Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging

Pretargeted imaging can be used to visualize and quantify slow-accumulating targeting vectors with short-lived radionuclides such as fluorine-18 – the most popular clinically applied Positron Emission Tomography (PET) radionuclide. Pretargeting results in higher target-to-background ratios compared to conventional imaging approaches using long-lived radionuclides. Currently, the tetrazine ligation is the most popular bioorthogonal reaction for pretargeted imaging, but a direct 18F-labeling strategy for highly reactive tetrazines, which would be highly beneficial if not essential for clinical translation, has thus far not been reported. In this work, a simple, scalable and reliable direct 18F-labeling procedure has been developed. We initially studied the applicability of different leaving groups and labeling methods to develop this procedure. The copper-mediated 18F-labeling exploiting stannane precursors showed the most promising results. This approach was then successfully applied to a set of tetrazines, including highly reactive H-tetrazines, suitable for pretargeted PET imaging. The labeling succeeded in radiochemical yields (RCYs) of up to approx. 25%. The new procedure was then applied to develop a pretargeting tetrazine-based imaging agent. The tracer was synthesized in a satisfactory RCY of ca. 10%, with a molar activity of 134 ± 22 GBq μmol−1 and a radiochemical purity of >99%. Further evaluation showed that the tracer displayed favorable characteristics (target-to-background ratios and clearance) that may qualify it for future clinical translation.


3-(3-fluoro-4-methoxyphenyl)-1,2,4,5-tetrazine (15-p)
The final compound was obtained from 3-Fluoro-4-methoxylbenzonitrile (0.60 g, 4.00 mmol) following General Procedure C. The crude was purified using flash chromatography (95/5 n-heptane/EtOAc) followed by recrystallization from n-heptane afforded 0.24 g (29%) of a red solid. Rf = 0.39 (n-heptane: The preparation of this intermediates, was performed using a previously described method. 4 To a solution of 4-hydroxybenzonitrile (4 g, 33.6 mmol) in 25% NH 4 OH (180 mL) was added a mixture of KI (27.31 g, 167.9 mmol), I 2 (9.38 g, 36.9 mmol) in H 2 O (40 mL). The reaction was allowed to stir at r.t. for 20 hours, in which time the mixture colour gradually turned from black to a white thick suspension. The precipitate formed was filtered off and the filtrate concentrated. The residue was then dissolved in DCM and washed with H 2 O, saturated aqueous Na 2 S 2 O 3 solution, and brine. The organic layer was dried over anhydrous MgSO 4 , filtered and concentratedunder reduced pressure. The crude was purified using flash chromatography (90/10 n-heptane/EtOAc) to yield the product (6.52 g, 79%) as a brown solid. 1 H NMR (400 Hz, Chloroform-d)

N-(4-cyano-2-fluorophenyl)acetamide (16a-p)
The preparation of this intermediate was performed using a previously described method. 6 To a solution of 4-amino-3fluorobenzonitrile (0.82 g, 6.00 mmol) in DCM (30.0 mL) was added acetic anhydride (0.80 mL, 8.40 mmol). The mixture was stirred at room temperature for 12 hours. The suspension was filtered, and the solvent removed under reduced pressure. The preparation of this intermediate, was performed using a method described previously. 6 To a solution of the corresponding aniline (1.5 g, 6.00 mmol) in DCM (30.0 mL) was added acetic anhydride (0.85 mL, 9 mmol). The mixture was stirred at room temperature for 12 hours. The suspension was filtered, and the solvent removed under vacuum. Purification by flash chromatography (70/30 nheptane/EtOAc) afforded 0.90 g (52%) of 31a as a white solid. Rf

