Chemical switch of a metabotropic glutamate receptor 2 silent allosteric modulator into dual metabotropic glutamate receptor 2/3 negative/positive allosteric modulators

Article abstract of DOI:10.1021/jm101069m

Using an mGluR2 FRET-based binding assay, binders of the transmembrane region devoid of functional activity were identified. It is reported that slight chemical modifications of these SAMs can dramatically change activity of the resulting analogues without altering their affinities. Starting from compound 1, three mGluR2 NAMs showing also mGluR3 PAM activities were obtained. SAMs therefore represent a useful approach to explore the chemical space for GPCR allosteric modulator identification.

Full text of DOI:10.1021/jm101069m

J. Med. Chem. 2010, 53, 8775–8779 8775
DOI: 10.1021/jm101069m
Chemical Switch of a Metabotropic Glutamate Receptor 2 Silent Allosteric Modulator into Dual
Metabotropic Glutamate Receptor 2/3 Negative/Positive Allosteric Modulators
ꢀ
Stephan Schann,* Stanislas Mayer, Christel Franchet, Melanie Frauli, Edith Steinberg, Mireille Thomas, Luc Baron, and
Pascal Neuville
Domain Therapeutics SA, Bioparc, Boulevard Sebastien Brant, F-67400 Strasbourg-Illkirch, France
Received August 17, 2010
Using an mGluR2 FRET-based binding assay, binders of the transmembrane region devoid of
functional activity were identified. It is reported that slight chemical modifications of these SAMs
can dramatically change activity of the resulting analogues without altering their affinities. Starting
from compound 1, three mGluR2 NAMs showing also mGluR3 PAM activities were obtained. SAMs
therefore represent a useful approach to explore the chemical space for GPCR allosteric modulator
identification.
Introduction
the standardscreening processin the pharmaceutical industry,
based on functional assays set up with specific conditions and
a single readout, undersamples the chemical space surround-
ing potential therapeutic candidates for a chosen GPCR.11
Screening of a diverse 10000 compound library with an
mGluR2 FRET-based binding assay led to the identification
of several micromolar mGluR2 binders, including 1, that
turned out to be inactive in a Ca2þ-based functional assay
(Table 1). Slight chemical modifications of 1 could dramatically
affect its functional activity changing it into NAMs for
mGluR2 and PAMs for mGluR3. To our knowledge, the
molecules described in this article are the first group II mGluR
AMs able to functionally distinguish between the two close
subtypes. They constitute novel pharmacological tools to study
the respective roles and therapeutic potentials of mGluR2 and
mGluR3. In addition, this is the first report describing the use
of a silentallosteric modulator (SAM) as a chemical source for
the identification of novel PAMs and NAMs.
