Glutamate is a major neurotransmitter in the central nervous system (CNS) and acts on diverse sets of receptors. The mGluRs are G-protein coupled receptors (GPCRs). Based on sequence similarity, signal transduction mechanism and pharmacological profiles, mGluRs are classified into three groups. Group II mGluRs (including mGluR2 and mGluR3 subtypes) modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Group II mGluRs have been implicated in a variety of neurological and psychiatric disorders in recent pathological studies, such as anxiety disorders1
, depression, schizophrenia2, 3
, chronic pain syndromes4
, seizure disorders5, 6
, Parkinson’s disease7
, and substance abuse8, 9
. Thus, compounds that are potent and selective for mGluR2/3 could provide a valuable tool to investigate the involvement of these receptors in various diseases.
A number of extremely potent group II mGluRs selective agonists and antagonists had been developed as competitive orthosteric ligands (examples in ).10
=11.1 nM for mGluR2; 38.0 nM for mGluR3)11
, LY379268 (EC50
=2.69 nM for mGluR2; 4.58 nM for mGluR3)12
, and MGS0028 (EC50
=0.57 nM for mGluR2; 2.07 nM for mGluR3)13
are the agonists for group II mGluRs, in which LY354740 (Eglumegad) was developed as a research drug for its potential in the treatment of anxiety and drug addiction.14
The cyclopropane amino-diacid structures were also further modified to generate selective antagonists for group II mGluR, such as LY341495 (IC50
=21 nM for mGluR2; 14 nM for mGluR3)15,16
and MGS0039 (IC50
=20 nM and Ki
=2.2 nM for mGluR2; IC50
=24 nM and Ki
=4.5 nM for mGluR3)17
. LY341495 has been studied for its antidepressant effect and other neurological effects in animal models.18, 19
Structures of selected agonists and antagonists for group II mGluRs.
To better understand group II mGluRs and its role in disease, we were interested in developing an mGluR2/3 selective radiotracer for in vivo
study. As a noninvasive medical imaging technique and a powerful tool in neurological research, PET offers the possibility to visualize and study mGluR2/3 under physiologic and pathologic conditions. Many PET radioligands have been developed for group I mGluRs (mGluR1 and mostly mGluR5),20
however, no PET tracer is available for imaging group II mGluRs. Here we report on the radiosynthesis of a prodrug, (S
C]methoxyphenethyl) glycine dimethyl ester ([11
C]CMGDE), as a PET tracer for group II mGluRs and its preliminary biological evaluation in Sprague-Dawley rats to imagine group II mGluRs.
Ornstein and coworkers had reported a series of aryl-substituted carboxycyclopropylglycine derivatives as the potent and selective antagonists of group II mGluRs,15
in which three of these compounds, 1
, and 3
( & ), were resolved into their four constituent isomers (R,S,S-, S,S,S-, S,R,R- and R,R,R
-isomer). They found that affinity and functional activity for group II mGluRs reside solely in the S,S,S
-isomers of compounds 1–3
, in which the binding affinity of the S,S,S
-isomer surpass that of the corresponding isomer mixture by factors of 3.4, 7.4 and 6.9, respectively. LY341495 (1-SSS
) was the most potent compound in this series and readily penetrated into the brain, however, it is impossible to introduce carbon-11 or fluorine-18 radiolabel into it (along with compounds 2
) without structural alterations. We therefore selected compounds 4
as a lead to PET ligand based on its affinity and easy radiolabelling, in which its S,S,S
-isomer will be radiolabelled with carbon-11. Although the IC50
value was available only for the mixture of 4
, it was anticipated the radiolabeled S,S,S
-isomer of 4
would be more potent than its isomeric mixtures by a similar factor as observed for compounds 1–3
. Moreover, since the amino diacid analog is very polar we also adopted a prodrug approach to improve CNS exposure and pharmaceutical properties of the PET tracer. As shows that the designed prodrug as methyl esters 5-SSS
(CMGDE) has better calculated physical properties compared to the parent compound 4-SSS
(CMG), in which the tPSA value drops from 109.85 to 87.85 and the cLogP value increase from −0.52 to 1.51.
The S,S,S,-isomer of compounds 1–4.
