Radiosynthesis of 3′-deoxy-3′-[18F]fluorothymidine: [18F]FLT for imaging of cellular proliferation in vivo

Nuclear Medicine & Biology, Vol. 27, pp. 143–156, 2000 Copyright © 2000 Elsevier Science Inc. All rights reserved.

ISSN 0969-8051/00/$–see front matter PII S0969-8051(99)00104-3

Radiosynthesis of 3⬘-Deoxy-3⬘-[18F]fluorothymidine: [18F]FLT for Imaging of Cellular Proliferation In Vivo John R. Grierson1 and Anthony F. Shields2 1

RESEARCH IMAGING LABORATORY, UNIVERSITY OF WASHINGTON MEDICAL CENTER, SEATTLE, WASHINGTON, USA AND 2

WAYNE STATE UNIVERSITY, KARAMANOS CANCER INSTITUTE, HARPER HOSPITAL, DETROIT, MICHIGAN, USA

ABSTRACT. A reliable radiosynthesis of 3ⴕ-deoxy-3ⴕ-[18F]fluorothymidine ([18F]FLT) has been developed based on [18F]fluoride displacement of a protected nosylate precursor. A simple three-step synthesis is described that is useful for preparing >10 mCi (370 MBq) of radiochemically pure [18F]FLT, with a specific activity >1 Ci/␮mol (37 GBq/␮mol) at EOS within 100 min and in 13% radiochemical yield (end of bombardment (EOB); 7% end of synthesis (EOS)). [18F]FLT has been designed as a new positron emission tomography imaging agent for visualizing cellular proliferation in vivo based on the metabolism of thymidine. NUCL MED BIOL 27;2:143–156, 2000. © 2000 Elsevier Science Inc. All rights reserved. KEY WORDS. [18F]FLT; 3⬘-deoxy-3⬘-fluorothymidine; DNA synthesis; PET imaging; Proliferation; Fluorine18; TK-1; Thymidine kinase; Thymidine metabolism; [11C]thymidine

INTRODUCTION Labeled thymidine (TdR) is the preferred radiopharmaceutical for indicating cellular proliferation because its specific incorporation into DNA is linked to the S phase of the cell cycle (18). The rapid labeling rate, along with the defined and durable nature of the intracellular label, had spurred the proposal that [11C]thymidine would be useful for imaging proliferation in vivo with positron emission tomography (PET) (5). Indeed, [11C]thymidine has worked well for PET imaging (7, 21–23, 35, 39, 42), despite the fact that only a portion of the injected dose gets incorporated into DNA. When thymidine’s systemic degradation (catabolism) is accounted for, retention of [11C]thymidine in normal proliferating tissues and most tumors can readily be imaged and quantitatively related to DNA synthesis (7, 21, 22, 34). Although [11C]TdR has proven useful as an imaging agent, construction of its input function from metabolite data is demanding and is an obstacle to wide acceptance of thymidine as a PET radiopharmaceutical. The difficult radiosynthesis of [11C]TdR is also a factor. As a practical alternative to [11C]thymidine, the analog 3⬘-deoxy-3⬘-[18F]fluorothymidine ([18F]FLT; Fig. 1) is being developed (11–16, 26, 30 –33). FLT is captured by proliferating cells, is stable to catabolism in vivo, and has a simpler radiosynthesis with a longer lived label (13, 19, 27, 33). FLT and thymidine undergo the same initial metabolism and are monophosphorylated by thymidine kinase-1, an enzyme that is expressed during the DNA synthesis phase (S phase) of the cell cycle (1, 19, 24, 25, 29). Whereas thymidine monophosphate is quickly fixed in cells by DNA synthesis, FLT monophosphate accumulates as a membrane impermeable metabolite and, from a PET imaging viewpoint, behaves as if it were in DNA (19, 24, 31, 37). For example, both [18F]FLT and 2-[11C]thymidine qualitatively Address correspondence to: John R. Grierson, Ph.D., University of Washington Medical Center, Research Imaging Laboratory, Box 356004, University of Washington, Seattle, WA 98195, USA; e-mail: grierson@ u.washington.edu. Received 10 September 1999. Accepted 7 November 1999.

imaged the same regions in a dog given both agents (31); in particular, uptake was seen in bone marrow, where there is a high rate of cellular proliferation. The fact that FLT is inert to catabolism plays a significant role in prolonging the input of the tracer and minimizing background activity. Indeed, a progressive increase in the standard uptake value (SUV) for [18F]FLT in dog marrow was still seen at late imaging times (20 – 60 min). This was not the case for 2-[11C]TdR, which reached an early plateau phase, and this difference accounts for the twofold higher SUV for [18F]FLT in marrow at 60 min (31). Although imaging with [18F]FLT in animals and humans has been successful (31), the yields of this promising tracer were too low for routine use (12–14). Here, we present a higher yielding and more reliable radiosynthesis of [18F]FLT that makes minimal use of specialized apparatus and materials. The improvements result from using a nosylate (4-nitrobenzenesulfonate) ester for nucleophilic displacement with [18F]fluoride. Within 2 h, the two-step process produces 10 –20 mCi of FLT (13% end of bombardment (EOB); 7% end of synthesis (EOS)) suitable for injection. METHODS AND MATERIALS

General Analytical Data Melting points are uncorrected. Elemental analyses (C, H, N) were performed by Galbraith Laboratories (Knoxville, TN USA). Fast atom bombardment mass spectrometry (MS) measurements were performed with a Micromass (Manchester, UK) 70SEQ tandem hybrid spectrometer and liquid chromatography (LC)-MS was performed with high performance liquid chromatography (HPLC) systems interfaced to a Micromass Quattro II tandem, or a Micromass ZMD, quadrapole spectrometer using electrospray ionization. Proton (1H) nuclear magnetic resonance (NMR) spectroscopy was performed with a GE (Fremont, CA USA) Omega 300 MHz spectrometer. Chemical shifts are reported in ppm (␦) relative to tetramethylsilane (TMS) in CDCl3. Optical rotation measurements were recorded with a JASCO (Easton, MD USA) DIP370 digital polarimeter. X-ray solid-state structure data for compound (5) was obtained with a Nonius (Bohemia, NY USA) KappaCCD instru-

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Synthesis of the FLT Labeling Precursor An overview of the synthesis of the FLT labeling precursor, compound (8) and the radiosynthesis is shown in Figure 2.