Metabotropic glutamate receptors (mGluRs)a belong to class
C of G-protein coupled receptors (GPCRs), the best family of
drug targets with nearly half of the registered therapeutic
agents. They have been divided into three groups according to
their sequence homologies, pharmacological properties, and
signal transduction pathways.1 Group II mGluRs (composed
of mGluR2 and mGluR3) are largely expressed in central
regions such as cortex, hippocampus, striatum, and amygdala.2
Their activation by group-selective orthosteric agonists leads to
anxiolytic-like and antipsychotic-like activities in rodents and is
currently under clinical evaluations for schizophrenia treat-
ment.3,4 However, due to high homology between mGluR2 and
mGluR3 orthosteric binding sites, subtype-selective agonists or
antagonists were never described. Current efforts toward the
selective regulation of group II mGluRs are focused on positive
allosteric modulators (PAMs) and negative allosteric modula-
tors (NAMs) for the treatment of schizophrenia and Alzheimer’s
disease respectively.5-8
Results and Discussion
Allosteric modulation represents an emerging and promis-
ing approach for the regulation of GPCRs. Compared with
activation or inhibition with orthosteric ligands, allosteric
modulation is associated with major advantages such as sub-
type selectivity, saturable effects, mimicking of physiological
response, and tractable chemistry. To date, two GPCR allo-
steric modulators(AMs) have reachedthe market:Cinacalcet,
a calcium-sensing receptor PAM for the treatment of hyper-
parathyroidism, and Maraviroc, a chemokine receptor CCR5
NAM for the treatment of HIV.9,10 One possible explanation
for this relatively low number of AM drugs lies in the lack of
technology specifically dedicated for their discovery. Indeed,
To selectively identify AMs of the human mGluR2, a
FRET-based binding assay was developed using a truncated
form of the receptor where the N-ter region was replaced by
green fluorescent protein (GFP).12 This truncation results in
a receptor that no longer contains the glutamate orthosteric
binding site (Figure 1). The fluorescent probe used was
derived from nanomolar ligands known to interact with the 7
transmembrane (7TM) region of group II mGluRs.13
Screening of this GFP-receptor with a diverse 10000 com-
pound library led to the identification of several 7TM binders
that were further characterized using a functional assay with
the native human mGluR2 cotransfected with an hybrid
G-protein coupling to the Ca2þ pathway.14 PAMs and NAMs
were identified together with binders exhibiting no functional
activity such as 1, a close derivative of the mGluR4 PAM
PHCCC,15 that was recently shown to exert weak mGluR4
PAM activity.16 Compound 1 showed micromolar affinity
for mGluR2, with a Ki value of 6.6 μM but no activity
*To whom correspondence should be addressed. Phone: þ33 3 9040
6150. Fax: þ33 3 9040 6155. E-mail: sschann@domaintherapeutics.
com.
aAbbreviations: AM, allosteric modulator; FRET, fluorescence
resonance energy transfer; GFP, green fluorescent protein; GPCR,
G-protein-coupled receptor; mGluR, metabotropic glutamate receptor;
NAM, negative allosteric modulator; PAM, positive allosteric modu-
lator; SAM, silent allosteric modulator; 7TM, 7 transmembrane.
r
2010 American Chemical Society
Published on Web 11/24/2010
pubs.acs.org/jmc

8776 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Schann et al.
Table 1. Binding Affinities on mGluR2 and Functional Activities on mGluR2 and mGluR3a
human mGluR2b
human mGluR3b
compd
R
binding affinity Ki (μM)
Ca2þ functional test IC50 (μM)
Ca2þ functional test EC50 (μM)
PHCCC
H
NA
6.6
1.0
0.6
0.8
NA
NA
NAc
NA
1
2
3
4
F
Cl
0.8 (NAM)
1.5 (NAM)
1.0 (NAM)
13.4 (PAM)
8.9 (PAM)
10.4 (PAM)
Me
OMe
a All values are the mean of three separate assay determinations performed in duplicate. Standard deviations for these values were within 15% of the
mean value. b NA: not active. c See ref 16.
the receptor with Ki of respectively 1, 0.6, and 0.8 μM (Table 1).
Functional activities of these compounds were evaluated
using the hybrid G-protein Gqi9 that couples mGluR2 with
the intracellular Ca2þ transduction pathway. Results show
that replacement of fluorine atom of 1 leads to dramatic
changes of functional activity. Analogues 2, 3, and 4 exert
NAM activities, with IC50 values in the low micromolar range
(Table 1 and Figure 2a). Compounds 2-4 were also effective
in right-shifting glutamate EC50 and decreasing glutamate
Emax onhuman mGluR2(see Figure3afor an illustrationwith
3). Very interestingly, it was recently reported that another
close analogue of 1, the (-) enantiomer derivative having a
4-fluoro-pyridin-2-yl moiety instead of the 4-fluorophenyl
moiety, showed weak NAM activity on mGluR2.16 This
further reinforces the observation that slight chemical modi-
fications can switch functional activity of mGluR2 ligands.
The switch in functional activity of 2-4 did not seem to
be electronically driven as it resulted from the replacement
of a fluorine atom by either electron-donating or electron-
withdrawing groups. It was more likely the result of the increased
lipophilicity found in 2, 3, and 4 and the accommodation of the
corresponding new substituents in a lipophilic pocket.
To determine their group II mGluR selectivity profile, 1-4
were also evaluated against mGluR3 in a functional assay
setup with GR15 to couple the Ca2þ pathway. Compound 1
was inactive up to 100 μM similarly to that on mGluR2, but
surprisingly analogues 2, 3, and 4 were shown to be PAMs
with respective EC50 of 13.4, 8.9, and 10.4 μM (Table 1 and
Figure 2b). Tested against a dose-response of glutamate, 2-4
also left-shifted EC50 and increased Emax of glutamate (see
Figure 3b for an illustration with 3). The interesting opposite
activities of 2-4 on mGluR2 and mGluR3 can be explained
by the difference in amino acid composition of the two close
group II mGluR 7TM regions. For instance, it was described
that the three amino acids involved in the active site of the
mGluR2 PAM LY487379 differ in mGluR3 demonstrating
that these sites, in opposition to the orthosteric binding site,
are subject to less evolutionary pressure.21
Figure 1. Description of the engineered mGluR2 used for the
FRET-based binding assay.
