Synthesis and SAR of a mGluR5 allosteric partial antagonist lead: Unexpected modulation of pharmacology with slight structural modifications to a 5-(phenylethynyl)pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmcl.2008.05.091

This Letter describes the synthesis and SAR, developed through an iterative analogue library approach, of a mGluR5 allosteric partial antagonist lead based on a 5-(phenylethynyl)pyrimidine scaffold. With slight structural modifications to the distal pheny

Full text of DOI:10.1016/j.bmcl.2008.05.091

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4098–4101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of a mGluR5 allosteric partial antagonist lead:
Unexpected modulation of pharmacology with slight structural modifications
to a 5-(phenylethynyl)pyrimidine scaffold
Sameer Sharma a,c,d, Alice L. Rodriguez a,c, P. Jeffrey Conn a,c, Craig W. Lindsley a,b,c,d,
*
a Department of Pharmacology, Vanderbilt University Medical Center, 12415D MRBIV, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt Institute of Chemical Biology, Nashville, TN 37232, USA
d Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the synthesis and SAR, developed through an iterative analogue library approach, of
a mGluR5 allosteric partial antagonist lead based on a 5-(phenylethynyl)pyrimidine scaffold. With slight
structural modifications to the distal phenyl ring, analogues demonstrated a range of pharmacological
activities from mGluR5 partial antagonism to full antagonism/negative allosteric modulation to positive
allosteric modulation.
Received 1 April 2008
Accepted 23 May 2008
Available online 29 May 2008
Keywords:
Allosteric
Ó 2008 Elsevier Ltd. All rights reserved.
mGluR5
Partial antagonism
Positive allosteric modulation
Antagonist
Glutamate is the major excitatory transmitter in the central
nervous system, exerting its effects through both ionotropic and
metabotropic glutamate receptors. The metabotropic glutamate
receptors (mGluRs) are members of the GPCR family C, character-
ized by a large extracellular amino-terminal agonist binding do-
main. To date, eight mGluRs have been cloned, sequenced, and
assigned to three groups (Group I, mGluR1 and mGluR5; Group
II, mGluR2 and mGluR3; Group III, mGluRs 4,6,7,8) based on their
sequence homology, pharmacology, and coupling to effector mech-
anisms.1 In preclinical models, studies with the non-competitive
antagonists MPEP (1) and MTEP (2) have demonstrated that selec-
tive antagonism of mGluR5 has therapeutic potential for chronic
disorders such as pain, anxiety, depression, cocaine addiction,
and Fragile X syndrome.2 Indeed, there is now direct clinical vali-
dation of anxiolytic activity by allosteric antagonism of mGluR5
in patients with fenobam (3).3
A major potential issue with MPEP (1), MTEP (2), and fenobam
(3) is that these non-competitive, allosteric antagonists function as
inverse agonists (Fig. 1).2,3 These compounds completely block all
activities of them GluR5 receptor, including constitutive activity.
Based on the close association of mGluR5 with its signaling partner
the NMDA receptor, there is growing concern that complete block-
ade may have several adverse affects commonly associated with
S
O
O
NH
N
N
N
N
N
H
Cl
N
MPEP, 1
MTEP, 2
fenobam, 3
Figure 1. Representative non-competitive mGluR5 antagonists that bind at an
allosteric site on the mGluR5 receptor engender mGluR subtype selectivity.
NMDAR antagonists such as cognitive deficits and psychotomi-
metic effects.3,4 These adverse effects could severely limit the clin-
ical utility of currently available mGluR5 antagonists for the
treatment of chronic disorders.
