Discovery of (R)-(2-fluoro-4-((-4-methoxyphenyl)ethynyl)phenyl) (3-hydroxypiperidin-1-yl)methanone (ML337), an mGlu3 selective and CNS penetrant negative allosteric modulator (NAM)

Article abstract of DOI:10.1021/jm400439t

A multidimensional, iterative parallel synthesis effort identified a series of highly selective mGlu₃ NAMs with submicromolar potency and good CNS penetration. Of these, ML337 resulted (mGlu₃ IC₅₀ = 593 nM, mGlu₂ IC₅₀ >30 μM) with B:P ratios of 0.92 (mouse) to 0.3 (rat). DMPK profiling and shallow SAR led to the incorporation of deuterium atoms to address a metabolic soft spot, which subsequently lowered both in vitro and in vivo clearance by >50%.

Full text of DOI:10.1021/jm400439t

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Brief Article
Discovery of (R)-(2-fluoro-4-((-4-methoxyphenyl)ethynyl)phenyl)
(3-hydroxypiperidin-1-yl)methanone (ML337), an mGlu3 Selective
and CNS Penetrant Negative Allosteric Modulator (NAM)
Cody J. Wenthur, Ryan Morrsion, Andrew S Felts, Katrina A. Smith, Julie L. Engers,
Frank W. Byers, J. Scott Daniels, Kyle A Emmitte, P. Jeffrey Conn, and Craig W Lindsley
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400439t • Publication Date (Web): 29 May 2013
Downloaded from http://pubs.acs.org on June 1, 2013
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Journal of Medicinal Chemistry
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Discovery of (R)-(2-fluoro-4-((-4-
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methoxyphenyl)ethynyl)phenyl) (3-hydroxypiperidin-1-
yl)methanone (ML337), an mGlu3 Selective and CNS Penetrant
Negative Allosteric Modulator (NAM)
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Cody J. Wenthur ‡,§,#, Ryan Morrison ‡,§,#, Andrew S. Felts‡,§,#, Katrina A. Smith‡,§,#, Julie L. Engers‡,§,#, Frank W.
,‡,§, ,#
Byers‡,§,#, J. Scott Daniels ‡,§,#, Kyle A. Emmitte‡,§, ,#, P. Jeffrey Conn‡,§,#, Craig W. Lindsley*
║
║
‡Department of Pharmacology, §Vanderbilt Center for Neuroscience Drug Discovery, #Vanderbilt Specialized
Chemistry for Accelerated Probe Development (MLPCN), Vanderbilt University Medical Center, Nashville, Tennes-
see 37232, ║Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232.
KEYWORDS. Metabotropic glutamate receptor, mGlu3, negative allosteric modulator (NAM), ML337, MLPCN probe
Supporting Information Placeholder
ABSTRACT: A multi-dimensional, iterative parallel synthesis effort identified a series of highly selective mGlu3 NAMs
with sub-micromolar potency and good CNS penetration. Of these, ML337 resulted (mGlu3 IC50 = 593 nM, mGlu2 IC50 >30
µM) with B:P ratios of 0.92 (mouse) to 0.3 (rat). DMPK profiling and shallow SAR led to the incorporation of deuterium
atoms to address a metabolic soft spot, which subsequently lowered both in vitro and in vivo clearance by >50%.
INTRODUCTION
To date, only three mGlu3 NAMs have been reported
(Figure 1).11-13 The first, RO4491533 (1), a dual mGlu2/mGlu3
NAM (mGlu2 IC50 = 296 nM, mGlu3 IC50 = 270 nM)
G-protein-coupled metabotropic glutamate receptors
(mGluRs) have emerged as new drug targets with poten-
tial for treatment of a range of CNS disorders.1-4 Highly
subtype-selective allosteric ligands have previously been
developed for mGlu1, mGlu4, mGlu5 and mGlu7.1-9 While
the group II mGluRs (mGlu2 and mGlu3) are among the
most highly studied of the mGluR subgroups, previous
efforts were limited to group II mGluR ligands that act at
both mGlu2 and mGlu3.1-4,6 Recently, selective positive
allosteric modulators for mGlu2 have emerged, and
demonstrated that mGlu2 activation is responsible for the
antipsychotic efficacy of mGlu2/3 agonists.6 However, de-
spite major advances in understanding the functions of
mGlu2, mGlu3 remains one of the least understood mGluR
subtypes, due in large part to the lack of selective lig-
ands.1-9 Despite this, numerous studies indicate that
mGlu3 is the key mGluR subtype involved in glial-
neuronal communication, and inhibition of mGlu3 is hy-
pothesized to have therapeutic utility in the treatment of
cognitive disorders, schizophrenia, depression and Alz-
heimer’s disease.1-12 Therefore, our laboratory focused
attention on the development of selective mGlu3 negative
allosteric modulators (NAMs) as probes to elucidate the
role of mGlu3 in vivo.
