Catalytic enantioselective synthesis of 18F-fluorinated α-amino acids under phase-transfer conditions using (s)-NOBIN

Nuclear Medicine and Biology 31 (2004) 597– 603

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Catalytic enantioselective synthesis of 18F-fluorinated ␣-amino acids under phase-transfer conditions using (S)-NOBIN R.N. Krasikovaa, V.V. Zaitseva, S.M. Ametameyb,*, O.F. Kuznetsovaa, O.S. Fedorovaa, I.K. Mosevicha, Y.N. Belokonc, Sˇ. Vyskocˇild, S.V. Shatike, M. Naderf, P.A. Schubigerb a

Institute of Human Brain, Russian Academy of Science, 9, Pavlov strasse, 197376, St.-Petersburg, Russia b Center for Radiopharmaceutical Science of ETH, PSI and USZ, CH-5232, Villigen PSI, Switzerland c A.N. Nesmeyanov Institute of Organo-Element Compounds, Russian Academy of Sciences, Vavilov 28, 119991, Moscow, Russia d Charles University, Department of Organic Chemistry, Hlavova 2030,12840, Prague 2, Czech Republic e Institute for Experimental Medicine RAMS, 12, Pavlov strasse, 197376, St. Petersburg, Russia f ARGOS Zyklotron GesmbH, Seilerstrasse 4, 4010 Linz, Austria

Abstract We describe a new method for the asymmetric synthesis of [18F]fluorinated aromatic ␣-amino acids (FAA) under phase transfer conditions using achiral glycine derivative NiPBPGly and (S)-NOBIN as a novel substrate/catalyst pair. The key alkylation step proceeds under mild conditions. Substituted [18F]fluorobenzylbromides were prepared using nucleophilic [18F]fluoride and were used as alkylation agents. Two important FAA, 2-[18F]fluoro-L-tyrosine (2-FTYR) and 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA), were synthesized with an ee of 92 and 96%, respectively. The total synthesis time was 110 –120 min and radiochemical yields (d.c.) were 25⫾6% for 2-FTYR and 16⫾5% for 6-FDOPA. © 2004 Elsevier Inc. All rights reserved. Keywords: 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine; 2-[18F]fluoro-L-tyrosine; PTC asymmetric synthesis; (S)-NOBIN

1. Introduction

proach is facile and easy to automate, a low cross section of Ne(d,␣)18F nuclear reaction and 50% theoretical yield of fluorination reaction limit the productivity of this method. The generation of electrophilic [18F]F2via high efficient 18 O(p,n)18F nuclear reaction in the 18O-oxygen gas target requires complex double-shoot irradiation protocols to recover the radionuclide [8]. This production method has been applied recently in the synthesis of 2-FTYR [7], but it has not been implemented into routine practice in most PET centers so far. In general, both methods of generating electrophilic [18F]F2 require the addition of carrier which allows to get FAA in low specific activity. The nucleophilic pathway based on no-carrier-added (NCA) [18F]fluoride which is available in large amounts from proton irradiation of 18 O-enriched water can be more advantageous. Recently, the direct nucleophilic radiofluorination method for O-(2[18F]fluoroethyl)-L-tyrosine has been reported [9]. As for the radiolabeling of amino acids in the aromatic moiety, the direct nucleophilic route generally fails if the phenyl ring lacks an electron-withdrawing group. The implementation of asymmetric amino acid synthesis via diastereoselective alkylation of chiral glycine enolates 20

A growing clinical application of [18F]fluorinated ␣-amino acids (FAA) using positron emission tomography (PET) (for review see [1]) has stimulated an interest in new synthetic strategies. The possibility of producing high amounts of radioactive FAA, coupled to the longer half-life of 18F (110 min), will make the shipping of these useful radiopharmaceuticals possible to satellite PET centers. 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA), a well-established tracer for Parkinson’s disease [2], has offered an enormous potential for diagnostic applications due to the possibility of its use as oncologic PET tracer [3]. Its structural analog, 2-[18F]fluoro-L-tyrosine (2-FTYR), was among the first FAA evaluated as tumor seeking agents [4], but its clinical application is still limited. Most of the routine production methods for the aromatic FAA labeled in benzene ring are based on electrophilic regiospecific fluoro-destannylation reactions [5–7]. Although the electrophilic ap-

