Synthesis of 2′-deoxy-2′-[¹⁸F]fluoro-β-D-arabinofuranosyl nucleosides, [¹⁸F]FAU, [¹⁸F]FMAU, [¹⁸F]FBAU and [¹⁸F]FIAU, as potential PET agents for imaging cellular proliferation

Abstract

An efficient and reliable synthesis of 2′-deoxy-2′-[¹⁸F]fluoro-β-D-arabinofuranosyl nucleosides is presented. Overall decay-corrected radiochemical yields of 35-45% of 4 analogs, FAU, FMAU, FBAU and FIAU are routinely obtained in >98% radiochemical purity and with specific activities of greater than 3 Ci/μmol (110 MBq/μmol) in a synthesis time of approximately 3 hours. When ∼220 mCi (8.15 GBq) of starting [¹⁸F]fluoride is used, 25 –30 mCi (0.93 –1.11 GBq) of product (enough to image two patients sequentially) is typically obtained.

Introduction

As part of an ongoing effort to develop PET imaging agents for use in tumor detection and/or to monitor response to chemotherapy, we chose to radiolabel and evaluate a series of compounds which exhibit potent in vitro antiviral and antineoplastic activity. These compounds, by virtue of their interactions with key enzymes involved in DNA synthesis, are thought to be preferentially accumulated by rapidly proliferating tissues. A number of these 2′-substituted arabinosyl nucleosides are of interest as potential therapeutic agents. Among these are 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)uracil (FAU), 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-methyluracil (FMAU), 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-bromouracil (FBAU), and 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). There are subtle differences among these compounds in terms of their interactions with key enzymes involved in DNA synthesis. The 5-substituted analogs of thymidine (FMAU, FBAU, FIAU) are substrates for thymidine kinase and are incorporated into DNA [5], [9], [18]. FAU, which lacks a methyl or halogen in the 5-ring position, requires methylation (to FMAU) by thymidylate synthase prior to its use as a DNA precursor [5]; it has been examined as a potential anti-neoplastic agent and demonstrates cytotoxic effects when it is incorporated into DNA [5]. FMAU and FIAU can be phosphorylated by the cytosolic form of mammalian thymidine kinase (TK1) [3], [8], [18], while FIAU is preferentially phosphorylated by the herpes simplex virus thymidine kinase (HSV-TK), rather than mammalian TK1 [8].

These fluoroarabinofuranosyl analogs of thymidine, appropriately radiolabelled with various isotopes useful for imaging, have been proposed as potential tracers to image cellular proliferation in tumors [6], [14]. FMAU and FBAU have been previously labeled with carbon-11 and bromine-76, respectively [6], [10], [11]. FIAU has been studied, when labeled with radioiodine, as a potential molecular probe to monitor gene therapy using HSV-TK as a marker gene [4], [16], [17]. Since a fluorine atom is already incorporated into the therapeutic compounds, the logical radiolabel would be the positron-emitting isotope fluorine-18. With its relatively long 110 min half life and its widespread availability, fluorine-18 is the preferred isotope as it allows for relatively longer scanning times (compared to carbon-11) to accommodate equilibration of the biodistribution and potential DNA incorporation. Although the four unlabelled target compounds, FAU, FMAU, FBAU and FIAU, have been extensively investigated for their therapeutic potential, at the time this study was undertaken, a suitable synthesis for the fluorine-18-labeled analogs was unavailable.

For this specific study, we chose to examine several 2′-deoxy-2′-[¹⁸F]fluoro-β-D-arabinofuranosyl nucleosides: 1a ([¹⁸F]FAU), 1b ([¹⁸F]FMAU), 1c ([¹⁸F]FBAU) and 1d ([¹⁸F]FIAU). (See Figure 1).

The overall synthetic approach to these compounds is based on a multi-step sequence published by Howell et al. [7], [15] for the unlabelled compounds (Figure 2). As part of this published work by Howell et al., each of the unlabelled intermediates depicted in Figure 2 were isolated and fully characterized, including the β and α isomers of the dibenzoyl arabinofuranosides 6a,b,d and 7a,b,d and the β and α isomers of the final deprotected compounds 1a,b,d and 8a,b,d. In addition, the authors provided detailed chromatographic (HPLC) profiles for each of the intermediates and isomers, where applicable. One drawback to this overall sequence, however, is that the fluorine-18 label is introduced into the molecule during the first reaction of this sequence thus compounding any losses which may occur at any subsequent step.