Figure 1. Structures and activities of reported mGlu3 NAMs 1-3.
was efficacious in cognition and depression models.11
About the same time, Lilly disclosed LY2389575 (2), dis-
playing ~4-fold selectivity for mGlu3 over mGlu2 (mGlu2
IC50 = 17 µM, mGlu3 IC50 = 4.2 µM).12 In 2012, we disclosed
a potent (IC50 = 649 nM), selective (>15-fold vs. mGlu2)
and CNS-penetrant mGlu3 NAM (3, ML289), derived from
a 0.37 µM mGlu5 positive allosteric modulator (PAM).13
Once again, a subtle ‘molecular switch’,15 in the form of a
p-methoxy moiety, conferred selective mGlu3 inhibition
over mGlu5 potentiation. While this was a notable ad-
vance, we continued to seek an mGlu3 NAM probe that
was devoid of mGlu2 activity (IC50 >30 µM) in order to
enable proof of concept studies.
RESULTS AND DISCUSSION
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Chemistry. 3 became our lead compound from which to
an essential component of the biarylacetylene pharmaco-
phore.
From the literature regarding acetylene replacements in
develop a more potent and selective mGlu3 NAM.13 As we
have previously reported, due to the steep nature of allo-
steric modulator SAR (especially in series prone to ‘mo-
lecular switches’), we pursued an iterative parallel synthe-
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related mGlu5 NAM biaryl acetylene ligands, we synthesi-
zed and screened a diverse array of reported bioisosteres
(Figure 4);8 unfortunately, only a few weak NAMs were
identified, with most inactive (mGlu3 IC50s >10 µM).
Therefore, the p-methoxy phenyl acetylene component
was crucial for mGlu3 activity.
2,12,13
sis approach for the chemical optimization of 3,
which was divided into five quadrants for SAR exploration
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Figure 2. Library optimization strategy for 3 to improve mGlu3 NAM
activity, eliminate mGlu2 activity and improve the DMPK profile.
(Figure 2). First, we wanted to identify replacements for
the metabolically labile p-OMe moiety to improve im-
prove disposition.13 Second, we hoped to employ the
wealth of acetylene replacements from previous mGlu5
NAM discovery efforts to replace this less than optimal
moiety.8 Third, we desired to perform a broader amide
scan to identify novel amide congeners that eliminate
mGlu2 activity. Finally, we wanted to see if the ‘fluorine
walk’ approach2 would offer advantages in terms of po-
tency, selectivity or DMPK profiles.
Figure 3. Representative Ar moieties surveyed to replace the p-OMe
phenyl group. All lost significant activity against mGlu3 (IC50s >10
µM).
Scheme 1. Synthesis of Aryl Analogues 6a
Figure 4. Representative acetylene biosiosteres surveyed to replace
the p-OMe phenyl acetylene group.8 All were weak to inactive on
mGlu3 (IC50s >10 µM).
Based on these data, we elected to survey alternative amide
moieties in an effort to improve mGlu3 NAM activity and
selectivity while holding the p-OMe phenyl acetylene phar-
macophore constant. Key acid 7 was readily prepared by
Sonogashira coupling as shown in Scheme 1, and amide ana-
logues were prepared in high yield under standard condi-
tions (Scheme 2).13,15 This library proved far more productive,
yielding a number of active analogues, and for the first time,
robust SAR and a general lack of activity at mGlu2 (Table 1).
aReagents and conditions: (a) (R)-3-hydroxymethyl piperidine, EDC,
DMAP, DCM, DIPEA, 95%; (b) 20 mol% CuI, 5 mol% Pd(PPh3)4,
arylacetylene (1.1 equiv.), DMF, DIEA, 60 oC, 1 h, 15-90%.