* Corresponding author. Tel.: ⫹41563104260; fax: ⫹41563103132. E-mail address: [email protected] (S.M. Ametamey). 0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2003.12.010

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as applied for radioactive synthesis has a number of difficulties. The extreme reaction conditions such as very strong bases (BuLi, LDA, LiHMDS, etc.), low temperatures and strict anhydrous conditions [10] are required for the deprotonation of most chiral glycine derivatives. These difficulties do not allow an easy automation of the synthetic procedure. The use of chiral inductors based on a nickel (II) complex of the Schiff base of (S)-O-[(N-benzylprolyl)amino]benzophenone and glycine (NiBPBGly) [11] has brought improvements since asymmetric alkylations could be carried out under mild reaction conditions. Our initial studies using NiBPBGly as chiral inductor have demonstrated that 6-FDOPA can be obtained in enantiomeric purity of more than 82% [12,13]. Modification of the structure by introducing two chlorine atoms into the N-benzyl moiety of the chiral inductor (NiCPBGly) [14] allowed to obtain 95% enantiomeric purity of 6-FDOPA [13] which is sufficient for clinical use. With this approach we were unable to achieve a high asymmetric induction for 2-FTYR. An alternate strategy to the asymmetric synthesis is the phase-transfer catalytic (PTC) reaction. Two PTC systems consisting of the C2-symmetric chiral quaternary ammonium salt or O-benzyl-N-[(9-anthracenyl)methyl]cinchonidinium bromide as catalyst and N-(diphenylmethylene)glycine esters as the chiral substrates were earlier employed in the synthesis of 6-FDOPA and 2-FTYR [15–17]. This approach has shown high asymmetric efficiency at 0°C using the substituted [18F]fluorobenzyl bromides as alkylating agents. A disadvantage of these catalyst/substrate pairs is that the temperature of alkylation reaction has to be maintained at 0°C very precisely to achieve high ee of the FAA thus making this approach inconvenient for a routine use. Later, the O-allyl-N-(9-anthracenyl)cinchonidinium bromide was used in alkylation with iodide derivative, 2-[18F]fluoro-4,5-dimethoxybenzyl iodide, allowing to obtain high ee of 6-FDOPA at room temperature alkylation [18]. The synthesis of iodide derivative, however, was based on the iodination reaction with diiodosilane (SiI2H2), a tricky step with a moisture sensitive reagent, which has to be used immediately on opening. This method is difficult to implement in fully automated modules where all the reagents have to be installed in advance before delivery of the [18F]fluoride from the target. Recently, a new PTC pair [19] including an achiral Ni(II) complex of a Schiff base of 2-benzoylphenylamide of pyridine-2-carboxylic acid (PBP) and glycine (NiPBPGly) and 2-amino-2’-hydroxy-1,1’-binaphtyl ((R)- or (S)-NOBIN) have been introduced (Fig.1). This substrate/catalyst pair is allowed to obtain high ee of amino acids via room temperature alkylations. In this report, we applied (S)-NOBIN for the PTC synthesis of 2-FTYR and 6-FDOPA in high enantiomerc enrichment, using [18F]fluorobenzyl bromide derivatives (RX) as the electrophile in the alkylation of Ni(II) complex of a

Fig. 1. Chemical structures of chiral catalyst (S)-NOBIN and NiPBPGly.

Schiff base of 2-benzoylphenylamide of pyridine-2-carboxylic acid (PBP) and glycine (NiPBPGly) [19].