Step A: Synthesis of O2,O3ⴕ-anhydro-1-(2-deoxy-␤-Dthreo-pentofuranosyl)thymine (2)

FIG. 1. Fluorothymidine (FLT) is a thymidine analog. ment, and structure solution and refinement were done with the SHELX-97 software package (available from Nonius).

Radioactivity Measurements F activity (⬎100 ␮Ci) was measured with an ion chamber Capintec (Pittsburgh, PA USA) CRC-12 Radioisotope Calibrator. 141 Ce (␥ 142 keV, 48%) activity was counted (⬎1–500 ␮Ci) with the Capintec instrument using the 99mTc (140 keV) channel or, for low count rate samples (⬍0.1 ␮Ci), with a Packard (Downers Grove, IL USA) Auto-Gamma 5000 series instrument. The measured Packard counter efficiency for 141Ce was 5.6%. Background and low count rate samples (25–50 cpm) were each counted for 4 h. Radio-HPLC and specific activity measurements were done as previously described (10). 18

High Performance Liquid Chromatography LUNA 5 ␮m particle/C18(2) phase HPLC columns were obtained from Phenomenex (Torrance, CA USA): column A/semi-preparative (250 ⫻ 10 mm inner diameter [i.d.]); column B/analytical (250 ⫻ 4.6 mm i.d.); column C/minibore (250 ⫻ 2 mm i.d.). Void volumes of columns A, B, and C (9.90, 2.15, and 0.53 mL, respectively) were measured at equivalent linear velocities by eluting fructose (1 ⫽ 10 ␮L, 20 mg/mL) with 90% acetonitile in water and detection at 190 nm. For sample analyses, columns were eluted with isocratic solvent systems.

Adsorption Chromatography Thin layer chromatography (TLC) was carried out on aluminumbacked silica gel plates from E. Merck (Darmstadt, Germany) (0.2 mm, F254). Filter column chromatography was carried out according to the method of Yau and Coward (41). Typical separations of nucleoside mixtures were done by pre-adsorption of the crude product onto silica gel, followed by its filter chromatography over E. Merck silica gel 60 (230 – 400 mesh). Alumina (neutral) SepPak cartridges were obtained from Waters Associates (Milford, MA USA).

Chemicals All reagents, including anhydrous solvents, were purchased commercially (Aldrich/Fluka, Milwaukee, WI USA) and used as received. However, commercial anhydrous acetonitrile, used for the preparation of compound (2), was further dried over 3A molecular sieves. 141Ce trichloride was purchased from NEN Life Sciences (Boston, MA USA).

This procedure was modified from the method reported by Balagopala et al. (2). A mixture of thymidine (25.18 g, 104 mmol, [␣]D ⫹20.2° [23°C, c ⫽ 1, H2O]; lit (Aldrich) [␣]D ⫹ 18.5° [25°C, c ⫽ 1, H2O]) and triphenylphosphine (54.5 g, 208 mmol) was azeotropically dried with portions of acetonitrile (2 ⫻ 150 mL). The residual mass was suspended in MeCN (400 mL) and then cooled to ⫺20°C. To the rapidly stirred mixture, a solution of diisopropylazodicarcarboxylate (42.0 g, 208 mmol) in MeCN (150 mL) was added, dropwise over 45 min, while maintaining the temperature between ⫺15° and ⫺20°C. After the addition was complete, the cold mixture was stirred for 1.5 h and then allowed to slowly warm to 10°C over 5 h. At this point a light brown solution was present. This solution was rapidly stirred at room temperature and then treated with water (10 mL) in one portion. The addition led to the quick formation (1–3 min) of a thick suspension that was allowed to stand for 30 min and then filtered. The collected white solid was washed (2 ⫻ 100 mL MeCN) and vacuum dried to yield 17.57 g (75% yield) of (2); mp 230 –231°C; lit. (2, 9) 230 –231°C. The product was homogeneous by TLC (SiO2: 10% MeOH/EtOAc) and was used without further purification. This material, however, can be recrystallized from MeOH (25 mL/g) to afford white needles, mp 239.5–240°C. 1H NMR (dimethyl sulfoxide [DMSO]-d6) ␦: 1.78 (3H, d, J ⫽ 3Hz), 2.46 (1H, tq, H-2⬘), 2.57 (1H, dd, H-2⬘⬘), 3.50 (2H, m, H-5⬘/5⬘⬘), 4.20 (1H, td, H-4⬘), 5.05 (1H, t, 5⬘-OH), 5.25 (1H, H-4⬘), 5.83 (1H, d, J ⫽ 4Hz, H-1⬘), 7.59 (1H, d, J ⫽ 3Hz, H-6).