The first libraries were aimed at identifying a replace-
ment for the p-methoxy moiety or electronically perturb-
ing the aryl ring, rendering P450-mediated O-dealkylation
less facile.13 Following the synthetic route depicted in
Scheme 1, a library of 24 analogs was readily prepared via
standard amide and Sonogashira couplings, and screened
against both mGlu3 and mGlu2 in kinetic assays (See sup-
plemental information). All compounds possessed purity
exceeding 95% as judged by 1H NMR and analytical LCMS
(214 nM, 254 nM and ELSD). SAR in this region was found
to be shallow, as all attempts to increase steric bulk on
the ether or electronically deactivate the aromatic ring
(Figure 3) led to a significant loss of mGlu3 activity (IC50s
>10 µM); thus the p-methoxy moiety was discovered to be
Scheme 2. Synthesis of Amide Analogues 8a
aReagents and conditions: (a) HNR1R2, EDC, DMAP, DIPEA, CH2Cl2,
rt, 16 h, 70-95%.
A racemic 3-hydroxy piperidine congener (8a) showed
significant activity (mGlu3 IC50 = 760 nM), and upon syn-
thesis of the pure enantiomers, enantioselective inhibi-
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Journal of Medicinal Chemistry
the ethyl and allyl congeners as well, and resolved the
tion was noted. Here, the (R)-enantiomer (8d) was more
enantiomers via chiral SFC.15 Only modest ~2-fold in-
creases in mGlu3 NAM potency were noted for the (+)-
enantiomers (Supplemental Figure 1). Finally, following
the synthetic routes depicted in scheme 1 and 2, we incor-
porated fluorine atoms into the benzoic acid moiety of
8d, and discovered two additional sub-micromolar mGlu3
NAMs 9 and 10 worthy of further profiling (Figure 5).15
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8
potent (mGlu3 IC50 = 650 nM) than the (S)-enantiomer
(8c, mGlu3 IC50 = 1.1 µM). When the hydroxy group was
capped as a methyl ether in 8b, mGlu3 NAM activity was
lost. Interestingly, the [3.3.0] piperidine mimetic was
active (8k and 8l), and was a reasonably effective surro-
gate for the piperidine ring. Contraction to a pyrrolidine
ring, as in 8g-i, led to a significant diminution in potency,
as did an acyclic congener 8f. Based on the potency of the
tertiary hydroxyl analogue 8j (IC50 = 711 nM), we prepared
9
Molecular Pharmacology. The four leading mGlu3
NAMs 8d, 8j, 9 and 10 proved to be potent and highly
selective versus mGlu2 (Figure 6). Based on DMPK and
ancillary pharmacology profiles (vide infra), 9 was favored
for further characterization. As shown in Figure 6C, 9
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Table 1. Structures and Activities of Analogues 8.
O
NR1R2
8
MeO
*
Entry
NR1R2
mGlu3 pIC50
Glu Min*
(%)
mGlu2 IC50
(µM)
N
8a
8b
8c
8d
8f
0.4±3.0
0.0±3.2
1.7±3.2
2.0±1.5
1.6±39.8
---
5.87±0.04
5.26±0.05
5.77±0.04
6.18±0.02
5.12±0.11
4.56±1.61
4.99±0.09
5.12±0.11
5.96±0.06
>30
>30
>30
>30
>30
>30
>30
>30
>30
OH
N
OMe
N
OH
N
OH
OH
N
H
N
8g
8h
8i
OH
OH
Figure 6. Molecular pharmacology profile of 9 and related mGlu3
NAMs. A) mGlu3 EC80 antagonist CRC. All four compounds are po-
tent and fully efficacious mGlu3 NAMs (n = 3). B) mGlu2 EC80 CRC.
