Synthesis and validation of [18F]mBPET-1, a fluorine-18 labelled mTOR inhibitor derivative based on a benzofuran backbone

Abstract

Background: Targeted therapy of HER2 positive breast cancer has led to clinical success in some cases with primary and secondary resistance being major obstacles. Due to the substantial involvement of mTOR kinase in cell growth and proliferation pathways it is now targeted in combination treatments to counteract HER2 targeted therapy resistance. However, the selection of receptive patient populations for a specific drug combination is crucial. This work aims to develop a molecular probe capable of identifying patients with tumour populations which are receptive to RAD001 combination therapy. Based on the structure of a mTOR inhibitor specific for mTORC1, we designed, synthesised and characterised a novel benzofuran based molecular probe which suits late stage fluorination via Click chemistry. Results: Synthesis of the alkyne precursor 5 proceeded in 27.5% yield over 7 linear steps. Click derivatisation gave the non-radioactive standard in 25% yield. Radiosynthesis of [ 18 F]1-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-((5-methoxy-2-phenylbenzofuran-4-yl) methyl) piperazine ([ 18 F]mBPET-1) proceeded over two steps which were automated on an iPhase FlexLab synthesis module. In the first step, 2-[ 18 F]fluoroethylazide ([ 18 F]6) was produced, purified by automated distillation in 60% non-decay-corrected yield and subjected to Click conditions with 5. Semipreparative RP-HPLC purification and reformulation gave [ 18 F]mBPET-1 in 40% ± 5% (n = 6) overall RCY with a process time of 90 min. Radiochemical purity was ≥99% at end of synthesis (EOS) and ≥ 98% after 4 h at room temperature. Molar activities ranged from typically 24.8 GBq/μmol (EOS) to a maximum of 78.6 GBq/μmol (EOS). Lipophilicity of [ 18 F]mBPET-1 was determined at pH 7.4 (logD 7.4 = 0.89). [ 18 F]mBPET-1 showed high metabolic stability when incubated with mouse S9 liver fractions which resulted in a 0.8% drop in radiochemical purity after 3 h. Cell uptake assays showed 1.3-1.9-fold increased uptake of the [ 18 F]mBPET-1 in RAD001 sensitive compared to insensitive cells across a panel of 4 breast cancer cell lines. Conclusion: Molecular targeting of mTOR with [ 18 F]mBPET-1 distinguishes mTOR inhibitor sensitive and insensitive cell lines. Future studies will explore the ability of [ 18 F]mBPET-1 to predict response to mTOR inhibitor treatment in in vivo models.

Click Chemistry and Radiochemistry: An Update

“…As its name suggests, the CuAAC reaction is predicated on the Cu­(I)-catalyzed union of an organic azide and a terminal alkyne to create a 1,4-disubstituted 1,2,3-triazole (Figure A). A wide variety of options exist for the catalyst, ranging from commercially available saltse.g., the Sharpless–Folkin catalyst (CuSO 4 ·5H 2 O reduced by sodium ascorbate) , to Cu­(I) complexes stabilized by ancillary ligands. , Interestingly, silver-, ruthenium-, iridium-, and nickel-catalyzed azide–alkyne cycloadditions have also been reported, but the copper-mediated flavor remains the gold standard (no pun intended!). CuAAC reactions are simple, rapid, and tolerant of oxygen and water .…”

“…In these scenarios, the CuAAC ligation can be particularly useful, as azide- or alkyne-bearing prosthetic groups can be radiolabeled and then clicked to vectors in a one-pot two-step schema. This approach is especially popular in the synthesis of 18 F-labeled agents, as evidenced by a large library of 18 F-labeled clickable groups that includes [ 18 F]­fluorobenzyl azides, , azide- and alkyne-modified [ 18 F]­fluoropyridines, [ 18 F]­fluoroalkyl azides, ,− [ 18 F]­fluorocarbohydrate azides, ,,− [ 18 F]­fluoropolyoxyethylene azides, ,,− 3-[ 18 F]­fluoropropyne, 5-[ 18 F]­fluoropentyne, , ethynyl-4-[ 18 F]­fluorobenzene, and [ 18 F]­AMBF 3 . , In one recent example, Wang et al reported the synthesis of a modular scaffold for combined near-infrared (NIR) fluorescence and PET imaging via the CuAAC-mediated modification of an azide-bearing NIR dye with an 18 F-labeled alkyne . The work of Walker et al provides a particularly fine example of the pros and cons of a two-step, CuAAC-driven approach to radiofluorination (Figure ).…”

“…In contrast, Daum and co-workers manually created ferrocene-based probes via the initial radiofluorination of an azide-modified sugar and its Cu-catalyzed reaction with an alkyne-containing partner (Figure D) . Ultimately, the complete automation of the CuAAC ligation in a two-step one-pot approach represents the most promising path to clinical production for a diverse array of 18 F-labeled, click-based radiopharmaceuticals. ,, …”

“…61 Ultimately, the complete automation of the CuAAC ligation in a two-step one-pot approach represents the most promising path to clinical production for a diverse array of 18 F-labeled, click-based radiopharmaceuticals. 22,59,78 Innovative work by Pisaneschi et al underscores yet another advantage afforded by this two-step approach to radiolabeling: an opportunity to improve specific activity (i.e., the amount of radioactivity per mole of compound). 79 Specifically, the team used an inexpensive azide-coated resin as a scavenger to rapidly remove excess precursor during the automated CuAAC ligation between 2-[ 18 F]fluoroethylazide and alkyne-bearing peptides (Figure 5).…”

Synthesis and fluorine-18 radiolabeling of a phospholipid as a PET imaging agent for prostate cancer

“…[ 18 F]-PC44:12 (2). Due to our extensive experience with [ 18 F]-fluoroethyl azide click chemistry as well as the compatibility of this strategy with a large number of functional groups, we chose a click chemistry approach to radiolabel the PC44:12 phospholipid at the N-methyl position [18,19]. To demonstrate its possible use as a radiotracer for prostate cancer, initial biodistribution and tumor uptake in LNCaP tumor-bearing mice was evaluated and compared to [ 18 F]-fluoromethylcholine via dynamic PET imaging.…”