A New Approach To Produce [ 18 F ] MC 225 Via One-Step Synthesis , A PET Radiotracer For Measuring Pgp Function

Lara Garcia Varela University Medical Centre Groningen: Universitair Medisch Centrum Groningen https://orcid.org/00000001-9803-4708 Khaled Attia Symeres, Kadijk 3,9747 AT Groningen John Carlo Sembrano Symeres, Kadijk 3, 9747 AT Groningen Olivier Jacquet Symeres, Kadijk 3, 9747 AT Groningen Inês farinha antunes University Medical Centre Groningen: Universitair Medisch Centrum Groningen Chantal Kwizera University Medical Centre Groningen: Universitair Medisch Centrum Groningen Ton J. Visser Symeres, Kadijk 3, 9747, AT Groningen Rudi A.J.O. Dierckx University Medical Centre Groningen: Universitair Medisch Centrum Groningen Philip H. Elsinga University Medical Centre Groningen: Universitair Medisch Centrum Groningen Gert Luurtsema (  g.luurtsema@umcg.nl ) University Medical Centre Groningen: Universitair Medisch Centrum Groningen

time in Synthera was 106 min and the product was obtained with a radiochemical purity higher than 95% and RCY of 6.5%, while the production in E&Z lasted 120 min and the product had a lower radiochemical purity (91%) and RCY (3.8%).
Conclusions: [ 18 F]MC225 was successfully produced via a 1-step reaction. The procedure is suitable for automation using commercially available synthesis modules. The automation of the radiosynthesis in the Synthera module allows the production of the [ 18 F]MC225 by a reliable and simple method.
Background [ 18 F]MC225 is a radiotracer for imaging P-glycoprotein (P-gp) function at the blood-brain barrier (BBB) 1,2 . P-gp is an e ux transporter located in the luminal side of the cerebral endothelial cells which constitute the main component of the BBB 3,4 . P-gp belongs to the ATP-Binding Cassette transporter family, their function is ATP-dependent and apart from the BBB, these transporters are also located in several tissues involved in absorption and excretion functions such as the intestine, testes, placenta, kidneys, and liver 5 .
In the BBB, the main function of P-gp is to transport a wide variety of substances out of the brain to the blood. Therefore, this transporter contributes to limit the permeability of the BBB and protects the Central Nervous System (CNS) from neurotoxic compounds 5 .
However, the P-gp function can be altered due to different factors. Many unrelated xenobiotic compounds can increase the P-gp expression and function which leads to reduced concentration of drugs in the desired target causing decreases in drug e cacy 6-10 . Moreover, dysfunctions in P-gp function have been observed in various disease conditions 5 . For instance, patients with intractable epilepsy, have shown increased P-gp expression and function in isolated brain capillaries 11,12 . Also, a decreased P-gp function has been reported in Alzheimer's and Parkinson's disease 13 . Therefore, the assessment of P-gp function using Positron Emission Tomography (PET) imaging can help to improve the diagnosis of certain CNS diseases where the P-gp function is altered. In addition, this technique can also evaluate treatments that affect P-gp function 14 .
[ 18 F]MC225 has been developed as a weak substrate of the P-gp transporter 1 . Thus, this radiotracer showed a higher brain uptake at baseline conditions 2 , which allows us to measure both increased and decreased P-gp function at the BBB in rats 2 . Previous evaluation of the radiotracer in vivo has shown good pharmacokinetic properties, an adequate signal-to-noise ratio, high sensitivity toward the target, and low levels of radio-metabolites inside the brain. All these characteristics make [ 18 F]MC225 a suitable radiotracer 2,15 and worthwhile to perform clinical studies to evaluate the P-gp function in the human brain. this relatively time-consuming and complex procedure results in low radiochemical yields 1 . Thus, it may be convenient to simplify the radiotracer synthesis via 1-step procedure to make the reaction simpler, and thus more robust.
Therefore, this study aims to produce [ 18 F]MC225 via a 1-step reaction using a new MC225 precursor compound which allows the production of the radiotracer by direct 18 F-uorination. Moreover, the study discusses the most appropriate conditions for the automation of the synthesis.

