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Bioorg Med Chem Lett. Author manuscript; available in PMC 2014 June 1.
Published in final edited form as:
PMCID: PMC3664928

Radiosynthesis and Evaluation of [11C]EMPA as a potential PET Tracer for Orexin 2 Receptors


EMPA is a selective antagonist of orexin 2 (OX2) receptors. Previous literature with [3H]-EMPA suggest that it may be used as an imaging agent for OX2 receptors; however, brain penetration is known to be modest. To evaluate the potential of EMPA as a PET radiotracer in non-human primate (as a step to imaging in man), we radiolabeled EMPA with carbon-11. Radiosynthesis of [11C]N-ethyl-2-(N-(6-methoxypyridin-3-yl)-2-methylphenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide ([11C]EMPA), and evaluation as a potential PET tracer for OX2 receptors is described. Synthesis of an appropriate non-radioactive O-desmethyl precursor was achieved from EMPA with sodium iodide and chlorotrimethylsilane. Selective O-methylation using [11C]CH3I in the presence of cesium carbonate in DMSO at room temp afforded [11C]EMPA in 1.5–2.5% yield (non-decay corrected relative to trapped [11C]CH3I at EOS) with ≥95 % chemical and radiochemical purities. The total synthesis time was 34–36 min from EOB. Studies in rodent suggested that uptake in tissue was dominated by nonspecific binding. However, [11C]EMPA also showed poor uptake in both rats and baboon as measured with PET imaging.

Keywords: orexin, EMPA, PET radiotracer, brain, carbon-11

The orexins are peptides that play an important role in the regulation of the sleep-wake cycle, modulating feeding behavior, and energy homeostasis.1 A wide range of the central nervous system (CNS) disorders are associated with orexin receptors including insomnia,2 cluster headache3 and drug abuse.4 Recent research also suggests that orexin receptors may be related or contribute to several disorders in peripheral organs.5 Although they have diverse functions, orexin receptors have been most extensively investigated for their roles in CNS and have been aggressively pursued as therapeutic targets for sleeping disorders.6 The two members of the family, orexin-A (33 amino acids) and orexin-B (28 amino acids), are derived from the same biological precursor.7 Two receptor subtypes, termed OX1 and OX2, have been identified.8 Orexin-A is a nonselective neuropeptide that binds with similar affinities to OX1 and OX2 receptors, while orexin-B is a selective neuropeptide and has a 10-fold higher affinity for OX2 over OX1 receptors. Both receptors belong to the superfamily of G-protein-coupled receptors (GPCRs).9

Several small molecules have been reported as dual-OX1 and OX2 antagonists or selective OX1 and OX2 antagonists.10 Due to the difference in affinity of OX1 and OX2 receptors over orexin-A and orexin-B, and the different distribution of OX1 and OX2 receptors, it is critically important to choose compounds that show a dramatic difference in affinity and efficacy between these subtypes. With this in mind, we selected EMPA for development of a PET radiotracer for orexin 2 receptors.10d EMPA is a selective antagonist at the OX2 receptor (Ki = 1.10 ± 0.24 nM, OX2: IC50 = 2 nM, OX1: IC50 > 10000 nM). Previously, quantitative autoradiography was been used to determine the distribution and abundance of OX2 receptors in rat brain (Bmax= 6.62 ± 0.60 pmol·mg-1 protein) with the tritium [3H]-labeled EMPA10d. Selective binding in vitro to rat brain sections and ex vivo receptor occupancy studies revealed [3H]-EMPA is a useful tool studying the pharmacology of OX2. A high density of OX2 receptors was observed in the CA3 region of the hippocampus, cortical layer 6, tuberomammillary nucleus, induseum griseum and nucleus accumbens in the rat brain using [3H]-EMPA. The brain/plasma concentration ratio of EMPA determined at 45 min after i.p. administration was modest (0.7 in mouse), but sufficient for us to believe that synthesis and evaluation of [11C]EMPA in rodents and non-human primates was warranted.

Syntheses of the nonradioactive standard, EMPA, and O-desmethyl precursor 1 were achieved in good yield starting from 6-methoxypyridin-3-amine (Scheme 1). 6-methoxypyridin-3-amine was treated with 2-methylbenzene-1-sulfonyl chloride in pyridine to obtain N-(6-methoxypyridin-3-yl)-2-methylbenzenesulfonamide (2) in 88% yield. Compound 2 was coupled with methyl 2-bromoacetate. The resulting compound was then hydrolyzed with 1M NaOH aq to form carboxylic acid 3, which was then coupled to N-(pyridin-3-ylmethyl) ethanamine in the presence of EDC·HCl providing EMPA11. EMPA was treated with sodium iodide and chlorotrimethylsilane, refluxing in acetonitrile to obtain 1 as the O-desmethyl precursor.12

Scheme 1
Synthesis of EMPA and its precursor (1). Reagents and conditions: (i) pyridine, 0 °C to rt, 1 h, 88 %; (ii) methyl bromoacetate, tBuOK, DMSO, 60 °C; 1 M NaOH aq., MeOH, 1 h, 80 °C, 63 %; (iii) N-(pyridin-3-ylmethyl)ethanamine, ...

