Supporting Information

Electronic Supplementary Material (ESI) for ACS Chemical Neuroscience Supporting Information Fluorine-18-labeled Antagonist for PET Imaging of Kappa Opioid Receptors Zhengxin Cai,*, Songye Li, Richard Pracitto, Antonio Navarro, Anupama Shirali, Jim Ropchan, Yiyun Huang PET Center, Department of Radiology and Biomedical Imaging, Yale University, New Haven, Connecticut 652, United States Eli Lilly and Company, Indianapolis, Indiana 46285, United States *Corresponding author. E-mail: zhengxin.cai@yale.edu; Fax: +1-(23)785-2994; Tel: +1-(23)785-7691.

Contents Page 1. Synthesis 1.1. Synthesis of compound 15 S1 2. Radiolabeling 2.1. Synthesis of 13 and 14 S1-S4 Scheme S1. Radiosynthesis of 18 F-LY2459989 (14) from 11 or 12 Table S1. Radiosynthesis of 18 F-LY2459989 (14) from iodonium ylide CN-precursor (16) Table S2. Optimization of labelling conditions using 16 in the NanoTek - Round 1 Table S3. Optimization of labelling conditions using 16 in the NanoTek - Round 2 Table S4. Optimization of the hydrolysis conditions converting 13 to 14. Table S5. Quality specifications of 18 F-LY2459989 (14) and test results from three validation runs. Figure S1. HPLC profile of 18 F-LY2459989 (14) co-injected with 19 F-LY2459989. S1 S2 S3 S3 S4 S4 S5

The auxiliary 6,1-Dioxaspiro[4.5]decane-7,9-dione (SPI-5, 15) was synthesized following published procedures with slight modifications: a mixture of malonic acid (1. g, 9.61 mmol), concentrated sulfuric acid (5 µl) and acetic anhydride (.91 ml, 9.63 mmol) was stirred at 6 o C for 15 min, then cooled to room temperature. Then, cyclopentanone (.85 ml, 9.6 mmol) was added dropwise over 3 min. The mixture was stirred for an additional 6 h at ambient temperature and solvent evaporated. The residue was re-solubilized in Et 2 O, and washed three times with water. The organic layer was dried with Na 2 SO 4, filtered, and concentrated. Precipitation with Et 2 O/hexanes (1:1) at -2 C provided compound 15 (538 mg, 33%) as a white solid. The 1 H-NMR is consistent with published data. m.p. 55-58 ºC; 1 H-NMR: (4 MHz, ppm, CDCl 3 -d): δ 3.62 (s, 2H), 2.34 2.11 (m, 4H), 2.3 1.75 (m, 4H). Radiosynthesis. Scheme S1. Radiosynthesis of 18 F-LY2459989 (14) from 11 or 12 General procedure: 18 F-Fluoride in [ 18 O]water was trapped with a QMA cartridge and then eluted off by a solution of K 222 /K 2 (1.4 ml, 1 mg of K 222 dissolved in 1. ml of acetonitrile and 2.6 mg of potassium carbonate dissolved in.4 ml of water) into a 2 ml v-vial. The solution was first dried at 15 for 1 min under a steam of argon, followed by azeotropic drying with two portions of MeCN (.4 ml each). A solution of the precursor (11 or 12, 1-5 mg) in DMSO (.2 ml) was added. Then, the mixture was heated at 2 for 1 min. The resulting solution was cooled down, and added with.1 ml of 5 N NaOH and.2 ml of H 2 O 2, and heated at 1 for 5 min. The crude mixture was diluted with 1.5 ml of 22% acetonitrile in.1 M ammonium formate with.5% acetic acid (ph = 4.2), and loaded onto a Phenomenex Genesis column (1 x 25 mm, 5 µm) eluting with a mobile phase of 22% acetonitrile in.1 M ammonium formate with.5% acetic acid (ph = 4.2) at a flow rate of 5 ml/min. Finally, 1.4 to 6.8 mci of 18 F-LY2459989 (14) was obtained, with S.A. ranging from.8 to 4.8 mci/nmol. Table S1. Radiosynthesis of 18 F-LY2459989 (14) from iodonium ylide CN-precursor (16):