All four compounds are inactive up to 30 µM. C) Progressive fold
shift analysis with 9 and glutamate displayed a non-competitive
decrease in the EC80, indicating 9 is acting allosterically. D) Evaluat-
ing probe dependence. 9 is equipotent and efficacious in inhibiting
mGlu3 activation by both glutamate and LY379268 (Supplemental
Figure 2).
N
N
0.0±16.1
0.0±10.3
-0.1±4.4
OH
N
8j
HO
N
8k
8l
5.56±0.07
5.26±0.11
1.6±5.9
>30
>30
O
N
1.7±11.2
HO
displayed classical non-competitive antagonism with re-
spect to the orthosteric agonist glutamate in a progressive
fold shift assay.2,3,13,15 For certain electrophysiology stud-
ies, an exogenous agonist may be required in order to
engender selective group II mGluR activation; we there-
fore examined the probe dependence of 9, and noted no
differences between glutamate and LY37926816 (Figure
6D). Considering 9 was inactive against the remaining
mGluRs (no activity at mGlu1,2,4,5,6,7,8 up to 30 µM) we de-
clared ML337 an MLPCN probe.17
*mGlu3 pIC50 and Glu Min data reported as averages ±SEM from
our calcium mobilization assay; n = 3
F
O
O
N
N
OH
OH
MeO
MeO
VU0468008, 8d
VU0469942, 9 (ML337)
mGlu3 IC50 = 593 nM
mGlu3 pIC50 = 6.22+0.03
Glu min = 1.12+2.29
mGlu3 IC50 = 654 nM
mGlu3 pIC50 = 6.18+0.02
Glu min = 1.968+1.52
µ
mGlu2 IC50 >30
M
µ
mGlu2 IC50 >30
M
O
O
F
N
N
OH
OH
MeO
MeO
VU0469946, 8j
mGlu3 IC50 = 711 nM
mGlu3 pIC50 = 5.96+0.06
Glu min = -0.13+4.42
VU0469941, 10
mGlu3 IC50 = 456 nM
mGlu3 pIC50 = 6.34+0.03
Glu min = 1.64+1.72
DMPK Disposition Attributes. 9 was subsequntly pro-
filed in a battery of in vitro and in vivo DMPK assays to
assess its utility as in vivo probe (Table 2). Although 9
was found to be unstable in rat and human microsomes,
µ
µ
M
mGlu2 IC50 >30
M
mGlu2 IC50 >30
Figure 5. Potent and selective mGlu3 NAMs for further profiling.
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it possessed free fractions in both mouse and human apparent kinetic isotope effect as a means to combat the
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8
plasma approaching 0.03 (97% PPB), as well as a favorable
P450 inhibition profile and solubility (7.8 µM in PBS). In a
Ricerca radioligand binding panel of 68 GPCRs, ion chan-
nels and transporter,18 displayed significant activity (>50%
inhibition @10 µM) at only 2 targets (DAT, 71% and 5-
HT2B, 74%), but no functional activity at these targets. To
rapidly assess the extent of CNS penetration, we per-
formed a mouse tissue distribution study in which 8b, 8j,
9, and 10 were administered as a cassette via an IP route,
followed by LC/MS/MS analysis of plasma and brain tis-
sue. All four compounds afforded acceptable CNS expo-
sure, producing brain-to-plasma ratios (B:P) ranging from
0.59 to 0.92 in mice (Supplemental Table 1). 9 demon-
strated a B:P ratio approaching unity (B:P, 0.92), with a
BrainAUC of 3.37 µM and a corresponding plasmaAUC of 3.71
µM. A subsequent rat study demonstrated a good overall
CNS exposure for 9, producing a B:P ratio of 0.3 with high
plasma exposures (Supplemental Table 2).
shallow SAR of these allosteric modulators led to im-
proved disposition in vivo. 19
Conclusion. In summary, we have developed the most
potent (mGlu3 IC50 = 593 nM, 1.9% Glu min) and selective
(>30 µM versus mGlu1,2,4,5,6,7,8) mGlu3 NAM, 9, described to
date. ML337 possesses a favorable DMPK and ancillary
pharmacology profile, and is centrally penetrant. The
major metabolic soft spot was identified to be P450-
mediated O-demethylation, a fate that could not be over-
come through standard steric or electronic perturbations,
due to extremely shallow allosteric ligand SAR. However,
9
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Table 3. Effect of deuterium incorporation on in vitro and in
vivo rat PK with 8d and 9.