(agonist, PAM or NAM) in the Ca2þ functional assay up to
100 μM (see Figure 2A).
Such silent binders were reported previously for another
mGluR subtype, mGluR5.17-19 Interestingly, these reports
also described very close analogues displaying PAM or NAM
activities. Key modifications distinguishing mGluR5 silent
binders from PAMs or NAMs were described as aromatic
substitutions on phenyl or pyridine rings. We therefore decided
to focus on fluorine replacements in 1 to modulate the func-
tional activity in this series of mGluR2 compounds. The corre-
sponding analogues of 1 were synthesized using the procedure
described in Scheme 1.
PHCCC’s analogues 1-4 were prepared following a syn-
thetic route that slightly differs from the one previously used
by Annoura et al.20 Indeed, in order to explore the phenyl
amide moiety, it was found more convenient to start with the
cyclopropanation of the chromene-methyl-ester 5 with tri-
methylsulfoxonium iodide. In situ saponification of the cor-
responding product leads to the carboxylic acid key inter-
mediate 6 that was used for the synthesis of all compounds
of this paper. Coupling reactions were done using EDCI in
DMF, and the corresponding amides were treated, without
further purification, with hydroxylamine using microwave
irradiations in pyridine to lead to compounds 1-4. All com-
The close structural relationship between silent binders and
PAMs/NAMs was previously described for class A musca-
rinic receptors,22 class C mGluR5,17-19 and was observed for
other GPCR members such as the mGluR4 subtype or the
peptidic class A Neurotensin NTSR1. We propose here to use
the term silent allosteric modulator or SAM to define this new
class of AMs that fulfill the following two criteria: (1) binding
to the receptor of interest, (2) being inactive in a given
1
pounds were characterized by H NMR and LCMS before
pharmacological evaluations.
We found that replacement of fluorine by bulkier chlorine
(2), methyl (3), or methoxy (4) has positive influence on
mGluR2 affinities as the corresponding compounds bind

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8777
Figure 2. Activities of compounds 1 and 3 tested in dose-response experiments in the presence of a glutamate EC80 on mGluR2 (a) and in the
presence of a glutamate EC10 on mGluR3 (b).
Scheme 1. Synthesis of Compounds 1-4a
a Reagents and conditions: (a) (1) Me3SOI, NaH, DMF, 0 °C to RT, 19 h, (2) HCl 1N; (b) aniline derivatives, EDCI-HCl, DMAP, DMF, 15 h;
(c) NH2OH-HCl, pyridine, 150 °C 5 min. (microwave).
Figure 3. Concentration-response curves for glutamate in absence or presence of 3. Compound 3 was tested at 40 μM on mGluR2 (a) and at
30 μM on mGluR3 (b).
functional test, in which close derivatives are active. The silent
feature of the ligand will therefore refer to a particular test in
which analogues of the same chemical series will behave either
as a PAM or a NAM. Moreover, as an AM can be qualified as
silent under specific functional test conditions, but positive or
negative under different conditions,23-26 we are recommend-
ing using the term SAM only if accompanied by details of the
functional tests performed to characterize it.
This SAM to PAM/NAM change seems to be a generic
phenomenon that can be exploited by medicinal chemists as a
new source of chemical diversity for GPCR modulator iden-
tification. Indeed, current screening technologies require an
active member for the identification of a chemical family.
Detecting SAMs can enlarge the chemical space available by
enabling the discovery of novel chemical scaffolds not acces-
sible through standard functionaltests. SAMs therefore repre-
sent a unique source of compounds that can be modified by
medicinalchemists toobtainactive AMsorthatcanbefurther
functionally characterized to unveil their activity.