Step B: Synthesis of 1-(2-deoxy-␤-D-threopentofuranosyl)thymine (3) To a boiling solution of (2) (17.57 g, 78 mmol) in water (200 mL) was added LiOH-hydrate (3.45 g, 82 mmol). The solution was refluxed for 10 min and then cooled to 10°C and treated in the cold with Dowex 50W ion exchange resin (H⫹ form, 50 g). The pH of the solution was neutral after 10 min of agitation. The resin was filtered off and then washed with water. Ethanol (5 mL) and concentrated aqueous NH4OH (few drops) were added to the combined filtrates and then the mixture was concentrated to a glass. The glass was further dried by azeotropic distillation with MeCN (2 ⫻ 200 mL) and the formed solid was vacuum-dried at room temperature to afford 16.37 g (86% yield) of (3); mp 163–165°C. The product was homogeneous by TLC (SiO2: EtOAc), but can be recrystallized from MeCN (50 mL/g) to afford a fluffy white solid (75% recovery, mp 172–174°C, lit. (9) 169 –170°C). 1H NMR (DMSO-d6) ␦: 1.75 (3H, d, J ⫽ 3Hz), 1.80 (1H, dd, J ⫽ 2, 15 Hz, H-2⬘), 2.50 (1H, m, H-2⬘⬘), 3.60 (2H, m, H-5⬘/5⬘⬘), 3.65 (1H, m, H-4⬘), 4.18 (1H, m, H-3⬘), 4.64 (1H, t, J ⫽ 5.5 Hz, 5⬘-OH), 5.20 (1H, d, J ⫽ 3.4 Hz, 2⬘-OH), 6.00 (1H, dd, J ⫽ 2.5, 8.5 Hz, H-1⬘), 7.78 (1H, s, H-6), 11.20 (1H, s, NH).

Step C: Synthesis of 1-(2-deoxy-O3,O5-isopropylidine-␤D-threo-pentofuranosyl)thymine (4) A mixture of the diol (3) (13.37 g, 55 mmol) and pyridinium p-toluenesulfonate (PPTS, 300 mg) in dry acetone (400 mL) was

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FIG. 2. Chemical synthesis of the labeling precursor (8). Reagents: (a) 2 equiv. DIAD/TPP, MeCN, <ⴚ15ⴗC, then water; (b) LiOH (1 equiv.)/water, then Hⴙ-resin; (c) acetone/PPTS (cat), reflux; (d) 2,4-DMBnCl, K2CO3/MEK, reflux, phase transfer catalyst; (e) EtOH-water, PPTS (cat), reflux; (f) DMTrCl, pyr, rt; (g) 4-NBS-Cl/AgOTf, pyr, 0ⴗC; (h) K2CO3/KRY (2.2.2)/[18F]fluoride (n.c.a.), MeCN, 100ⴗC, 10 min; (i) CAN, MeCN-EtOH-water (4:1:1), 100ⴗC, 3 min; and (j) C-18 chromatography. refluxed under argon for 20 h. After 3 h, the solids had dissolved and the reaction was followed by TLC (SiO2: EtOAc). After 18 h, the cooled reaction mixture was concentrated to a translucent stiff gel. This material was dissolved in a minimum of hot EtOAc and then the warm solution was rapidly filtered through a column of silica gel (10 ⫻ 5 cm) in EtOAc. The column was eluted with EtOAc to obtain the acetonide (4). Concentration of selected column fractions afforded a colorless gel. A crystalline product was obtained by dissolving the gel in 100 mL of hot EtOAc, followed by slow (20 min) atmospheric distillation of the solvent until the first sign of crystalline material. After crystallization was complete at room temperature, the stiff white paste was triturated with hexanes and then the solid was collected to obtain 13.73 g (88% yield) of (4) as a white fluffy solid (mp 167–169°C; lit. (36) 169 –170°C). The product was homogeneous by TLC and NMR. 1H NMR (CDCl3) ␦: 1.37 (3H, s), 1.49 (3H, s), 1.95 (3H, br d, C-5 Me), 2.19 (1H, dd, H-2⬘), 2.60 (1H, ddd, H-2⬘⬘), 3.82 (1H, br dd), 4.20 (2H, m, H-5⬘,5⬘⬘), 4.46 (1H, dd), 6.18 (1H, dd, H-1⬘), 8.0 (1H, s, H-6), 8.98 (1H, br s, NH).

Step D: Synthesis of 1-(2-deoxy-O3,O5-isopropylidine-␤-Dthreo-pentofuranosyl)-3-(2,4-dimethoxybenzyl)thymine (5) To a cold (0°C) stirred mixture of phosgene in toluene (13 mL, 20% w/w) under argon was added, dropwise over 10 min, a solution of

2,4-dimethoxybenzyl alcohol (1.5 g, 8.9 mmol) in dry CH2Cl2 (20 mL). After the addition was complete, the mixture was stirred at room temperature and then concentrated (vacuum line) to yield approximately 5 mL of a colorless viscous solution. (The solution must not be freed of all solvent, because spontaneous polymerization will occur to yield a violet solid. In that event the reaction should be abandoned.) The solution of the crude chloroformate was diluted with methyl ethyl ketone (MEK, 10 mL) and then the mixture was added to a hot suspension of powdered K2CO3 (12 g), compound (4) (1.00 g, 3.5 mmol) and benzyltributylammonium chloride (BTBACl, 0.1 g) in MEK (60 mL). The mixture was rapidly stirred and refluxed under argon overnight. Two new materials were identified (TLC: SiO2, EtOAc) in this reaction, which had Rf’s higher and lower than the alcohol. The lower Rf product was compound (5) and the higher Rf material was not a nucleoside. The cooled reaction was filtered and the combined filtrates were treated with 1:1 pyridine water (4 mL) for 4 h, then the volatile materials were removed and the residue was azeotropically dried with toluene. The crude product was filter chromatographed (SiO2: 80% EtOAc/ Hex) to afford a white solid, which was recrystallized from EtOAc to afford 0.96 g (63% yield) of (5); mp 164 –165°C. This method has been repeated several times with equivalent results. Pure (5) exhibits mp 167–168°C. 1H NMR (CDCl3) ␦: 1.40 (3H, s), 1.50 (3H, s), 1.98 (3H, d), 2.16 (1H, dd, H-2⬘), 2.57 (1H, ddd, H-2⬘⬘), 3.76 (3H, s), 3.80 (1H, dd), 3.84 (3H, s), 4.19 (2H, m, H-5⬘,5⬘⬘),

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4.44 (1H, dd), 5.12 (2H, AB quartet, benzylic), 6.19 (1H, dd, H-1⬘), 6.35– 6.95 (3H, aromatic), 8.02 (1H, d, H-6); Anal Calcd for C22H28N2O7: C, 61.10; H, 6.53; N, 6.48 Found: C, 61.28; H, 6.63; N, 6.36.

