Synthesis, in vitro and in vivo evaluation of [11C]MMTP: A potential PET ligand for mGluR1 receptors

Article abstract of DOI:10.1016/j.bmcl.2010.04.146

Synthesis, in vitro and in vivo evaluation of [O-methyl- ¹¹C]dimethylamino-3(4-methoxyphenyl)-3H-pyrido[3′,2′:4,5] thieno[3,2-d]pyrimidin-4-one (1), a potential imaging agent for mGluR1 receptors using PET are described. Synthesis of the corresponding desmethyl precursor 2 was achieved by demethylation of the methoxyphenyl compound 1 in 90% yield. Methylation using [¹¹C]MeOTf in presence of NaOH afforded [ ¹¹C]1 in 30% yield (EOS) with >99% chemical and radiochemical purities and with a specific activity of 3-5 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. The radiotracer selectively labeled mGluR1 receptors in slide-mounted sections of postmortem human brain containing cerebellum, hippocampus, prefrontal cortex and striatum as demonstrated by in vitro autoradiography using phosphor-imaging. PET studies in anesthetized baboon show that [¹¹C]1 penetrates the BBB and accumulates in cerebellum, a region reported to have higher expression of mGluR1. These findings suggest [¹¹C]1 is a promising PET radiotracer candidate for mGluR1.

Full text of DOI:10.1016/j.bmcl.2010.04.146

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3499–3501
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, in vitro and in vivo evaluation of [11C]MMTP: A potential PET
ligand for mGluR1 receptors
Jaya Prabhakaran a, Vattoly J. Majo a, Matthew S. Milak a,b, Suham A. Kassir b, Mikael Palner a,
Lyudmila Savenkova a, Pratap Mali b, Victoria Arango a,b, J. John Mann a,b,c, Ramin V. Parsey a,b
,
J. S. Dileep Kumar a,b,c,
*
a Division of Molecular Imaging and Neuropathology, Department of Psychiatry, Columbia University, United States
b New York State Psychiatric Institute, New York, NY 10032, United States
c Department of Radiology, Columbia University, New York, NY 10032, United States
a r t i c l e i n f o
a b s t r a c t
Synthesis, in vitro and in vivo evaluation of [O-methyl-11C]dimethylamino-3(4-methoxyphenyl)-3H-pyr-
ido[30,20:4,5]thieno[3,2-d]pyrimidin-4-one (1), a potential imaging agent for mGluR1 receptors using PET
are described. Synthesis of the corresponding desmethyl precursor 2 was achieved by demethylation of
the methoxyphenyl compound 1 in 90% yield. Methylation using [11C]MeOTf in presence of NaOH affor-
ded [11C]1 in 30% yield (EOS) with >99% chemical and radiochemical purities and with a specific activity
Article history:
Received 12 April 2010
Revised 28 April 2010
Accepted 30 April 2010
Available online 5 May 2010
of 3–5 Ci/lmol (n = 6). The total synthesis time was 30 min from EOB. The radiotracer selectively labeled
Keywords:
mGluR1 receptors
Autoradiography
Positron emission tomography
Biological imaging
Human postmortem
mGluR1 receptors in slide-mounted sections of postmortem human brain containing cerebellum, hippo-
campus, prefrontal cortex and striatum as demonstrated by in vitro autoradiography using phosphor-
imaging. PET studies in anesthetized baboon show that [11C]1 penetrates the BBB and accumulates in cer-
ebellum, a region reported to have higher expression of mGluR1. These findings suggest [11C]1 is a prom-
ising PET radiotracer candidate for mGluR1.
Ó 2010 Elsevier Ltd. All rights reserved.
Glutamate is the most abundant excitatory amino acid (EAA) in
the mammalian brain and is involved in several pathological and
physiological conditions.1 Metabotropic glutamate receptors
(mGluRs) are G-protein coupled receptors (GPCR) involved in pain
perception and are potential therapeutic targets for multiple dis-
eases due to their ability to modulate excitatory glutamate trans-
mission and postsynaptic signaling,2,3 mGluRs are considered to be
drug targets in order to modulate glutamate transmission in the
treatment of various neurological and psychiatric diseases. Activa-
tion of group I receptors enhances the excitatory effects of gluta-
mate by modulation of ion channel activity.4 Among the eight
subtypes of mGluRs, mGluR1s are implicated in neuroprotection,
pain, multiple sclerosis, motor dysfunction, epilepsy, cerebral
ischemia and cerebellar long term depression (LTD).5 Hence mon-
itoring mGluR1, in vivo and non-invasively would lead to a better
understanding of the role of this receptor in the pathophysiology
of biological processes mediated through glutamergic pathways.