Methods
Synthesis of phenol precursor ( Figure 2) 5-[3-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydro-naphthalen-1-ol, called the phenol precursor was synthesized as previously described 16 but with some modi cations on the synthesis route ( Figure 2). 5-Hydroxy-1-tetralone is commercially available (Combi Blocks) and was protected with a benzyl group as follow: 5-Hydroxy tetralone (1, 5.0 g, 30.9 mmol, 1.0 eq.) and potassium carbonate (8.5 g, 61.7 mmol, 2.0 eq.) dissolved in ACN were stirred for 15 minutes at room temperature under N 2 -atmosphere. Benzyl bromide (4.04 mL, 33.9 mmol, 1.1 eq.) was added and the resulting beige suspension was stirred overnight at room temperature under N 2 -atmosphere. The reaction mixture was concentrated in vacuum and the crude material was partitioned between water (50 mL) and DCM (50 mL). The layers were separated, and the organic layer was successively washed with water (50 mL), a saturated aqueous solution of NaHCO 3 (50 mL), and brine (50 mL). The organic layer was collected, dried with anhydrous Na 2 SO 4 , ltered, and concentrated in vacuum to give a beige oil which slowly solidi ed on standing. The solids were triturated with heptane (30 mL) to give 5-(benzyloxy)-3,4-dihydronaphthalen-1(2H)-one (2) as a crystalline white solid (6.5 g, 83% of yield). 1  For the next step, the benzylated product (compound 2) was reacted with freshly prepared Grignard reagent. Magnesium turnings (0.96 g, 39.7 mmol, 2.0 eq.), anhydrous THF (30 mL), and iodine (1 grain, catalytic) were combined in a ame-dried three-necked ask under N 2 -atmosphere. Cyclopropyl bromide (2.4 mL, 39.7 mmol, 2.0 eq.) was added portion-wise (total addition time = 15 minutes). After the complete addition of the bromide, the mixture was slowly heated to 55°C. The color of the mixture turned from brown to colorless accompanied by slight effervescence. The resulting mixture was stirred at 55°C for 2h at room temperature under N 2 -atmosphere. At this point, the color of the reaction turned yellowish and the magnesium turnings were completely consumed. The Grignard reagent (CypPrMgBr) was cooled to 0°C by using an ice bath and compound 2 (5.0 g, 19.8 mmol, 1.0 eq.) dissolved in anhydrous THF (30 mL) was added. The resulting mixture was stirred at re ux overnight under N 2 -atmosphere. The mixture was then quenched with a saturated solution of NH 4 Cl (100 mL). Diethyl ether (100 mL) was added, and the mixture was stirred for 15 minutes at room temperature. The layers were separated, and the aqueous layer was extracted with diethyl ether (1× 25 mL). The organic layers were combined, washed with brine (50 mL), collected, dried with anhydrous Na 2 SO 4 , ltered, and concentrated in vacuum to give a yellow oil (6.4 g). The crude was used as such in the next step without puri cation.
After the Grignard reaction, the cyclopropyl ring was opened by using hydrochloric acid in acetic acid. Thus, the crude material was dissolved in glacial acetic acid (100 mL) and a 20% aqueous solution of HCl (100 mL) was added. The resulting solution was stirred for 2h at room temperature. The acidic mixture was then concentrated in vacuum and the crude was dissolved in DCM (100 mL). The solution was washed with a saturated solution of NaHCO 3 (3× 100 mL), water (100 mL), and brine (25 mL). The organic layer was collected, dried with anhydrous Na 2 SO 4 , ltered, and concentrated in vacuum to give 8- (3) as a brown oil (5.4 g, 87% of yield). No puri cation was performed regarding the presence of isomers and thus, the isolated material was used as such in the next step.
Formulation of the product Formulation of the product was done as previously described 1 . Brie y, the desired product eluted at 12 min from the Prep-HPLC was collected in an 80 ml sterile H 2 O bottle. The mixture was mixed with helium and the mixture was passed through an Oasis HLB 1 cm 3 (30 mg) extraction cartridge where the product was trapped. The cartridge was washed twice with 8 ml sterile H 2 O and the product was eluted with 1 ml of ethanol. Next, 4 ml of 0.9% NaCl 2 was passed through the cartridge to formulate the nal product. The solution was ltered through a Millipore Millex LG Filter (0.2 µm) before collection in a sterile vial.

Quality control methods
Quality control was executed with a Waters Acquity H-class UPLC system (Milford, CT, USA) as previously described 2 . The system used a Berthold FlowXStar LB 513 as a radioactivity detector (Bad Wildbad, Germany) and a Waters Acquity UPLC BEH Shield RP18 1.7µm (3.0mmx50mm) column. The product eluted after 3.5 minutes using ACN / 10Mm NH 4 CO 3 (pH=9.5) (50/50) at a ow rate of 0.8 ml/min. The UV detection was set to 215 nm. The reference compound (non-labelled MC225) was used to prepare a calibration curve to know the amount of non-labelled compound and thus the molar activity (A m ) of the nal product at the end of the radiosynthesis.