Steps E, F, and G: Syntheses of 1-(2-deoxy-␤-D-threopentofuranosyl)-3-(2,4-dimethoxybenzyl)thymine (6); 1(2-deoxy-5-O- (4,4ⴕdimethoxytrityl)-␤-D-threopentofuranosyl)-3-(2,4-di-methoxybenzyl)thymine (7); and 1-(2-deoxy-3-O-(4-nitrobenzenesulfonyl)-5-O-(4,4ⴕdimethoxy-trityl)-␤-D-threo-pento-furanosyl)-3-(2,4dimethoxybenzyl)thymine (8) (Nosylate Labeling Substrate) A mixture of (5) (1.26 g, 2.9 mmol) and PPTS (50 mg) in EtOH-water (6:1, 70 mL) was refluxed for 9 h. The volatile materials were removed and the residue was dried by azeotropic distillation with MeCN and then toluene to afford crude compound (6). This material was dissolved in dry pyridine (5 mL) and then treated with DMTrCl (1.13 g, 3.3 mmol) at room temperature. After an overnight reaction, the mixture was partitioned between EtOAc and saturated aqueous NaHCO3 and then the organic extract was washed with brine. The extract was dried (MgSO4) and concentrated and then the residue was freed of pyridine by coevaporation with portions of toluene. The crude product was adsorbed onto silica, then filter chromatographed (SiO2: 60% EtOAc/Hex) to afford 1.47 g of (7) as a rigid yellow foam, after vacuum drying. A portion of isolated (7) (0.74 g, approximately 1.1 mmol) was dissolved in dry pyridine at 0°C and then treated successively with 4-nitrobenzenesulfonyl chloride (0.705 g, 3.2 mmol) and silver trifluoromethanesulfonate (0.818 g, 3.2 mmol). The reaction was kept in the cold for 30 min and then warmed to room temperature for 1 h. The reaction was quenched by adding water (few drops) and then diluted with EtOAc (150 mL), followed by filtration with diatomaceous earth. The filtrate was washed with water and then brine, followed by drying (MgSO4) and concentration. Pyridine was removed by co-evaporation with toluene and the residue was filter chromatographed (SiO2: 40%, 50% EtOAc/Hex) to afford (8) (0.60 g, 48% overall yield from (5)) as a rigid bright yellow foam, after vacuum drying. Solutions of (8) in MeCN (26 mg/mL) are practically colorless. 1H NMR (CDCl3) ␦: 1.77 (3H, d, J ⫽ 1.5 Hz, C-5 Me), 2.50 (1H, dd, J ⫽ 2.6, 16.2 Hz, H2⬘), 2.73 (1H, ddd, J ⫽ 5.2, 7.5, 16.2 Hz, H2⬘⬘), 3.19 (1H, dd, J ⫽ 5.5, 10.3 Hz, H5⬘), 3.54 (1H, dd, J ⫽ 6.3, 10.3 Hz, H5⬘⬘), 3.75 (3H, s, MeO), 3.81 (6H, s, 2 ⫻ MeO), 3.83 (3H, s, MeO), 4.17 (1H, m, J ⫽ 3.5, 5.5, 6.3 Hz, H4⬘), 5.07 (2H, center of AB quartet, J ⫽ 14.8 Hz, benzylic), 5.17 (1H, br t, J ⫽ 3.9 Hz, H-3⬘), 6.22 (1H, dd, J ⫽ 2.6, 7.5 Hz, H1⬘), 6.39 (1H, dd, J ⫽ 2.4, 8.4 Hz, aromatic-H5 of 2,4-DMBn), 6.44 (1H, d, J ⫽ 2.4 Hz, aromatic-H3 of 2,4-DMBn), 6.82 (4H, d, J ⫽ 8.7 Hz, aromatic), 6.94 (1H, d, J ⫽ 8.4Hz, aromatic-H6 of 2,4-DMBn); 7.20 –7.38 (10H, overlapping multiplets, aromatic and pyrimidineH6), 7.83 (2H, d, J ⫽ 8.7 Hz, aromatic-H2/6 of nosylate), 8.17 (2H, d, J ⫽ 8.7 Hz, aromatic-H3/5 of nosylate); LRMS (ESI⫹) m/z Calcd for C46H46N3O13S (M⫹H)⫹ 880.3. Found: 880.3; Anal Calcd for C46H45N3O13S: C, 63.79; H, 5.15; N, 4.78. Found: C, 63.72; H, 5.39; N, 4.61. From other experiments purified samples of (6) and (7) were isolated. Compound (6) was purified by filter chromatography (SiO2: EtOAc) to afford a rigid white foam after vacuum drying of the isolated oil. 1H NMR (CDCl3) ␦: 1.90 (3H, d, C-5 Me), 2.18

J. R. Grierson & A. F. Shields

(1H, dd, H-2⬘), 2.55 (1H, ddd, H-2⬘⬘), 3.18 (1H, br s), 3.75 (3H, s), 3.82 (3H, s), 3.87 (1H, dd), 4.03 (1H, br s), 4.24 (1H, br d), 4.46 (1H, br dd), 5.08 (2H, br s, benzylic), 6.10 (1H, dd, H-1⬘), 6.35– 6.90 (3H, multiplets, aromatic), 7.80 (1H, d, H-6). Compound (7) was purified by filter chromatography (SiO2: 60% EtOAc/Hex) to afford a rigid white foam after vacuum drying of the isolated oil. 1H NMR (CDCl3) ␦: 1.84 (3H, d, C-5 Me), 2.09 (1H, dd, H-2⬘), 2.57 (1H, dd, H-2⬘⬘), 3.13 (1H, d, 3⬘-OH), 3.52 (1H, dd, H-5⬘), 3.62 (1H, dd, H-5⬘⬘), 3.79 (3H, s), 3.77 (6H, s, 2 ⫻ MeO), 3.82 (3H, s), 4.00 (1H, m , H-4⬘), 4.44 (1H, m H-3⬘), 5.10 (2H, AB quartet, benzylic), 6.21 (1H, dd, H-1⬘), 6.35– 6.44 (2H, aromatic), 6.81–7.47 (14H, aromatic), 7.67 (1H, d, H-6).