The bulk of this work was presented as an abstract at the 14^th International Symposium on Radiopharmaceutical Chemistry [12]. Another literature report by Alauddin et al on the synthesis of [¹⁸F]FMAU has been recently published [1] in which the initial labeling with fluorine-18 is performed using n-Bu₄N¹⁸F [2], while the remaining steps of bromination, condensation, and deprotection are similar to the procedures we previously reported [12].

[¹⁸F]Fluoride was produced by the (p, n) nuclear reaction on [¹⁸O]water (Isotec; >95% isotopic purity) with 11 MeV protons using an RDS-112 cyclotron (CTI). All other common solvents and reagents were obtained from Aldrich Chemical Co. Solvents used for water-sensitive reactions (i.e. MeCN, DMF, CH₂Cl₂) were anhydrous grade and were stored over molecular sieves prior to use. The CHCl₃, also obtained from Aldrich, was HPLC grade and was stabilized with amylenes instead of ethanol. It was also stored over molecular sieves. The labeling precursors, 2-O-(imidazolylsulfonyl)-1,3,5-tri-O-benzoyl-α-D-ribofuranose (2) and 2-O-[(trifluoromethyl)sulfonyl]-1,3,5-tri-O-benzoyl-α-D-ribofuranose (3) [15] were obtained from Dr. Kenneth Snader, NCI. An authentic unlabelled sample of the first intermediate, 2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofuranose (4) was obtained from Sigma Chemical Co. 2,4-Bis-O-(trimethylsilyl)pyrimidines were synthesized according to Howell, et al. [7]. Authentic unlabelled samples of the final products 1-(2-deoxy-2-fluoro-β−D-arabinofuranosyl)uracil (1a) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymine (1b) were obtained from Dr. Kenneth Snader, NCI. An authentic unlabelled sample of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil (1d) was obtained from Moravek Biochemicals.

The apparatus used for all reactions described is depicted in Figure 3. The valves (V1-V4) are all plug-type from Hamilton Co., Reno NV and are equipped with actuators for remote operation. The reagent inlet valve (V1) is a double 6-way HVX distribution valve in which there is one common port for each of 2 separate banks of 6 distribution ports. Each bank is actuated concurrently allowing reagents to be delivered to the reaction vial from each of the distribution ports (vials 2-5) through a common delivery line using only one argon pressurization (“push”) line. The reaction vessel isolation valve (V2), reaction vessel vent valve (V3) and the chromatography collection valve (V4) are all 4-port, 2 adjacent-position HVP switching valves. The 1.7 mL reaction vial is a 12 X 32mm disposable glass screw-cap (8/425) vial to which a small “v” has been incorporated through the use of a glass-blowing torch. The vial is capped with a 2-port reaction cap fabricated from PEEK and sealed with an EPDM O-ring. The cap ports are attached to the isolation valve V2 through PEEK or PFA lines (1/16“ O.D. X 0.030” I.D.). The acetonitrile solution of kryptofix 2.2.2 (KRP)/K₂CO₃ containing [¹⁸F]fluoride which is eluted off a small anion exchange trapping column and small volumes (< 100μL) of any other reagents are added via port 1 of the distribution valve V1 through a PEEK septum adapter. This adapter allows a slow argon flow to carry the reagent into the reaction vial during its introduction. With the length of the inlet line sufficient to reach the bottom of the reaction vial, the vial contents can then be removed through V1 port 6 and loaded onto a separation column via pressure introduced through valve V3. The column can then be eluted with solvent introduced through V3 after passing through the reaction vial. The column outlet line is passed over a small ionization chamber radiation detector (LND) to monitor the radioactivity in the eluant. The reaction vial is contained within an aluminum block (2.5 x 2.5 x 3.0 cm) which incorporates a small temperature-controlled cartridge heater for heating and a thermocouple for temperature monitoring. The block is ported for cooling by a regulated flow of liquid carbon dioxide.

The progress of the reaction sequence was followed at each step by radiochromatographic analysis of a diluted solution of each of the isolated intermediates. Radio-HPLC was performed on a Waters HPLC system consisting of 2 Model 510 pumps and a Rheodyne 7125 injector. For mass determination, a Waters PDA detector operating at 230 and 254 nm was used. Radiation detection was performed with a NaI scintillation detector (Biocron; Ortec). For analytical radio-HPLC, an Inertsil 5-ODS2 (C18) column (3.2 x 250mm) + precolumn (3.2 x 30mm) was used at a flow rate of 1 mL/min using the following solvents: Solvent A: 70/30 Acetonitrile/Water; Solvent B: 60/40 Acetonitrile/Water; Solvent C: 90/10 10mM Sodium Acetate (pH 5.0)/Acetonitrile; Solvent D: 94/6 10mM Sodium Acetate (pH 5.0)/Acetonitrile. For semi-preparative HPLC purification of the final products 1a-d, an Econosil C18 column (10μm; 10 x 250mm) employing 10% EtOH (5% EtOH for 1a) at 8 mL/min was used. Additionally, each of the final four compounds, 1a –1d, after sufficient time for decay of the fluorine-18, were subjected to HPLC-MS using a Zorbax Rx-C18 column (2.1 x 150mm) with 5/95 MeCN/TEA formate buffer (0.1mL TEA, 0.4 mL formic acid/L water) at 0.3 mL/min. The LC-MS was run in negative ion APCI mode. The mass spectrum obtained in each case was consistent with that of the final products.

