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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Bioorg Med Chem Lett. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2743750

Fully automated synthesis and initial PET evaluation of [11C]PBR28


Fully automated synthesis and initial PET evaluation of a TSPO radioligand, [11C]PBR28 (N-(2-[11C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide), are reported. These results facilitate the potential preclinical and clinical PET studies of [11C]PBR28 in animals and humans.

Keywords: Translocator protein (TSPO), [11C]PBR28, Radiosynthesis, Positron emission tomography (PET), Traumatic brain injury (TBI), Brain imaging

Translocator protein 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor)1 is a protein found in lung, liver, heart, spleen, kidney, adrenals, brain, glial cells, masts cell and macrophages, and is implicated in numerous nervous system disorders such as epilepsy, cerebral ischemia, nerve injury and neurodegenerative diseases, and immune system diseases such as cancer.2 Brain TSPO density increases in several neuropathological conditions and after experimental injuries to the central nervous system as well.3 TSPO is an attractive target for molecular imaging of neuroinflammation like Alzheimer's disease and tumor progression using the biomedical imaging technique positron emission tomography (PET).4 The prototypical TSPO-selective PET radioligand is [11C]PK11195; however, it is reported to have many limitations such as low brain uptake and low sensitivity.4 These limitations have motivated investigators to search for new TSPO PET radioligands. Promising candidates progressing to human PET studies include [11C]DAA1106, [18F]FEDAA1106 and [11C]PBR286-8 as indicated in Figure 1. [11C]PBR28 (N-(2-[11C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide, IC50 0.658 nM) was originally developed by Innis and Pike et al. at the National Institute of Mental Health (NIMH).8-11 Wishing to study this compound in our laboratory, we investigated a fully automated synthesis of [11C]PBR28 using [11C]methyl triflate ([11C]CH3OTf)12,13 and performed initial PET imaging in an animal model of traumatic brain injury (TBI), which overexpresses TSPO.

Figure 1
Chemical structures of PET TSPO radioligands.

The precursor N-(2-hydroxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide (desmethyl-PBR28, 4a) and reference standard N-(2-methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide (PBR28, 4b) were prepared according to the procedures outlined in Scheme 1. The route taken was generally based on the literature methods with slight modifications.11,14,15 Displacement of 4-chloride by phenol was readily achieved by treatment of 4-chloro-3-nitropyridine with phenol in the presence of K2CO3 to give 3-nitro-4-phenoxypyridine (1) in 97% yield. Reduction of nitro group of compound 1 was performed efficiently with SnCl2 and concentrated HCl instead of 6 N HCl in MeOH to afford 4-phenoxy-3-pyridinamine (2)16 in 92% yield. Condensation of compound 2 with o-salicylaldehyde or o-anisaldehyde in MeOH, followed by reduction with NaBH4 afforded 2-((4-phenoxypyridin-3-ylamino)methyl)phenol (3a) and N-(2-methoxybenzyl)-4-phenoxypyridin-3-amine (3b) in 91% and 90% yield, respectively. PBR28 (4b) was obtained directly by acetylation of the amine 3b with acetyl chloride in CH2Cl2 in 84% yield. Acetylation of the amine and phenolic hydroxyl groups of compound 3a with acetyl chloride, subsequent hydrolysis of its acetate with LiOH in MeOH provided desmethyl-PBR28 (4a) in 78% yield. As depicted in Scheme 2 PBR28 (4b) can be achieved by direct O-methylation of desmethyl-PBR28 (4a) involving anion formation with NaH, followed by CH3I in DMF in 36% yield.

Scheme 1
Synthesis of desmethyl-PBR28 and PBR28. Reagents, conditions, and yields: (a) phenol, K2CO3, DMF, 80°C, 97%; (b) SnCl2, conc. HCl, MeOH, 80°C, 92%; (c) for 3a: o-salicylaldehyde, 90°C, then NaBH4, MeOH, room temperature (rt), 91%; ...
Scheme 2
Alternate synthetic approach for PBR28. Reagents, conditions, and yields: (a) NaH, CH3I, DMF, rt, 36%.

