6.92 (m, 3 H), 5.24 (s, 2 H), 4.83 (q, J = 7.2 Hz, 1 H), 3.59−3.43 (m,

2 H), 3.06−2.93 (m, 1 H), 2.93−2.81 (m, 1 H), 1.21 (d, J = 6.9 Hz, 3

H), 0.99 (s, 9 H).

1

= 1.36 min, > 98% at 215 and 254 nm, m/z = 255.1 [M + H]. H

NMR (500 MHz, CDCl₃) δ 8.74 (d, J = 1.4 Hz, 1 H), 7.73 (br. s, 1

H), 7.39−7.29 (m, 2 H), 7.24 (br. s, 1 H), 7.05−6.94 (m, 3 H), 5.14

(s, 2 H), 3.64 (td, J = 6.6, 2.9 Hz, 2 H), 3.09 (t, J = 6.6 Hz, 2 H).

7-Methyl-3-(phenoxymethyl)-6,7-dihydro-1,7-naphthyridin-

8(5H)-one (14b). Methylamine (2.0 M in THF) (2 mL) was added to

a stirred solution of 3-(2-oxo-ethyl)-5-phenoxymethyl-pyridine-2-

carboxylic acid methyl ester (40 mg, 0.14 mmol) in CH₂Cl₂(2

mL). The mixture was stirred at rt for 1 h. Then, sodium

triacetoxyborohydride (45 mg, 0.21 mmol) was added and the mixture

was stirred at rt for 16 h. The mixture was diluted with CH₂Cl₂and

washed with a saturated solution of NaHCO₃and brine. The organic

layer was separated, dried (Na₂SO₄), ﬁltered, and the solvents

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica; 7N solution of ammonia in methanol in

CH₂Cl₂0/100 to 4/96). The desired fractions were collected and the

solvents evaporated in vacuo to yield an impure fraction which was

repuriﬁed by RP-HPLC on (C18 XBridge 19 mm × mm 100 5 μm).

Mobile phase (gradient from 80% 0.1% NH₄CO₃H/NH₄OH pH 9

solution in water, 20% CH₃CN to 0% 0.1% NH₄CO₃H/NH₄OH pH 9

solution in water, 100% CH₃CN) to yield 14b as a white solid (5.8 mg,

15%). LCMS t_R= 1.59 min, > 98% at 215 and 254 nm, m/z = 269.1

1

[M + H]. H NMR (500 MHz, CDCl₃) δ 8.73 (d, J = 1.7 Hz, 1 H),

7.68 (s, 1 H), 7.36−7.28 (m, 2 H), 7.08−6.90 (m, 3 H), 5.13 (s, 2 H),

3.62 (t, J = 6.6 Hz, 2 H), 3.22 (s, 3 H), 3.08 (t, J = 6.8 Hz, 2 H). .

3-Phenoxymethyl-7R-(1,2,2-trimethyl-propyl)-6,7-dihydro-5H-

[1,7]naphthyridin-8-one (14c). Starting from 3-(2-oxo-ethyl)-5-

phenoxymethyl-pyridine-2-carboxylic acid methyl ester (40 mg, 0.14

mmol) and (R)-(+)-3,3-dimethyl 2-aminobutane 0.039 mL, 0.34

mmol) following the procedure described for compound 14a−b,

compound 14c was obtained as a beige solid (24 mg, 25%). LCMS t_R

1

= 2.82 min, > 98% at 215 and 254 nm, m/z = 339.1 [M + H]. H

NMR (500 MHz, CDCl₃) δ 8.73 (d, J = 1.7 Hz, 1 H), 7.68 (s, 1 H),

7.28−7.37 (m, 2 H), 6.92−7.04 (m, 3 H), 5.13 (s, 2 H), 4.92 (q, J =

7.2 Hz, 1 H), 3.62−3.46 (m, 2 H), 3.10−2.98 (m, 1 H), 2.98−2.84 (m,

1 H), 1.22 (d, J = 7.2 Hz, 3 H), 1.00 (s, 9 H); α_D²⁰= −15.7° (c = 0.55

w/v%, MeOH).

