[11C]GR103545: novel one-pot radiosynthesis with high specific activity
Introduction
Two and a half decades have passed since it was shown that opioid receptors (ORs) could be labeled in vivo, with the first successful positron emission tomography (PET) study done in 1985 using the mu-OR agonist [11C]carfentanil [1]. ORs are widely distributed in the central nervous system and peripheral sensory and autonomic nerves, and PET studies offer a non-invasive method to investigate in vivo the function or dysfunction of the different ORs in relation to neurobiology and neuropsychiatric disorders [2]. Several selective kappa-OR (κ-OR) agonist and antagonist ligands have since been developed [3], [4], [5], but only a few have advanced to clinical trials in humans [6], [7]. GR89696, (±)-methyl 4-[(3,4-dichlorophenyl)acetyl]-3-(pyrrolidin-1-ylmethyl)piperazine-1-carboxylate, is a potent κ-OR agonist, and its 11C-labeled form, [11C]GR89696, has been assessed as a potential PET imaging agent [8], [9]. Furthermore, [11C]GR103545, the active R-enantiomer of [11C]GR89696, has also been synthesized and shown to display favorable properties as a PET imaging radiotracer for κ-OR in the brain of non-human primates [10], [11]. It has exhibited high binding affinity with fast uptake and clearance rates. Its binding distribution is similar to the known central nervous system regional distribution of κ-ORs in humans [10]. However, its advancement to imaging applications in humans was hampered by the radiolabeling method, which generally gives [11C]GR103545 not only in low and variable radiochemical yields (RCYs) but also in relatively low specific activity (SA) (69.3–75.5 MBq/nmol; 2–2.6 mCi/nmol) [9], [11]. Following the published procedure (Scheme 1), we were unsuccessful in labeling [11C]GR103545 in SA high enough for imaging studies in humans.1 It is well established that opioid agonists are powerful analgesics and can induce undesirable physiological effects even when given at doses of micrograms that are relevant in PET imaging experiments. For example, in the case of [11C]carfentanil, undesirable physiological effects were noted in early imaging experiments in humans, and injected mass of carfentanil has been limited to <0.03 μg/kg in subsequent human PET studies (Frost JJ. Personal communication) [1]. Similarly, GR103545, a potent, selective κ-OR agonist, has been shown to cause dysphoria and sedation at the microgram dose level [7], [12]. Thus, it is essential to find a reliable radiosynthetic method that produces [11C]GR103545 in acceptable RCY and, more importantly, in high SA, in order to successfully advance [11C]GR103545 to imaging applications in humans, so that the mass associated with an injected amount of radioactivity can be held to a level that does not elicit undesirable physiological side effects. We report here our findings in the search for such a method.
GR103545 contains a methyl carbamate group, and the original radiosynthesis of [11C]GR103545 employed a three-step procedure, starting with [11C]CO2, to construct the methyl carbamate functionality (Scheme 1). We envisioned an alternative synthetic approach toward this functionality by using carbon-11 methylation of a carbamic acid intermediate (Scheme 2).
In PET radiochemistry, carbon-11 methylation by nucleophilic substitution with [11C]methyl iodide ([11C]CH3I) or [11C]methyl trifluoromethanesulfonate ([11C]methyl triflate; [11C]CH3OTf) is a favored method for incorporating a carbon-11 nuclide into a molecule. The simplicity of this reaction has rendered it the most widely used procedure for the synthesis of a large number of 11C-labeled radiotracers via N-, O-, or S-methylation with carbon-11 [13], [14]. In our laboratory, we routinely carry out these carbon-11 methylations to produce a wide range of radiopharmaceuticals in good yields and high SA by the gas-phase transformation of [11]CO2 via [11C]CH4 to [11C]CH3I (or onward to [11C]CH3OTf) [15]. Therefore, it was expected that the approach depicted in Scheme 2 would lead to high-SA [11C]GR103545. The requisite carbamic acid intermediate in turn can be constructed through two ways: direct carboxylation by CO2 [16], [17] or transcarboxylation via fixated CO2. Transcarboxylation with the 1,8-diazabicyclo[5.4.0]undec-7-ene–carbon dioxide (DBU–CO2) zwitterionic carbamic complex has been used to prepare urethanes [18], carbonates and carbamates [19], [20], as well as carboxylation of active methylene compounds [21].
Section snippets
Materials and methods
Reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Fisher Scientific, Merck or J.T. Baker) and were used without further purification. Both the desmethoxycarbonyl precursor (1) and reference standard (GR103545) were obtained from the National Institute of Mental Health's Chemical Synthesis and Drug Supply Program. The DBU–CO2 carbamic complex was prepared according to the literature [20].
Radiosynthesis of [11C]GR103545 was carried out using a TRACERLab FXC automated
Results
Scheme 3 outlines our new and efficient approach for radiolabeling [11C]GR103545. All radiosyntheses were carried on a TRACERLab FXC automated synthesizer. In total, 26 experiments were performed under two radiolabeling conditions: about half of the syntheses (n=15) were carried out in the presence of Cs2CO3/TBATf, and the remainder (n=11) was carried out without Cs2CO3/TBATf. The combined SA for both labeling conditions was 290.45±99.9 MBq (7.85±2.7 mCi/nmol) at the end of synthesis.
Discussion
Organic carbamates are used in many applications, ranging from synthesis of polyurethanes, pesticides and fungicides, in addition to medicinal drugs and other important synthetic intermediates [30]. They have traditionally been prepared using hazardous and toxic reagents, such as phosgene and isocyanate intermediates, as well as carbon monoxide [31]. We first carried out the radiosynthesis of [11C]GR103545 using the phosgene approach as reported in the original literature. As shown in Table 1,
Conclusions
We have developed a one-pot method for the automated radiosynthesis of the κ-OR tracer [11C]GR103545 with high SA under mild conditions and in good yield via O-methylation with carbon-11 of compound 2. This carbamic intermediate was prepared in situ by a transcarboxylation reaction between compound 1 and DBU–CO2 zwitterionic carbamic complex. The method is highly efficient and reproducible, yielding [11C]GR103545 in approximately nine times greater SA and four times higher yield than the
Acknowledgments
We thank Rachel Hull, Soheila Najafzadeh, Joseph Olsen and Mike Tabriz of the radiochemistry laboratories of the Yale University PET Center for their expert technical assistance.
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