Conclusion
In summary, using a FRET-based binding assay, we have
revealed a novel category of ligands for mGluR2, silent
allosteric modulators that bind the receptor but exert no
activity in a calcium-based functional test. Such SAMs repre-
sent a unique source of novel chemical diversity for identifica-
tion of GPCR modulators as they can be transformed into
PAMs or NAMs by simple chemical modifications. Moreover,
we have obtained compounds that differentiate the close
mGluR2 and mGluR3 in functional tests. This is, to our
knowledge, the first report of allosteric modulators functionally
displaying opposite activities on the two group II mGluRs.
Experimental Section
Chemistry. General. All reagents and starting materials were
commercial grade and used without further purification. Com-
mercially available anhydrous solvents were used for reactions
conducted under inert atmosphere (argon). Microwave assisted

8778 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Schann et al.
reactions were run in a Biotage microwave initiator. Silica gel
used for chromatography columns was SDS silica gel (60AAC
40-63 μM). All reactions were followed by thin layer chromatog-
raphy (TLC plates: precoated silica gel F-254) or liquid chro-
matography mass spectometry (LCMS). Concentration referred
to evaporation under vacuum using a rotary evaporator. The
reaction yields were not optimized. 1H NMR spectra were
recorded on a Bruker 400 MHz spectrometer. Proton chemical
shifts (δ in ppm) are listed relative to residual CDCl3 (7.27 ppm)
or DMSO (2.50 ppm). Splitting patterns are designated as
s (singlet), d (doublet), dd (double-doublet), t (triplet), q (quartet),
m (multiplet), or br (broad). Electrospray mass spectometry
spectra were obtained on a Waters micromass platform LCMS
spectrometer. All mass spectra were full-scan experiments (mass
range 100-1500 amu) obtained using electrospray ionization.
The HPLC system was a Waters platform with a 2767 sample
manager, a 2525 pump, a photodiode array detector (190-400
nM). The column used was an Xterra C18 3.5 μM (4.6 m ꢀ
50 mm). The mobile phase consisted in an appropriate gradient
of A and B. A was water with 0.05% of TFA, and B was
acetonitrile with 0.05% of TFA. Flow rate was 1 mL per min. All
LCMS were performed at room temperature. The purities of
final compounds 1-4 were measured by HPLC and were found
to be above 98%.
7-Oxo-7,7a-dihydrocyclopropa[b]chromene-1a(1H )-carboxylic
Acid (6). To a suspension of trimethylsulfoxonium iodide
(2.85 g, 1.5 equiv) in DMSO (30 mL) cooled at 15 °C, NaH
(500 mg, 1.4 equiv) was added portion by portion over 10 min.
This suspension was stirred 1 h, and then a solution of chrome-
none 5 (1.766 g) in DMSO (15 mL) was added dropwise over
10 min. Reaction mixture was allowed to warm to room tem-
perature and was stirred 19 h. The deep-yellow mixture was
hydrolyzed with aqueous HCl solution (1N, 200 mL) and was
extracted with EtOAc (200 mL). Organic layer was washed with
aqueous NaOH solution (1N, 100 mL). This basic aqueous layer
was acidified to pH 1 by addition of concentrated aqueous HCl
and was extracted with EtOAc (200 mL). This organic layer was
washed with brine, dried over MgSO4, and was concentrated
under reduce pressure to afford 6 as a beige solid in 30% yield.
1H NMR (400 MHz, DMSO): 1.69 (t, J = 6.7 Hz, 1H), 2.12
(dd, J = 11.1 Hz, J = 6.7 Hz, 1H), 2.64 (dd, J = 11.1 Hz, J =
6.7 Hz, 1H), 7.14-7.18 (m, 2H), 7.63-7.67 (m, 1H), 7.79 (d, J =
8.2 Hz,1H).
General procedures for amidation of (6) with anilines and for
reaction of corresponding amides with hydroxylamine. To a
solution of 7-oxo-7,7a-dihydrocyclopropa[b]chromene-1a(1H )-
carboxylic acid (40 mg, 0.19 mmol) in DMF (1 mL), EDCI-HCl
(58 mg, 1.6 equiv), DMAP (35 mg, 1.5 equiv), and the selected
amine (2.0 equiv) were added. The resulting mixture was stirred
at R.T. until carboxylic acid 6 was consumed base on LCMS
analysis (around 16 h). Reaction mixture was then hydrolyzed
with aqueous HCl (1N, 25 mL) and extracted with EtOAc
(25 mL). Organic layer was washed with brine (20 mL), aqueous
NaOH (1N, 20 mL), brine (15 mL), dried over MgSO4, and
concentrated. Residue was used in the next step without further
characterization.