Steps H, I, and J: Synthesis and Purification of [18F]FLT [18F]Fluoride in enriched 18O water was produced as previously described (10) and then separated from recovered target water (3 mL) by passing the solution through a short plug of ion exchange resin (28) (BioRad AG 1-X8, 12–15 mg, OH⫺ form, 200 – 400 mesh). Resin-bound activity was eluted with aqueous 0.1 M potassium carbonate (200 ␮L). The eluate was collected in a new borosilicate test tube (100 ⫻ 16 mm i.d., Kimax 51) containing Kryptofix [2.2.2] (15 mg, 40 ␮mol). The test tube containing the solution of [18F]fluoride, potassium carbonate, and Kryptofix [2.2.2] was placed in an aluminum dry block at 100°C and then water was removed by azeotropic distillation with MeCN (4 ⫻ 1 mL) under argon. The final residue was solubilized in 2 mL of dry MeCN, and then compound (8) (25 mg, 28 ␮mol) in dry MeCN (1 mL) was added. Within seconds the solution became intensely blue; the reaction was continued for 10 min. (This same color also develops in the absence of [18F]fluoride) During this reaction, no attempt was made to protect the solution from atmospheric moisture, but MeCN gently distilled from the open test tube while the solution was reduced to approximately 2 mL. With the aid of the apparatus shown in Figure 3 (left), the cooled (to room temperature) reaction mixture was loaded in the opened syringe on top of the alumina (neutral) SepPak, and then the solution was pulled through by suction (large syringe). The alumina was washed with MeCN (2 ⫻ 1.5 mL) and then the collection tube was opened and the eluate concentrated at 100°C under argon. The oily dark blue residue was treated at 100°C with ceric ammonium nitrate (CAN; 40 mg, 73 ␮mol) in 4:1:1 MeCN/EtOH/water (600 ␮L). After 3 min the hot mixture (orange) was quenched with 1 mL of 4% aqueous NaHCO3, forming a thick brown suspension. The mixture was concentrated at 100°C under argon to ⬍1 cc and then diluted with water (2.5 mL). With the aid of the micron filtration apparatus shown in Figure 3 (right), the reaction mixture was loaded into the top syringe, and then forced downward through the filter (0.2 ␮m) and alumina SepPak. The solution path was rinsed in two portions, with 1 mL of water held in the side-mounted syringe on the upper three-way stopcock. With the upper stopcock closed to the lower position, air in the lower side-mounted syringe was used to blow out the SepPak. The collected solution in the tube (approximately 4 mL) was loaded into the injector of a semi-preparative C-18 HPLC system (5 mL sample loop previously loaded with water [USP]). The column (column A) was eluted with 10% EtOH in sterile, pyrogen-free water (USP) at 2 mL/min for 2 min and then at 6 mL/min. The column eluate was monitored, in line, by serial ultraviolet (UV; 267 nm) and gamma-ray detectors and the stream was routed through micron filters (0.2 ␮m) into a septum sealed, sterile, pyrogen-free

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FIG. 3. Apparatus used in the processing of [18F]fluorothymidine (FLT). glass collection vial, or similar waste vial. [18F]FLT is typically isolated in ⬍10 mL of solution after 10 –13 min (Fig. 4A). Purified [18F]FLT solution was made isotonic by adding enough 23.4% saline (USP) to achieve a 0.9% concentration. Postchromatography, the column was rinsed with 50% EtOH-water (USP) for 15 min at 3 mL/min. The column rinse was sufficient to remove residual UV active materials. When not in use the column was stored in 50% EtOH-water.

Optimization of FLT’s HPLC Purification (Fig. 5) FLT (40 ␮L, 1 mg/mL) was injected undiluted or diluted (4 mL) in either water or 10% EtOH-water. Each of these solutions was loaded (5 mL sample loop) onto the HPLC column used to purify [18F]FLT and then eluted at 6 mL/min with 10% EtOH-water. The chromatograms for these injections (A, 40 ␮L undiluted injection; B, FLT in 4 mL 10% EtOH-water; and C, FLT in 4 mL water) demonstrate that the C18 HPLC column has the ability to pre-concentrate FLT from a large injection volume of water.

