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

OBJECTIVE

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

METHODS

Benzyl-14-(R,S)-tosyloxy-6-thiaheptadecanoate and methyl 16-bromo-4-thia-palmitate were used as precursors for the synthesis of [F]FTHA and [F]FTP, respectively. For comparison, a fatty acid analog lacking a thia-substitution, 16-[F]fluoro-palmitic acid ([F]FP), was synthesized from the precursor methyl 16-bromo-palmitate. A standard nucleophilic reaction using cryptand (Kryptofix/K222, 8.1 mg), potassium carbonate (KCO, 4.0 mg) and F-fluoride were employed for the F-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 [F]fluorothia fatty acids and [F]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.

RESULTS

Radiosyntheses of [F]FTHA, [F]FTP and [F]FP were achieved in moderate (8-20% uncorrected) yields. However, it was observed that the HPLC-purified [F]fluorothia fatty acids, [F]FTHA and [F]FTP at higher radioactivity concentrations (>1.11 GBq/mL, 30 mCi/mL) underwent formation of F-labeled side products over time but [F]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 [F]FTHA and [F]FTP synthesis but showed only limited to no effect. Analysis of the side products by LCMS showed formation of sulfoxides of both [F]FTHA and [F]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).

CONCLUSIONS

Successful automated synthesis of [F]fluorothia fatty acids were carried out in cGMP facility for their routine production and clinical applications. Instability of [F]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.