7-(4-Fluoro-phenyl)-3-phenoxymethyl-6,7-dihydro-5H-[1,7]-

naphthyridin-8-one (14d). 4-Fluoroaniline (0.032 mL, 0.34 mmol)

was added to a stirred solution of 3-(2-oxo-ethyl)-5-phenoxymethyl-

pyridine-2-carboxylic acid methyl ester (80 mg, 0.28 mmol) in CH₂Cl₂

(4 mL). The mixture was stirred at rt for 1 h. Then, sodium

triacetoxyborohydride (89 mg, 0.42 mmol) was added and the mixture

was stirred at rt for 16 h. Then, acetic acid (0.040 mL) was added and

the mixture was stirred at rt for additional 20 h. The mixture was

2-(4-Fluoro-phenyl)-6-phenoxymethyl-3,4-dihydro-2H-[2,7]-

naphthyridin-1-one (15d). Starting from 6-phenoxymethyl-4-vinyl-

nicotinic acid methyl ester (33, 30 mg, 0.11 mmol) and 4-ﬂuoroaniline

(0.044 mL, 0.47 mmol) following the procedure described for

compound 15c, title compound 15d was obtained as a white solid (24

mg, 62%); mp 198.8 °C. LCMS t_R= 2.66 min, > 98% at 215 and 254

nm, m/z = 349.1 [M + H]. ¹H NMR (500 MHz, CDCl₃) δ 9.24 (s, 1

M

dx.doi.org/10.1021/jm500259z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

H), 7.46 (s, 1 H), 7.39−7.29 (m, 4 H), 7.17−7.08 (m, 2 H), 7.04−6.96

(m, 3 H), 5.27 (s, 2 H), 3.99 (t, J = 6.5 Hz, 2 H), 3.17 (t, J = 6.4 Hz, 2

H)..

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

basal was calculated before normalization to the maximal response to

glutamate. A 2-fold enhancement over basal response at the maximum

concentration was suﬃcient criteria for weak PAM activity (e.g., 40%

Glu max at an EC₂₀add of glutamate; the basal EC₂₀response range

allowed was 10−30%).

Selectivity Screening. mGlu₁. To assess the eﬀect of test

compounds at mGlu₁, Ca²⁺mobilization assays were performed as

described previously.^46,47Brieﬂy, HEK293 cells stably expressing rat

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

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

medium at a density of 20000 cells/well. Calcium ﬂux was measured

over time as an increase in ﬂuorescence of the Ca²⁺indicator dye,

Fluo-4AM using a FDSS 6000. Either vehicle or a ﬁxed concentration

of test compound (10 μM, ﬁnal concentration) was added, followed

140 s later by a CRC of glutamate. Data were analyzed as described

above.

Group II and Group III mGlus. The functional activity of the

compounds of interest was assessed at the rat group II and III mGlu

receptors by measuring thallium ﬂux through GIRK channels as

previously described.⁶²Brieﬂy, HEK293-GIRK cells expressing mGlu

subtypes 2, 3, 4, 6, 7, or 8 were plated into 384-well, black-walled,

clear-bottom poly-^D-lysine coated plates at a density of 15000 cells/

well in assay medium. A single concentration of test compound (10

μM) or vehicle was added, followed 140 s later by a CRC of glutamate

(or L-AP4 for mGlu₇) diluted in thallium buﬀer (125 mM NaHCO₃, 1

mM MgSO₄, 1.8 mM CaSO₄, 5 mM glucose, 12 mM thallium sulfate,

10 mM HEPES), and ﬂuorescence was measured using a FDSS 6000.

Data were analyzed as described previously.⁶²

Cyclopropyl(3-((3-ﬂuorobenzyl)oxy)-7,8-dihydro-1,6-naphthyri-

din-6(5H)-yl)methanone (20d). 3-(3-Bromo-7,8-dihydro-1,6-naph-

thyridin-6(5H)-yl)(cyclopropyl)methanone (35a, 35 mg, 0.125

mmol), 3-ﬂuorobenzyl alcohol (15 mg, 0.125 mmol), cesium

carbonate (59 mg, 0.18 mmol), CuI (3 mg, 0.01 mmol), and 1,10-

phenanthroline (3.9 mg, 0.02 mmol) were combined in a 2.0 mL

microwave vial and placed under an argon atmosphere. Degassed

toluene was added and the mixture heated at 110 °C for 4 h. The

mixture was cooled to rt, ﬁltered over Celite, and the ﬁltrate

concentrated to dryness. RP-HPLC puriﬁcation aﬀorded 20d as an oﬀ-

white powder (15 mg, 33%). LCMS t_R= 0.79 min, > 98% at 215 and

254 nm, m/z = 328.1 [M + H]. HRMS (ES+, M + H) calcd for

C₂₂H₂₀FN₂O₂, 327.1509; found, 327.1512.