2. Materials and Methods The commercially available reagents and solvents were used without further purification unless stated otherwise. 18 O-enriched water (95%) was purchased from the Global Scientific Technologies, Sosnovy Bor, Russia; anhydrous potassium carbonate, sodium boronhydride, magnesium sulphate, 3,4-methoxy-6-nitrobenzaldehyde (nitropiperonal), 4,7,13,16,20,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K2.2.2) were obtained from Aldrich; triphenyldibromophosphorane, dimethylsulfoxide, dichloromethane were supplied by Fluka; methanol, acetone and anhydrous acetonitrile were bought from Merck; hydroiodic acid (Chimex Ltd, Russia) was distilled and packed into sealed tubes (1 mL volume); solid-phase extraction columns and cartridges (Supelclean LC-18, Alumina-B, SepPak Silica plus, QMA SepPak Light) were purchased from Supelco or Waters. 2-Fluoro-L-p-tyrosine was provided by ABX, Germany; 6-fluoro-DL-DOPA was obtained from the RBI. 2-Nitro-4methoxybenzaldehyde was synthesized at the IHB using slightly modified literature procedure [20]; achiral nickel (II) complex of Schiff base of 2-benzoylphenylamide of pyridine-2-carboxylic acid and glycine and 2-amino-2’hydroxy-1,1’-binaphtyl ((S)-NOBIN), were synthesized as described elsewhere [19,21] Irradiations were performed on the cyclotron MC17, Scanditronix, Sweden. Radioactivity was measured with a dose calibrator PTW Curiementor-2 (Germany). HPLC analysis was performed using the following system: Gilson Pump 305, a Rheodyne injector (20 ␮L loop), Gilson 116 UV absorbance detector in series with a Beckman 170 radiodetector. Peak analysis was done with a 4400 integrator (Varian, USA). For radiochemical purity and monitoring of the different reactions steps analytical HPLC was used under following conditions: Lichrospher 100 RP-18, 5 ␮m, endcapped (Merck), 4.6⫻250 mm, mobile phase CH3COOH, pH 4/CH3OH (35/65 by volume), 0.7 mL/min. The course of reaction was also followed using TLC coupled with radioactivity measurements (Silica plates, Sorbfil,

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Russia, CH2Cl2as a solvent). The enantiomeric purity of the final products was checked by chiral HPLC (Crownpak (⫹), Daicel, eluent HClO4, pH 2, flow 0.8 mL/min). The analysis of final preparations for residual amounts of nickel, phosphorus, bromine and boron was performed by the inductively coupled plasma technique using ICP-MS PQ-3 (Plasma Quad, VG, UK). 2.1. Radiochemical synthesis 2.1.1. Preparation of the [18F]fluoride and [K/K2.2.2]⫹ 18 ⫺ F complex No carrier added (NCA) [18F]fluoride was prepared by the 18O(p,n)18F reaction via 17 MeV proton irradiation of [18O]H2O in a low pressure small volume water target. The separation of [18F]fluoride from 18O-enriched water (allowing its recycling use) was performed using QMA SepPak Light cartridge (Waters). The QMA resin was transformed into carbonate form by passing 10 mL of 0.5 M aqueous potassium carbonate followed by 10 mL of water. The 18F trapped on the QMA resin was recovered by elution with 2 mL of solution which contained 9.5⫾0.4 mg (0.025 mmol) of K2.2.2 and 1.7⫾0.2 mg (0.012mmol) of potassium carbonate dissolved in 2 mL of acetonitrile/water (96/4 v/v). The solvents were evaporated at 130°C under nitrogen flow of 200 mL/min until dryness. The [K/K2.2.2]⫹ 18F⫺ complex thus obtained was used directly in the next step without usual azeotropic drying procedure. 2.1.2. 8F-labeled fluorobenzaldehyde derivatives Ia and Ib A solution of 7– 8 mg of 2-nitro-4-methoxybenzaldehyde or 6-nitro-3,4-methylendioxybenzaldehyde (nitropiperonal) in 0.5 mL of anhydrous DMSO was added to the vial containing dry [K/K2.2.2]⫹ 18F⫺ complex. Reaction was performed at 180°C for 5 min. After cooling reaction mixture was diluted with 4.5 mL of water and the resulting solution was passed through a 6 mL column Supelclean LC-18 preconditioned by ethanol (2⫻5 mL) and water (3⫻5 mL). A small amount of diluted reaction mixture was analyzed by radio TLC to determine the incorporation yield of 18F into benzaldehydes. The Rf values for [18F]fluoride, 2-[18F]fluoro-4-methoxybenzaldehyde (Ia) and 6-[18F]fluoro3,4-methylendioxybenzaldehyde (Ib) were 0.05, 0.53, and 0.50, respectively. 2.1.3. 18F-labeled fluorobenzyl alcohol derivatives IIa and IIb LC-18 column retaining Ia and Ib was rinsed with water (2⫻5 mL). Aqueous solution of sodium borohydride (25-28 mg in 1 mL of water) was introduced on the top of the column and slowly pushed by flow of nitrogen (10 mL/ min). The column was rinsed with 5 mL of water and flushed by nitrogen flow for 5 min. Dichloromethane (0.3 mL) was passed through the column to remove the residual water. The [18F]fluorobenzylalcohol derivatives 2-[18F]fluoro4-methoxybenzylalcohol (IIa) and 6-[18F]fluoro-3,4-meth-

