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Radiosynthesis of Calcitonin Calcitonin Gene-Related Peptide Receptor (CGRP) imaging Radioligand [¹¹C]MK4232, through In-loop Chemistry

Tanpreet Kaur,¹ Allen F. Brooks, ¹ and Peter Scott¹

¹Department of Radiology, University of Michigan Medical School, Ann Arbor, Michigan, USA

Introduction:  An improved method for the synthesis of CGRP receptor antagonist (CGRP-RA), 2-((R)-8-(3,5-difluorophenyl)-8-methyl-6-(methyl-11C)-10-oxo-6,9-diazaspiro[4.5]decan-9-yl)-N-((R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl)acetamide ([¹¹C]MK4232) is presented.^1-2 This ligand displays excellent overall selectivity. The overall objective of the present study was to develop an improved route for the in-loop synthesis of [¹¹C]MK4232. This improved method enables a reliable cGMP production of [¹¹C]MK4232 using commercially available synthesis module to facilitate future clinical research studies in understanding role of CGRP receptor in migraine treatment. Three validation batches have been successfully prepared and that they passed all QC criteria confirming suitability for human use.

Scheme 1: Automated synthesis of [¹¹C]MK4232.

Methods: Cyclotron produced [¹¹C]CO₂ (111 GBq, 3 Ci) was generated using the ¹⁴N(p,α)¹¹C nuclear reaction using a GE PETTrace cyclotron (60 μA beam for 30 min). [¹¹C]CO₂ was transferred to a GE TRACERlab FX_C-Pro in loop methylation configuration and reacted with H₂ gas in the presence of Ni to generate [¹¹C]CH₄. The resulting [¹¹C]CH₄ was exposed to iodine vapors at 720°C to afford [¹¹C]MeI which was then passed through a silver triflate-graphpac column to furnish the desired electrophilic reagent [¹¹C]CH₃OTf (~37 GBq, 1 Ci). [¹¹C]CH₃OTf was passed through the loop loaded with the desmethyl MK-4232 precursor (1) (1.0 ± 0.2 mg in 100 µL of 3-pentanone). The crude mixture was diluted with 1 mL of HPLC buffer (60% ethanol in 30 mM NH₄OAc, pH 4.6), and then purified using semi-preparative reverse phase HPLC (C18, 10m, 150 x 10 mm (P/No. 00F-4253-N0), 60% ethanol in 30 mM NH₄OAc, pH 4.6, UV: 254 nm) at a flow rate of 3 mL/min (for a representative trace, see Figure 2). The product peak around [¹¹C]MK4232, 1 (t_R = 7 - 8 min) was collected for 60s directly into the round bottom flask containing 50 mL of water. For reformulation, the purified [¹¹C]MK4232 was trapped on a C18 (Waters, 1cc vac) cartridge, which was then rinsed with water (5 mL) to remove the buffer salts and eluted with ethanol (0.5 mL) and diluted with saline for injection (9.5 mL). The final product was filtered with 0.22 mm sterile filter.

Results:  The synthesis was attempted using both in reactor and in-loop. The current in-loop method resulted in the synthesis of [¹¹C]MK4232 in decay corrected product yield: 429 ± 49.7 mCi, RCY = 14.3 ± 49.7%, n = 3, Specific activity = 25,225 ± 1638 Ci/mmol, pH = 5, Purity = 100%. Synthesis time was 35-37 minutes. 

Conclusions:  In summary, a fully automated method for the radiosynthesis of [¹¹C]MK4232 has been developed. The synthesis method provides a straightforward chemistry, leading to good radiochemical yields and purity. This improved method enables a reliable cGMP production of [¹¹C]MK4232 using a commercially available radiosynthesis module. Future testing of [¹¹C]MK-4232 will be carried out in both healthy controls and subjects with migraine disorders.

References:  1). Bell, I.M., Gallicchio, S.N., Stump, C.A., Bruno, J.G., Fan, H., Gantert, L.T., Hostetler, E.D., Kemmerer, A.L., McWherter, M., Moore, E.L., Mosser, S.D., Purcell, M.L., Riffel, K., Salvatore, C.A., Sanabria-Bohórquez, S., Staas, D.D., White, R.B., Williams, M., Zartman, C.B., Cook, J.J., Hargreaves, R.J., Kane, S.A., Graham, S.L., Selnick, H.G., ACS Med. Chem. Lett. 2013, 4, 863–868. 2). Hostetler, E.D., Joshi, A.D., Sanabria-Bohórquez, S., Fan, H., Zeng, Z., Purcell, M., Gantert, L., Riffel, K., Williams, M., O’Malley, S., Miller, P., Selnick, H.G., Gallicchio, S.N., Bell, I.M., Salvatore, C.A., Kane, S.A., Li, C.-C., Hargreaves, R.J., Groot, T. de, Bormans, G., Hecken, A.V., Derdelinckx, I., Hoon, J. de, Reynders, T., Declercq, R., Lepeleire, I.D., Kennedy, W.P., Blanchard, R., Marcantonio, E.E., Sur, C., Cook, J.J., Laere, K.V., Evelhoch, J.L., J Pharmacol Exp Ther 2013, 347, 478–486.

Acknowledgements: This work was supported by NIH Grant.