Based on our experience in the development of allosteric mod-
ulators of mGluRs with a broad range of activities including nega-
tive allosteric modulators, positive allosteric modulators, and
neutral allosteric site ligands at the MPEP (1) binding site together
with theoretical models of allosteric function, we postulated that it
might be possible to develop ‘partial antagonists’.5 In this case, a
‘partial antagonist’ would fully occupy the MPEP (1) binding site
on the mGluR5 receptor but only partially block agonist response,
resulting in partial mGluR5 inhibition.4 To test this hypothesis, 200
analogues of MPEP (1) were synthesized and evaluated for dis-
placement of [3H]methoxy-PEPy. Compounds that fully displaced
the radioligand were then screened in a calcium fluorescence
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.091

S. Sharma et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4098–4101
4099
assay, which determined the ability of these analogues to block
N
N
N
N
(a)
Br
H
functional responses of mGluR5 to glutamate. The goal was to
identify compounds that only partially block mGluR5 at concentra-
tions that fully occupy the allosteric site. As reported by Rodriguez
et al. in 2005, this effort identified three compounds that only par-
tially inhibited or had no functional effects on mGluR5 response.4
M-5MPEP (4) and Br-5MPEPy (5) represented the first ‘partial
antagonists’ of mGluR5 and induced a maximal blockade of
ꢀ50%, while 5MPEP (6) acted as a neutral allosteric site ligand
(Fig. 2).4
Based on these data, we performed a high-throughput func-
tional screen of our 160,000 compound screening collection to
identify novel compounds with a range of activities as full and par-
tial mGluR5 antagonists. This effort identified a number of novel
full mGluR5 antagonists as well as a small number of mGluR5 par-
tial antagonists. As shown in Figure 3, we identified several com-
pounds, such as 7 (EC50 = 297 nM) and 8 (EC50 = 62 nM), that
fully occupied the MPEP binding site, but induced a maximal 58%
or 82.1% blockade, respectively, in the primary HTS. In this Letter,
we describe the synthesis and SAR developed around partial antag-
onist lead 8, and the unexpected modulation of pharmacology that
resulted.
We began our lead optimization campaign around partial
antagonist 8 (IC50 = 62 nM, 17.9% response, 82.1% partial antago-
nism), a 5-(phenylethynyl)pyrimidine scaffold. Lead 8 was resyn-
thesized (Scheme 1) employing a microwave-assisted organic
synthesis (MAOS) Sonogashira protocol with commercially avail-
able 5-bromopyrimidine 9 and phenyl acetylene 10 and catalytic
CuI/Pd(PPh3)4 to deliver 50 mg quantities of 8 in >90% yield.6 After
screening in triplicate with full concentration response curves, the
activity of 8 was right-ward shifted ꢀ8-fold (IC50 = 486 nM), but
data indicated that 8 was clearly a partial antagonist (29% re-
sponse, 71% partial antagonism).7 In radioligand binding assays
with [3H]methoxy-PEPy, 8 was found to possess a Ki of 125.9 nM,
indicating it binds to the MPEP site.
+
9
10
8
Scheme 1. Reagents and conditions: (a) 9/10 (1:1), 10 mol% Pd(PPh3)4, 20% CuI, 20
equiv diethylamine, DMF, mw, 70 °C, 10 min, >90% yield.
It was unclear from the outset if the degree of mGluR5 partial
antagonism would be maintained within this chemical series, or
if the degree of partial antagonism would vary over a dynamic
range to include complete blockade as seen with MPEP (1). Our
optimization effort relied on an iterative library synthesis approach
in which we employed MAOS to rapidly prepare two, 24-mem-
bered analogue libraries.8 In the event, commercially available 5-
bromopyrimidine 9 was subjected to a microwave-assisted Sono-
gashira coupling protocol (10% mol Pdo catalyst, 20 mol% CuI, 20
equiv diethylamine, 1:1 ratio of 9/11, 70 °C, 10 min) with a library
of functionalized alkynes 11 to deliver diverse 5-ethynylpyrimi-
dine analogues 12 (Scheme 2). By application of MAOS and the
Fu catalyst (Pd(tBuP)2), the palladium precipitated upon rapid
cooling, and no Pdo scavenging was required prior to mass-directed
preparative HPLC purification, unlike in the scale-up of 8 which
employed Pd(PPh3)4. Moreover, excellent chemical yields (70–
95%) were obtained for a diverse range of functionalized alkynes,
suggesting this new protocol is quite general and broad in scope.