(3-(Benzyloxy)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)(4-

ﬂuorophenyl)methanone (20g). 3-Bromo-7,8-dihydro-1,6-naphthyr-

idin-6(5H)-yl)(4-ﬂuorophenyl)methanone (35c, 28 mg, 0.09 mmol),

benzyl alcohol (9.6 mg, 0.09 mmol), cesium carbonate (43 mg, 0.13

mmol), CuI (2 mg, 0.009 mmol), and 1,10-phenanthroline (3.2 mg,

0.018 mmol) were combined in a 2.0 mL microwave vial and placed

under an argon atmosphere. Degassed toluene was added and the

mixture heated at 110 °C for 4 h. The mixture was cooled to rt, ﬁltered

over Celite, and the ﬁltrate concentrated to dryness. RP-HPLC

puriﬁcation aﬀorded the 20g as an oﬀ-white powder (6.9 mg, 22%).

LCMS t_R= 0.85 min, > 98% at 215 and 254 nm, m/z = 363.1 [M +

H]. HRMS (ES+, M + H) calcd for C₂₂H₂₂FN₂O₂, 363.1509; found,

363.1503.

(4-Fluorophenyl)(3-(phenoxymethyl)-7,8-dihydro-1,6-naphthyri-

din-6(5H)-yl)methanone (20a). (4-Fluorophenyl)(3-(hydroxymeth-

yl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)methanone (39, 40 mg,

0.14 mmol), phenol (40 mg, 0.43 mmol), and polystyrene supported

triphenylphosphine (150 mg, 0.45 mmol) were combined in THF (6

mL) and diisopropyl azodicarboxylate (42 mg, 0.21 mmol) was added,

and the reaction was allowed to stir overnight at rt. The reaction

mixture was ﬁltered through Celite and concentrated in vacuo. The

residue was puriﬁed via RP-HPLC (10−90% gradient of CH₃CN in

water with 0.1% TFA modiﬁer) to aﬀord title compound as an oﬀ-

white powder (35 mg, 69%). LCMS t_R= 0.75 min, > 98% at 215 and

In Vivo Pharmacology. Animal Husbandry. Animals were

housed in the animal care facility certiﬁed by the American Association

for the Accreditation of Laboratory Animal Care (AAALAC) under a

12 h light/dark cycle (lights on, 7 a.m.; lights oﬀ, 7 p.m.) and had free

access to food and water. The animals used in these experiments were

food-deprived the evening before experimentation for oral admin-

istration of test compound. The experimental protocols performed

during the light cycle were approved by the Institutional Animals Care

and Use Committee of Vanderbilt University and conformed to the

guidelines established by the National Research Council Guide for the

Care and Use of Laboratory Animals.

Preparation of Test Article. 12c was formulated in volumes speciﬁc

to the number of animals dosed each day. The solutions were

formulated so that animals were injected with a maximal dosing

volume of 10 mL/kg. The appropriate amount according to the dosage

was mixed into a 20% 2-(hydroxypropyl)-β-cyclodextrin in sterile

water (HP-β-CD; Sigma, catalogue no. C0926-10G) solution. Each

mixture was ultrahomogenized on ice for 2−3 min using a hand-held

tissue homogenizer. Next, the pH of all solutions was checked using

0−14 EMD pH strips and adjusted to a pH of 6−7 if necessary with

1N NaOH. The mixtures were then vortexed and stored in a

sonication bath at 40 °C until time of injection. d-Amphetamine

hemisulfate (AMP) was obtained from Sigma (catalogue no. A5880-

1G; St. Louis, MO). Salt-correction was used to determine the correct

amount of the d-amphetamine hemisulfate form in mg to add to sterile

water in order to yield a 1 mg/mL solution, injected with a maximal

dosing volume of 10 mL/kg.

Reversal of Amphetamine-Induced Hyperlocomotion. Stud-

ies were conducted using Smart Open Field activity chambers (27 cm

× 27 cm × 20 cm) (Kinder Scientiﬁc, Poway, CA) equipped with 16

horizontal (x- and y-axes) infrared photobeams. Changes in locomotor

activity were measured as the number of photobeam breaks over time

and were recorded with a Pentium I computer equipped with rat

activity monitoring system software (Motor Monitor, Kinder

Scientiﬁc, Poway, CA). Male Harlan Sprague−Dawley rats (Harlan

Laboratories, Indianapolis, IN) weighing 250−375 g were used.