Entry Solvent T ( C) Time (min) Base/Additive RCY 13 (%) RCY 14 (%) 1 MeCN 8 5 TEAB, 4.8 mg 3.3 2 MeCN 1 5 TEAB, 4.8 mg 1 3 MeCN 12 5 TEAB, 4.8 mg 1 4 DMF 12 5 TEAB, 1 mg 19 1 5 DMF 12 1 TEAB, 1 mg 8 11 6 DMF 12 2 TEAB, 1 mg 8 12 7 DMF 8 15 K 222 (3.5 mg), K 2 (.35 mg) 14 8 DMF 12 15 K 222 (3.5 mg), K 2 (.35 mg) 13 9 DMF 8 5 TEAB, 2 mg 49.5 1 DMF 8 1 TEAB, 2 mg 44.7 11 DMF 8 2 TEAB, 2 mg 43 1.1 12 DMF 1 5 TEAB, 2 mg 44.7 13 DMF 1 1 TEAB, 2 mg 45 1.9 14 DMF 1 2 TEAB, 2 mg 42 3.2 15 DMF 12 5 TEAB, 2 mg 47 1.5 16 DMF 12 1 TEAB, 2 mg 45 2.7 17 DMF 12 2 TEAB, 2 mg 42 3.2 General optimization procedure using 16 with conventional heating: 18 F-LY2459989 (14) was synthesized using a homemade semi-automated module. 18 F-Fluoride in [ 18 O]water was trapped with a Chromafix H cartridge and then eluted with a solution of K 222 /K 2 (3.5 mg of K 222 and.35 mg of potassium carbonate) or tetraethylammonium bicarbonate (TEAB, 2 mg, 4.8 mg, or 1 mg) in MeCN/H 2 O (7:3) into a 2 ml v-vial. The solution was first dried at 15 for 1 min under a steam of argon, followed by the addition of two portions of 1 ml MeCN each. Azeotropic drying continued for another 5-1 min. A solution of the precursor (1-5 mg) in DMF or MeCN (.2 to.4 ml) was added and the mixture heated at 8-12 for 5-2 min. An aliquot of the crude mixture was diluted with 1 ml of 2/8 MeCN/.1 M ammonium formate with.5% acetic acid (ph = 4.2), and analyzed on a radio-hplc system. Radiochemical yield (RCY) was calculated based on the integration of product peaks over the total activity. General optimization procedure using 16 in a microfluidic reactor: 18 F-LY2459989 (14) was synthesized using a continuous flow microfluidic reactor, NanoTek (Advion, Ithaca, NY, USA). 18 F-Fluoride in [ 18 O]water was trapped with a Chromafix H cartridge and then eluted with a solution of tetraethylammonium bicarbonate (TEAB,.5 M or.2 M) in MeCN/H 2 O (7:3). The solution was azeotropically dried. A solution of the precursor (1 mm) in MeCN was used. Reactions were carried out

from 8 to 18, at flow rate of 5-5 µl/min. An aliquot of the crude mixture was diluted with 1 ml of 2% MeCN in.1 M ammonium formate and.5% acetic acid (ph = 4.2), and analyzed on a radio- HPLC or radio-tlc. RCY was calculated based on the integration of product peaks over the total activity. Table S2. Optimization of labelling conditions using 16 in NanoTek- Round 1. # Entry T ( C) Volume (µl) Flow (µl/min) Radio-TCL (%) Radio-HPLC 18 F Prec. * 18 F Prec. * 13 14 13 14 1 8 1 1 2 2 5.6 - - 2 1 1 1 2 2 31 - - 3 12 1 1 2 2 46 6 4 14 1 1 2 2 48 47 5 16 1 1 2 2 4 4 6 18 1 1 2 2 47 52 7 12 1 1 5 5 46 - - 8 12 1 1 1 1 26 - - 9 12 1 1 3 3 23 - - 1 12 1 1 5 5 16 - - 11 12 1 1 15 15 18 - - 12 12 2 1 2 1 16 - - 13 12 5 1 25 5 3.6 - - 14 12 1 2 1 2 46 - - 15 12 1 5 5 25 26 - - # Eluting solution: TEAB (.5 M), reactor volume: 15.7 µl (1 µm 2 m loop). * Prec.: precursor solution (16, 1 mmin MeCN). Table S3. Optimization of labeling conditions using 16 in NanoTek-Round 2. # Entry T ( C) Volume (µl) Flow (µl/min) Radio-TCL (%) Radio-TCL (%) 18 F Prec. * 18 F Prec. * 13 14 13 14 1 8 2 2 2 2 2.8 4.9 2 1 2 2 2 2 21 24 3 12 2 2 2 2 76 77 4 14 2 2 2 2 76 59 5 16 2 2 2 2 54 45 6 12 2 2 5 5 59 41 7 12 2 2 1 1 42 42 8 12 2 2 3 3 38 38 9 12 2 2 5 5 28 2 # Eluting solution: TEAB (.2 M), reactor volume: 15.7 µl (1 µm 2 m loop) * Prec.: precursor solution (16, 1 mmin MeCN). Routine production procedure of 18 F-LY2459989 (14) in a semi-automated synthesis module: 18 F- LY2459989 (14) was synthesized using a homemade semi-automated module. 18 F-Fluoride in [ 18 O]water was trapped with a Chromafix H cartridge, and then eluted with a solution of tetraethylammonium