We anticipated two methylation sites on precursor 1 and indeed found that methylation with CH3I in the presence of various bases in DMSO afforded a mixture of the N-methylated and O-methylated products. Detection conditions to optimize for radiochemical O-methylation were evaluated using stochiometric amounts of CH3I (1 eq), precursor 1 (1 mg), and several bases. We found that the use of K2CO3 at 50 °C predominantly formed the N-methylated compound and anticipated that O-methylation would occur preferentially at lower temperatures. Based on our small screen of reaction conditions with precursor 1 and various bases at room temperature, we found Cs2CO3 yielded the greatest proportion of O-methylated product (Table 1). The N-methylated and O-methylated products could be separated by HPLC, which we used as our strategy to isolate [11C]EMPA described below.

Table 1
Assessment of N-methylation to O-methylation selectivity for various reaction conditions.

As shown in Scheme 2, precursor 1 reacted with [11C]CH3I in 0.3 ml DMSO in the presence of cesium carbonate at room temperature to provide [11C]EMPA. The radiolabelled product was purified via semi-preparative C-18 HPLC (70% H2O + TFA (0.1% v/v)/ 30% CH3CN + TFA (0.1% v/v), 5.0 mL/min, the desired product was collected between 11 min and 11.5 min) and The final product was reformulated by loading onto solid-phase exchange (SPE) C-18 cartridges rinsed with H2O (5 mL), eluted with EtOH (1 mL), and diluted with saline (0.9%, 9 mL).13 The average radiochemical yield was 1.5–2.5% (decay-uncorrected 1.5–2.5% yield at the end of synthesis (EOS, non-decay corrected relative to trapped [11C]CH3I) to trapped [11C]CH3I; n = 3). Chemical and radiochemical purities were ≥ 95 % with a specific activity ranged from 100 ± 20 mCi/μmol (end of bombardment, EOB).

Scheme 2
Radiosynthesis of [11C]EMPA. Reagents and conditions: (i) 11CH3I, Cs2CO3, DMSO, rt, 3 min. RCY: 1.5–2.5% (decay-uncorrected to trapped [11C]CH3I). Specific acitivity:100 ± 20 mCi/μmol.

With [11C]EMPA available, we examined its lipophilicity14 and plasma protein binding 15 in vitro. Both the log D (1.94, n = 2) and plasma protein binding (PPB, 11.33% unbound, n = 3) appeared suitable for blood-brain barrier (BBB) penetration. Given the literature precedence and our analysis of the physical properties of EMPA, including its molecular weight, lipophilicity, and PPB character, we anticipated that [11C]EMPA may suitably cross the BBB for evaluation as an OX2 imaging agent.

We first examined the BBB permeability of [11C]EMPA by PET-CT scans in anesthetized male Sprague-Dawley (SD) rats.16 Anesthesia was performed with isoflurane. For saturability determination, we co-administered [11C]EMPA and EMPA (2 mg/kg) to rats intravenously. The radiotracer was injected at the dose of 1.0–1.2 mCi for each study and emission data were collected for 60 min. A concentration of less than 0.1% of the injected dose per cubic centimeter was distributed in the brain tissue (Figure 1). The organs of greatest accumulation were the liver and kidneys at 90 min (Figure 1B), which is expected after metabolism and excretion of the radiotracer. We noted that whole brain uptake was not different with unlabeled EMPA pretreatment indicating [11C]EMPA has high nonspecific binding in the rat brain.

Figure 1
(A) Whole-brain time-activity curves generated from rodent PET imaging data (0–60 min). (B) Biodistribution of [11C]EMPA in rats at 90 min post injection (gray) and following pretreatment with unlabeled EMPA (black). Data are normalized to blood ...

To determine if the [11C]EMPA has species-dependent brain permeability, we conducted the [11C]EMPA PET-MR imaging in a papio anubis baboon. [11C]EMPA exhibited very poor BBB penetration and low brain uptake over the scanning time (80 min),17 Figure 3. Coregistration of the PET image with an MRI of the same animal indicated that the areas appearing to have modest levels of radioactive uptake are in blood pool regions outside of the BBB18, and less than 0.01% of the injected dose per cubic centimeter was distributed in the brain tissue. One explanation of the failure of brain penetration of EMPA is that EMPA maybe a permeability glycoprotein (P-gp) substrate that limit BBB penetration. Further in vitro and in vivo experiments will be required to investigate mechanisms, however this limits the potential of [11C]EMPA as an OX2 receptor imaging agent in humans.