Animals were habituated in the locomotor activity test chambers for 30

min. Animals were next pretreated for an additional 30 min with either

vehicle or a dose of 12c po, followed by a subcutaneous injection of 1

mg/kg amphetamine or vehicle and monitored for an additional 60

min.

1

254 nm, m/z = 363.1 [M + H]. H NMR (400 MHz, CDCl₃) δ 8.77

(s, 1H), 8.06 (s, 1H), 7.53−7.49 (m, 3H), 7.36 (t, J = 8.0, 2H), 7.18 (t,

J = 8.0, 2H), 7.06 (t, J = 8.2, 1H), 6.87 (d, J = 8.0, 1H), 5.18 (s, 2H),

4.98 (s, 2H), 3.92 (s, 2H), 3.36 (t, J = 6.0, 2H). HRMS (ES+, M + H)

calcd for C₂₂H₁₉FN₂O₂, 362.1431; found, 362.1434.

Fluorescence-Based Calcium Flux Assays (Concentration−

Response Curves (Potency) and Glutamate Fold Shifts

(Eﬃcacy)). For measurement of compound-evoked increases in

intracellular calcium, HEK293 cells stably expressing rat mGlu₅were

plated in 384-well,⁴⁴poly-D-lysine coated, black-walled, clear-bottomed

plates in 20 μL of assay medium (DMEM supplemented with 10%

dialyzed fetal bovine serum, 20 mM HEPES, and 1 mM sodium

pyruvate) at a density of 15000 cells/well. Cells were grown overnight

at 37 °C/5% CO₂. The next day, medium was removed and cells were

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

California) prepared as a 2.3 mM stock in dimethyl sulfoxide (DMSO)

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

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

Invitrogen, Carlsbad, CA) supplemented with 20 mM HEPES and 2.5

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

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

PAM potency curves, mGlu₅compounds were diluted in calcium assay

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

concentration of glutamate 140 s later and then an EC₈₀concentration

of glutamate 90−120 s later. For fold-shift experiments, either a single

concentration (10 μM) or multiple ﬁxed concentrations (55 nM to 30

μM) of mGlu₅compound or vehicle was added, followed by the

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

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

ﬂuorescence using a Functional Drug Screening System 6000 (FDSS

N

dx.doi.org/10.1021/jm500259z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Treatment Groups. Dose group 1, VAMP = 20% β-CD (12c

vehicle), po + 1 mg/kg AMP, sc (n = 8). Dose group 2, 3AMP = 3

mg/kg 12c, po + 1 mg/kg AMP, sc (n = 6). Dose group 3, 10AMP =

10 mg/kg 12c, po + 1 mg/kg AMP, sc (n = 7). Dose group 4, 30AMP

= 30 mg/kg 12c, po + 1 mg/kg AMP, sc (n = 8). Dose group 5,

56.6AMP = 56.6 mg/kg 12c, po + 1 mg/kg AMP, sc (n = 8). Dose

group 6, 100AMP = 100 mg/kg 12c, po + 1 mg/kg AMP, sc (n = 8).

Dose group 7, 100V = 100 mg/kg 12c, po + sterile water (AMP

vehicle), sc (n = 8). Changes in locomotor activity were recorded for a

total of 120 min. Data were expressed as changes in ambulation

deﬁned as the total number of photobeam breaks per 5 min interval.

At the end of this behavioral study, each animal was euthanized, then

decapitated, and the plasma and brain tissues were collected for the

evaluation of exposure levels of 12c.

