Semi-Automated Synthesis of [ F-18 ] FBAM , a Thiol Reactive Prosthetic Group , Using Continuous Flow Chemistry

[F-18]FBAM, a thiol reactive bifunctional agent, was successfully synthesized using continuous flow chemistry in a micro reactor that is part of Advion NanoTek Microfluidic Synthesizer. As the radiofluorination was carried out microfluidically, a very small amount of precursor was used and over all radiochemical yield was 38% ± 4% (n = 8, decay corrected) and the radiochemical purity was ≥98% with specific activity of 430 mC/μmol. The total reaction time including HPLC purification was 55 min that is 14 min more than manual synthesis and 6 min less than fully automated synthesis.


Introduction
Bioactive peptides and proteins are important key regulators in cell growth and cellular functions in living organisms [1]- [3].Various peptides, proteins, antibodies, antibody fragments and nucleotides have been radiolabeled and used to image tumors and inflammatory processes [4].Among these tracers, [F-18] labeled molecular probes are increasingly popular because of the ease of production of 18 F and its favorable properties.However, harsh reaction conditions required to directly radiofluorinate these sensitive biomolecules hampered the preparation of the requisite tracers in good yields and high specific activity.To address these problems, radiochemists have taken advantage of bifunctional agents, also known as prosthetic groups.These prosthetic groups are catego-rized into three classes amine, thiol and carboxylic reactive.Notable thiol reactive bifunctional agents include [ 18 F] FBAM [5] [6], [ 18 F]FBOM [7], [ 18 F]FBABM [8], [ 18 F]FPyAM [9] and [ 18 F]FPyMe [10] (Figure 1).
Microfluidics represents a useful approach to conduct the reactions with minimal quantities of expensive precursors; other advantages include reduced reaction times and increased radiolabeling yields.These effects are realized due to the high surface to volume ratio encountered while the reagents are flowing through the microchannel which is accompanied by rapid mixing of the reagents leading to increased heat transfer between the reactants.One of the commercially available microfluidic devices with micro-channel system (MCS) is the Advion NanoTek Microfluidic Synthesizer.This unit consists of three modules called the reagent, reactor and concentrator modules.The isotope is dried and dissolved in the concentrator module and transferred to a loop attached to the reactor module while the precursor is stored in a second loop attached to reagent module.The reagents are then meter delivered and passed through the microreactor consisting of a 100 μm channel made of quartz.We wish to report an improved synthesis of [ 18 F] FBAM utilizing this microfluidic system involving continuous flow.