Table 1 Binding Affinity of the Substituted Carboxycyclopropylglycines.15
Structures & calculated properties of CMG and CMGDE.
Radiosynthesis of [11
C]CMG and [11
C]CMGDE is shown in . [11
C]CMG was synthesized in one pot.21
Carbon-11 methylation of the phenolic hydroxyl in 622
was achieved by heating 6
C]methyl iodide at 100 °C in DMSO and 5N NaOH, in which two ester groups were also hydrolyzed. In situ
addition of hydrochloric acid removed the Boc group, followed by neutralization with NaOH. The crude product was purified by semi-preparative HPLC23
to give 76 mCi of pure [11
C]CMG (radiochemical purity > 95%). Total synthesis time was about 45 min. Identity of the labeled compound was confirmed by co-injection of the product [11
C]CMG with the cold compound 2-SSS
(CMG). Radiosynthesis of the prodrug [11
C]CMGDE was carried out in two steps.24
was reacted with [11
C]methyl iodide in DMSO at 120 °C in the presence of aqueous K2
to afford the labeled intermediate 7
. It was critical to use K2
as the base in this reaction, which promoted the reaction but not hydrolyzed the ester groups. The labeling reaction was monitored by the analytical HPLC.23
The intermediate 7
was purified by semi-preparative HPLC, which was then deprotected with trifluoroacetic acid (TFA) in CH2
at room temperature for 12 min. After neutralized by K2
solution, the solvent was removed under reduced pressure. The reaction mixture was purified by the semi-preparative HPLC to give 4.4 mCi of [11
C]CMGDE with a radiochemical purity of more than 96%. exhibits the HPLC chromatogram of the purified product [11
C]CMGDE. The two-steps synthesis took about 70
Reagents and conditions: (a) 1. [11C]CH3I, DMSO, 5N NaOH, 100 °C; 2. 6N HCl, 100 °C; 3. 5N NaOH. (b) [11C]CH3I, aq. K2CO3, DMSO, 120 °C; (c) 1. TFA, CH2Cl2, rt; 2. aq. K2CO3.
HPLC chromatogram of [11C]CMGDE.
MicroPET imaging studies in the male Spague Dawley rats25
showed that there was no brain penetration when injected with [11
C]CMG (). However, the studies showed that the prodrug [11
C]CMGDE readily penetrated into the brain and upon entering into brain the radiotracer26
generated from [11
C]CMGDE had fast reversible binding in several cortical areas, hippocampus and striatum, the sites, which are known to express group II mGluRs27
( & ). The maximum accumulation (2.5–3.5% of the injected dose per cm3
) was observed 2 min after administration (). To confirm the binding specificity, a selective antagonist for group II mGluRs, LY 34149528
, was used as a block agent. The results showed 20–30% decrease of [11
C]CMGDE binding in several brain areas excluding olfactory area, which might be affected by the nonspecific accumulation in the Harderian glands (). The highest activity averaged at the time interval 20–40 min was in the striatum, where the highest “blocking” of 31.5 ± 3.2% was also observed. Pre-injection of LY341495 induced 29.5 ± 4.2 % decrease in the accumulation of [11
C]CMGDE in the whole brain determined at 20–40 min after administration of radioactivity.
Distribution of [11C]CMG (0.4 mCi) in a Sprague-Dawley rat at 20–25 min after administration.
Figure 6 Coronal and axial slices of [11C]CMGDE (0.4 mCi) distribution in the rat brain from 1min till 40 min after administration. Color-coded images are normalized to each other and correspond the acquisition time of 1 min at the same midbrain level (coronal (more ...)
Figure 7 Control PET imaging studies illustrate high accumulation of [11C]CMGDE in several areas of rat brain. Blocking studies with LY341495 verify specific binding. Mean and SEM of the accumulation are presented for 6 control rats and 5 rats after blocking with (more ...)
In summary, this is the first successful approach to develop PET imaging ligand for group II mGluRs. The prodrug approach had greatly improved brain penetration and the receptor targeting. These data might provide a foundation for future development of specific PET imaging ligands for group II mGluRs and other subtype selective radioligands. Optimization of the reaction conditions for preparation of [11C]CMGDE is current under way to reduced total synthesis time.