Data Analysis. Behavioral data were analyzed using a one-way

ANOVA with main eﬀects of treatment and time. Post hoc analyses

were performed using a Dunnett’s t-test with all treatment groups

compared to the VAMP group using JMP 8.0 (SAS Institute, Cary,

NC) statistical software. Data were graphed using SigmaPlot for

Windows version 11.0 (Saugua, MA). A probability of p ≤ 0.05 was

taken as the level of statistical signiﬁcance. Percent eﬀect and reversal

calculations for the 3AMP, 10AMP, 30AMP, 56.6AMP, and 100AMP

treatment groups were performed with the following formula relative

to the VAMP treatment group: (1) Total number of photobeam

breaks in the time interval from t = 60 to t = 120 was calculated for

each rat in each treatment group, (2) mean total number of

photobeam breaks in the time interval from t = 60 to t = 120 was

calculated for the VAMP group, (3) percent eﬀect = ratio of the total

number of photobeam breaks for each rat in each treatment group

divided by the mean total number of photobeam breaks in the time

interval from t = 60 to t = 120 of the VAMP group multiplied by 100,

(4) percent reversal = 100 − the percent eﬀect for each rat in each

treatment group, (5) ﬁnally, the mean SE percent reversal for each

treatment group was calculated from the individual percent reversal

values. Percent eﬀect and change calculations for the VAMP and 100 V

treatment groups relative to the VV treatment group were also

performed with the following formula: (1) total number of photobeam

breaks in the time interval from t = 60 to t = 120 was calculated for

each rat in each treatment group, (2) mean total number of

photobeam breaks in the time interval from t = 60 to t = 120 was

calculated for the VV group; (3) percent eﬀect = ratio of the total

number of photobeam breaks for each rat in each treatment group

divided by the mean total number of photobeam breaks in the time

interval from t = 60 to t = 120 of the VV group multiplied by 100, (4)

percent change = the percent eﬀect for each rat in each treatment

time per sample. HPLC ﬂow rate was 0.5 mL/min, source temperature

was 500 °C, and mass spectral analyses were performed using the

following MRM transitions: 353.5 → 259.2 and 353.5 → 123.2 m/z.

ESI was achieved by a turbo-ionspray source in positive ionization

mode (5.0 kV spray voltage). The raw plasma and brain concentration

data obtained by LC-MS/MS were analyzed by Analyst software (AB

Sciex, version 1.5.1), which used the ratio of peak area responses of

drug relative to internal standard to construct a standard curve with a

dynamic range covering the concentrations found in the samples. Free

plasma and brain concentrations were calculated by multiplication of

the total concentration values by f_u,plasmaand f_u,brain, respectively, based

on data from in vitro rat plasma protein and rat brain homogenate

binding experiments.

Rotarod. The eﬀects of 12c on motor performance were evaluated

using an automated rotarod setup with a rotarod (7.0 cm in diameter)

rotating at a constant speed of 20 rotations/min (MedAssociates, Inc.,

St Albans, CA). Male Harlan Sprague−Dawley rats (Harlan

Laboratories, Indianapolis, IN) weighing 250−300 g were used.

Animals were given two training trials of 120 s on the rotarod with a

10 min interval between trials, followed by baseline assessment of

performance with a 120 s trial, and any animals that did not reach a

performance criteria of 85 s were excluded from the study. Animals

were next pretreated for 30 min with vehicle (20% HP-β-CD) or a

dose of 12c (n = 6 each group) and then tested on the rotarod using a

120 s trial. The amount of time in seconds that each animal remained

on the rotarod was recorded; animals not falling oﬀ of the rotarod

were given a maximal score of 120 s. Data were expressed as the mean

latency to fall oﬀ the rotarod in seconds for each treatment group.

Data Analysis. Behavioral data were analyzed using a one-way

ANOVA with main eﬀects of treatment. Post hoc analyses were

performed using a Dunnett’s t test with all treatment groups compared

to the vehicle group using JMP 8.0 (SAS Institute, Cary, NC)

statistical software. Data were graphed using SigmaPlot for Windows

Version 11.0 (Saugua, MA). A probability of p ≤ 0.05 was taken as the

level of statistical signiﬁcance.

Modiﬁed Irwin Neurological Test Battery. Male Harlan

Sprague−Dawley rats (Harlan Laboratories, Indianapolis, IN)

weighing 250−300 g were used. Animals were evaluated in the

modiﬁed Irwin neurological test battery at t = 0 to provide a baseline

measurement across each functional end point. Animals were next

administered either vehicle (20% HP-β-CD) or a 100 mg/kg po dose

of 12c (n = 6 each group) and then assessed after a 30 min, 1 h, and 4

h pretreatment interval in the modiﬁed Irwin neurological test battery.

Changes in the diﬀerent functional end points of the Irwin test battery

were given a score of 0, 1 or 2, with 0 = no eﬀect, 1 = moderate eﬀect,

and 2 = robust full eﬀect. All data were collected blinded to treatment

for each animal. The following functional end points were scored.