In summary, we have successfully synthesized [11C]EMPA by O-methylation and evaluated the potential for its use in human PET imaging. The total time required for the radiosynthesis was 34–36 min from EOB using [11C]CH3I and cesium carbonate in DMSO. Radiolabelled product was obtained in 1.5–2.5% yield (EOS) with excellent purity in the formulation. However, in vivo PET studies in rats and baboon showed poor uptake in brain. Our studies suggest that EMPA would not be a good PET tracer for neurological disorders diagnosis by targeting OX2 receptor in the CNS and that new orexin imaging probes designed specifically for the CNS are needed.

Figure 2
(A) [11C]EMPA PET-MRI Imaging (baboon brain). Summed PET images (0–80 min) superimposed with a MEMPRAGE-MRI of the brain from the same baboon, following injection of [11C]EMPA (4.49 mCi). (B) Whole brain time-activity curve for [11C]EMPA in baboon. ...


This research was carried out at the Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided by the Center for Functional Neuroimaging Technologies, P41EB015896, a P41 Regional Resource supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health. This work also involved the use of instrumentation supported by the NIH Shared Instrumentation Grant Program and/or High-End Instrumentation Grant Program; specifically, grant numbers: S10RR017208, S10RR026666, S10RR022976, S10RR019933, S10RR029495. We would like to thank members of the Hooker laboratory for helpful discussions. The authors are grateful to Joseph Mandeville, Grae Arabsz, Shirley Hsu, and Helen Deng for assistance during NHP imaging.