Chemistry
The reported procedure for preparing requisite precursor 16 required synthesizing  Attempts to directly N-akylate maleimide 8 with various alkyl bromides failed or produced very poor yields (Scheme 2).We then treated maleimide 8 with either NaH or K 2 CO 3 in THF or DMF at room temperature followed by the addition of 1,6-dibromohexane, 9; after, stirring the reaction mixture at reflux only trace amounts of the desired product 11 were produced.Adding sodium iodide to catalyze the reaction did not improve the yield.Further experiments using the bromide 10 as an alkylating agent also did not result in the desired product 7.It is possible that the N-Alkylation of maleimide does not proceed as expected because the maleimide anion underwent a 1,4-addition to another molecule of maleimide instead of reacting with alkyl bromide.
Precursor 16 was successfully prepared from 6-bromohexanol, 12, in six steps in an overall yield of 24.6% (Scheme 3).tert-Butyl-N-hydroxycarbamate, 13, was then O-alkylated with 6-bromohexanol, 12, to obtain alcohol 6, using 1,8-diazabicyclo[5.4.0] undecene (DBU) as the base in DMF at room temperature.Appel reaction [11] of alcohol 6 to bromide 10 was achieved with carbon tetrabromide and triphenylphosphine in CH 2 Cl 2 by stirring the reaction mixture at room temperature.Bromide 10 was allowed to react with sodium azide in DMF at 80˚C to obtain azide 14 in nearly quantitative yield.The crude azide 14 was then hydrogenated with 5% Pd-C in EtOAc to obtain amine 15 [12].Reaction of amine 15 with N-methoxycarbonyl-maleimide in thepresence of NaHCO 3 bin refluxing in dioxane afforded 7. The Boc-protecting group was readily removed with HCl/EtOAC at room temperature to obtain 16 as the HCl salt.The condensation of 16 with 4-fluorobenzaldehyde in DMF at room temperature gave the 17 as a white solid.The two step radiosynthesis of [ 18 F]FBAM, 1, was performed in the Advion NanoTek Microfludic Synthesizer (Scheme 4).Using the drying macros of NanoTek LF 1.4 software, a complex of Kryptofix 222/K 2 CO 3 /[ 18 F]fluoride was thoroughly dried and allowed to react with 4-N,N,N-trimethylamino-benzaldehyde triflate, 18, in a microreactor (2 m × 100 μ) at 120˚C to obtain 4-[ 18 F]fluorobenz-aldehyde, 19.The labeling efficiency under microfluidic conditions was compared with the previously reported procedures (Table 1).The outlet tube from the reactor was immersed in a reaction vial containing precursor 16 (8 mg) dissolved in a 1:1 mixture of 1 N HCl:MeOH which was then heated at 75˚C for 10 min to obtain [ 18 F]FBAM, 1, in higher overall radiochemical yield (38% ± 4%) when compared to the earlier reports (29% ± 4%).The crude product 1 was subjected to C 18 Sep-Pak solid phase extraction before purifying on semi preparative HPLC.[ 18 F]FBAM (29 mCi) was obtained from 100 mCi of [ 18 F] fluoride in 55 min, including HPLC purification, in a radiochemical purity of ≥98%.The identity of the product was confirmed using analytical HPLC by co-elution with 17.

Materials and Methods
All reagents and solvents were purchased from Acros or Aldrich and were used as received.Flash column chromatography was performed using silica gel (60 Å, 230 -400 mesh, Sorbent Technologies, USA) [13].Analytical thin-layer chromatography (TLC) was performed using 250 μm silica plates (Analtech, Inc., Newark, DE) with a visualization by UV (254 nm) or phosphomolebdic acid spray. 1 H and 13 C-nuclear magnetic resonance spectra (NMR) were recorded at 300 or 125 MHz, respectively.Chemical shifts for 1 H-NMR and 13 C-NMR spectra were referenced to the residual protons of the deuterated solvents or to TMS. High resolution mass spectrometry was performed using a  (7).
After cooling, the mixture was diluted with water (15 mL) and passed through C 18 Sep-Pak cartridge to eliminate water soluble precursor and any unreacted isotope.The product was eluted with acetonitrile (3 mL).The pure product was isolated by semipreparative HPLC column (Phenomenex Luna reverse phase column, 250 × 10 mm, 10 µ), using gradient elution (A: CH 3 CN, B: 0.1 M ammonium formate, 0.5 min 40% A and 60% B; 5 -15 min 40%A -70% A and 15 -30 min 70%A, flow rate 4 mL/ min).A fraction (16 -18 min) was collected, diluted with water (10 mL), and passed through a C 18 Sep-Pak cartridge to trap the desired product.The cartridge was then washed with diethyl ether (2 mL) and the solvent evaporated under a stream of dry N 2 to afford 29 mCi (38% decay corrected) of [ 18 F]FBAM.The identity of the product was confirmed using analytical HPLC by co-injection with a reference standard, 17, R t = 6.4 min.

Conclusion
[ 18 F]FBAM was successfully synthesized by continuous flow chemistry using an Advion NanoTek Microfludic Synthesis System in high radiochemical yield (38% ± 4%, n = 4; previously reported 29% ± 4%) and radiochemcial purity of ≥98%.The requisite key precursor N-(6-amino-oxyhexyl)maleimide .HCl, 16, was prepared by a different method then that previously reported.Smaller quantities of expensive precursors were used for the synthesis under microfluidic conditions.The overall time for the synthesis was 55 min and the specific activity was determined to be 430 mCi/µmol.

Table 1 .
Comparison of amount of precursor 18 used to obtain compound 1.