Autonomic nervous system functions: ptosis, exophthalmos, miosis,

mydriasis, corneal reﬂex, pinna reﬂex, piloerection, respiratory rate,

writhing, tail erection, lacrimation, salivation, vasodilation, skin color,

irritability, and rectal temperature. Somatomotor nervous system

functions: motor activity, ataxia, arch/roll, tremors, leg weakness, rigid

stance, spraddle, placing loss, grasping loss, righting loss, catalepsy, tail

pinch reaction, escape loss, and physical appearance.

Data Analysis. Mean change values for each functional end point in

the Irwin test battery of each treatment group were calculated using

Microsoft Excel. Behavioral data were then analyzed using a one-way

ANOVA with main eﬀect of treatment. Post hoc analyses were

performed using a Dunnett’s t test with all treatment groups compared

to the vehicle group using JMP 8.0 (SAS Institute, Cary, NC)

statistical software. Data were graphed using SigmaPlot for Windows

version 11.0 (Saugua, MA). A probability of p ≤ 0.05 was taken as the

level of statistical signiﬁcance.

group − 100, (5) ﬁnally, the mean

SE percent change for each

treatment group was calculated from the individual percent change

values.

LC-MS/MS Sample Preparation and Analysis Methodology. Brain

samples were homogenized in 3 mL of 70:30 2-propanol:water and

then centrifuged at 3500 rpm for 5 min. Resulting brain sample

supernatant and plasma samples were transferred into a 96-well plate

containing plasma blanks, plasma double blank, a standard curve of the

test article, and quality control samples for subsequent LC-MS/MS

analysis. Each sample was diluted with acetonitrile containing 50 nM

carbamazepine (internal standard), centrifuged at 3500 rpm, and the

resulting supernatant was transferred to a new 96-well plate. After an

equal volume of water was added to each sample (to provide 50:50

acetonitrile:water solution), the plate was analyzed via ESI on a triple-

quadrupole mass spectrometer (AB Sciex API-4000) coupled with an

autosampling liquid chromatography system (Shimadzu LC-10AD

pumps, Leap Technologies CTC PAL autosampler). Analyte (test

article) was separated by gradient elution using a C18 3.0 mm × 50

mm, 3 μm column (Fortis Technologies) thermostated at 40 °C.

HPLC mobile phase A was 0.1% formic acid in water (pH

unadjusted), mobile phase B was 0.1% formic acid in acetonitrile

(pH unadjusted), and the gradient used was 30−90% mobile phase B

with 0.2 min hold at 30% B, linear increase to 90% B over 0.8 min,

hold at 90% B for 0.6 min, and a return to 30% B over 0.1 min

followed by a re-equilibration (0.5 min) to provide a 2.0 min total run

ASSOCIATED CONTENT

■

S

* Supporting Information

mGlu selectivity for 12c, 13g, and 14d; operational model

aﬃnity and cooperativity calculations (12c), DMPK procedures

and methods, rotarod, modiﬁed Irwin, Euroﬁns ancillary

O

dx.doi.org/10.1021/jm500259z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(12) Williams, D. L., Jr.; Lindsley, C. W. Discovery of positive

allosteric modulators of metabotropic glutamate receptor subtype 5

(mGluR5). Curr. Top. Med. Chem. 2005, 5, 825−846.

pharmacology, compound characterization, NMR spectra for

representative compounds, and subseries 19. This material is

available free of charge via the Internet at http://pubs.acs.org.

(13) Shipe, W. D.; Wolkenberg, S. E.; Williams, D. L., Jr.; Lindsley,

C. W. Recent advances in positive allosteric modulators of

metabotropic glutamate receptors. Curr. Opin. Drug Discovery Dev.

2005, 8, 449−457.

AUTHOR INFORMATION

Corresponding Author

■

*Phone: (615)-936-8407. Fax: (615)-343-3088.E-mail: shaun.

stau ﬀ er@vanderbilt.edu. Address: Vanderbilt University Med-

ical Center, Vanderbilt Center for Neuroscience Drug

Discovery, 1205 Light Hall, Nashville, Tennessee 37232-0697,

United States.

(14) Marino, M. J.; Conn, P. J. Direct and indirect modulation of the

N-methyl ^D-aspartate receptor. Curr. Drug Targets: CNS Neurol. Disord.

2002, 1, 1−16.