Synthesis of the target tracer [11C]PBR28 ([11C]4b) is indicated in Scheme 3. The phenolic precursor 4a was labeled using [11C]CH3OTf12,13 through O-[11C]methylation17 under basic conditions (NaH) and isolated by a semi-preparative HPLC method18 to produce the corresponding pure radiolabeled compound [11C]4b in 70-80% radiochemical yield, decay corrected to end of bombardment (EOB), based on [11C]CO2. In comparison with the results reported in the literature,11 several significant improvements in the radiosynthesis have been made. [11C]CH3OTf was used as a radiolabeled precursor, which is a proven methylation reagent with greater reactivity than commonly used [11C]methyl iodide ([11C]CH3I).11 NaH was used as a strong base instead of (tBu)4NOH, and CH3CN was used as the reaction solvent instead of MeOH. The reaction temperature 80 °C was higher than room temperature, and the reaction time was only 3 min, shorter than 7 min in the literature. We also used a “vial” method instead of the reported “loop” method. Therefore, the radiochemical yields for [11C]PBR28 in our method is much higher than that reported previously (26%).11 The radiosynthesis was performed in an in-house automated multi-purpose 11C-radiosynthesis module, allowing measurement of specific radioactivity during synthesis.19,20 The overall synthesis, purification and formulation time was 25-30 min from EOB. The specific radioactivity was in a range of 5-15 Ci/μmol at EOB. Chemical purity and radiochemical purity were determined by analytical HPLC.21 The chemical purity of the precursor and reference standard was > 96%. The radiochemical purity of the target tracer was > 99% determined by radio-HPLC through γ-ray (PIN diode) flow detector, and the chemical purity of the target tracer was >93% determined by reverse-phase HPLC through UV flow detector.

Scheme 3
Synthesis of [11C]PBR28. Reagents, conditions, and yields: (a) NaH, [11C]CH3OTf, CH3CN, 80 °C, 3 min, 70-80%.

The characterization data for compounds 1-4 and experimental details for the tracer [11C]4b are given.22

Initial PET evaluation of [11C]PBR28 was performed in rats. Three female Sprague-Dawley rats were imaged. Two animals received a moderate controlled cortical impact (2 mm deformation) to the left parietal cortex (TBI). The third animal received a sham surgery (SHM; incision, skull preparation with no impact). Animals were scanned seven days after surgery. Animals were anesthetized with isoflurane and placed on a stereotaxic-like head-holder.23 The PET scan was performed right after an intravenous (IV) tail vein injection of 0.31 ± 0.07 mCi [11C]PBR28 (0.58 ± 0.35 nmol/kg). Dynamic data were acquired for 90 minutes on the IndyPET III scanner, a small animal PET scanner designed and developed in the Department of Radiology at Indiana University School of Medicine.24-26 The raw data collected from 40-90 min was used to generate the images for analysis. Standardized uptake value (SUV) images were created by normalizing intensity values at each voxel by body weight and injected dose. Regions of interest (ROIs) indicated by the green arrows, approximating the left and right parietal cortices, were drawn on each SUV image. An ellipse (superior-inferior radius = 3 mm; anterior-posterior radius = 4.5 mm) was placed on the approximate anterior-posterior location of the parietal cortex. The ellipse was placed on sequential sagittal slices so that the final ROI spanned the entire lateral to medial extent of each hemisphere. Across all three animals (2 TBI, 1 SHM), SUV values were 16.9 ± 2.23 % higher in the left ROI relative to the right. [11C]PBR28-PET images in 2 TBI and 1 SHM rats are shown in Figure 2.

Figure 2Figure 2Figure 2
[11C]PBR28-PET images in 2 TBI and 1 SHM rats.

In conclusion, an efficient and convenient automated synthesis of [11C]PBR28 was developed, and PET evaluation of [11C]PBR28 was performed in a rat model of TBI. These results warrant that the automated preparation of [11C]PBR28 is suitable for preclinical and clinical studies in animals and humans using PET.


This work was supported in part by the Indiana Genomics Initiative (INGEN) of Indiana University, as well as by grants from the NIH 5R01AG019771, 3P30AG010133-18S1, 5P50NS052606, and Indiana Economic Development Corporation (IEDC 87884). We gratefully acknowledge Dr. Robert B. Innis at the NIMH for his assistance. The referees' criticisms and editor's comments for the revision of the manuscript are greatly appreciated.