599

ylendioxybenzylalcohol (IIb) were recovered from the column by elution with 2 mL of dichloromethane. The eluate was dried “on-line” by passing through the small column (3 mL Supelco extraction tube filled with 1 g of potassium carbonate in its upper part and 0.8 g of magnesium sulfate at the bottom end). The reduction efficiency was evaluated by radio TLC or analytical HPLC. The Rf values were 0.2 for both. Using analytical HPLC described above the retention times for IIa and IIb were 7.3 and 7.1 min, respectively. 2.1.4. 18F-labeled fluorobenzylbromide derivatives IIIa and IIIb The eluate containing IIa and IIbwas collected in a vial filled with 80-100 mg of triphenyldibromophosphorane (Ph3PBr2). The bromination was performed at room temperature for 5 min under vortexing. 18F-labeled fluorobenzyl bromide derivatives 2-[18F]fluoro-4-methoxybenzylbromide (IIIa) and 6-[18F]fluoro-3,4-methylendioxybenzylbromide (IIIb) were purified by passing of the reaction mixture through SPE. Conversion rates into bromides were evaluated by TLC and HPLC analysis of the samples of the crude reaction mixture. Rf values were 0.70 and 0.65 for IIIa and IIIb. Using analytical HPLC described above the retention times for IIaand IIb were 13.2 and 12.5 min, respectively. 2.1.5. Alkylation of achiral nickel (II) complex of Schiff base of 2-benzoylphenylamide of pyridine-2-carboxylic acid and glycine (NiPBPGly) with IIIa and IIIb in the presence of (S)-NOBIN and recovery of the FAA The solution of purified bromide derivatives IIIa and IIIb in 1 mL of dichloromethane was collected in an alkylation vial which contained 20 mg of solid sodium hydroxide, 20 mg of NiPBPGly and 2 mg of the (S)-NOBIN. The reaction mixture was stirred using Vortex at room temperature or at 0°C for 3– 6 min. In some experiments the alkylation was performed in different solvents as it is shown in Table 1. Dichloromethane solution containing purified IIIa and IIIb was evaporated to dryness whereas the residual was redissolved in a suitable solvent. On completion of the alkylation step the reaction mixture was quenched by addition of 0.5 mL of hydroiodic acid (57%, not stabilized), the organic solvent was evaporated under vacuum and nitrogen flow at room temperature. The residue was heated for 10 min at 170°C in a sealed vial to accomplish hydrolysis of the alkylated products and deprotection of the hydroxyl groups. Cooled solution was diluted with 0.5 mL of water and partly neutralized by addition of 6 M NaOH (0.3 mL). The precipitate obtained was removed by filtration through Millex GV SLGV013OS filter, 22 ␮m. The resulting slightly red transparent solution was injected into a semipreparative HPLC column (C18 Nucleosil 100-10, 250⫻10 mm, eluent 0.1% CH3COOH (pH 4), flow rate 5 mL/min. Retention times for 2-18F-fluoro-L-tyrosine (2-FTYR) and 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA) were 17 and 10 min, respectively. Using analytical chiral