Synthesis of Calibration Standards for Chromatography A C18 HPLC chromatogram representing a mixture of the authentic compounds prepared below appears in Figure 6 (top). SYNTHESIS OF 5ⴕ-O-DIMETHOXYTRITYL-FLT (11) (FIG. 7, SCHEME 2). A solution of FLT (39 mg, 0.16 mmol) and 4,4⬘-dimethoxytrityl

chloride (DMTrCl, 65 mg, 0.19 mmol) in dry pyridine (3 mL) was stirred at room temperature overnight and then partitioned in 1:1 brine:water. The organic extract was washed with brine, dried, and concentrated. The residue was dried further by azeotropic distillation of toluene and then filter chromatographed (SiO2: 30, 50, 70, 100% EtOAc/Hex) to afford compound (11) (65 mg, 75% yield) as a faint yellow rigid foam after vacuum drying. 1H NMR (CDCl3) ␦: 1.42 (3H, d, J ⫽ 1.1 Hz, C5-Me), 2.33 (1H, dddd, JHF ⫽ 39.6 Hz, H-2⬘), 2.67 (1H, ddd, JHF ⫽ 21 Hz, H-2⬘⬘), 3.38 (1H, dd, H-5⬘), 3.53 (1H, br dd, H-5⬘⬘), 3.80 (6H, s, 2 ⫻ MeO), 4.33 (1H, br dt, JHF ⫽ 28.1 Hz, H-4⬘), 5.30 (1H, dd, JHF ⫽ 53.9 Hz, H-3⬘), 6.48 (1H, dd, H-1⬘), 6.82 (4H, d, Ar-H), 7.20 –7.40 (9H, multiplets, Ar-H), 7.60 (1H, br q, J ⫽ 1.5 Hz, H-6), 8.55 (1H, br s, N-H). SYNTHESIS OF 1-(2,3-DIDEOXY-3-FLUORO-5-O-(4,4ⴕ-DIMETHOXYTRITYL)␤-D-ERYTHRO-PENTOFURANOSYL)-3-(2,4-DIMETHOXYBENZYL)THYMINE (9) (FIG. 7, SCHEME 1). A stirred room temperature solution of compound (7) (200 mg, 0.29 mmol) and pyridine (35 ␮L, 0.43 mmol) in dry CH2Cl2 (3 mL) under argon was treated with DAST in one portion. The reaction was followed by TLC (SiO2: 60% EtOAc/ Hex) and then quenched after 1 h with saturated aqueous NaHCO3 (2 mL). Ethyl acetate (50 mL) was added and then the organic solution was dried and concentrated. The residue was filter chromatographed (SiO2: 30, 50, 100% EtOAc/Hex) to isolate compound (9) (89 mg, 44% yield) as a rigid colorless foam after vacuum drying. Compound (9) appeared on TLC as the more mobile of the

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FIG. 4. Chromatographic data for the purification and analysis of [18F]fluorothymidine (FLT). (A) Semi-preparative purification of [18F]FLT, column A, 10% EtOH eluted at 3 mL/min (2 min) then 6 mL/min. (B) Analytical high performance liquid chromatography (HPLC) of purified [18F]FLT, column B, 10% EtOH at 1 mL/min. (C) HPLC calibration curve of curve used for specific activity calculations. two reaction products. 1H NMR (CDCl3) ␦: 1.50 (3H, d, J ⫽ 1.5 Hz, C5-Me), 2.31 (1H, dddd, JHF ⫽ 38.7 Hz, H-2⬘), 2.66 (1H, ddd, JHF ⫽ 16.3 Hz, H-2⬘⬘), 3.37 (1H, dd, H-5⬘), 3.52 (1H, dd, H-5⬘⬘), 3.76 (3H, s, MeO), 3.78 (6H, s, 2 ⫻ MeO), 3.84 (3H, s MeO), 4.32

(1H, br dt, JHF ⫽ 28.4Hz, H-4⬘), 5.12 (2H, center of AB quartet, benzylic), 5.28 (1H, dd, JHF ⫽ 53.6 Hz, H-3⬘), 6.40 (1H, dd, aromatic-H5 of 2,4-DMBn), 6.45 (1H, d, aromatic-H3 of 2,4DMBn), 6.53 (1H, dd, H-1⬘), 6.84 (4H, d, aromatic), 6.97 (1H, d,

FIG. 5. Optimization of the purification of fluorothymidine (FLT). Same mass of FLT injected onto column A as a solution in (A) 40 ␮L water, (B) 4 mL of 10% EtOH, and (C) 4 mL of water.

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course of the reaction. The volatile materials were removed and then the residue was adsorbed onto silica, followed by filter chromatography (SiO2: 30, 100% EtOAc/Hex) to afford compound (12) (46 mg, 87% yield) as a colorless glass after vacuum drying. 1H NMR (CDCl3) ␦: 1.31 (3H, d, J ⫽ 2 Hz, C5-Me), 3.38 (2H, center of AB quartet of d, H-5⬘/5⬘⬘), 3.76 (3H, s, MeO), 3.767 (3H, s, MeO), 3.771 (3H, s, MeO), 3.82 (3H, s, MeO), 4.97 (1H, diffuse m, vinyl), 5.14 (2H, AB quartet, benzylic), 5.89 (1H, dq, vinyl), 6.34 (1H, dt, H-1⬘), 6.40 (1H, dd, aromatic-H5 of 2,4-DMBn), 6.44 (1H, d, aromatic-H3 of 2,4-DMBn), 6.81 (4H, d, aromatic), 6.96 (1H, d, aromatic-H3 of 2,4-DMBn), 7.10 (1H, dt), 7.24 –7.32 (6H, multiplets, aromatic), 7.38 –7.44 (3H, multiplets, aromatic).

Conversion of FLT into a Mixture of Its Nucleoside Isomers with Iodotrimethylsilane

FIG. 6. High performance liquid chromatography of calibration standards and the label incorporated product pro[18F]fluorothymidine (FLT). Column B: 70% MeCN-water, 1 mL/min. aromatic-H6 of 2,4-DMBn), 7.20 –7.40 (9H, multiplets, aromatic), 7.6 (1H, br s, H-6). SYNTHESIS