All 48 analogues 12 were screened in our calcium fluorescence
‘triple add’ assay that can identify agonists, antagonists and posi-
tive allosteric modulators (potentiators). Only 11 of the 48 ana-
logues (23%) demonstrated any measurable effect on calcium
transients, and these effects were unexpected (Table 1) providing
only one partial antagonist (PA), but six full antagonists (A)
and four positive allosteric modulators (PAM). This dynamic
Rn
N
N
Rn
N
N
(a)
Br
H
+
9
11
12
Scheme 2. Reagents and conditions: (a) 9/11 (1:1), 10 mol% Pd(t-BuP)2, 20% CuI, 20
equiv diethylamine, DMF, mw, 70 °C, 10 min, 70–95% yield.
Figure 2. MPEP analogues with partial antagonists activity 4 and 5, as well as a
neutral allosteric site ligand 6. mGluR5 partial antagonists 4 and 5 completely
occupy the MPEP binding site, but induce a maximal blockade of approximately 50%
without further blockade at higher concentrations.
Table 1
Structures and allosteric activities of analogues 12
N
N
R
12
Antag%a
Fold
a
Compound
R
Allosteric
activity
IC50/EC50
(l
M)
shifta
8
H
3-Me
3-Cl
3-CF3
3-Me, 4-F
3,5-diF
4-Ph
2,5-diMe
4-Me
4-Et
PA
A
A
A
A
0.48
0.007
0.034
0.45
0.35
6.4
7.8
11.2
3.3
71
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
4.2
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
100
100
100
100
100
100
58
N/A
N/A
N/A
N/A
A
A
PA
PAM
PAM
PAM
PAM
18.8
3.9
7.9
3.1
2.3
2.0
4-Cl
4-CHO
Figure 3. MPEP (10 lM) blocks calcium fluorescence response to glutamate,
a
whereas saturating concentrations of representative mGluR5 partial antagonists
from the HTS screen only partially inhibit calcium fluorescence response to
glutamate.
IC50s, EC50s, Antag%, and fold shift are the average of at least three determi-
nations. N/A, not applicable; PA, partial antagonist; A, antagonist; PAM, positive
allosteric modulator.

4100
S. Sharma et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4098–4101
Cl
O
H
N
N
O
N
F
N
CN
F
N
N
HN
OH
O
O
13, DFB
14
, CPPHA
15, CDPPB
Figure 4. Structures of mGluR5 PAMs previously reported from our laboratory: DFB (13), CPPHA (14), and CDPPB (15).
modulation of pharmacological response was unanticipated. More-
over, despite a rather ‘flat’ SAR, trends emerged that identified
‘chemical switches’ to modulate modes of mGluR5 pharmacology.
In general, incorporation of chemical moieties into the 3-posi-
tion of the distal phenyl ring afforded full mGluR5 antagonists with
IC50s ranging from 7.5 nM (12a) to 7.8 lM (12f). Small groups like
3-Me (12a, IC50 = 7.5 nM) and 3-Cl (12b, IC50 = 33.8 nM) provided
the most potent non-competitive antagonists, while larger groups
such as 3-CF3 (12c, IC50 = 454 nM) and 4-Ph (12f, IC50 = 7.8 lM)
saw a diminution in inhibitory activity. Within this library, only
a 2,5-dimethyl congener (12g) retained partial mGluR5 antagonist
activity (IC50 = 11.2 lM, 42% response), but suffered a 23-fold loss
in activity. Interestingly, the concept of partial antagonism is not
conserved within a chemical series. More surprising was the obser-
vation that substituents in the 4-position of the distal phenyl ring
of 8 produced mGluR5 positive allosteric modulators (Table 1,
12h–12k), and the first examples of mGluR5 PAMs based on an
MPEP-like scaffold. Even though these analogues possess EC50s in
the micromolar range, they afford significant potentiation of
mGluR5. As in the case of the non-competitive antagonists 12a–
12f, small groups in the 4-position provided the most efficacious
PAMs. Unlike mGluR5 PAMs reported previously from our labora-
tory, DFB (13), CPPHA (14), and CDPPB (15), which achieved
<70% of maximal glutamate response (Fig. 4), PAMs such as 12h
Figure 5. Compound 12h potentiates mGluR5 activation by glutamate. In the
absence of glutamate, 12h does not activate mGluR5. In the presence of a sub-
threshold quantity of glutamate, 12h potentiates mGluR5 in
a concentration-
dependent manner.