Results And Discussion
This study aimed to develop a new synthesis method to produce the PET radiotracer [ 18 F]MC225 via a 1step synthesis. To this purpose, a mesylate precursor has been developed to facilitate direct 18 Fuorination yielding [ 18 F]MC225.
This mesylate precursor (8) was synthesized from the phenol precursor (5) which was previously used in the 2-step synthesis of [ 18 F]MC225 1 . Phenol precursor (5) was produced via an alternative synthesis which uses a benzyl-protected tetralone (2). The benzyl protecting group was chosen to reduce both the double bonds of both isomers (4) and to cleave the benzyl protecting group in one step. Palladium on carbon (Pd/C) was used as a catalyst and the hydrogenation was performed at atmospheric pressure. Full reduction of the double bonds was observed after stirring the mixture for 2 h at room temperature.
The reaction was run for 48h to remove the benzyl group. Moreover, the benzylated species were more stable than the unprotected phenol. At the end of the synthesis, the phenol precursor (5) was isolated and puri ed via column chromatography and obtained in a 25% yield (calculated from intermediate 2, Figure  2).
The development of the mesylate precursor (8) was challenging. Firstly, the deprotection of the benzyl group of compound 6 was facing di culties. Compound 6 seems to be unstable on silica thus reaction needs to achieve quantitative conversion to continue with the next step without further puri cation. The reaction was slow and different batches of Pd/C and Pd/C hydroxide were added to speed up the reaction and to obtain a maximum conversion. The reaction took more than one week to reach full conversion. Moreover, the reaction mixture needed to be slightly acidic to be able to reach completion, thus acetic acid was added. In the following step, compound 7 was used without further puri cation, and therefore, it was dissolved in dioxane and reacted with methanesulfonic anhydride at re ux. However, the mesylate precursor was not stable. Especially basic workup of the reaction mixture resulted in the elimination of the mesylate. Several strategies were explored to stabilize compound 8. Eventually, it was observed that the mesylate salt formed during reaction with methanesulfonic anhydride was reasonably stable and helped to stabilize the precursor for a longer time. However, the nal mesylate precursor showed several impurities (purity 75%). Therefore, several attempts were made to improve purity e.g., via RP-automated column chromatography, prep HPLC and precipitation. All these attempts were unsuccessful leading to the deterioration of the product on the column or the isolation of impure product in very low yield after precipitation. Thus, the crude reaction mixture was evaporated and used as such for the labeling experiments.
The radiolabeling was performed by adding the mesylate precursor to the dried [ 18 F]-KF/kryto x 222 complex and heating at 140 ˚C for 30 minutes. After the radiochemistry reaction, the crude mixture was puri ed using semi-preparative HPLC (as described in the methods) (Figure 7).
The automated synthesis was performed using two modules: IBA Synthera Synthesis Module and the Modular Lab PharmTracer (Eckert & Ziegler). The best results were obtained with IBA Synthera module. The nal product produced by both modules was analyzed by UPLC. Table 1 shows the results of both synthesis modules. The total synthesis time was 20 min shorter in the Synthera module than with E&Z module, mainly because of the faster drying process of the 18 F-KF/Krypto x 222 complex. The radiochemical purity of [ 18 F]MC225 and the radiochemical yield (RCY) corrected for decay was higher in the IBA module. However, a high A m of the nal product was obtained with both modules. Regarding the preparation for the synthesis, the set-up of the synthesis was easier and quicker in the IBA Synthera than in the E&Z module. Although the Modular Lab PharmTracer from E&Z allows the con guration of multistep and complicated synthesis by the combination of various disposable components, the amount of tubing and valves make the preparation more laborious. In the case of Synthera, the cartridge provides the connections and only the reagents must be placed in the right position during the preparation, thus the time spent in the preparation is shorter. For this reason, Synthera may be a preferable module to perform this synthesis. The nal product collected from the semi-preparative HPLC was injected onto the UPLC system to perform quality control. Figure 8 shows an example of UPLC chromatography that shows the desired product appearing at 3.5 min after the injection.

Conclusion
Overall, the production of [ 18 F]MC225 by the 1-step synthesis using the mesylate precursor and the IBA Synthera module seems to be the most successful automated method. The highest radiochemical yield and the adequate purity and A m of the nal product will enable the use of this procedure for GMP productions.
This study provides an alternative method for the production of [ 18 F]MC225 using a 1-step approach. However, to obtain a GMP compliant synthesis a suitable puri cation of the mesylate precursor is still warranted. Nevertheless, an automated synthesis using the mesylate precursor and the IBA Synthera module produced [ 18 F]MC225 in a reliable and simple manner. Quality control performed with UPLC The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
LGV performed the automated radiosynthesis, analyzed the data, and wrote the manuscript. KA performed the synthesis of the phenol and mesylate precursor, contributed to the automation of the radiosynthesis, the interpretation of the data, and the preparation of the manuscript. JCS and OJ helped in the synthesis of the phenol and mesylate precursor and commented on the nal manuscript, IFA commented on the nal manuscript, CK helped in the automation of the radiosynthesis and the UPLC analysis, TJV supervised the phenol and mesylate precursor synthesis, helped in the interpretation of the data and commented on the nal manuscript, RAJOD commented on the nal manuscript and PHE and GL supervised and designed the study, contributed to the interpretation of the data and in the preparation of the manuscript.  Alternative route for synthesizing the phenol precursor. Synthesis of the MC225 mesylate precursor as its mesylate salt.