For radio-TLC analysis, Whatman K6F Silica Gel glass plates, 250μm thick, 5 x 20cm were used and were developed with either EtOAc or CHCl₃ (0.75% v/v EtOH) as the mobile phase. Radiation detection was performed with a Bioscan System 200 radio-TLC scanner.

For each reaction, radio-HPLC and/or radio-TLC, employing the conditions listed, could clearly separate the desired product from the starting compound as well as other radiolabelled side-products. Additionally, HPLC separation of the β and α isomers of the dibenzoyl arabinosides (6a-d and 7a-d respectively) and the final products (1a-d and 8a-d respectively) is easily achieved (see Table 1).

The target [¹⁸O]water containing the [¹⁸F]fluoride (∼300 μL) was collected directly into a 1.7 mL v-vial containing 50 μL of butanediol and 10 μL of aqueous K₂CO₃ (6.5mg/mL). To this mixture was added 1 mL of anhydrous MeCN and the water was removed by azeotropic distillation at 120°C aided with a stream of argon gas. Two additional 0.8 mL aliquots of MeCN were added and also removed at 120°C with Ar flow to assure complete azeotropic removal of water. To the resulting oily residue was added 2 (10 mg, 17 μmol) in 60μL of MeCN and KHF₂ (1.33 mg, 17 μmol) in 10 μL of 1% AcOH. The mixture was initially heated with an Ar flow at 120°C for 1 min and then at 160°C for 20 min with no Ar flow. The cooled reaction mixture was dissolved in 0.8 mL of CH₂Cl₂ and the resulting solution was loaded onto a silica gel Sep-Pak (Waters Associates), along with an additional 0.8 mL rinse of CH₂Cl₂. The product was then eluted from the silica gel Sep-Pak with 1-2 mL of Et₂O. The Et₂O was collected and evaporated at 80°C with an Ar flow; last traces of ether were removed by the addition of CH₂Cl₂ followed by evaporation. The resulting oil (4) could then be used in the next reaction (bromination) of the sequence.

Cyclotron-produced [¹⁸F]fluoride (175-250 mCi) contained in 300-350 μL of [¹⁸O]water was trapped directly (with >99.6% efficiency) from the cyclotron target on a small anion exchange column (Waters QMA, bicarbonate form, 1.5 x 20 mm). The [¹⁸F]fluoride was then released (typically >99% of that trapped) from this column by eluting with a solution of KRP (6.0 mg, 16 μmol), K₂CO₃ (1.0 mg, 7 μmol) and water (15 μL) in 1 mL of anhydrous MeCN into a dry 1.8 mL v-vial. The solvent was removed from the resulting mixture (along with traces of water as a MeCN azeotrope) by heating the vial at 125°C with the aid of a 200-300 cc/min flow of argon. To the [¹⁸F]fluoride/KRP/K₂CO₃ residue was added 100 μL of anhydrous DMF and the resulting solution was heated at 150°C for 30 sec with a slow Ar flow. 2-O-[(Trifluoromethyl)sulfonyl]-1,3,5-tri-O-benzoyl-α-D-ribofuranose (3) (9 mg, 15 μmol) in 150 μL of anhydrous DMF was then added and the vial was closed and heated at 150°C for 5 min. The volume of DMF was reduced to <50 μL by heating the now open vial at 150°C under a 200-300 cc/min Ar flow. The resulting dark oil was cooled and dissolved in 0.5 mL of CH₂Cl₂. This solution was then applied to a silica gel Sep-Pak Light (Waters Associates; previously equilibrated with CH₂Cl₂) for removal of the KRP and any unreacted [¹⁸F]fluoride. While monitoring the eluant with a radiation detector, the silica gel Sep-Pak was eluted with CH₂Cl₂ (passed through the reaction vial) and the major radioactive peak containing the desired 2-deoxy-2-[¹⁸F]fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofuranose (4) was collected in a 1.7 mL v-vial and assayed for radioactivity in a dose calibrator (Capintec 712M). A small aliquot (<1 μL) was collected and dissolved in MeCN for radio-HPLC/radio-TLC analysis. TLC Analysis: Rf = 0.30 (CHCl₃); Rf = 0.85 (EtOAc). HPLC Analysis (Solvent A): k′ =8.3.