(EC50 = 3.3 l
M) afforded 99% of maximal glutamate response.5 In
the absence of glutamate, 12h and related congeners had no effect
on receptor response, but in the presence of a sub-threshold con-
centration of glutamate, a concentration-dependent potentiation
of mGluR5 response was observed (Fig. 5). In addition, compound
12h displayed a robust 4.2-fold left-ward shift of the glutamate
agonist response curve with an increase in glutamate max (Fig. 6).
The analogues of HTS partial antagonist lead 8 in this small li-
brary elucidated a ‘molecular switch’ to modulate pharmacological
activity. Lead 8, with an unsubstituted distal phenyl ring, fully
occupied the MPEP binding site, possessed an IC50 of 486 nM, but
only afforded partial response (29% response, 71% partial antago-
nism), that is, an allosteric partial antagonist.
Incorporation of small chemical moieties in the 3-position of
the distal phenyl ring, such as a 3-methyl group delivers 12a, a full
non-competitive mGluR5 antagonist (IC50 = 7.5 nM). When the
methyl group is moved from the 3-position to the 4-position as
in 12h, an efficacious (99% of glutamate max) mGluR5 positive
Figure 6. Compound 12h potentiation of response to glutamate is manifested as
increased mGluR5 agonist sensitivity. The glutamate EC50 value is shifted from
389 nM to 93 nM, or a 4.2-fold shift.
ric modulation. Moreover, this study demonstrates that allosteric
partial antagonism is neither conserved within a series nor an
inherent property of a given chemical scaffold. Further studies in
this arena are in progress and will be reported in due course.
Acknowledgments
allosteric modulator (EC50 = 3.3 lM, 4.2-fold shift) results. The
observation of a conserved ‘molecular switch’, accessed by toggling
between 3- and 4-substitution on the distal phenyl ring, within
this chemical series is unprecedented and once again, highlights
the complexities involved in the optimization and development
of allosteric ligands.
In summary, a lead optimization campaign based on mGluR5
allosteric partial antagonist lead, 8, resulted in relatively ‘flat’
SAR (only 23% of analogues possessed any mGluR5 activity), and
subtle structural variants, that is, ‘molecular switches’, gave rise
to a range of mGluR5 pharmacological activities from partial
antagonism to full non-competitive antagonism to positive alloste-
The authors thank the National Institute of Mental Health the
Vanderbilt Department of Pharmacology and the Vanderbilt Insti-
tute of Chemical Biology for support of this research.
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20 mM Hepes, 10% dialyzed FBS, and 1 mM sodium pyruvate). The plates were
incubated overnight at 37 °C in 5% CO2. Media were removed and assay buffer
(Hanks Balanced Salt Solution, 20 mM Hepes, 2.5 mM Probenecid, pH 7.4)
containing 4.0
lM Fluo4-AM dye (Invitrogen) was added. Cells were incubated
for 45 min (37 °C, 5% CO2) to allow for dye loading. Dye was removed, 20
lL
assay buffer was added, and the cell plate was allowed to incubate for 10 min.