OF

1-(2,3-DIDEOXY-3-FLUORO-␤-D-ERYTHRO-PENTO-

A stirred room temperature solution of FLT (40 mg) in dry degassed MeCN (5 mL) under argon was treated with neat iodotrimethylsilane (TMSI, 10 ␮L). On contact with the reagent, a faint orange-brown solution formed. The mixture was stirred overnight and then solid NaHCO3 (0.5 g) was added, followed by saturated aqueous NaHCO3 (0.1 mL). After stirring vigorously for 10 min, the mixture was allowed to stand and then the MeCN solution was decanted. The solution was concentrated to dryness and then the residue was dissolved in 1:1 MeCN-water. An analysis of this mixture by HPLC (Fig. 8) revealed the presence of thymine (3.8 min), FLT (10.0 min), and three other components (A [5.4 min], B [6.5 min], and C [10.9 min]) in the ratio of 0.1:0.5:1.5:1.0:0.9 for Thy/A/B/FLT/C. This mixture was further subjected to LC-MS analysis (column C: 10% EtOH-water, 0.24 mL/min, UV 267 nm; MS: ESI⫺) that showed that compounds A, B, and C and FLT had the same molecular weight (m/z 243.2 [M-H]) and produced thymine as a fragment ion (m/z 124.9 [M-H]). Based on a presumed similarities in HPLC retention times in Figure 8, compounds A and B were tentatively assigned as the pair of 3⬘-deoxy-3⬘-fluoronucleoside ␣/␤-pentopyranoside isomers (12/13) and compound C as the 3⬘-deoxy-3⬘-fluoronucleoside ␣-pentofuranoside isomer (14) (Fig. 9).

FURANOSYL)-3-(2,4-DIMETHOXYBENZYL)-THYMINE (10) (FIG. 7, SCHEME 1).

A solution of compound (9) (31 mg) and PPTS (16 mg) in 5:1 EtOH:water (12 mL) was refluxed for 10 min, then a few drops of diisopropylamine was added and the mixture was concentrated to a moist film. (The addition of diisopropylamine is necessary to avoid reformation of compound (9) during solvent and water removal.) This material was dried by co-evaporation with MeCN and then filter chromatographed (SiO2: 30, 100% EtOAc/Hex) to afford compound (10) (15 mg, 83% yield) as a colorless glass after vacuum drying. 1H NMR (CDCl3) ␦: 1.93 (3H, d, J ⫽ 1.2 Hz, C5-Me), 2.42–2.60 (2H, multiplets, H-2⬘/2⬘⬘), 3.00 (1H, br m, 5⬘-OH), 3.76 (3H, s, MeO), 3.82 (3H, s, MeO), 3.80 –3.88 (2H, diffuse multiplet, H-5⬘/5⬘⬘), 4.29 (1H, br d, JHF ⫽ 25.5 Hz, H-4⬘), 5.10 (2H, center of AB quartet, benzylic), 5.30 (1H, br d, JHF ⫽ 54Hz, H-3⬘), 6.17 (1H, dd, H-1⬘), 6.38 (1H, dd, aromatic-H5 of 2,4-DMBn), 6.42 (1H, d, aromatic-H3 of 2,4-DMBn), 6.93 (1H, d, aromatic-H6 of 2,4-DMBn), 7.40 (1H, d, H-6). SYNTHESIS OF 1-(2,3-DIDEOXY-5ⴕ-O-(4,4ⴕ-DIMETHOXYTRITYL)-␤-D-GLYCERO-PENT-2-ENOFURANOSYL)-3-(2,4-DIMETHOXYBENZYL)THYMINE

(12)

A solution of compound (8) (74 mg, 0.083 mmol) in dry MeCN (3 mL) was treated with excess TBAF/THF (1 M, 200 ␮L) for 20 min at room temperature, and then at 100°C for 2 min. On contact with TBAF the solution of (8) turned deep red and this color changed to green over the

(ELIMINATION PRODUCT; FIG. 7, SCHEME 3).

Sensitivity of FLT to Oxidation by CAN FLT (10 mg) was treated with CAN (23 mg, 1 equiv.) in 4:1:1 MeCN:EtOH:water (1 mL) at room temperature and then refluxed for 10 min. In addition, an identical sample of FLT was treated with 3 equivalents of CAN at reflux for 10 min. The room temperature reaction was analyzed by LC-MS (as above) after 15 and 60 min, and after refluxing the solution. Similarly, the reaction of FLT with excess CAN was analyzed after refluxing. From these reactions, three new compounds (D, E, and F) were observed and gave mass ions (M-H) that were assigned to compounds resulting from the oxidation of FLT’s C-5 methyl group, first to the hydroxymethyl (compound E) and then to the aldehyde (compound F), as well as some sugar-base cleavage to give compound D (uracil-5-carboxyaldehyde). Figures 10 and 11 summarize the HPLC and MS data.

Kryptofix and Cerium Salt Removal from Mixtures with FLT Passage of Kryptofix [2.2.2] and cerium salts through the FLT synthesis process was examined in modeled reactions. Removal of Kryptofix by neutral alumina, prior to the deprotection of pro-FLT and subsequently during the HPLC purification of FLT, was determined by 1H NMR.

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FIG. 7. Syntheses of some authentic fluorothymidine (FLT) standards KRYPTOFIX-ALUMINA TREATMENT. Kryptofix [2.2.2] (16.8 mg) was dissolved in 0.1 M K2CO3 (0.2 mL) and then dried by azeotropic distillation with MeCN (4 ⫻ 1 mL). The residual film was dissolved in MeCN (3 mL, 100°C) and then the cooled (room temperature) solution was passed through a neutral alumina SepPak. The SepPak was washed with dry MeCN (2 ⫻ 1 mL). The

combined eluates were concentrated under argon and then the residue was dissolved in D2O (1.68 mL). A portion of this solution (600 ␮L) was diluted with sodium 3-(trimethylsilyl)-1-propanesulfonic acid (TPS) in D2O (200 ␮L, 0.05 M). A second solution of Kryptofix [2.2.2] in D2O (16.8 mg/1.68 mL) was prepared, and then sampled and diluted with the standard TPS solution, as before. Comparison of these two Kryptofix [2.2.2] solutions by 1H NMR revealed 10-fold less (92:8) Kryptofix [2.2.2] in the SepPak treated sample. HPLC REMOVAL OF KRYPTOFIX. Kryptofix [2.2.2] (20 mg) in water (3.5 mL) was diluted with a solution of FLT (0.2 mL, 1 mg/mL). The mixture was injected onto column C, and the column was eluted (10% EtOH) in the manner used to purify [18F]FLT. Three eluate fractions were collected: 1) material eluting prior to FLT, 2) a FLT fraction, and 3) a post-FLT/column wash fraction (obtained with 90% MeCN-water). Kryptofix was only found in the post-FLT fraction (1H NMR). Moreover, LC-MS analysis of the FLT fraction determined ⬍1 ␮g of contaminating Kryptofix.