3-Iodo-5-(methoxycarbonyl)benzoic acid (37a)
I I The preparation of this intermediate, was performed using a previously described method. 7 To a solution of dimethyl 5iodoisophthalate (12.8 g, 40 mmol), methanol (80 mL), and DCM (40 mL) was added NaOH (1.68 g, 42 mmol). The mixture was allowed to stir at room temperature for 24 hours. The solvents were removed under reduced pressure. Lots of white precipitate formed when water (9 mL), dichloromethane (10 mL), and ethyl acetate (10 mL) were added while stirring, which was collected by filtration, well washed with a mixture of dichloromethane (10 mL) and ethyl acetate (10 mL), and then with water (10 mL). After transferring the solid (mono sodium salt) to a separatory funnel, ethyl acetate (80 mL) and concentrated HCl (3 mL) diluted with water (20 mL) were successively added. The mixture was vigorously shaken until the solid was disappeared. Then the organic layer was separated, and the aqueous layer was extracted by ethyl acetate (25 mL    3-(4-iodophenyl)-6-methyl-1,2,4,5-tetrazine (15 mg, 0.05 mmol, 1 equiv) is dissolved DCM (1 mL/1mmol) in a sealed tube before adding mCPBA (13.8 mg, 0.06 mmol, 1.2 equiv), the mixture is sealed and allowed to stir at room temperature for 3 hours. A solution of 6,10-dioxaspiro[4.5]decane-7,9-dione (9.4 mg, 0.05 mmol,1.1 equiv) in Na 2 CO 3 10% (2.86 mL/mmol) is prepared and then added dropwise to the mixture in the sealed tube. The mixture was stirred at room temperature for additional 2 hours. To the reaction mixture 5 mL of water is added and is extracted by DCM , dried over MgSO 4 , filtered and concentrated in vacuo. The crude was submited to combi flash from 100% DCM to DCM/10% EtOH. All fractions containing compound were concentrated, disolved in warm methanol and left to crystalized at 4 °C, which afforded pink crystals (5.4 mg, 15%  In a sealed tube m-Chloroperbenzoic acid (11.3 mg, 0.05 mmol) and 3-(4-iodophenyl)-6-methyl-1,2,4,5-tetrazine (1) (10 mg, 0.03 mmol) were dissolved in DCM (1 mL/0.23 mmol) and stirred at r.t. during 3 hours. Mesitulene (5.1 µL, 0.04 mmol) is added and the mixture is cooled to 0ºC followed by dropwise addition of TfOH (8.9µL, 0.10mmol). The reaction mixture was stirred at r.t during 10 minutes. The crude reaction was concentrated under vacuum. Diethyl ether was added and the mixture was stirred at r.t. during 20 minutes and then stored in the freezer during 1 hour for ensure complete precipitation, before filtered and washed with diethyl ether. The resulting solid was collected with methanol and dried under vacuum (12mg, 71% KOAc Compound 3a was synthesized according a previously reported method with minor modifications. 10 An oven-dried MW vial was charged with Pd 2 dba 3 (6.7 mg, 0.007 mmol), XPhos (6.9 mg, 0.01 mmol), 3-(4-iodophenyl)-6-methyl-1,2,4,5-tetrazine (1) (50 mg, 0.242 mmol), bis(pinacolato)diboron (123 mg, 0.484 mmol) and KOAc (72 mg, 0.726 mmol). The MW vial was sealed with a septum and then evacuated and backfilled with argon (this sequence was carried out two times). 1,4-Dioxane (0.50 mL) was added via syringe, through the septum. The reaction mixture was refluxed at 110°C for 24 h, then NaNO2 and AcOH was added to oxidise back the tetrazine core. The solution was extracted with DCM washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The tetrazine was then purified via automatic flash chromatography utilising n-heptane:EtOAc (80/20), resulting in 45 mg of a pink solid (63% The preparation of these intermediates, was performed using a method described previously with minor modifications. 11 Palladium acetate (4.5 mg, 12%) and 1,3,5,7-tetramethyl-2,4,8-trioxa-(2,4-dimethoxyphenyl)-6-phosphaadamantane (PA-Ph) (9.8 mg, 20%) dry THF (1.5 mL) and hexamethylditin (75 µL, 137 mg, 0.42 mmol, 2.5 equiv.) were successively added to a microwave vial equipped with a stir bar which was then sealed and purged with N 2 . A solution of the appropriate iodo-phenyl-1,2,4,5-tetrazine (0.17 mmol) in dry THF (1 mL) was added via a syringe and the reaction allowed to stir at 70 o C in a microwave for 45 minutes. The reaction was allowed to cool to room temperature and unsealed before being quenched with saturated aqueous KF (1 mL). The solution was extracted with DCM washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The tetrazine was then purified via automatic flash chromatography utilising n-heptane and EtOAc as the eluent.