(15) Stauffer, S. R. Progress toward positive allosteric modulators of

the metabotropic glutamate receptor rubtype 5 (mGlu5). ACS Chem.

Neurosci. 2011, 2, 450−470.

Notes

(16) Lindsley, C. W.; Stauffer, S. R. Metabotropic glutamate receptor

5-positive allosteric modulators for the treatment of schizophrenia

(2004−2012). Pharm. Pat. Analyst 2013, 2, 93−108.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

(17) Kinney, G. G.; O’Brien, J. A.; Lemaire, W.; Burno, M.; Bickel, D.

J.; Clements, M. K.; Chen, T. B.; Wisnoski, D. D.; Lindsley, C. W.;

Tiller, P. R.; Smith, S.; Jacobson, M. A.; Sur, C.; Duggan, M. E.;

Pettibone, D. J.; Conn, P. J.; Williams, D. L., Jr. A novel selective

positive allosteric modulator of metabotropic glutamate receptor

subtype 5 has in vivo activity and antipsychotic-like effects in rat

behavioral models. J. Pharmacol. Exp. Ther. 2005, 313, 199−206.

(18) Le Poul, E.; Bessis, A. S.; Lutgens, R.; Bonnet, B.; Rocher, J. P.;

Epping-Jordan, M. P.; Mutel, V. In vitro pharmacological character-

ization of selective mGluR5 positive allosteric modulators. 5th

International Metabotropic Glutamate Receptors Meeting, Taormina,

Italy, 2005.

■

Vanderbilt Center for Neuroscience Drug Discovery

(VCNDD) research was supported by grants from Janssen

Pharmaceutical Companies of Johnson & Johnson and in part

by the NIH (NS031373, MH062646).

ABBREVIATIONS USED

■

mGlu, metabotropic glutamate receptor; PAM, positive

allosteric modulator; NAM, negative allosteric modulator;

LTD, long-term depression; MPEP, 2-methyl-6-

(phenylethynyl)pyridine; CPPHA, N-(4-chloro-2-((1,3-dioxoi-

soindolin-2-yl)methyl)phenyl)-2-hydroxybenzamide; CDPPB,

3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; AHL,

amphetamine-induced hyperlocomotion

(19) Bessis, A.-S.; Bonnet, B.; Le, P. E.; Rocher, J.-P.; Epping-Jordan,

M. Preparation of piperidine derivatives as modulators of metabotropic

glutamate receptors (mGluR5). WO 2005044797, 2005.

(20) Sharma, S.; Kedrowski, J.; Rook, J. M.; Smith, R. L.; Jones, C. K.;

Rodriguez, A. L.; Conn, P. J.; Lindsley, C. W. Discovery of molecular

switches that modulate modes of metabotropic glutamate receptor

subtype 5 (mGlu5) pharmacology in vitro and in vivo within a series of

functionalized, regioisomeric 2- and 5-(phenylethynyl)pyrimidines. J.

Med. Chem. 2009, 52, 4103−4106.

(21) Rodriguez, A. L.; Grier, M. D.; Jones, C. K.; Herman, E. J.; Kane,

A. S.; Smith, R. L.; Williams, R.; Zhou, Y.; Marlo, J. E.; Days, E. L.;

Blatt, T. N.; Jadhav, S.; Menon, U. N.; Vinson, P. N.; Rook, J. M.;

Stauffer, S. R.; Niswender, C. M.; Lindsley, C. W.; Weaver, C. D.;

Conn, P. J. Discovery of novel allosteric modulators of metabotropic

glutamate receptor subtype 5 reveals chemical and functional diversity

and in vivo activity in rat behavioral models of anxiolytic and

antipsychotic activity. Mol. Pharmacol. 2010, 78, 1105−1123.

(22) Gastambide, F.; Cotel, M. C.; Gilmour, G.; O’Neill, M. J.;

Robbins, T. W.; Tricklebank, M. D. Selective remediation of reversal

learning deficits in the neurodevelopmental MAM model of

schizophrenia by a novel mGlu5 positive allosteric modulator.

Neuropsychopharmacology 2012, 37, 1057−1066.

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dx.doi.org/10.1021/jm500259z | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Tetrahydronaphthyridine and dihydronaphthyridinone ethers as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu 5)

DOI: 10.1021/jm500259z

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Article abstract of DOI:10.1021/jm500259z

Full text of DOI:10.1021/jm500259z

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