Table 2. DMPK Characterization of 9
Parameter
9
MW
TPSA
cLogP
353.38
59.7
3.51
In Vitro Pharmacology
CYP (1A2, 2C9, 3A4, 2D6)
In Vitro PK
IC50 (µM)
>30, >30, >30, >30
by exploiting apparent kinetic isotope effects, we were
able to combat the shallow SAR within this allosteric
modulator series and discover an mGlu3 NAM with im-
proved disposition. Electrophysiology and in vivo studies
with 9, and its deuterated analogue 12, are in progress and
will be reported in due course.
Rat CLHEP (mL/min/kg)
Human CLHEP (mL/min/kg)
Rat PPB (fu)
54.1
18.9
0.005
0.027
mPPB (fu)
In Vivo Rat PK (IP, 10 mg/kg, 0-6 h)
Plasma AUC0-6 (µM*h)
Brain AUC0-6 (µM*h)
Brain:Plasma
33.1
9.6
0.3
The major metabolite of 9, as with 3, was P450-mediated
O-demethylation.14 As mentioned above, all efforts to re-
place this group synthetically proved futile, resulting in
inactive compounds. In an attempt to improve the PK in
rodents, we elected to introduce deuterium atoms into
the methoxy substituent (D3) of both 8d and 9 in order to
increase the metabolic stability of these mGlu3 NAMs
(providing 11 and 12, respectively).19 As shown in Table 3,
introduction of the D3CO moitety led to an analog with a
substantially lower intrinsic clearance (CLint) and predict-
ed hepatic clearance value (CLhep) in vitro. Indeed, the
deuteration strategy resulted in an approximate 50% low-
ering of the plasma clearance (CLp) in rats while provid-
ing mGlu3 NAMs of comparable potency and selectivity
(Supplemental Figure 3). Importantly, identification of
the principal metabolites of the deuterated analogs re-
vealed there to be no metabolic shunt from P450-mediated
O-demethylation (data not shown). Thus, employing the
EXPERIMENTAL SECTION
Chemistry. The general chemistry, experimental infor-
mation, and syntheses of all other compounds are sup-
plied in the Supporting Information. (R)-(2-Fluoro-4-((4-
methoxyphenyl)ethynyl)(3-hydroxypiperdin-1-
yl)methanone, 9: To
a solution of 2-fluoro-4-((4-
methoxyphenyl) ethynyl) benzoic acid (675 mg, 2.5
mmol) in 20 mL DMF, was added DIPEA (1.07 g, 8.25
mmol) while stirring. EDC (560 mg, 3 mmol), HOBt (337
mg, 2.5 mmol), and (R)-3-hydroxypiperidine hydrochlo-
ride (342 mg, 2.5 mmol) were then added. The reaction
was allowed to stir for 4 hours at room temperature, then
quenched with a solution of saturated NaHCO3 (20 mL),
washed with 5% LiCl (aqueous, 2 x 20 mL), and brine (20
mL). The reaction was extracted into dichloromethane
(50 mL), and solvent was removed under vacuum. HPLC
purification afforded 9 as an ivory solid (420 mg, 47%). 1H
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Journal of Medicinal Chemistry
of Group II Metabotropic Glutamate Receptor Allosteric Modu-
NMR (500 MHz, d6-DMSO, 75⁰ C) δ (ppm): 7.50 (m, 2H);
1
2
3
4
lators. ACS Chem. Neurosci. 2011, 2, 382-393.
7.39 (m, 3H); 6.99 (m, 2H); 4.06 (s, 1H); 3.82 (s, 3H); 3.53
(s, 1H); 3.29 (m, 2H); 2.93 (m, 1H); 1.87 (m, 1H); 1.74 (s,
(7) Emmitte, K.A. Recent advances in the design and development
of novel negative allosteric modulators of mGlu5. ACS Chem.
Neurosci. 2011, 2, 411-432.