After incubation in assay buffer, cell plates were loaded into Flexstation II
(Molecular Devices Corp.). Test compound in assay buffer was added 19 s into
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6. Representative experimental for the synthesis of 8 (5-(phenylethynyl)pyrimidine).
To a solution of 5-bromopyrimidine (50 mg, 0.32 mmol) in DMF (4 mL) were
added phenylacetylene (32 mg, 0.32 mmol), Pd(PBu3)2 (16 mg, 0.03 mmol), CuI
(12 mg, 0.06 mmol) and diethylamine (1 mL). The reaction vessel was sealed
and heated at 70 °C for 10 min in a microwave reactor. The reaction was cooled
to room temperature, diluted with EtOAc/hexanes (2:1) and washed with water.
The crude product was purified on the Agilent 1200 preparative LC/MS.
Concentration of purified fractions afforded 5-(phenylethynyl)pyrimidine as
an orange solid. 1H NMR (CDCl3, 400 MHz) d 9.15 (s, 1H), 8.86 (s, 2H), 7.59-7.54
(m, 2H), 7.42-7.36 (m, 3H); 13C NMR (CDCl3, 100 MHz) d 158.6, 156.7, 131.8,
131.8, 131.7, 129.3, 128.6, 128.5, 121.8, 119.9, 96.3, 82.3; LC–MS (214 nm) >99%,
single peak, 2.98 min, m/e 181.8 (M+1); HR-MS 181.0760 (calc. 181.0766),
(3
antagonists, respectively. Controls included compound vehicle (0.3% DMSO)
plus assay buffer, ECmax (100 glutamate), and submaximal or nearly
maximal concentration of glutamate. Compounds were tested in
concentrations ranging from 46 pM to 100 M. Fold shifts were determined
using the same functional assay by varying the amount of glutamate in the
presence of either a fixed concentration of compound (30 M) or vehicle. A
concentration response curve was generated using glutamate concentrations
ranging from 4.6 nM to 10 M. Controls included compound vehicle (0.3%
DMSO) plus assay buffer, ECmax (100 glutamate), and glutamate
lM) amount of glutamate was added 109 s into the assay for potentiators and
l
M
l
l
l
l
M
concentration response curve. Assays were performed in triplicate on three
different days. Concentration response curves were generated using GraphPad
Prism 4.0. Radioligand binding assay. The allosteric antagonist MPEP analogue
[3H]methoxy-PEPy was used to evaluate the interaction of the test compounds
with the allosteric MPEP site on mGluR5. Membranes were prepared from
HEK293A cells containing rat mGluR5. Compounds were diluted in assay buffer
(50 mM Tris, 0.9% NaCl, pH 7.4) to a 5Â stock and 100
added to each well of a 96-well assay plate. Aliquots of membranes (300
diluted in assay buffer (40
g/well) were added to each well. [3H]methoxy-PEPy
(100 L) (2 nM final concentration in assay buffer) was added, and the reaction
l
L of test compound was
lL)
l
l
mixture was incubated at room temperature for 60 min with shaking. After the
incubation period, the membrane-bound ligand was separated from free ligand
by filtration through glass fiber 96-well filter plates (Unifilter-96, GF/B). The
contents of each well were transferred simultaneously to the filter plate and
washed four times with assay buffer (Brandel cell harvester). Scintillation fluid
(40
determined by scintillation counting (TopCount). Non-specific binding was
estimated using 5 M MPEP. Assays were performed in triplicate on three
lL) was added to each well, and the membrane-bound radioactivity was
l
different days. Concentration response curves were generated using GraphPad
Prism 4.0.
8. Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.; Weaver, D.; Lindsley, C.
W. J. Comb. Chem. 2008, 10, 345–354.
C
12H9N2.
7. Functional assay. HEK293A cells expressing rat mGluR5 receptor were plated (BD
Falcon poly- -lysine Cellware) at 50,000 cells/well in assay media (DMEM,
D