Novel Allosteric Potentiator of mGluR5
569
Eight mGluRs have been identified and cloned and have
been assigned to three groups based on structural similarity,
primary coupling to intracellular signaling pathways, and
pharmacology. Group I (mGluR1 and mGluR5) mGluRs are
coupled through G␣q/11 to increases in inositol phosphate
Materials and Methods
Compounds
A synthetic scheme for CPPHA is shown in Fig. 1.
2-{[(4-Chloro-2-methylphenyl)amino]carbonyl}phenyl acetate (3) was
synthesized as follows: to a stirred solution of 4-chloro-2-methylaniline,
1 (10.0 g, 0.071 mol) in toluene was added (24.7 ml, 0.014 mol) N,N-
diisopropylethylamine, followed by slow addition of acetylsalicyloyl
chloride 2. The mixture was stirred until complete by thin layer chro-
matography. Reaction was filtered and dried under vacuum to afford
9.4 g of 3. Analytical LC/MS: (CH3CN/H2O/1% TFA, 4-min gradient),
88% pure, M ϩ 1 peak m/e 304.
2-({[2-(Bromomethyl)-4-chlorophenyl]amino}carbonyl)phenyl acetate
(4) was synthesized as follows: 3 (9.4 g, 0.031 mol) was immediately
taken up in a solution of CCl4 with recrystallized N-bromosuccinimide
(5.5 g, 0.031 mol) and benzoyl peroxide (Bz2O2) (0.75 g, 3.10 mmol). The
reaction was heated at 90°C, along with a light source, until complete by
thin layer chromatography. Upon completion, the solvent was reduced
metabolism and resultant increases in intracellular Ca2ϩ
.
Group I mGluRs are mostly located postsynaptically and
have a modulatory effect on postsynaptic signaling. Group II
(mGluR2 and mGluR3) and group III (mGluR4, mGluR6,
mGluR7, and mGluR8) mGluRs are coupled through G␣i/o to
decreased cAMP synthesis. Group II and group III mGluRs
are mostly located presynaptically and modulate (inhibit)
neurotransmitter release. The group II and group III
mGluRs can be distinguished functionally by group-selective
pharmacological tools (for review, see Conn and Pin, 1997).
The group I receptor mGluR5 may play a role in a
number of disease states, including anxiety (Spooren et al., by two-thirds and filtered through a small plug of silica gel, yielding
10.5 g of 4. Analytical LC/MS: (CH3CN/H2O/1% TFA, 4-min gradient),
85% pure, M ϩ 1 peak m/e 384.
2000; Tatarczynska et al., 2001), pain (Salt and Binns,
2000; Bhave et al., 2001), addiction to cocaine (Chiamulera
et al., 2001), and schizophrenia (for review, see Chavez-
Noriega et al., 2002). We are interested in studying the
function of mGluR5 and initiated an effort to discover and
develop novel pharmacological tools specific for this recep-
tor. Although a number of mGluR agonists and antagonists
have previously been discovered using traditional ap-
proaches, these compounds are mostly analogs of gluta-
mate, quisqualate, or phenylglycine (for review, see Scho-
epp et al., 1999) that bind to the agonist binding site
(orthosteric site) in the amino-terminal domain of the re-
ceptor. Although group-selective agonists and antagonists
were obtained with these approaches, it has been much
more challenging to develop compounds that are subtype-
selective, probably because the structure of the orthosteric
agonist binding site is well conserved within an mGluR
group. Recently, it has become possible to use functional
assays to search for compounds that interact with recep-
tors at allosteric sites that are separate from the orthos-
teric site. For the mGluRs, the first compounds clearly
shown to interact with a allosteric sites were 7-(hydroxy-
limino) cyclopropa[b]chromen-1a-carboxamide ethyl ester
(mGluR1-selective; Litschig et al., 1999) and 2-methyl-6-
CPPHA (6) was synthesized as follows: compound 4 (6.0 g, 0.015 mol)
was dissolved in 50 ml of dimethylformamide. Then, pthalimide 5
(3.31g, 0.023 mol), K2CO3 (6.2 g, 0.045 mol), and a catalytic amount of
KI were added and allowed to stir at 50°C overnight. Upon completion
the reaction was diluted with ethyl acetate and then washed with brine
(6 ϫ 25 ml) to afford 2.9 g of 6 in a crude mixture, which was then
purified by normal phase chromatography. 1H NMR (300 MHz, CDCl3):
␦ 4.83 (S, 2H), 7.05 (m, 2H), 7.36 (dd, J ϭ 2.4 Hz, 8.6 Hz, 2H), 7.50 (dt,
J ϭ 1.5 Hz, 8.5Hz, 1H), 7.59 (d, J ϭ 2.4Hz, 1H), 7.75 ppm (m, 3H), 7.91
(m, 2H), 8.18 (d, J ϭ 7.2 Hz, 2H); 10.17 (s, 1H), 12.27 (s, 1H) Analytical
LC/MS: single peak (214 nm) at 3.633 min (CH3CN/H2O/1% TFA, 4-min
gradient), high-resolution mass spectrometry calculated for
C22H15N2O4Cl (M ϩ H), 407.0799; found 407.0793 (M ϩ H).
