*3.2. Assessment of the MCT1 Inhibition by [14C]lactate Uptake Assay*

Inhibition of the MCT1-mediated lactate transport was determined using the rat brain endothelial cell line (RBE4, a gift from F. Roux research group [40]) as previously described [26]. PCR analysis and Western Blot demonstrated that only the MCT1 isoform is expressed by RBE4 cells.

#### *3.3. Radiochemistry*

#### 3.3.1. General Methods

No-carrier-added [18F]fluoride was produced via the [18O(p,n)18F] nuclear reaction by irradiation of an [18O]H2O target (Hyox 18 enriched water, Rotem Industries Ltd., Arava, Israel) on a Cyclone 18/9 (IBA RadioPharma Solutions, Lourain-La-Neuve, Belgium) with fixed energy proton beam using Nirta [18F]fluoride XL target. Radio thin layer chromatography (radio-TLC) was performed on silica gel (ALUGRAM SIL G/UV254, Machery-Nagel, Düren, Germany) pre-coated plates with a mixture of dichloromethane (DCM)/MeOH (4:1) as eluent. The plates were exposed to storage phosphor screens (BAS IP MS 2025 E, GE Healthcare Europe GmbH, Freiburg, Germany) and recorded using the Amersham Typhoon RGB Biomolecular Imager (GE Healthcare Life Sciences). Images were quantified with the ImageQuant TL8.1 software (GE Healthcare Life Sciences).

Analytical chromatographic separations were performed on a JASCO LC-2000 system, incorporating a PU-2080Plus pump, AS-2055Plus auto injector (100 μL sample loop), and a UV-2070Plus detector coupled with a gamma radioactivity HPLC flow detector (Gabi Star, raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). Data analysis was performed with the Galaxy chromatography software (version 1.10.0.5590, Agilent Technologies Deutschland GmbH, Waldbronn, Germany) using the chromatograms obtained at 254 and 370 nm. Reprosil-Pur C18-AQ column (250 × 4.6 mm; 5 μm; Dr. Maisch HPLC GmbH; Ammerbuch-Entingen, Germany) with an eluent mixture of ACN/20 mM NH4OAc (aq., pH 6.8) and a flow of 1.0 mL/min were used (gradient: eluent A 10% ACN/20 mM NH4OAc (aq.); eluent B 90% ACN/20 mM NH4OAc (aq.); 0–5 min 100% A, 5–20 min up to 62% B, 20–21 min up to 100% B, 21–26 min 100% B, 26–27 min up to 100% A, 27–35 min 100% A; isocratic: 30% MeOH/20 mM NH4OAc (aq.), flow: 0.75 mL/min, UV detection at 370 nm).

Semi-preparative HPLC separation was performed by using the HPLC system implemented in the TRACERlab FX2 N synthesizer (GE Healthcare). A Reprosil-Pur 120 CN column (250 × 20 mm, 10 μm, Dr. Maisch HPLC GmbH, Germany) was used with 45% MeOH/20 mM NH4OAc (aq.) as eluent mixture and a flow rate of 7.0 mL/min. The ammonium acetate concentration stated as aq. 20 mM NH4OAc, corresponds to the concentration in the aqueous component of an eluent mixture.

The molar activities were determined based on a calibration curve carried out under isocratic HPLC conditions (Reprosil-Pur C18-AQ, 250 × 4.6 mm, 30% MeOH/20 mM NH4OAc (aq.), flow: 0.75 mL/min) using chromatograms obtained at 370 nm as an appropriate maximum of UV absorbance.