bicarbonate (TEAB, 2 mg) in MeCN/H 2 O (7:3) into a 2 ml v-vial. The solution was first dried at 15 for 1 min under a steam of argon, followed by the addition of two portions of 1 ml MeCN each. Azeotropic drying continued for another 5-1 min. A solution of the precursor (2 mg) in DMF (.4 ml) was added and the mixture heated at 8 for 5 min. The crude mixture was diluted with 1 ml of HPLC mobile phase, and loaded on a reversed phase SemiPrep-HPLC. The product was eluted out with MeCN/.1M ammonium formate (v/v, 2/8, ph = 4.2) at flow rate of 5 ml per minute, into a reservoir containing 5 ml DI water. Then, it was passed through a C18 SepPak cartridge, washed with diluted HCl aqueous solution, and eluted with USP absolute ethanol. The final dose was diluted with USP sterile saline to ensure that the ethanol concentration is less than 1%, and sterilized via filtration through.22 μm sterile filter. The radiochemical purity of the final product was more than 98%. The formulated final product was stable at room temperature for up to 8 hours post synthesis. Table S4. Optimization of the hydrolysis conditions converting 13 to 14. Entry T( C) Time (min) Base/Additive Conversion (%) 1 8 5 H 2 O 2, NaOH 52 2 rt 1 K 2, DMSO, H 2 O 2 93 3 8 5 K 2, DMSO, H 2 O 2 85 4 rt 1 K 2, H 2 O 2 72 5 8 5 K 2, H 2 O 2 94 Table S5. Quality specifications of 18 F-LY2459989 (14) and test results from three validation runs. Description Specifications Results (n = 3) Isolated product 1 mci at EOS 33-86 mci at EOS Visual inspection Clear and free of particulates Clear and free of particulates Radiochemcial identity 1% of standard retention time.5 3.9 % of standard retention time Radiochemical purity 9% 96 99% Chemical purity 1 µg of carrier mass/dose & Meet specifications 1 µg of non-carrier mass/dose Residual solvent MeCN 4 ppm; DMF 88 ppm MeCN 4 ppm; DMF 88 ppm ph assay 4.5 8.5 7. Sterile filter integrity 5 psi 5 psi Radionuclide half-life T 1/2(calc) = 98.8 12.8 min T 1/2(calc) = 14.6-16.1 min Endotoxin analysis < 17.5 EU/mL < 5. EU/mL Product stability Stable for 8 h Stable for 8 h Sterility testing No aerobic or anaerobic growth after 14 days of incubation No growths

7.183 mau mau 7.278 8681 1. mvolts mvolts 5 Analog - Analog Board 1 215924_F3PB_FP_ID Retention Time Area Area Percent Radio 19 F-LY2459989 5 4 4 3 3 2 2 1 1 5 1 2 3 4 5 6 7 8 9 1 11 12 Minutes SPD-2A Ch1-254nm 215924_F3PB_FP_ID Retention Time Area Area Percent UV 254 18 F-LY2459989 5 4 4 3 3 2 2 1 6935474 1. 1 1 2 3 4 5 6 7 8 9 1 11 Figure S1. HPLC profile of 18 F-LY2459989 (14) co-injected with 19 F-LY2459989. Minutes