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Table 1 Results of the PTC synthesis of 2-FTYR mediated by (5)-NOBIN under various alkylation conditions Solvent

Alkylation temperature

Time (min)

Purification of RX (IIIa)

Quenching of reaction

% of L isomer in the final product

Acetone (CH2)2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2

Room 0°C 0°C Room Room Room

3 6 5 5 5–7 5

No Yes Yes No Yes Yes

50 89.7 98 89.7 95.3 ⫾ 0.5 (n ⫽ 7) 98.2 ⫾ 0.5 (n ⫽ 6)

CH2Cl2*

Room

5

AIB/SiO2 AIB/SiO2 AIB/SiO2 AIB/SiO2 AIB/SiO2 SepPak SilicaPlus SepPak SilicaPlus

Yes

96.4 ⫾ 0.5 (n ⫽ 5)

* Values for 6-FDOPA.

HPLC described above, the retention times for D- and L2-FTYR were 6 and 7.6 min; for D- and L- 6-FDOPA they were 8.1 and 9.8 min, respectively.

regulations governing the safe and humane use of laboratory animals in research.

2.2. Biodistribution of 2-[18F]-fluoro-L-tyrosine in rats

3. Results and discussion

The biodistribution studies were performed in a Wistar rats bearing glioma-35-derived rats tumors (homografts). The adult male Wistar rats weighing 150 –200 g were used. For the induction of the tumor, 10% suspension of glioma-35 tumor cells in 5% dimethylsulfoxide solution in saline, stored in liquid nitrogen, was thawed quickly at 37°C and injected subcutaneously into the experimental animal (0.5 mL per animal). When the tumor size reached the weight of about 50 g, the rats were killed by cervical dislocation, tumor tissues were taken, pulverized in saline (1:10 w/v) by passing through the sieve with 1 mm pore size and pumping through the injection needle with 1 mm inner diameter. This suspension (0.5 mL per animal) was injected subcutaneously or into the muscle of the right hind limb of the rats. The tumors were allowed to grow for 14 days. The rats were injected with 80 –90 ␮Ci (2.5–-3 MBq) of 2-FTYR in 0.3 mL of the isotonic solution via tail vein. They were sacrificed at 60 min postinjection; organs and tissues of interest were dissected, isolated, wiped of excess blood, weighed and counted in a gamma counter. The percentage of injected dose per gram (% ID/g) was than calculated using a stored sample of the injection solution as a reference. The animal distributions experiments were performed in the Institute of Experimental Medicine RAMS, St. Petersburg under Institutional Animal Care and National

3.1. Radiochemical synthesis The radioactive synthesis comprises two major synthetic stages: preparation of the alkylating agents RX (Fig. 2) and alkylation of the achiral NiPBPGly complex under the PTC conditions with (S)-NOBIN (Fig. 2) as the catalyst. The alkylating agents 2-[18F]fluoro-4-methoxy-benzyl bromide (IIIa) and 3,4-methylendioxy-6-[18F]fluorobenzyl bromide (IIIb) were prepared in three synthetic steps starting from the corresponding nitrobenzaldehydes (2-nitro-4-methoxybenzaldehyde and 6-nitropiperonal). The first step (Fig. 2), a classical nucleophilic substitution on a nitro leaving group using reactive [K/K2.2.2]⫹18F⫺ complex, was performed in analogy to reported methods [20] but with some modifications [22]. In our procedure, 18F-fluoride retained on the QMA cartridge was eluted with a solution of potassium carbonate and kryptofix dissolved in the water-acetonitrile mixture which contained only 4% of water [22]. With this composition a single evaporation of the eluate allowed to obtain [K/K2.2.2]⫹18F⫺ complex reactive in the following nucleophilic fluorination. This procedure gave efficient and reproducible fluorination yields using a simplified automation and a shorter synthesis time. The incorporation rate of the 18 F into the benzaldehydes evaluated by TLC analysis was