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12. Precursor 1: 1H-NMR (500 MHz, DMSO-d6): δ 8.73 (d, J = 8.5 Hz, 2H), 8.11 (d, J = 7.0 Hz, 1H), 7.83 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.56 (m, 1H), 7.37 (m, 2H), 7.28 (m, 2H), 6.21 (d, J = 10 Hz, 1H), 4.69 (s, 2H), 4.63 (s, 2H), 3.36 (q, 2H), 2.51 (s, 3H), 1.10 (t, J1 = J2 = 7.0 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6): δ 168.3, 161.7, 143.3, 143.1, 142.1, 138.2, 137.7, 137.4, 133.7, 133.2, 130.3, 126.9, 126.8, 126.3, 119.6, 119.5, 119.0, 52.5, 46.1, 42.2, 20.6, 14.3. LC-MS calculated for C22H24N4O4S expected (M): 440.2; Found (M+1): 441.3.
13. 11CO2 was obtained via the 14N (p, α) 11C reaction on nitrogen with 2.5% oxygen, with11 MeV protons (Siemens Eclipse cyclotron), and trapped on molecular sieves in a TRACERlab FX-MeI synthesizer (General Electric). 11CH4 was obtained by the reduction of 11CO2 in the presence of Ni-hydrogen at 350 °C and passed through an oven containing I2 to produce 11CH3I via a radical reaction. 11CH3I was trapped in a TRACERlab FX-M synthesizer reactor (General Electric) preloaded with a solution of precursor (3) (1.0 mg) and Cs2CO3 (6.0 mg) in dry DMSO (300 μL) that had stirred at rt for 5 min prior to trapping. The solution was stirred at rt for 3 min and water (1.2 mL) was added. The reaction mixture was purified by reverse phase semi-preparative HPLC (Phenomenex Gemini NX-C18, 250 mm x 10 mm, 5 μm, 5.0 mL/min, 70% H2O + TFA (0.1% v/v)/ 30% CH3CN + TFA (0.1% v/v)) and the desired fraction was collected. The final product was reformulated by loading onto a solid-phase exchange (SPE) C-18 cartridge rinsing with H2O (5 mL), eluting with EtOH (1 mL), and diluting with saline (0.9%, 9 mL). The chemical and radiochemical purity of the final product was tested by analytical HPLC (Agilent Eclipse XDB-C18, 150 mm x 4.6 mm, 1.0 mL/min, 70% H2O + TFA (0.1% v/v)/ 30% CH3CN + TFA (0.1% v/v)). The identity of the product was confirmed by analytical HPLC with additional co-injection of EMPA reference standard. The average time required for the synthesis from end of cyclotron bombardment to end of synthesis was 34–36 min. The average radiochemical yield was 1.5–2.5% (decay-uncorrected to trapped [11C]CH3I; n = 3). Chemical and radiochemical purities were ≥ 95 % with a specific activity range from 100 ± 20 mCi/μmol (EOB).
14. log D Determination: An aliquot (~50 μL) of the formulated [11C]EMPA was added to a test tube containing 2.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH7.4). The test tube was mixed by vortex for 2 min and then centrifuged for 2 min to fully separate the aqueous and organic phase. A sample taken from the octanol layer (0.1mL) and the aqueous layer (1.0mL) was saved for radioactivity measurement. An additional aliquot of the octanol layer (2.0 mL) was carefully transferred to a new test tube containing 0.5 mL of octanol and 2.5 mL of phosphate buffer solution (pH 7.4). The previous procedure (vortex mixing, centrifugation, sampling, and transfer to the next test tube) was repeated until six sets of aliquot samples had been prepared. The radioactivity of each sample was measured in a WIZARD Automatic Gamma Counter (Perkin-Elmer, Waltham, MA). The log D of each set of samples was derived by the following equation: log D = log (decay-corrected radioactivity in octanol sample x 10/decay-corrected radioactivity in phosphate buffer sample).
15. Plasma Protein Binding Assay: An aliquot of [11C]EMPA in saline (10 μL) was added to a sample of baboon plasma (0.8 mL, pooled from two separate animals). The mixture was gently mixed by repeated inversion and incubated for 10 min at room temperature. Following incubation a small sample (20 μL) was removed to determine the total radioactivity in the plasma sample (AT; AT=Abound+Aunbound). An additional 0.2 mL of the plasma sample was placed in the upper compartment of a centrifree tube (Amicon, Inc., Beverly, MA) and then centrifuged for 10 min. The upper part of the centrifree tube was discarded, and an aliquot (20 μL) from the bottom part of the tube was removed to determine the amount of radioactivity that passed through the membrane (Aunbound). Plasma protein binding was derived by the following equation: %unbound= Aunbound x 100/AT.
16. Rodent PET/CT acquisition. Male Sprague-Dawley rats were utilized in pairs, anesthetized with inhalational isoflurane (Forane) at 3% in a carrier of 2 L/min medical oxygen and maintained at 2% isoflurane for the duration of the scan. The rats were arranged head-to-head in a Triumph Trimodality PET/CT/SPECT scanner (Gamma Medica, Northridge, CA) featuring a PET resolution of approximately 1 mm. Rats were injected standard references or vehicle via a lateral tail vein catheterization at the start of PET acquisition. Dynamic PET acquisition lasted for 60–90 min and was followed by computed tomography (CT) for anatomic coregistration.
17. Baboon MR-PET acquisition. A female Papio Anubis baboon, deprived of food for 12 h prior to the study, was administered intramuscular ketamine (10 mg/kg) and intubated. For maintenance of anesthesia throughout the study, the baboon was provided 1%–4% isoflurane (Forane) in a mixture of medical oxygen and nitrogen. The baboon was catheterized antecubitally for radiotracer injection and a radial arterial line was placed for metabolite analysis. MR-PET images were acquired in a Biograph mMR scanner (Siemens, Munich, Germany), with a PET resolution of 5 mm and field of view of 59.4 cm and 25.8 cm (transaxial and axial, respectively). Dynamic PET image acquisition was initiated followed by administration of the radiotracer in a homogenous solution of 10% ethanol and 90% isotonic saline. An MEMPRAGE sequence began after 30 min of the baseline scan for anatomic coregistration. 4–5 mCi of [11C] EMPA was administered to the baboon for each scan.
18. Image reconstruction and analysis. Dynamic data from the PET scans were recorded in list mode and corrected for attenuation. Baboon data were reconstructed using a 3D-OSEM method resulting in a full width at half-maximum resolution of 4 mm. Rat PET data were reconstructed using a 3D-MLEM method resulting in a full width at half-maximum resolution of 1 mm. Reconstructed images were exported from the scanner in DICOM format along with an anatomic CT for rodent studies and MRI for baboon scans. These files were imported to PMOD (PMOD Technologies, Ltd.) and manually coregistered using six degrees of freedom. Volumes of interest (VOIs) were drawn manually as spheres in brain regions guided by high resolution structural images (MRI for baboon and CT for rats) and summed PET data, with a radius no less than double that of the PET voxel size to minimize partial volume effects (4 mm for baboon and 1 mm for rodent scans). A common VOI mask was applied to both baboon scans. Time-activity curves (TACs) were exported in terms of decay corrected activity per unit volume at specified time points with gradually increasing intervals. The TACs were expressed as percent injected dose per unit volume for analysis.