3.4.3. Analysis of Radiometabolites of [18F]**1** in Mouse

The radiotracer [18F]**1** (32.84 MBq in 200 μL isotonic saline) was injected in an awake female CD-1 mouse (12 weeks old, 31 g) via the tail vein. After 30 min, the animal was slightly anesthetized with isoflurane, and blood was collected from retro-orbital bleeding followed by cervical dislocation. Blood plasma was separated by centrifugation of the blood sample at 10,000 rpm for 2 min. Brain was also isolated, cleaned by pouring PBS and homogenized in demineralized water (~2 mL/g tissue) in a borosilicate glass with a PTFE plunger by 10 strokes at 1000 rpm (POTTER S, Homogenizer, B. Braun Biotech, Sartorius AG, Göttingen, Germany). Blood plasma and brain homogenate were provided for preparation of samples for radio-HPLC analyses as described in the following.

RP-HPLC: Protein precipitation was performed by addition of an ice-cold ACN/H2O mixture (9:1) to the plasma and brain samples in a ratio of 4:1 (*v*/*v*: solvent/tissue sample, n = 2). The samples were vortexed for 2 min, incubated on ice for 3 min, and the suspensions were centrifuged at 10,000 rpm at 4 ◦C for 5 min. For the second extraction, the precipitates were re-dissolved in 100 μL of the solvent mixture, vortexed for 3 min, incubated on ice for 5 min and subjected to the same centrifuging procedure. The combined supernatants (total volume between 1–2 mL) were concentrated at 70 ◦C under argon flow to a final volume of approximately 100 μL and were analyzed by analytical radio-HPLC. A Reprosil-Pur C18-AQ column (250 mm × 4.6 mm; 5 μm) was used as stationary phase and elution was done using the gradient mode as described in Section 3.3.1. To determine the percentage of radioactivity in the supernatants compared to total activity, aliquots of each step as well as the precipitates were quantified by gamma counting.

MLC: Preparation of the plasma and brain samples was performed as already described [47,48]. A Reprosil-Pur C18-AQ column (250 × 4.6 mm, particle size: 10 μm) coupled with a pre-column of 10 mm length was used. Separations were performed by using an isocratic mode with an aqueous eluent containing 50 mM sodium dodecyl sulfate and 10 mM Na2HPO4 at a flow rate of 1.0 mL/min.

#### 3.4.4. Small Animal Micro-PET/MR Studies

The dynamic biodistribution of the radiotracers was assessed by small animal PET (nanoscan, Mediso, Hungary) over 60 min PET with a subsequently T1 weighted MR. Anaesthetized (2% isofluran, carrier gas mixture of 40% air and 60% O2) female CD-1 mice (bodyweight = 30.3 ± 1.1 g) were kept during imaging on a heated animal bed to sustain body temperature and were pretreated with vehicle (0.9% saline) and <sup>α</sup>-CHC-Na (25 mg/kg bodyweight) prior to tracer application ([18F]**1**: 5.8 <sup>±</sup> 0.2 MBq, 1.1 fmol/g bodyweight and [18F]FACH: 5.9 <sup>±</sup> 0.5 MBq, 3.7 <sup>±</sup> 1.8 fmol/g), whereby all injections were administered intravenously. The acquisitions were performed in normal mode and a coincidence Mode 1–5. For subsequent dynamic reconstructions, list mode data were sorted into sinograms (12 × 10 s, 6 × 30 s, 5 × 60 s, and 10 × 300 s). The frames were reconstructed by Ordered Subset Expectation Maximization applied to 3D sinograms (OSEM3D) with an attenuation correction with 4 iterations, 6 subsets and a voxel size of 0.4 mm<sup>3</sup> (Nucline v2.01, Mediso, Hungary). Analyses of reconstructed studies were performed with PMOD software (v4.005, PMOD Technologies LLC, Zurich, Switzerland) and results are expressed in Standardized Uptake Value (SUV).

#### **4. Conclusions**

In summary, two new analogs of FACH, **1** and **2**, were synthesized and the former, with moderate MCT1 inhibition, was regarded to be a good PET candidate and therefore chosen for labeling with fluorine-18. Although the partition coefficient log D7.4 of [18F]**1** was 2-fold higher than the one of [18F]FACH, the brain accumulation of both radiotracers was in a similar moderate range. This demonstrates that log D7.4 alone does not govern passive diffusion into the brain, which is also reflected by comparing log D7.4 with calculated log *K*BB (brain-blood partition coefficient). Nevertheless, the high uptake of [18F]**1** in kidney and other peripheral MCT-expressing organs together with the strong inhibition by specific drugs provide evidence that this radiotracer is suitable for future investigation of MCT imaging with PET. Moreover, these results suggest that further structural modifications towards improving MCT-mediated transport might result in higher brain uptake in vivo.