The structure of DFB is shown in Fig. 1. Synthesis of DFB was
detailed in O’Brien et al. (2003).
Stable Cell Lines
CHO cells were transfected with cloned human mGluR5 (human
mGluR5 CHO cells) as follows: pCMV-T7-hmGluR5 (Daggett et al.,
1995) was digested with HpaI and EcoRI (New England Biolabs,
Beverly, MA) and the isolated human mGluR5 fragment was sub-
cloned into pIRESpuro2 (BD Biosciences Clontech, Palo Alto, CA).
Stable cell lines were established after transfection of CHONFAT--
lactamase cells with LipofectAMINE 2000 (Invitrogen, Carlsbad,
CA) and drug selection with 10 g/ml puromycin (BD Biosciences
(phenylethynyl)-pyridine (MPEP, mGluR5-selective; Gasparini Clontech). Clonal cell lines were generated by limited dilution. Cells
were grown in Dulbecco’s modified Eagle’s medium (11960; Invitro-
gen) containing 10% dialyzed fetal bovine serum (26400; Invitrogen),
2 mM L-glutamine (25030; Invitrogen), 100 units/ml penicillin/strep-
tomycin (15070; Invitrogen), nonessential amino acids (11120; In-
vitrogen), 25 mM HEPES (15630; Invitrogen), 55 M -mercapto-
ethanol (21985; Invitrogen), and 10 g/ml puromycin (8052-2; BD
Biosciences Clontech). Positive expression was determined by mea-
suring Ca2ϩ flux using a FLIPR384, fluorometric imaging plate
reader (Molecular Devices Corp., Sunnyvale, CA). Cloned rat
mGluR5a receptors were transfected, and the resulting cells (rat
mGluR5 CHO cells) were grown in the same manner. Cell lines
expressing mGluR1b, 4, 7, and 8 were developed that were compat-
ible with Ca2ϩ-sensitive fluorescence assays. Cells expressing
mGluR4 were coexpressed with the chimeric G protein G␣qi5 (Con-
klin et al., 1993), and cells expressing mGluR7 and mGluR8 were
et al., 1999). These compounds do not bind to the orthos-
teric binding sites of their respective receptors, but rather
they act as negative allosteric modulators, binding to sites
in the seven-strand transmembrane spanning domain of
their cognate receptors to exert their inhibitory effects.
Several other examples of negative allosteric modulators of
mGluRs have since been reported. Subsequently, positive
allosteric modulators were reported for mGluR1 (Knoflach
et al., 2001), mGluR2 (Schaffhauser et al., 2003), and
mGluR4 (Marino et al., 2003), and we have recently re-
ported 3,3Ј difluorobenzaldazine (DFB) as a selective pos-
itive allosteric modulator of mGluR5 (O’Brien et al., 2003).
We have continued with this approach of using functional
assays to identify and characterize allosteric modulators,
and now report N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-
isoindol-2-yl)methyl] phenyl}-2-hydroxybenzamide (CP-
PHA), a novel mGluR5-selective positive allosteric modu-
lator of a different structural class from DFB.
coexpressed with the promiscuous G protein G␣15
.
Fluorometric Imaging Plate Reader
Methods used for Ca2ϩ flux measurements using FLIPR384, flu-
orometric imaging plate reader (Molecular Devices Corp.) have been