However, in vivo quantification of the mGluR1 has been hindered
by the lack of a successful PET tracer. Although the methyl ana-
logue of the low affinity nonselective mGluR1 ligand CPCCO2Et,
CPCCO2Me, is reported its pharmacological data as well as biolog-
ical studies with [11C]CPCCO2Me have not been revealed.6 In a pat-
ent application, [3H]-radiolabeling of a variety of quinolines and
quinolinones bearing mGluR1 antagonists were proposed for use
in PET, but no data is available on their binding potential to
mGluR1.7
[
11C]labeled mGluR1 antagonist, (3-ethyl-2-[11C]-
methyl-6-quinolinyl)(cis-4-methoxycyclohexyl)-methanone [11C]-
JNJ16567083), is a PET radioligand that was used for in vivo
imaging in rats, however, no further data is available to support
its effectiveness as an imaging agent.8 We selected dimethyl-
amino-3(4-methoxyphenyl)-3H-pyrido[30,20:4,5]thieno[3,2-d]pyrimi-
din-4-one 1) as a candidate PET ligand for mGluR1 due to its high
affinity (Ki = 7.9 nM) and selectivity over mGluR5 (>1000 times).
9–11
Compound 1 was reported to be active in an in vivo model
for pain.12 Moreover, presence of potent C-11 labeling site and a
log P value 3.15, which is within the range for passive brain entry
prompted us to develop [11C]1 as a PET ligand for mGluR1.13
Demethylation of compound 1, which is available through com-
mercial vendors, using 1 M BBr3 in DCM afforded the desmethyl
precursor
2
in 90% yield (Scheme 1).14 Methylation using
[
11C]MeOTf in presence of NaOH afforded [11C]1 in 30% radiochem-
ical yield (EOS) with >99% chemical and radiochemical purities.15
The specific activity was determined based on a standard mass
curve of the cold standard and typical specific activity ranged from
* Corresponding author. Tel.: +1 212 543 1163; fax: +1 212 543 1054.
E-mail address: dk2038@columbia.edu (J.S. Dileep Kumar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.146

3500
J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3499–3501
N
N
N
N
N
a
N
O
N
OH
O
O
S
S
N
b
1
2
11CH3
O
N
N
N
N
O
S
[
11C]1
Scheme 1. Synthesis of [11C]1. Reagents and conditions: (a) BBr3, DCM, rt, 90%; (b)
11C]MeOTf, NaOH, rt, 30% (EOS).
[
Figure 2. PET and MRI co-registered images of [11C]1 in baboon. First row: baboon
PET image; second row: MRI; First column: axial, middle column: coronal, last
column: sagittal views.
0.2
0.15
0.1
CEREBELLUM
CAUDATE
HIPPOCAMPUS
FRONTAL CORTEX
PUTAMEN
AMYGDALA
0.05
0
0
20
40
Time (minutes)
60
80
Figure 3. Time activity curves of [11C]1 in baboon.
Figure 1. Phosphor images of [11C]1 in postmortem human brain sections. Top
row-total and bottom row-nonspecific. First column: cerebellum; second column:
hippocampus; third column: frontal cortex; fourth column: striatum.
85 fmol per sq cm) and striatum (Bmax = 20 fmol per sq cm) human
brain sections.16 High density of mGluR1 as evident from phosphor
image study is adequate for quantifying the receptor by PET.
Subsequently, we performed PET studies of [11C]1 in anesthe-
tized baboons.17 PET images shows that [11C]1 penetrates the
BBB and accumulates in brain (Fig. 2).
3 to 5 Ci/l
mol (n = 6). Yield and specific activity of [11C]1 are suffi-
ciently high to carry out routine imaging studies.
After achieving the radiolabeling of ligand 1 in high yield and
specific activity, [11C]1 was tested to obtain its binding to mGluR1
in human brain postmortem using phosphor image autoradiogra-
Time activity curves (TACs) indicate rapid washout of radioli-
gand in almost all brain regions except cerebellum where mGluR1
is abundant (Fig. 3). The radioactivity levels of most of the regions
apart from cerebellum reached baseline at 35 min. Ratio of most of
the other brain regions to cerebellum was 2, 1.65, and 1.6 at 35, 65,
and 85 min, respectively. Metabolite analyses show 45% and 30%
unmetabolized [11C]1 at 30 and 90 min. The radioligand did not ex-
hibit considerable protein binding in human and baboon plasma.