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References and notes

1. Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere J-J, Lindemann P, Norenberg MD, Nutt D, Weizman A, Zhang M-R, Gavish M. Trends Pharmacol.Sci. 2006;27:402. [PubMed]
2. Wang M, Gao M, Hutchins GD, Zheng Q-H. Eur. J. Med. Chem. 2009;44:2748. [PubMed]
3. Benavides J, Fage D, Carter C, Scatton B. Brain Res. 1987;421:167. [PubMed]
4. Probst KC, Izquierdo D, Bird JLE, Brichard L, Franck D, Davis JR, Fryer TD, Richards HK, Clark JC, Davenport AP, Weissberg PL, Warburton EA. Nucl. Med. Biol. 2007;34:439. [PubMed]
5. Venneti S, Lopresti BJ, Wiley CA. Progr. Neurobiol. 2006;80:308. [PMC free article] [PubMed]
6. Yasuno F, Ota M, Kosaka J, Ito H, Higuchi M, Doronbekov TK, Nozaki S, Fujimura Y, Koeda M, Asada T, Suhara T. Biol. Psychiatry. 2008;64:835. [PubMed]
7. Fujimura Y, Ikoma Y, Yasuno F, Suhara T, Ota M, Matsumoto R, Nozaki S, Takano A, Kosaka J, Zhang MR, Nakao R, Suzuki K, Kato N, Ito H. J. Nucl. Med. 2006;47:43. [PubMed]
8. Brown AK, Fujita M, Fujimura Y, Liow JS, Stabin M, Ryu YH, Imaizumi M, Hong J, Pike VW, Innis RB. J. Nucl. Med. 2007;48:2072. [PubMed]
9. Imaizumi M, Kim HJ, Zoghbi SS, Briard E, Hong J, Musachio JL, Ruetzler C, Chuang DM, Pike VW, Innis RB, Fujita M. Neurosci. Lett. 2007;411:200. [PubMed]
10. Imaizumi M, Briard E, Zoghbi SS, Gourley JP, Hong J, Fujimura Y, Pike VW, Innis RB, Fujita M. Neuroimage. 2008;39:1289. [PMC free article] [PubMed]
11. Briard E, Zoghbi SS, Imaizumi M, Gourley JP, Shetty HU, Hong J, Cropley V, Fujita M, Innis RB, Pike VW. J. Med. Chem. 2008;51:17. [PubMed]
12. Jewett DM. Int. J. Radiat. Appl. Instrum. A. 1992;43:1383. [PubMed]
13. Mock BH, Mulholland GK, Vavrek MJ. Nucl. Med. Biol. 199926:467. [PubMed]
14. Wilson AA, Garcia A, Parkes J, McCormick P, Stephenson KA, Houle S, Vasdev N. Nucl. Med. Biol. 2008;35:305. [PubMed]
15. Okubo T, Yoshikawa R, Chaki S, Okuyama S, Nakazato A. Bioorg. Med. Chem. 2004;12:3569. [PubMed]
16. Elslager EF, Clarke J, Werbel LM, Worth DF. J. Med. Chem. 1972;15:827. [PubMed]
17. Zheng Q-H, Liu X, Fei X, Wang J-Q, Ohannesian DW, Erickson LC, Stone KL, Hutchins GD. Nucl. Med. Biol. 2003;30:405. [PubMed]
18. Wang M, Lacy G, Gao M, Miller KD, Sledge GW, Zheng Q-H. Bioorg. Med. Chem. Lett. 2007;17:332. [PubMed]
19. Mock BH, Zheng Q-H, DeGrado TR. J. Label. Compd. Radiopharm. 2005;48:S225.
20. Mock BH, Glick-Wilson BE, Zheng Q-H, DeGrado TR. J. Label. Compd. Radiopharm. 2005;48:S224.
21. Zheng Q-H, Mock BH. Biomed. Chromatogr. 2005;19:671. [PubMed]
22. (a). Compound 1, a pale yellow solid, mp 70-71 °C (lit.10 75 °C). 1H NMR (CDCl3, 500 MHz): δ 9.14 (s, 1H), 8.55 (d, J = 6.0 Hz, 1H), 7.52-7.48 (m, 2H), 7.37-7.34 (m, 1H), 7.17-7.15 (m, 2H), 6.79 (d, J = 6.0 Hz, 1H). Compound 2, a red oil. 1H NMR (CDCl3, 500 MHz): δ 8.17 (s, 1H), 7.89 (d, J = 5.5 Hz, 1H), 7.43-7.39 (m, 2H), 7.24-7.21 (m, 1H), 7.10-7.07 (m, 2H), 6.58 (d, J = 5.5 Hz, 1H), 3.95 (br s, 2H). Compound 3a, a white solid, mp 193-194 °C (lit.10 180-182 °C). 1H NMR (DMSO-d6, 500 MHz): δ 9.62 (br s, 1H), 7.86 (s, 1H), 7.70 (d, J = 5.5 Hz, 1H), 7.47-7.44 (m, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.07-7.04 ( m, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.74 (t, J = 7.5 Hz, 1H), 6.54 (d, J = 5.5 Hz, 1H), 5.93 (t, J = 6.5 Hz, 1H), 4.35 ( d, J = 6.5 Hz, 2H). LC/MS (m/z, ESI): [M+H]+ calcd for C18H17N2O2 293.1, found 293.0. Compound 3b, a pale yellow oil. 1H NMR (CDCl3, 500 MHz): δ 8.09 (s, 1H), 7.84 (d, J = 5.5 Hz, 1H), 7.41-7.37 (m, 2H), 7.32-7.31 (m, 1H), 7.28-7.24 (m, 1H), 7.22-7.19 (m, 1H), 7.07-7.04 ( m, 2H), 6.94-6.88 (m, 2H), 6.54 (d, J = 5.5 Hz, 1H), 4.73 (t, J = 5.5 Hz, 1H), 4.45 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H). LC/MS (m/z, ESI): [M+H]+ calcd for C19H19N2O2 307.1, found 307.1. Compound 4a, a white solid, mp 123-124 °C (lit.10 123-125 °C). 