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 modifications 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 N2-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 N2-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 NaHCO3 (50 mL), and brine (50 mL). The organic layer was collected, dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give a beige oil which slowly solidified 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). 1H-NMR (300 MHz, CDCl3) δ 7.68 (dd, J = 7.9, 1.1 Hz, 1H), 7.54 – 7.18 (m, 7H), 7.08 (dd, J = 8.1, 1.2 Hz, 1H), 5.11 (s, 2H), 2.98 (t, J = 6.2 Hz, 2H), 2.64 (dd, J = 7.5, 5.7 Hz, 2H), 2.27 – 1.97 (m, 2H).
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 flame-dried three-necked flask under N2-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 N2-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 reflux overnight under N2-atmosphere. The mixture was then quenched with a saturated solution of NH4Cl (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 Na2SO4, filtered, and concentrated in vacuum to give a yellow oil (6.4 g). The crude was used as such in the next step without purification.
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 NaHCO3 (3× 100 mL), water (100 mL), and brine (25 mL). The organic layer was collected, dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give 8-(benzyloxy)-4-(3-chloropropyl)-1,2-dihydronaphthalene and (E)-5-(benzyloxy)-1-(3-chloropropylidene)-1,2,3,4-tetrahydronaphthalene (3) as a brown oil (5.4 g, 87% of yield). No purification was performed regarding the presence of isomers and thus, the isolated material was used as such in the next step.
The crude mixture containing compound 3 (5.4 g, 19.8 mmol, 1.0 eq.) was dissolved in DMF (100 mL). Anhydrous Na2CO3 (6.3 g, 59.4 mmol, 3.0 eq.) was added along with commercially available (Combi Blocks) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (5.75 g, 29.7 mmol, 1.5 eq.). The mixture was stirred overnight at 100°C under N2-atmosphere. The mixture was allowed to cool down to room temperature. The mixture was partitioned between ethyl acetate (250 mL) and water (250 mL). The organic layer was collected, washed with water (3× 100 mL), collected, dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give (Z)-2-(3-(5-(benzyloxy)-3,4-dihydronaphthalen-1-yl)propyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline and (E)-2-(3-(5-(benzyloxy)-3,4-dihydronaphthalen-1(2H)-ylidene)propyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline mixture (4) compounds as a brown oil (8.2 g, quantitative). No purification was performed due to the presence of isomers and thus, the isolated material was used as such in the next step.
Subsequently, a hydrogenation step was carried out to reduce double bonds of both isomers and to cleave the benzyl protecting group in one step. Therefore, the isomeric mixture 4 (8.2 g, 17.5 mmol, 1.0 eq.) was dissolved in a mixture of absolute ethanol (100 mL) and DMF (100 mL). This mixture was stirred under N2-atmosphere for 15 minutes. To this mixture was added, palladium on carbon (Pd/C) (10wt% loading, 1.86 g, 1.75 mmol, 0.1 eq.). The resulting mixture was stirred for 48h at room temperature under H2-atmosphere (atmospheric pressure) to remove the benzyl group. The mixture was filtered over a thin path of Celite and the filtrate was concentrated in vacuum, dissolved in EtOAc (100 mL), washed with water (4× 100 mL), and brine (100 mL). The organic layer was collected, dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give the crude containing the phenol. The crude was purified via silica column chromatography (DCM: MeOH, 95:5). The fractions that contain the pure compound were concentrated in vacuum to afford 5-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5,6,7,8-tetrahydronaphthalen-1-ol (5) compound as an off-white foam (1.82 g, 25% yield calculated from benzylated intermediate (2)). 1H NMR (300 MHz, CDCl3) δ 6.98 (t, J = 7.8 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.62 – 6.49 (m, 3H), 3.84 (d, J = 1.6 Hz, 6H), 3.57 (s, 2H), 2.88 – 2.45 (m, 9H), 1.92 – 1.57 (m, 8H).
Compound 5 (50.0 mg, 1 eq, 131 µmol) was dissolved in DMF (3 mL) and sodium hydride (15.7 mg, 5 eq, 655 µmol) was added to the dissolved compound. The resulting mixture was stirred for 20 min until no further gas evolution was observed. 1-Bromo-2-fluoroethane (33.3 mg, 20 µL, 2 eq, 262 µmol) was added drop-wise to the reaction mixture. The reaction was monitored by LC-MS. After 20 min of stirring at room temperature, the reaction was complete. The reaction mixture was quenched by addition to a mixture of water/ ethylacetate (50 mL/50 mL). The organic layer was separated and the aqueous layer extracted with ethyl acetate (2x100 mL). The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure. The crude material was purified by automated column chromatography yielding non labelled MC225 (35.0 mg, 62.5 %).1H NMR (299 MHz, CDCl3) δ 7.08 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.59 (s, 1H), 6.52 (s, 1H), 4.90 – 4.77 (m, 1H), 4.75 – 4.57 (m, 1H), 4.24 (dd, J = 5.0, 3.4 Hz, 1H), 4.20 – 4.10 (m, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.56 (s, 2H), 2.93 – 2.76 (m, 3H), 2.76 – 2.63 (m, 3H), 2.58 – 2.46 (m, 2H), 1.98 – 1.52 (m, 9H).
