Radiation induced oxidation of [¹⁸F]fluorothia fatty acids under cGMP manufacturing conditions

The objectives of the present work were to optimize and validate the synthesis and stability of 14(R,S)-[¹⁸F]fluoro-6-thia-heptadecanoic acid ([¹⁸F]FTHA) and 16-[¹⁸F]fluoro-4-thia-palmitic acid ([¹⁸F]FTP) under cGMP conditions for clinical applications.

Benzyl-14-(R,S)-tosyloxy-6-thiaheptadecanoate and methyl 16-bromo-4-thia-palmitate were used as precursors for the synthesis of [¹⁸F]FTHA and [¹⁸F]FTP, respectively. For comparison, a fatty acid analog lacking a thia-substitution, 16-[¹⁸F]fluoro-palmitic acid ([¹⁸F]FP), was synthesized from the precursor methyl 16-bromo-palmitate. A standard nucleophilic reaction using cryptand (Kryptofix/K222, 8.1 mg), potassium carbonate (K₂CO₃, 4.0 mg) and ¹⁸F-fluoride were employed for the ¹⁸F-labeling and potassium hydroxide (0.8 M) was used for the post-labeling ester hydrolysis. The final products were purified via reverse phase semi-preparative HPLC and concentrated via trap and release on a C-18 plus solid phase extraction cartridge. The radiochemical purities of the [¹⁸F]fluorothia fatty acids and [¹⁸F]FP were examined over a period of 4 h post-synthesis using an analytical HPLC. All the syntheses were optimized in an automated TRACERlab FX-N Pro synthesizer. Liquid chromatography mass spectrometry (LCMS) and high resolution mass spectrometry (HRMS) was employed to study the identity and nature of side products formed during radiosynthesis and as a consequence of post-synthesis radiation induced oxidation.

Radiosyntheses of [¹⁸F]FTHA, [¹⁸F]FTP and [¹⁸F]FP were achieved in moderate (8-20% uncorrected) yields. However, it was observed that the HPLC-purified [¹⁸F]fluorothia fatty acids, [¹⁸F]FTHA and [¹⁸F]FTP at higher radioactivity concentrations (>1.11 GBq/mL, 30 mCi/mL) underwent formation of ¹⁸F-labeled side products over time but [¹⁸F]FP (lacking a sulfur heteroatom) remained stable up to 4 h post-synthesis. Various radiation protectors like ethanol and ascorbic acid were examined to minimize the formation of side products formed during [¹⁸F]FTHA and [¹⁸F]FTP synthesis but showed only limited to no effect. Analysis of the side products by LCMS showed formation of sulfoxides of both [¹⁸F]FTHA and [¹⁸F]FTP. The identity of the sulfoxide side product was further confirmed by synthesizing a non-radioactive reference standard of the sulfoxide analog of FTP and matching retention times on HPLC and molecular ion peaks on LC/HRMS. Radiation-induced oxidation of the sulfur heteroatom was mitigated by dilution of product with isotonic saline to reduce the radioactivity concentration to <0.518 GBq/mL (14 mCi/mL).

Successful automated synthesis of [¹⁸F]fluorothia fatty acids were carried out in cGMP facility for their routine production and clinical applications. Instability of [¹⁸F]fluorothia fatty acids were observed at radioactivity concentrations exceeding 1.11 GBq/mL (30 mCi/mL) but mitigated through dilution of the product to <0.518 GBq/mL (14 mCi/mL). The identities of the side products formed were established as the sulfoxides of the respective thia fatty acids caused by radiation-induced oxidation of the sulfur heteroatom.