The reason for the faster washout of the tracer from other regions
apart from cerebellum is not clear. However, the radioligand shows
specific binding in in vitro phosphor image studies. A species dif-
ference may attribute to this discrepancy.
phy (Fig. 1). Frozen brain sections (20 lm) of cerebellum, striatum,
hippocampus and prefrontal cortex were used for the phosphor
image study. Four sections each for total and nonspecific binding,
per brain region, were assayed. Slide-mounted sections were
brought to 22 °C and pre-incubated in 50 mM Tris–HCl, 1.2 mM
MgCl2, 2 mM CaCl2 and 0.1% BSA (pH 7.4) for 45 min at room tem-
perature. This was followed by incubation in 12 nM of radioligand
(SA = 1.88 Ci/micromole) for 60 min at room temperature. Adja-
cent sections were incubated with JNJ16259685 a known selective
mGluR1 antagonist (10 lM) to determine nonspecific binding.
After the incubation, sections were washed with ice-cold buffer
containing 50 mM Tris-buffer (pH 7.4) at 4 °C and briefly dipped
in ice-cold water to remove salts. Slides were quickly dried under
a stream of cold air and exposed to a phosphor-imaging screen
(Packard, ST screen wrapped in Mylar film) with high- and low-
activity standards (American Radiolabeled Chemicals) for 90 min.
Screens are scanned with a Packard Cyclone phosphor-imaging
system and analyzed with OptiQuant Acquisition and Analysis
software (Packard). Figure 1 illustrates specific binding of [11C]1
to mGluR1 and reveal specific binding and presence of high density
of mGluR1in cerebellum (Bmax = 140 20 fmol per sq cm), hippo-
campus (Bmax = 110 25 fmol per sq cm), frontal cortex (Bmax
In summary, we successfully synthesized [11C]1, a potential
imaging agent for mGluR1 receptors. The total time required for
the radiosynthesis was 30 min from EOB using [11C]methyl triflate
in acetone. [11C]1 was obtained in 30% yield (EOS) with >99%
chemical and radiochemical purities with a specific activity ranged
from 3 to 5 Ci/lmol (n = 6). Phosphor image studies indicate that
this newly developed [11C]1 ligand binds to mGluR1 enriched brain
regions. PET studies in baboon show that [11C]1 penetrates BBB and
is retained in cerebellum, a region with higher expression of
mGluR1. Hence [11C]1 has the potential for the quantification of
mGluR1 receptors in brain in vivo using PET. Further detailed
in vitro and in vivo investigation of [11C]1 are in progress.

J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3499–3501
3501
desmethyl compound 2.
Acknowledgment
1HNMR (CDCl3, 400 MHz) d ppm 8.40 (d, 1H), 7.58 (s, 1H), 7.10 (m, 2H), 6.85
(m, 3H), 2.47 (s, 6H).
Financial support by the National Institute of Mental Health is
gratefully acknowledged (R21 MH081801).
HRMS calcd for C17H15N4O2S; 338.0840: Found; 338.0838.
15. Radiosynthesis of [11C]1: The precursor desmethyl-1 (0.5–1.0 mg) was dissolved
in 500 lL of acetone in a capped 5 mL V-vial. Sodium hydroxide (10 lL, 5 M)
was added and the resultant solution was allowed to stand for 2 min. High
11C]CO2 produced from RDS112 Cyclotron (ꢀ37 GBq) was
References and notes
specific active
[
subsequently converted into [11C]CH3OTf and is transported by a stream of
argon (20–30 mL/min) into the vial over approximately 5 min at room
temperature. At the end of the trapping, the product mixture was diluted
with 0.5 mL of acetonitrile and is directly injected into a semi preparative RP-
1. Hinoi, E.; Takarada, T.; Tsuchihashi, Y.; Yoneda, Y. Curr. Drug Targets: CNS
Neurol. Disord. 2005, 4, 211.
2. Schoepp, D. D.; Monn, J. A. P. Glutamate and GABA Receptors and Transporters
2002, 151.
3. Swanson, C. J.; Bures, M.; Johnson, M. P.; Linden, A.-M.; Monn, J. A.; Schoepp, D.
D. Nat. Rev. Drug Disc. 2005, 4, 131.
4. Moghaddam, B. T. Psychopharmacology 2004, 174, 39.
HPLC (Phenomenex C18, 10 ꢁ 250 mm, 10
lm) and eluted with acetonitrile/
0.1 M ammonium formate solution (40:60) at a flow rate of 10 mL/min. The
precursor eluted after 3–4 min during the HPLC analysis. The product fraction
with a retention time of 7–8 min based on c-detector was collected, diluted
with 100 mL of deionized water, and passed through a classic C-18 Sep-PakÒ
cartridge. Reconstitution of the product in 1 mL of absolute ethanol afforded
5. Gasparini, F.; Kuhn, R.; Pin, J.-P. Curr. Opin. Pharmacol. 2002, 2, 43.
6. Yu, M.; Brownell, A. -L. Mol. Imaging (Symposium) 2002, 230.
7. Lesage, A. S. J.; Bischoff, F. P.; Janssen, C. G. M.; Lavreysen, H. PCT Int. Appl.
2003, WO 2003082350.