Synthesis mesylate precursor (Figure 3)
5-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5,6,7,8-tetrahydronaphthalen-1-ol (5) (150.0 mg, 1 eq, 393.2 µmol) (phenol precursor or 5) was dissolved in DMF (12 mL) and sodium hydride (47.18 mg, 5 eq, 1.97 mmol) was added to the dissolved compound. The resulting mixture was stirred for 2 hours until no gas evolution appeared anymore. ((2-Bromoethoxy)methyl)benzene (169 mg, 0.124 mL, 1.99 eq, 784 µmol) was added drop-wise to the reaction, and the progress of the reaction was monitored by LC-MS. After 20 min of stirring the reaction was completed according to LC-MS. The reaction mixture was quenched by adding to a mixture of water/ ethyl acetate (50 mL/50 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2x100 mL). The combined organic layers were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The compound was analyzed by HPLC-MS. The crude material was purified by automated column chromatography (Biotage Purification System, Uppsala, Sweden) with a gradient of 0% to 50% ethyl acetate in heptane yielding 2-(3-(5-(2-(benzyloxy)ethoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)propyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (6) (165 mg, 84.4%). 1H NMR (300 MHz, CDCl3) δ 7.43 – 7.28 (m, 5H), 7.07 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.67 – 6.61 (m, 1H), 6.59 (s, 1H), 6.52 (s, 1H), 4.65 (s, 2H), 4.14 (dd, J = 5.8, 4.1 Hz, 2H), 3.90 – 3.78 (m, 8H), 3.55 (s, 2H), 2.91 – 2.76 (m, 3H), 2.76 – 2.62 (m, 4H), 2.58 – 2.44 (m, 2H), 1.91 – 1.60 (m, 8H).
A solution of 6 (150.00 mg, 1 eq., 290.87 µmol) in methanol (12 mL) and acetic acid (0.5 mL) was stirred under N2 for 20 min. Pd/C (20.00 mg, 0.5 Eq, 29.09 µmol) was added to the solution and the reaction was stirred overnight at 5 bar H2 pressure. After 3 days of stirring full conversion was observed. The mixture was filtered through a pad of Celite and the organic solvents were removed by rotary evaporation. The residue was dissolved in ethyl acetate and washed with a potassium bicarbonate solution. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure yielding 2-((5-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5,6,7,8-tetrahydronaphthalen-1-yl)oxy)ethan-1-ol (7) (100.20 mg, 81 %). 1H NMR (299 MHz, cdcl3) δ 7.08 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.59 (s, 1H), 6.52 (s, 1H), 4.07 (dd, J = 5.2, 3.7 Hz, 2H), 3.97 (dd, J = 5.2, 3.6 Hz, 2H), 3.84 (d, J = 2.1 Hz, 6H), 3.56 (s, 2H), 2.89 – 2.76 (m, 3H), 2.76 – 2.65 (m, 3H), 2.57 – 2.46 (m, 2H), 1.93 – 1.51 (m, 9H).
Compound 7 (105.00 mg, 1 eq., 246.73 µmol) was used without further purification and was dissolved in 1,4-dioxane (8 mL) and methanesulfonic anhydride (45.13 mg, 1.05 eq., 259.06 µmol) was added to the mixture. The resulting mixture was heated to reflux during one week until the reaction was complete. The reaction mixture was evaporated under reduced pressure, and 6,7-dimethoxy-2-(3-(5-(2-((methylsulfonyl)oxy)ethoxy)-1,2,3,4-tetrahydronaphthalen-1-yl)propyl)-1,2,3,4-tetrahydroisoquinolin-2-ium methanesulfonate (8) MC225 precursor was isolated as its mesylate salt. At this stage, we could not isolate intermediate 8 in pure form (93 mg, 75 % pure,) and decided to use it as such for the radiolabeling.
[18F]Fluoride production
[18F]Fluoride was produced using a cyclotron (IBA Cyclone, Louvain-la-Neuve, Belgium) by the nuclear reaction 18O(p,n)18F.
Automated synthesis (Figure 4)
The synthesis was automated using two different modules the IBA Synthera module (Figure 5) and the Modular Lab PharmTracer from Eckert & Ziegler (Figure 6). Aqueous [18F]fluoride was trapped on Sep-Pak Light Accell Plus QMA anion exchange cartridge (Waters, Milford, USA) previously conditioned with 5 ml of Na2CO3 1.4%, washed with 10 ml of H2O for injection, and then dried with a nitrogen stream. [18F]Fluoride was eluted using a solution of 1.3±0.12 mg K2CO3 and 7.1±0.26 mg Kryptofix 222 in 1 ml of ACN/H2O (8:2). Azeotropic drying of the [18F]KF/Kryptofix222 complex was performed at 110°C under an argon flow and adding 1 ml of ACN. After the evaporation of the solvent, the procedure was repeated twice using 0.5 ml of ACN.
Immediately after the azeotropic drying, 1 mg of mesylate precursor dissolved in 1 ml DMF (or 0.5 ml in the case of E&Z) was added to the reactor vial containing the dried [18F]-KF/krytofix222 complex. The reaction was heated at 140˚C for 30 min and after cooling down the solution 1 ml of H2O was added (1.3 ml in case of E&Z). The crude reaction was injected onto the semi-preparative HPLC and the product was collected after 12 min.
Purification of the crude reaction was done in a semi-preparative HPLC system using a Symmetryshield RP8 5µm 7.8 x 300 mm column. 0.1 M NaOAc/ACN (5.5/4.5) (v/v) (pH = 4.7) were used as eluent at a flow of 3 ml/min. The UV signal was measured at a wavelength of 215 nm.
Formulation of the product
Formulation of the product was done as previously described 1. Briefly, the desired product eluted at 12 min from the Prep-HPLC was collected in an 80 ml sterile H2O bottle. The mixture was mixed with helium and the mixture was passed through an Oasis HLB 1 cm3 (30 mg) extraction cartridge where the product was trapped. The cartridge was washed twice with 8 ml sterile H2O and the product was eluted with 1 ml of ethanol. Next, 4 ml of 0.9% NaCl2 was passed through the cartridge to formulate the final product. The solution was filtered 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 NH4CO3 (pH=9.5) (50/50) at a flow 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 (Am) of the final product at the end of the radiosynthesis.