To a solution of ketone 7-10 (0.19 mmol) in pyridine (1.0 mL),
hydroxylamine hydrochloride (25 mg, 1.9 equiv) was added. The
resulting mixture was heated through microwave irradiation for
5 min at 150 °C. Reaction mixture was then hydrolyzed with
aqueous HCl (1N, 25 mL) and extracted with EtOAc (25 mL).
Organic layer was washed with brine (15 mL), dried over MgSO4,
and concentrated. Residue was triturated in Et2O to afford 1-4
in 21% to 51% yields.
10.19 (s, 1H, D2O exchange), 11.46 (s, 1H, D2O exchange). MS
m/z (M þ H)þ = 313.
N-(4-Chlorophenyl)-7-oxo-7,7a-dihydrocyclopropa[b]chromene-
1a(1H )-carboxamide (2). 2 was obtained as a beige solid in 51%
yield. 1H NMR (400 MHz, DMSO): 1.45 (dd, J = 7.0 Hz, J =
5.9 Hz, 1H), 1.97 (dd, J = 10.8 Hz, J = 5.9 Hz, 1H), 3.08 (dd,
J = 10.8 Hz, J = 7.0 Hz, 1H), 7.04-7.07 (m, 1H), 7.17 (dd, J =
8.3 Hz, J = 1.1 Hz, 1H), 7.39-7.43 (m, 3H), 7.73-7.77 (m, 3H),
10.24 (s, 1H, D2O exchange), 11.47 (s, 1H, D2O exchange). MS
m/z (M [35Cl] þ H)þ = 329.
N-(4-Methylphenyl)-7-oxo-7,7a-dihydrocyclopropa[b]chromene-
1a(1H )-carboxamide (3). 3 was obtained as an orange solid in
31% yield. 1H NMR (400 MHz, DMSO): 1.42 (dd, J = 7.0 Hz,
J = 5.8 Hz, 1H), 1.95 (dd, J = 10.8 Hz, J = 5.8 Hz, 1H), 2.28 (s,
3H), 3.07 (dd, J = 10.8 Hz, J = 7.0 Hz, 1H), 7.03-7.07 (m, 1H),
7.14-7.19 (m, 3H), 7.38-7.43 (m, 1H), 7.57-7.60 (m, 2H), 7.75
(dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 10.03 (s, 1H, D2O exchange),
11.45 (s, 1H, D2O exchange). MS m/z (M þ H)þ = 309.
N-(4-Methoxyphenyl)-7-oxo-7,7a-dihydrocyclopropa[b]chromene-
1a(1H )-carboxamide (4). 4 was obtained as a white solid in 34%
yield. 1H NMR (400 MHz, DMSO): 1.41 (dd, J = 6.8 Hz, J =
5.8 Hz, 1H), 1.94 (dd, J = 10.7 Hz, J = 5.8 Hz, 1H), 3.06 (dd,
J = 10.7 Hz, J = 6.9 Hz, 1H), 3.74 (s, 3H), 6.90-6.93 (m, 2H),
7.02-7.07 (m, 1H), 7.16-7.18 (m, 1H), 7.38-7.42 (m, 1H),
7.57-7.61 (m, 2H), 7.74 (dd, J = 7.9 Hz, J = 1.7 Hz, 1H), 10.0
(s, 1H D2O exchange), 10.38 (bs, 1H, D2O exchange). MS m/z
(M þ H)þ = 325.
FRET Assay on Human mGluR2 Receptor. Compounds were
evaluated on human mGluR2 receptor using a fluorescence
resonance energy transfer (FRET) binding assay, enclosing an
appropriate dyed probe and a HEK cellular model stably
expressing the human mGluR2 receptor truncated from its
amino terminal extremity and fused to the enhanced green
fluorescent protein (eGFP; GE HealthCare). The HEK GFP-
hmGluR2 cells were seeded onto poly-D-lysine precoated black-
walled 96-well plates in Modified Eagle’s Medium (MEM)
supplemented with 10% FCS (0.8 ꢀ 105 cells per well). After
24 h of culture at 37 °C, the cell media was removed and cells
were washed with assay buffer (137.5 mM NaCl, 1.25 mM
MgCl2, 1.25 mM CaCl2, 6 mM KCl, 5.6 mM glucose, 10 mM
HEPES, 0.4 mM NaH2PO4, 0.1% bovine serum albumin (w/v),
pH 7.4). The tested compounds were coapplied with the probe
on cells by the FLIPRTETRA (Molecular Devices). Binding of a
tested compound on the receptor is detected by a modification of
the FRET signal between the dyed probe and the GFP-fused
receptor, as measured by the variation of GFP fluorescence at
510 nm. Time curves of ligand binding were recorded during
2000 s (excitation: light emitting diode 470-495 nm, emission:
510 ( 10 nm). Dose-dependent FRET inhibition curves were
fitted with variable slope, using GraphPad Prism software, in
order to determine IC50 and Ki values. Experiments were all
performed in duplicate, three times independently.