13
1H); 1.44 (m, 2H). C NMR (125 MHz, d6-DMSO, 75⁰ C) δ
(ppm): 163.3, 159.7, 158.0, 156.0, 132.7, 127.2, 125.2 (d, J = 9.3
Hz), 124.3 (d, J = 16.7 Hz), 117.7 (d, J = 22.7 Hz), 114.2, 113.4,
91.1, 85.9, 64.7, 55.0, 53.2, 48.2, 32.2, 28.9. [α]D23 = -27.6o (c
= 1, MeOH). LC (254 nm) 0.704 min (>99%); MS (ESI)
m/z = 354.1. HRMS (TOF, ES+) C21H20FNO3.[M+H]+ calc.
mass 354.1505, found 354.1507.
5
6
7
8
(8) Stauffer, S.R. Progress toward Positive Allosteric Modulators of
the Metabotropic Glutamate Receptor Subtype 5 (mGlu5). ACS
Chem. Neurosci. 2011, 2, 450-470.
9
(9) Suzuki, G.; Tsukamoto, N.; Fushiki, H.; Kawagishi, A.; Nakamu-
ra, M.; Kurihara, H.; Mitsuya, M.; Ohkubo, M.; Ohta, H In vitro
pharmacological characterization of novel isoxazolopyridone
derivatives as allosteric metabotropic glutamate receptor 7 an-
tagonists.. J. Pharmacol. Exp. Ther. 2007, 323, 147-156.
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ASSOCIATED CONTENT
Supporting Information. Experimental procedures and
spectroscopic data for selected compounds, detailed phar-
macology and DMPK methods. This material is available free
of charge via the Internet at http://pubs.acs.org.
(10) Harrision, P.J.; Lyon, L.; Sartorius, L.J.; Burnet, P.W.J.; Lane, T.A
The group II metabotropic glutamate receptor
3 (mGlu3,
GRM3): expression, function and involvement in schizophrenia.
J. Psychopharm. 2008, 22, 308-322.
Corresponding Author
(11) Campo, B.; Kalinichev, M.; Lambeng, N.; El Yacoubi, M.; Royer-
Urios, I.; Schneider, M.; Legarnd, C.; Parron, D.; Girard, F.; Bes-
sif, A.; Poli, S.; Vaugeois, J-M.; Le Poul, E.; Celanire, S. Charac-
terization of an mGluR2/3 negative allosteric modulator in ro-
dent models of depression. J. Neurogenetics 2011, 24, 152-166.
*Phone:
615-322-8700.
Fax:
615-343-3088.
E-mail:
craig.lindsley@vanderbilt.edu.
Funding Sources
This work was generously supported by the NIH/MLPCN
U54 MH084659 (C.W.L.) and NIMH R01MH099269 (K.A.E).
(12) Caraci, F.; Molinaro, G.; Battaglia, G.; Giuffrida, M.L.; Riozzi, B.;
Traficante, A.; Bruno, V.; Cannella, M,.; Mero, S.; Wang, X.;
Heinz, B.A.; Nisenbaum, E.S.; Britton, T.C.; Drago, F.; Sortino,
M.A.; Copani, A.; Nicoletti, F. Targeting group II metabotropic
glutamate (mGlu) receptors for the treatment of psychosis as-
sociated with Alzheimer's disease: selective activation of mGlu2
receptors amplifies beta-amyloid toxicity in cultured neurons,
whereas dual activation of mGlu2 and mGlu3 receptors is neu-
roprotective. Mol. Pharm. 2011, 79, 618-626.
ABBREVIATIONS USED
mGlu3, metabotropic glutamate receptor subtype 3; CRC,
concentration-response-curve;
IP,
intra-peritoneal;
MLPCN, Molecular Libraries Probe Production Centers
Network; RCF, relative centrifugal force
(13) Sheffler, D.J.; Wenthur, C.J.; Bruner, J.A.; Carrington, S.J.S.;
Blobaum, A.L.; Morrison, R.D.; Daniels, J.S.; Niswender, C.M.;
Conn, P.J.; Lindsley, C.W. Development of a novel, CNS pene-
trant metabotropic glutamate receptor 3 (mGlu3) NAM probe
(ML289) derived from a closely related mGlu5 PAM. Bioorg.
Med. Chem. Lett. 2012, 22, 3921-3925.
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