N Sn
The final compound was obtained from 50 mg (0.17 mmol) of the starting material, following the General Procedure D.1. The crude was purified using flash chromatography (90/10 n-heptane/EtOAc) to yield 53 mg (95%) of a pink solid.   PIDA The 1,2-dihydro-1,2,4,5-tetrazine stannane was dissolved in dry DCM and cooled to 0 °C, followed by the portion wise addition of (Diacetoxyiodo)benzene (1.2 equiv.). The reaction was allowed to warm to r.t. and stirred for 3h. The crude was purified using flash chromatography.  The preparation of these intermediates, was performed using a method described previously with minor modifications. 11 Pd(PPh 3 ) 4 (19.4 mg, 10%) and Hexamethylditin (87 µL, 0.42 mmol, 2.5 equiv.) were successively added to a microwave vial equipped with a stir bar which was then sealed and purged with N 2 . A solution of the appropriate iodo-phenyl-1,2,4,5-tetrazine (0.17 mmol) in dry THF (2.5 mL) was added via a syringe and the reaction allowed to stir at 65 o C in a microwave for 3 hours. The reaction was allowed to cool to room temperature and unsealed before being quenched with saturated aqueous KF (1 mL). The solution was extracted with DCM washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The tetrazine was then purified via automatic flash chromatography utilising n-heptane and EtOAc as eluent.      -5-(1,2,4,5-tetrazin-3-yl)   MeV (CTI siemens) or 16 MeV (Scanditronix) proton beam. Automated syntheses were performed on a Scansys Laboratorieteknik synthesis module housed in a hot cell. Analytical HPLC was performed on a Dionex system connected to a P680A pump, a UVD 170U detector and a Scansys radiodetector. The system was controlled by Chromeleon software. Semipreparative HPLC was performed on the built-in HPLC system in the synthesis module and the flow rate was set to 4 mL/min at all times.

Sn
Radiochemical conversion (RCC) of all radiolabeled compounds was determined by analysing a labeled aliquot of the reaction mixture by radio-HPLC and analyzed by integrating the radioactive peaks from the reaction solution. 12 The products were characterized by comparing the radio-HPLC trace of the reaction mixtures with the HPLC UV traces of the authentic 19 F-reference samples, respectively. The radiochemical yield (RCY) was determined using the activity of [ 18 F]fluoride received from the cyclotron at the beginning of the synthesis and that of the formulated product at the end of the synthesis, the decomposition was corrected and have been decay corrected (d.c.). The molar activity (A m ) was determined by integrating the area of the UV absorbance peak corresponding to the radiolabeled product on the HPLC chromatogram. This area was converted into a molar mass by comparison with an average of integrated areas (triplet) of a known concentration for the corresponding reference compounds. The values for radiochemical yield (RCY), radiochemical purity (RCP) and molar activity (A m ) are given as mean values. This applies for all radiolabeled compounds described below.

Determination of Sn and Cu-Content.
The contents of tin and copper were determined by PerkinElmer Elan 6100DRC ICP-MS. Solutions of the formulated tracer [ 18 F]21 in PBS were measured. The sample was analyzed with quantitative method of Cu and Sn. Standard: multielement standard Merck VI and Sn standard for ICP-MS. Software for instrument control, data collection, calibration and quantification: PerkinElmer Elan version 3.3.
The residual amounts of Cu and Sn in the final solution were well below the allowed limits specified in the ICH Guidelines 20 (41-60 and 2.3-3.0 µg/L vs. 300 and 600 µg/day, respectively) (n=4).

