Development of fluorine-18 labeled peptidic PET tracers for imaging active tissue transglutaminase  Berend van der Wildt, Micha M.M. Wilhelmus, Esther J.M. Kooijman, Cornelis A.M. Jongenelen, Robert C. Schuit, Christian Büchold, Ralf Pasternack, Adriaan A. Lammertsma, Benjamin Drukarch, Albert D. Windhorst  Nuclear Medicine and Biology  Volume 44, Pages 90-104 (January 2017) DOI: 10.1016/j.nucmedbio.2016.10.002 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 A) General irreversible inhibition of human TG2 by DON inhibitor; B) structure of the well-known TG2 inhibitor Z006. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Ex vivo biodistribution of [18F]6f and [18F]6g in healthy Wistar rats following intravenous administration of 10–20MBq of [18F]6f and [18F]6g under isoflurane anesthesia. Activity concentrations are expressed as % ID/g±standard error of the mean and are the averages of four rats per time point. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 Blood plasma metabolite analysis following intravenous administration of 10–20MBq of [18F]6f or [18F]6g in healthy Wistar rats under isoflurane anesthesia (A). Blood plasma was separated in polar and non-polar fractions using a solid phase extraction procedure. The percentage of intact tracer and metabolites was determined by HPLC analysis of the non-polar fractions. Data are expressed as percentage of total activity in blood plasma ± standard deviation and are the averages of three rats per time-point. (B) Representative on-line HPLC radiodetection chromatograms of non-polar fraction from blood plasma obtained at 5min (left) and 60min (right) after intravenous administration of [18F]6g. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 LC–MS/MS analysis following [18F]6g incubation in rat plasma or administration to Wistar rats. A: Neutral loss ion scan (−28, loss of N2) LC–MS/MS chromatogram of rat blood plasma (in vitro). B: Product ion scan of peak corresponding to m/z: 682.2. C: Multiple reaction monitoring LC–MS/MS chromatogram of blood plasma (ex vivo) obtained after administration of carrier added [18F]6g to rats. D: Multiple reaction monitoring LC–MS/MS chromatogram of radiometabolite [18F]M1 after administration of [18F]6g to rats and HPLC purification of metabolite [18F]M1 from blood plasma (ex vivo). E) Synthesis of metabolite M1 by saponification of 6g. Reagents and conditions: i) 1M NaOH in MeOH, 15min, rt, 26%. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 [18F]6g binding to TG2 under activating, baseline and blocked conditions. Results are expressed as percentage of the residual activity in the spin filter compared to the total activity ± standard deviation (n=3). Incubation buffers as follows: Baseline: 50mM Tris–HCl, pH7.4; Activated: 50mM Tris–HCl, pH7.4, 5mM CaCl2, 1mM DTT; Z006: 50mM Tris–HCl, pH7.4, 5mM CaCl2 and 1mM DTT and inhibitor Z006 at 100μM; GDP: 50mM Tris–HCl, pH7.4, 0.5mM GDP; EDTA: 50mM Tris–HCl, pH7.4, 1mM EDTA; No TG2: Baseline conditions with omission of TG2. Each buffer contained 0.1MBq of [18F]6g and each buffer with the exception of ‘No TG2’ contained 10μg·mL−1 TG2. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 6 Representative autoradiography images of MDA-MB-231 tumor sections incubated with [18F]6g (0.1MBq·mL−1) and corresponding quantification (n=4). Incubation buffers as follows: Baseline: 5mM Tris–HCl, pH7.4; Activated: 5mM Tris–HCl, pH7.4, 5mM CaCl2, 1mM DTT; Blocked 6g: 5mM Tris–HCl, pH7.4, 5mM CaCl2 and 1mM DTT and inhibitor 6g at 100μM; Blocked 4l: 5mM Tris–HCl, pH7.4, 5mM CaCl2, 1mM DTT and inhibitor 4l at 100μM. Errors bars indicate standard deviation. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 1 Attempted synthesis of [11C]2, the position of the carbon-11 label is depicted with *. Reagents and conditions: i) dichloromethyl methylether, THF, 30min, 60°C ii) [11C]CH2N2, N-methylmorpholine, trace amounts of H2O, THF, 0°C – rt, 5min. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 2 Synthesis of Z006 analogues 6a–h. Reagents and conditions: i) H2, Pd/C, MeOH, 2h, rt, 93% ii) for 5a–d, 5g and 5h: respective carboxylic acid (commercially available or synthesized as described in Supplementary data), EDC, DMAP, DMF, 2h, rt or for 5e–f: respective carboxylic acid, BOP, DiPEA, DCM, 16h, rt Then, toward 5h: CH3I, K2CO3, DMF, 16h, 40°C, 39–94% iii) TFA/DCM, rt, 1h iv) isobutyl chloroformate, N-methylmorpholine, THF, 10min, −10°C, then CH2N2 (0.5M solution in ether), 3h, −10°C to rt, 43–88%. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 3 Synthesis of labeling precursor 9. Reagents and conditions: i) Fmoc-OSu, DiPEA, DCM, 16h, rt, 82% ii) TFA/DCM, 1h, rt iii) isobutyl chloroformate, N-methylmorpholine, THF, 10min, −10°C, then CH2N2 (0.5M solution in diethylether), 3h, −10°C to rt, 60% iv) piperazine, DCM, 1h, rt, 91%. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 4 Radiosynthesis of [18F]6f. Reagents and conditions: i) K2CO3, [18F/K222], DMF, 15min, 100°C, 60% ii) NaOH, MeOH/DMSO, 15min, 50°C, quant. Iii) 9, BOP, DMSO, 30min, rt, 44%. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 5 Synthesis of labeling precursor 14. Reagents and conditions: i) (boc-aminooxy)acetic acid, EDC, DMAP, DMF, 2h, rt, 91% ii) TFA/DCM, 1h, 0°C, 77%. Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Scheme 6 Radiosynthesis of [18F]6g. Reagents and conditions: i) K2CO3, [18F/K222], DMF, 15min, 100°C, 60% ii) 14, TFA, DMF/THF, 30min, 50°C, 60% iii) isobutylchloroformate, N-methylmorpholine, DMF/THF, 5min, −10°C iv) CH2N2 (0.5M in Et2O), 30min, −10°C to rt, 30% (2 steps). Nuclear Medicine and Biology 2017 44, 90-104DOI: (10.1016/j.nucmedbio.2016.10.002) Copyright © 2016 Elsevier Inc. Terms and Conditions