The CH₂Cl₂ was evaporated from the 2-deoxy-2-[¹⁸F]fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofuranose product (4) solution at 110°C with an argon gas flow (100-500 cc/min). To the residual oil was added 30 μL of a solution of HBr (30 wt% in acetic acid) (111 μmol) (contained in a closed loop attached to a 4-port, 2 adjacent-position HVP switching valve) followed by 300-400 μL of CH₂Cl₂ (directed through the HBr loop to rinse it out thoroughly). The reaction vial was closed and heated at 125°C for 12-15 min. This was followed by evaporation of the CH₂Cl₂, HBr and AcOH at 125°C with argon gas flow to <100 μL total volume. Toluene (0.5 mL) was then added and evaporated also at 125°C to aid in the azeotropic removal of traces of HBr and acetic acid. The residual brown oil (2-deoxy-2-[¹⁸F]fluoro-3,5-di-O-benzoyl-α-D-arabinofuranosyl bromide) (5) was then dissolved in 300 μL of alcohol-free CHCl₃, assayed for radioactivity and a small portion was removed and dissolved in MeCN for radio-HPLC and/or radio-TLC analysis. TLC Analysis: Rf = 0.40 (CHCl₃); Rf = 0.80 (EtOAc). HPLC Analysis (Solvent A): k′ = 6.1.

To the CHCl₃ (alcohol-free) solution of 2-deoxy-2-[¹⁸F]fluoro-3,5-di-O-benzoyl-α-D-arabinofuranosyl bromide (5) was added bis(trimethylsilyl)trifluoroacetamide (20μL). A solution of a 2,4-bis-O-(trimethylsilyl)pyrimidine (200 μL of 0.24M in the form of 2,4-bis-O-(trimethylsilyl)uracil, 2,4-bis-O-(trimethylsilyl)thymine, 2,4-bis-O-(trimethylsilyl)-5-iodouracil, or 2,4-bis-O-(trimethylsilyl)-5-bromouracil in CHCl₃) [7], [15] was then added and the resulting solution was heated in a closed vial at 150°C for 30 min. After cooling to room temperature, 1 mL of CH₂Cl₂ was added and, after assaying for radioactivity, a small aliquot was removed and dissolved in 70% aqueous MeCN for radio-HPLC analysis. The reaction solution was then passed through a silica gel column (3 x 10 cm, preequilibrated with CH₂Cl₂) while monitoring the eluant with a radiation detector. The silica gel column was washed with an additional 10 mL of CH₂Cl₂ and the main product fraction (as a mixture of β and α isomers 6a-d and 7a-d) was eluted with CHCl₃ containing 1-2% EtOH. The CHCl₃/EtOH from this main product fraction was evaporated (110°C, Ar flow) and the residue was dissolved in 0.5 mL of anhydrous MeCN. TLC Analysis: Rf = 0.04 (CHCl₃); Rf = 0.72 (EtOAc).

To the β/α isomeric mixture of the 1-(2′-deoxy-2′-[¹⁸F]fluoro-3,5-di-O-benzoyl-D-arabinofuranosyl)pyrimidine (6 + 7) solution in 0.5 mL of MeCN, cooled to room temperature, was added 0.5M sodium methoxide in MeOH (150 μL, 75 μmol). After 10 min at 20°C, 500 μL of 1% (w/v) acetic acid solution was added. The MeCN was then removed by evaporation (125°C, Ar flow) and the residue was dissolved in 2.5 mL of water. After assaying for radioactivity and performing radio-HPLC analysis, the desired 1-(2′-deoxy-2′-[¹⁸F]fluoro-β-D-arabinofuranosyl)pyrimidines (1a-d) were isolated from the unwanted α-isomers 8a-d by semi-prep HPLC (C18, 5% EtOH for 1a, 10% EtOH for 1b-d). TLC Analysis: Rf = 0.23 (EtOAc).

For in vivo imaging studies, the aqueous ethanolic solutions of the final products 1a-d were made isotonic by addition of concentrated saline and were sterilized through a 0.2μm sterile filter (Millipore, Millex-GS). The pH of the final product solutions were 5.0 to 6.0 and the absence of KRP was confirmed by the method of Mock et al. [13].