Synthesis and Preclinical Evaluation of 22-[18F]Fluorodocosahexaenoic Acid as a Positron Emission Tomography Probe for Monitoring Brain Docosahexaenoic Acid Uptake Kinetics

Docosahexaenoic acid [22:6(n-3), DHA], a polyunsaturated fatty acid, has an important role in regulating neuronal functions and in normal brain development. Dysregulated brain DHA uptake and metabolism are found in individuals carrying the APOE4 allele, which increases the genetic risk for Alzheimer’s disease (AD), and are implicated in the progression of several neurodegenerative disorders. However, there are limited tools to assess brain DHA kinetics in vivo that can be translated to humans. Here, we report the synthesis of an ω-radiofluorinated PET probe of DHA, 22-[18F]fluorodocosahexaenoic acid (22-[18F]FDHA), for imaging the uptake of DHA into the brain. Using the nonradiolabeled 22-FDHA, we confirmed that fluorination of DHA at the ω-position does not significantly alter the anti-inflammatory effect of DHA in microglial cells. Through dynamic PET-MR studies using mice, we observed the accumulation of 22-[18F]FDHA in the brain over time and estimated DHA’s incorporation coefficient (K*) using an image-derived input function. Finally, DHA brain K* was validated using intravenous administration of 15 mg/kg arecoline, a natural product known to increase the DHA K* in rodents. 22-[18F]FDHA is a promising PET probe that can reveal altered lipid metabolism in APOE4 carriers, AD, and other neurologic disorders. This new probe, once translated into humans, would enable noninvasive and longitudinal studies of brain DHA dynamics by guiding both pharmacological and nonpharmacological interventions for neurodegenerative diseases.


General Chemistry
All reagents and solvents were obtained from various commercial sources and used without further purification unless otherwise noted.All reactions were performed under nitrogen gas unless otherwise mentioned.Thin-layer chromatography (TLC) was performed on precoated silica gel 60 Å plates with UV 254 indicator and visualized with either UV light or a KMnO4 stain.Automated flash chromatography was performed using a Teledyne ISCO CombiFlash Rf 200 automatic chromatography system with RediSep prepacked gel cartridges.Nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Oxford NMR AS400, a 400 MHz Varian Mercury, or a 500 MHz Varian VNMRS NMR spectrometer.Proton and 13 C NMR chemical shifts are reported in ppm (δ) downfield from TMS using internal TMS or residual protonated solvent as reference (CHCl3: 7.26 ppm for 1 H and 77.16 ppm for 13 C).Fluorine-19 NMR chemical shifts are reported in ppm downfield from CFCl3 using the deuterium lock channel as a reference.NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dt = triplet of doublets, dq = quartet of doublets, tt = triplet of triplets, m = multiplet), coupling constant (J in Hz), and integration.Low-resolution mass spectra were obtained on an MSQ Plus single quadrupole mass spectrometer in positive mode with an electrospray ionization (ESI) ion source.High-resolution mass spectra were obtained on a Thermo Fisher Q-Exactive Orbitrap system in positive mode with an ESI ion source.
Radioactivity was recorded using a Model 105 radio-detector (Carroll & Ramsey Associates, Berkeley, CA).The mobile phases were A, H2O with 0.1% TFA and B, MeCN with 0.1% TFA.
Stirring was continued overnight, then the mixture was diluted with ethyl acetate (EtOAc, 40 mL).
To this was then added a mixture of saturated aq.NH4Cl (40 mL) and saturated aq.Na2S2O3 (10 mL).The biphasic mixture was separated, and the aqueous layer was then extracted with EtOAc (2 × 25 mL) and the combined organic layers were washed with water (20 mL), then saturated aq.
A procedure from the literature 1 was modified as follows: to a flask charged with 9 (130.5 mg, 0.38 mmol) was added a mixture of methanol (4 mL), EtOAc (4 mL), and 2-methyl-2-butene (8 mL).The solution was immediately purged with N2 and to it was then added Lindlar's catalyst (130.2 mg) and pyridine (2 mL).H2 gas was bubbled through the suspension for 1 min, which was then kept stirring under a constant positive-pressure atmosphere of H2 (balloon pressure, with a 26G needle through a rubber septum to vent) while monitoring the progress of the hydrogenation by TLC (1:1 EtOAc/hexanes).After 2 h, the mixture was filtered, and the filtrate was then partitioned between EtOAc (30 mL) and 1 M aq.HCl (25 mL).The aqueous layer was further extracted with EtOAc (3 ×15 mL), and the pooled organic solutions were washed with water (20 mL) and then brine (15 mL).The organic phase was then dried over anhyd.Na2SO4 and evaporated under reduced pressure yielding a brown oil.The residue was loaded onto silica gel and after flash chromatography (4 g silica, 0-25% EtOAc in hexanes), the hydrogenated product was obtained as a light-yellow oil (79.2 mg, 59% crude yield).Analytical HPLC of the isolated products showed that it was a mixture of the intended hexaene product 10 (ca.70% by UV at 250 nm) with overreduced and under-reduced derivatives; the mixture was used in the next step without further purification.In smaller-scale reactions, the crude product may be isolated from strongly colored impurities by taking the residue up in 1:4 EtOAc/hexanes, passing the solution through a short silica plug, and eluting with 1:4 EtOAc/hexanes.
A procedure was adapted from the literature 2 as follows: to a solution of the partially purified 10 (17.5 mg) in anhyd.DCM (3 mL), cooled in an ice-water bath, was added triethylamine (14 µL) and 4-(dimethylamino)pyridine (0.8 mg).Then, a solution of tosyl chloride (34.7 mg) in DCM (0.5 mL) was added to the mixture.After slowly warming to room temperature and then continuing stirring for 2 days, the solution was diluted with DCM (10 mL).The solution was then washed with cold water (10 mL), then with brine (5 mL).The organic solution was then dried over anhyd.