Fig 2. NCA radiosynthesis of 2-[18F]fluoro-4-methoxy-benzyl bromide (IIIa) and 3,4-methylendioxy-6-[18F]fluorobenzyl bromide (IIIb) from substituted nitrobenzaldehydes.

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601

Fig. 3. Asymmetric alkylation of NiPBPGly with 2-[18F]fluoro-4-methoxy-benzyl bromide (IIIa) under PTC conditions mediated by (S)-NOBIN.

87⫹1% for 2-nitro-4-methoxy benzaldehyde (n⫽15) and 53⫹6% for nitropiperonal (n⫽39) [23,24]. The following steps, on-line reduction of the methoxy substituted [18F]fluorobenzyl aldehydes Ia and IIa with an aqueous solution of NaBH4 and bromination of the alcohol derivatives using solid commercially available Ph3PBr2 were performed according to the procedure reported for the synthesis of 4-[18F]fluoro benzylbromide [25]. Careful removal of traces of water from the crude alcohols IIa and IIb eluted from C18 cartridge by methylene chloride was needed in order to achieve a high transformation of the alcohols into their corresponding bromides [24]. The drying procedure was, however, accompanied with a considerable loss of the radioactivity (up to 30% of the total radioactivity of IIa, IIb). Isolated radiochemical yields (d.c.) were 68⫾8% for 2-[18F]fluoro-4-methoxy-benzyl bromide (IIIa) and 41⫾9% for 6-[18F]fluoropiperonyl bromide (IIIb) within 45– 48 min. For the asymmetric alkylation (Fig. 3) the glycine derivative NiPBPGly and (S)-NOBIN) as a substrate/catalyst pair was employed. Both the substrate and the catalyst are readily prepared from cheap commercially available reagents [19,21,26]. Previous work has demonstrated that the alkylation of NiPBPGly with 3,4-dimethoxy benzyl bromide promoted by (R)-NOBIN gave D-dihydroxyphenyl alanine (D-DOPA) in 98.5% ee (6 min alkylation, room temperature, solid NaOH as a base). Therefore, for the radiosynthesis of 2-FTYR and 6-FDOPA we applied similar reaction conditions using (S)-NOBIN to obtain the desired biologically active L-isomers. As for the recovery of the product from the alkylated complex, a multistep time-consuming procedure described in [19] was not suitable for operating with short- lived isotopes and therefore was modified. The results of the alkylation of NiPBPGly with substituted [18F]-fluorobenzyl bromides IIIa and IIIb in the presence of the (S)-NOBIN in various solvents are shown in Table 1. Although dichloroethane was recommended as the best solvent for PTC alkylation in “cold” experiments [19], we were not able to achieve high asymmetric induction with this solvent even at 0°C. In our hands, the highest ee of

2-FTYR was obtained in dichloromethane at room temperature alkylation, which is advantageous for automation. Our data show that the quenching of the reaction mixture with an acid (CH3COOH or 57% HI) before solvent removal was necessary to prevent heating-induced racemization of the (S)-complex (Fig. 3). Another important factor has been the careful removal of excess of Ph3PBr2 and nonlabeled by-products. Low ee was obtained when this purification step was not carefully done. For this purification step different packing materials and cartridges were tested [23]. When purification of IIIa was performed with hand-packed combined AluminaB/SiO2 column or SepPak Silica Plus cartridge (Table 1), the highest ee for 2-FTYR was obtained. Optimal alkylation and purification procedures for 2-FTYR were applied to the synthesis of 6-FDOPA. The enantiomeric purity obtained for 6-FDOPA was 95–96%. Radiochemical yields (EOB) were 25⫾6 % (n⫽9) for 2-FTYR and 16⫾5% (n⫽5) for 6-FDOPA within 110 –120 min synthesis time including semipreparative HPLC purification. The radiochemical purity was determined by HPLC and was always ⬎99% for both 2-FTYR or 6-FDOPA (Fig. 4). Automation of the synthetic steps was accomplished on