1H NMR (CDCl3, 500 MHz): δ 9.07 (br s, 1H), 8.48 (d, J = 5.5 Hz, 1H), 8.43 (s, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.30 (t, J = 7.5 Hz, 1H), 7.23-7.20 (m, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.79-6.72 (m, 5H), 4.94 (d, J = 14.5 Hz, 1H), 4.79 (d, J = 15.0 Hz, 1H), 2.05 (s, 3H). LC/MS (m/z, ESI): [M+H]+ calcd for C20H19N2O3 335.1, found 335.1. Compound 4b, a white solid, mp 84-85 °C (lit.9 89-91 °C). 1H NMR (CDCl3, 500 MHz): δ 8.28 (d, J = 5.5 Hz, 1H), 8.21 (s, 1H), 7.44-7.40 (m, 2H), 7.39-7.37 (m, 1H), 7.29-7.28 (m, 1H), 7.24-7.20 (m, 1H), 6.95-8.86 (m, 3H), 6.74 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 5.5 Hz, 1H), 5.14 (d, J = 14.5 Hz, 1H), 4.89 (d, J = 14 Hz, 1H), 3.58 (s, 3H), 1.99 (s, 3H). LC/MS (m/z, ESI): [M+H]+ calcd for C21H21N2O3 349.1, found 349.0. (b). Production of the tracer [11C]PBR28 ([11C]4b). [11C]CO2 was produced by the 14N(p,α)11C nuclear reaction in small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV proton cyclotron on research purity nitrogen (+1% O2) in a Siemens radionuclide delivery system (Eclipse RDS-111). In a small reaction vial (5 mL), the precursor 4a (0.5-1.0 mg) was dissolved in CH3CN (300 μL). To this solution was added NaH (1 mg). No carrier-added (high specific activity) [11C]CH3OTf that was produced by the gas-phase production method13 from [11C]CO2 through [11C]CH and [11C]CH3Br with silver triflate (AgOTf) column was passed into the reaction vial at rt, until radioactivity reached a maximum (~2 min), and then the reaction vial was isolated and reacted at 80 °C for 3 min. The contents of the reaction vial were diluted with NaHCO3 (1 mL, 0.1 M), and injected onto the semi-preparative HPLC column with 2 mL injection loop for purification, which we used a Prodigy (Phenomenex), S-5 μm, 12 nm, 10 × 250 mm i.d. C-18 column; 52% CH3CN/H2O mobile phase; flow rate 5.0 mL/min; and UV (254 nm) and γ-ray (PIN diode) flow detectors. The product fraction was collected, the solvent was removed by rotatory evaporation under vacuum, and the final product, [11C]PBR28 ([11C]4b), was formulated in saline, sterile-filtered through a sterile vented Millex-GS 0.22 μm cellulose acetate membrane, and collected into a sterile vial. Total radioactivity was assayed and total volume was noted for dose dispensing. The overall synthesis, purification and formulation time was 25-30 min from EOB. Retention times in the analytical HPLC, which we used a Prodigy (Phenomenex) 5 μm C-18 column, 4.6 × 250 mm; 52% CH3CN/H2O mobile phase; flow rate 1.0 mL/min; and UV (254 nm) and γ-ray (PIN diode) flow detectors, were: tR 4a = 6.51 min, tR 4b = 7.57 min, tR [11C]4b = 7.57 min. Retention times in the semi-preparative HPLC were: tR 4a = 6.50 min, tR 4b = 7.98 min, tR [11C]4b = 7.98 min. The radiochemical yields were 70-80% decay corrected to EOB, based on [11C]CO2.
23. Cheng TE, Yoder KK, Normandin MD, Risacher SL, Converse AK, Hampel JA, Miller MA, Morris ED. J. Neurosci. Methods. 2009;176:24. [PMC free article] [PubMed]
24. Frese T, Rouze NC, Bouman CA, Sauer K, Hutchins GD. IEEE Trans. Med. Imaging. 2003;22:258. [PubMed]
25. Rouze NC, Hutchins GD. IEEE Trans. Nucl. Sci. 2003;50:1491.
26. Rouze NC, Soon VC, Young JW, Siegel S, Hutchins GD. IEEE Nucl. Sci. Symposium Conference Record. 2005;4:2394.