8. Huang, Y.; Narendran, R.; Bischoff, F.; Guo, N.; Zhu, Z.; Bae, S.-A.; Lesage, A. S.;
Laruelle, M. A. J. Med. Chem. 2005, 48, 5096.
9. Matasi, J. J.; Tulshian, D.; Burnett, D. A.; Wu, W.-L; Korakas, P.; Silverman, L. S.;
Sasikumar, T. K.; Qiang, L.; Domalski, M. S. U.S. Patent 2006, 152,535.
10. Sasikumar, T. K.; Li, Q.; Burnett, D. A.; Greenlee, W. J.; Li, C.; Heimark, L.;
Pramanik, B.; Grilli, M.; Bertorelli, R.; Lozza, G.; Reggiani, A. Bioorg. Med. Chem.
Lett. 2009, 19, 3199.
11. Wu, W.-L.; Burnett, D. A.; Domalski, D.; Greenlee, W. J.; Li, C.; Bertorelli, R.;
Fredduzzi, S.; Lozza, G.; Veltri, A.; Reggiani, A. J. Med. Chem. 2007, 50, 5550.
12. Varty, G. B.; Grill, M.; Forlani, A.; Fredduzzi, S.; Grzelak, M. E.; Guthrie, D. H.;
Hodgson, R. A.; Sherry, X. L.; Nicolussi, E.; Pond, A. J.; Parker, E. M.; Hunter, J. C.;
Higgins, G. A.; Reggiani, A.; Bertorelli, R. Psychopharmacology 2005, 179, 207.
13. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Radiat. Isot. 2001,
54, 203.
14. O-Methyl compound 1 (140 mg, 0.4 mmol) is dissolved in methylene chloride
(5 mL) and BBr3 (1 M, solution in methylene chloride, 0.6 mL, 0.44 mmol) was
added to it. The solution was stirred for 1 h at room temperature. The reaction
mixture was cooled in an ice-bath and quenched by adding methanol. The
mixture was then poured into 5 mL NH4OH solution and extracted with
methylene chloride. The combined organic extracts were dried over anhydrous
MgSO4 and the solvent was removed under vacuum to provide the crude
product. The product was chromatographed over silica gel using a mixture of
1:1 methylene chloride and ethyl acetate to obtain 120 mg (90%) of the
[
11C]1 (30% yield, based on [11C]CH3I at EOS). A portion of the ethanol solution
was analyzed by analytical RP-HPLC (Phenomenex, Prodigy ODS(3)
m; mobile phase: acetonitrile/0.1 M ammonium formate;
4.6 ꢁ 250 mm, 5
l
45:55, flow rate: 2 mL/min, retention time: 8 min, wavelength: 230 nm) to
determine the specific activity and radiochemical purity. Then the final
solution of the [11C]1 in 10% ethanol-saline was analyzed to confirm the
chemical and radiochemical purities.
16. The autoradiograms were quantified using a dilution series (each 1:10) of
droplets (5
lL) made from the incubation solution, these were added to a TLC
plate and exposed together with the autoradiograms. The pixel intensity of the
droplets were fitted by
a power equation using counted samples as
concentration references. Individual ROI0s were thereby quantified for the
total amount of compound using the specific activity.
17. Baboon PET scanning with [
11C]1: Fasted animals were immobilized with
ketamine (10 mg/kg, im), and anesthetized with 1.5–2.0% isoflurane via an
endotracheal tube. Core temperature was kept constant at 37 °C with a heated
water blanket. An iv infusion line with 0.9% NaCl was maintained during the
experiment and used for hydration and radiotracer injection. An arterial line is
placed for obtaining arterial samples for the input function. After a 10 min
transmission scan, 5 0.5 mCi of [11C]1 (S.A. of 2.5 0.5 Ci/
lmol) was injected
as an i.v. bolus and emission data were collected for 120 min in 3-D mode in a
Siemens ECAT EXACT HR+ (CPS/Knoxville, TN). The head was positioned at the
center of the field of view as defined by imbedded laser lines. Regions of
interest drawn on the animal’s MRI scan were transferred to co-registered
(AIR) frames of PET data.