Fig. 4. Analytical radiochromatogram of purified 2-FTYR (Crownpak (⫹), Daicel, eluent HClO4, pH 2, flow 0.8 mL/min).

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Table 2 ICP analysis of 2-[18F]Fluoro-L-tyrosine (product fraction from the semipreparative HPLC purification, concentrations in parts per million) Entry

Nickel

Phosphorus

Boron

Bromine

1 2 3

0.53 0.91 0.73

0.47 0.28 0.47

0.065 0.086 0.068

⬍0.01 ⬍0.01 ⬍0.01

a commercially available Anatech-RB86 laboratory robot. The final preparations were analysed for residual amounts of nickel, phosphorus, bromine and boron, which may originate from potentially toxic reagents, used during the different synthesis steps. The results (Table 2) demonstrated that with the applied SPE techniques and final semipreparative purification procedures the product quality meets clinical demands in term of the total amounts of these elements. No other impurities were detected in the final preparations using HPLC analysis with UV detector in the wide range of the UV wavelengths, except those originating from the solvents used during semipreparative HPLC purification.

4. Conclusion A general method of synthesis for highly enantiomerically enriched [18F]-fluorinated alpha amino acids, labeled in the aromatic portion, has been developed. 2-FTYR and 6-FDOPA were obtained in more than 96% enantiomerical purity for clinical application using a new PTC system based on (S)-NOBIN and achiral NiPBPGly complex. The accomplishment of the key alkylation step at room temperature allows implementation of this method into modern automated modules for PET radiopharmaceuticals.

Acknowledgments This work was supported by the Swiss National Science Foundation, Grant No. JO62161.00/1, the research initiative grants of the Scientific Center of Russian Academy of Science, St. Petersburg, ISTC Grant No. A-356, ISTC Grant No. 2780, Russian Grant for Fundamental Research No.0203-3209.

References 3.2. Biodistribution studies The results of the biodistributions studies of 2-FTYR using the glioma35 rats model in Wistar rats are shown in Table 3. The tissue uptake of radioactivity is expressed as the percent injected dose per gram of tissue (%ID/g). Tumor uptake and normal tissue distribution were measured at 60 min postinjection. Among different organs the greatest uptake was observed in the liver (up to 4.3% ID/g), which is typical for labelled amino acids. At 60 min postinjection tumor to muscle ratios (TMR) ranged from 3.5 to 4.3. In one rat subcutaneous tumor growth resulted in brain metastasis. The uptake of the tracer in this lesion was 4.2% ID/g. The results indicated that FTYR obtained via the newly developed asymmetric PTC synthesis is potentially useful as a tumor seeking agent with PET.

Table 3 Tissue distribution of radioactivity in 5 Wistar rats bearing glioma-35 rat tumor after injection of 2.5–3 MBg of 2-FTYR at 60 min post-injection Blood Kidney Heart Liver Pancreas Brain Muscle Subcutaneous TMR tumor 0.89 1.25 1.55 1.46

0.39 2.5 1.86 2,11 2.25

0.41 0.90 0.88 0.90 1.23

1.59 2.58 4.99 4.83 4.31

1.07 2.59 1.44 1.523 1.79

0.56 0.77 0.74 0.82 1.50

TMR: tumor to muscle ratio. * Brain metastases uptake is 4.2% ID/g.

0.23 0.43 0.48 0.56 0.61

0.87 1.84 1.76 2.40 2.13

3.74 4.22 3.67 4.28 3.49*

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