Ca2þ Functional Assays on Human mGluR2 and mGluR3.
HEK cells were cultured in Modified Eagle’s Medium (MEM)
supplemented with 10% fetal calf serum (FCS) and transfected
by electroporation as previously described.27,28 Plasmids encod-
ing mGlu receptors were constructed from a pRK backbone and
human mGluR2 (cloned from SK-NSH mRNA extract) or
mGluR3 (purchased from BioXTal) cDNAs. Construction of
the plasmids encoding the chimeric Gqi9 protein or the pro-
miscuous G protein GR15 (used to deviate the natural coupling
of the receptors from inhibition of cAMP production to Ca2þ
release pathway) has been described previously.14,27 Receptor
activity was detected by changes in intracellular calcium, as
measured using the fluorescent Ca2þ sensitive dye, Fluo4AM
(Molecular Probes). Cells were plated after transfection onto
poly ornithine coated, clear bottom, black-walled, 96-well plates
and cultured for 24 h. The day of the screening, cells were
washed with freshly prepared buffer (1ꢀ HBSS supplemented
with 20 mM HEPES, 1 mM MgSO4, 3.3 mM Na2CO3, 1.3 mM
N-(4-Fluorophenyl)-7-oxo-7,7a-dihydrocyclopropa[b]chromene-
1a(1H )-carboxamide (1). 1 was obtained as a beige solid in 21%
yield. 1H NMR (400 MHz, DMSO): 1.44 (dd, J = 7.0 Hz, J =
5.8 Hz, 1H), 1.96 (dd, J = 10.8 Hz, J = 5.8 Hz, 1H), 3.07
(dd, J = 10.8 Hz, J = 7.0 Hz, 1H), 7.03-7.08 (m, 1H),
7.16-7.22 (m, 3H), 7.39-7.44 (m, 1H), 7.70-7.77 (m, 3H),

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8779
CaCl2, 2.5 mM Probenecid, and 0.5% BSA) and loaded at 37 °C,
5% CO2 for 1 h 30 min with buffer containing 1 μM Fluo4AM
and 0.1 mg/mL pluronic acid. After washing, cells were incu-
bated with 50 μL of buffer; 50 μL of 3ꢀ drug solution (prepared
in buffer) and then 50 μL of 3ꢀ glutamate solution (prepared in
buffer) were successively added by the fluorescence microplate
reader FlexStation II384 (Molecular Devices) in each well after
20 s of recording. Fluorescence signals (excitation, 485 nm; emission,
525 nm) were then measured at sampling intervals of 1.5 s for 60 s
after each injection. Dose-inhibition curves were fitted with variable
slope, using GraphPad Prism software, in order to determine EC50
or IC50 values. Compounds 1-5 were tested in positive allosteric
modulators (PAM) conditions in the presence of a glutamate EC10
concentration and in negative allosteric modulators (NAM) con-
ditions in presence of a glutamate EC80 concentration. Experi-
ments were all performed in triplicate, three times independently.
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subtype5(mGlu5) pharmacologyinvitroandinvivowithinaseriesof
functionalized, regioisomeric 2- and 5-(phenylethynyl)pyrimidines.
J. Med. Chem. 2009, 52 (14), 4103–4106.
Acknowledgment. Plasmids encoding the chimeric Gqi9
protein or the promiscuous G protein GR15 were a kind gift
of Dr Jean-Philippe Pin (IGF, Montpellier, France).
Supporting Information Available: 1H NMR of compounds
1-4 and 6, and LCMS analyses of final compounds 1-4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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