Reaction kinetics
Reactivities of the fluoro-Tz 6-13 (reference compounds for the radiolabeled analogs) in the IEDDA reaction with TCO were determined by pseudo-first order measurements in 1,4-dioxane and/or acetonitrile (depending on stability, solubility and availability) at 25 °C and in Dulbecco's phosphate buffered saline (DPBS) at 37 °C by stopped flow spectrophotometry.
Solutions of TCO 24 in anhydrous 1,4-dioxane or acetonitrile and axTCO-PEG 4 25 (S1) in DPBS (10 mM) were prepared at an approximate concentration above 2 mM. Note: The axially configured axTCO is also used as TCO tag on modified CC49. 23 The exact concentration was determined by absorbance titration with 3,6-dimethyltetrazine 26 (S2) (extinction coefficient 510 M -1 cm -1 at 520 nm), quantifying the decrease in tetrazine absorbance upon reaction with TCO or S1. These initial stock solutions were diluted before stopped-flow analysis to reach a final TCO concentration of 2 mM.
Stock solutions of tetrazines were prepared in DMSO at a concentration of 10 mM. Serial dilution into 1,4-dioxane or acetonitrile (TCO) or DPBS (S1) was used to prepare solutions for stopped-flow analysis at a Tz concentration of 100 µM.
Stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535nm LED (optical pathlength 10mm, full width half-maximum 34nm) to monitor the characteristic tetrazine visible light absorbance (520-540 nm). The reagent syringes were loaded with solutions of the Tz and TCO or S1 and the instrument was primed. Subsequent data were collected in triplicate to sextuplicate for each tetrazine. Reactions were conducted at 25 °C (1,4-dioxane or acetonitrile) or 37 °C (DPBS) and recorded automatically at the time of acquisition. Data sets were analyzed by fitting an exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo-first order rate constants that were converted into second order rate constants by dividing through the concentration of excess TCO compound.   [a] calculated based on k 2 (dioxane) for 6 [b] calculated based on k 2 (acetonitrile) and a relative reactivity of 1.4 for Tz 7 Ex vivo biodistribution data for all evaluated Tzs (13, 14-18-m, 19, 21) are displayed below -Ability of 19 F-tetrazines to block 111 In-DOTA-tetrazine from the tetrazine ligation. The relationship between cLogD 7.4 and blocking effect was analysed using Persson's correlation. Results were considered significant when p < 0.05.

Pretargeted PET imaging of TCO-modified mAb (CC49-TCO) with [ 18 F]21
LS174T tumor xenografts in mice were established using the same procedure as for the blocking assay and ex vivo studies (see previous section). Tumor-bearing animals were matched into 2 groups based on their tumor volume (tumor volumes of ~ 60-180 mm 3 , n = 3-4 in each group) and were administered either with CC49-TCO (100 µg/100 µL, ~7 TCO/mAb) or CC49 (100 µg/100 µL). After 72 h, the animals were injected via the tail vein with [ 18 F]21 (2.86 ± 0.99 MBq /100 µL). The tracer was allowed to distribute and the mice were PET/CT scanned (Inveon, Siemens Medical Solutions) after 1 h (PET acquisition: 5 min, energy window of 350-650 KeV and a time resolution of 6 ns; CT scan: 360 projections, 65 kV, 500 μA and 400 ms). During scans the animals were placed on a heating pad to avoid temperature chenges and anaesthetized by breathing sevoflurane (3%). After the scan animals were euthanized and ex vivo biodistribution performed as described previously for the ex vivo blocking assay (see previous section).
Signograms from PET scans were reconstructed using a 3-dimensional maximum a posteriori algorithm with scatter correction and CT-based attenuation correction. PET and CT images were co-registered and tissue uptake analyzed using Inveon software (Siemens). The mean percentage of injected dose per grams (%ID/g) was extracted by manually creating regions of interest (ROI) on fussed PET/CT images. Differences in tumor uptake between mice pretreated with CC49-TCO and CC49 was analysed using Welch's t-test. Results were considered significant when p < 0.05.