FIG. 8. High performance liquid chromatography of fluorothymidine (FLT) nucleoside isomers: products from the isomerization of FLT with iodotrimethylsilane (see Fig. 9 for product structures). Column B: 10% EtOH-water, 1 mL/min.

PRECIPITATION OF CERIUM SALTS. Passage of cerium salts through the FLT synthesis process was determined using tracer 141Ce. A full-scale mock labeling reaction was performed (26 mg labeling substrate, 18 mg Kryptofix, 200 ␮L 0.5 M carbonate, 40 mg unlabeled CAN). During the deprotection step, however, the CAN

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FIG. 9. Structural assignments for compounds A, B, and C in Figure 8. Fluorothymidine (FLT) nucleoside isomers from the reaction of FLT with iodotrimethylsilane.

reaction was first concentrated to a residue and then 141Ce trichloride (0.49 mCi, 50 ␮L in 0.5 M HCl) and water (1 mL) were added and the turbid mixture was allowed to stand for a few minutes before adding bicarbonate (1 mL, 4% solution). Addition of the bicarbonate precipitated cerium salts from solution. The whole mixture was processed using the filtration apparatus shown in Figure 3. Breakthrough of cerium through the filter/alumina SepPak unit was 0.2 ␮g (6 ppm, based on 40 mg CAN) and ⬎99% of the radioactivity was found on the micron filter unit. HPLC REMOVAL OF CERIUM SALTS. To see how easily soluble cerium salts in the filtrate solution would subsequently pass through a HPLC column, a pair of C18 SepPaks in tandem were treated with the filtrate solution. The loaded SepPaks were washed with water and then purged with air. The SepPaks were cut open and the media was extruded and counted. The C18 media retained 66% of the initial cerium load. The interpretation is that the SepPak columns capture most of the sub-micron sized Ce(OH)3 particles by a sieving process.

FIG. 10. High performance liquid chromatography of fluorothymidine (FLT) oxidation products derived from ceric ammonium nitrate (see Fig. 11 for product structures). Column B: 10% EtOHwater, 1 mL/min.

Solid State Structure of (5) A colorless, plate shaped crystal 0.39 ⫻ 0.34 ⫻ 0.17 mm was mounted on a glass capillary in epoxy. Data was collected at ⫺112°C with three sets of exposures (Table 1). The crystal-to-detector distance was 27 mm and the exposure time was 30 sec for all sets. The scan width was 1 degree. Data collection was 95.9% complete to 30.51 degrees in ␪. A total of 72,848 partial and complete reflections were collected, covering indices h ⫽ ⫺11–11, k ⫽ ⫺34 –33, and l ⫽ ⫺14 –14. There were 6,289 symmetry independent reflections and the Rint ⫽ 0.037 indicated that the quality of the data was good. Indexing and unit cell refinement, based on 1,358 reflections, indicated a monoclinic lattice. The spacegroup was found to be P21 (No. 4) based on symmetric absences 0k0; k ⫽ 2n. The spacegroup P21/m has the same symmetric absences; however, this space group is not possible because the compound is chiral. Solution by direct methods produced a complete heavy atom-phasing model consistent with the proposed structure. There are two independent molecules in the

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FIG. 11. Structural assignments for fluorothymidine (FLT) oxidation products D, E, and F in Figure 10 with mass spectral and ultraviolet correlations.

asymmetric unit giving Z ⫽ 4. All hydrogen atoms were located by difference Fourier synthesis and refined with a riding model. Uiso values were fixed such that they were 1.1 Ueq of their parent atom and 1.5 Ueq for the methyl Hs. All other nonhydrogen atoms were refined anisotropically by full-matrix least squares. There were no heavy atoms present and none of the reflections were sensitive to anomalous dispersion. Therefore, it was not possible to confirm the absolute configuration. In fact, refinement of the inverted structure led to the same R-value. The ORTEP diagram for compound (5) is shown in Figure 12. Empirical formula: C22H28N2O7 Formula weight: 432.46 Temperature: 161(2) K Wavelength: 0.71070 Å (MoK␣) Crystal system, spacegroup: Monoclinic, P21 (No. 4) Unit cell dimensions: a ⫽ 8.32240 (10) Å; b ⫽ 24.2481(4) Å; c ⫽ 10.42400 (10) Å; ␤ ⫽ 91.0860 (1) deg Volume: 2,103.21 (5) Å3 Z, Calculated density: 4, 1.366 mg/mm3 Reflections used for indexing: 1,358 Absorption coefficient: 0.102 mm⫺1 F(000): 920 Crystal description/color: plate/colorless Crystal size: 0.39 ⫻ 0.34 ⫻ 0.17 mm Theta range for data collection: 2.13–30.51 degrees Index ranges: ⫺11 ⬍ h ⬍ 11, ⫺34 ⬍ k ⬍ 33, ⫺14 ⬍ l ⬍ 14 Reflections collected/unique: 72,848/6,289 [Rint ⫽ 0.037] Completeness to 2␪ ⫽ 30.51: 95.9% Absorption correction: none Refinement method: Full-matrix least-squares on F2 Data/restraints/parameters: 6,289/0/559 Goodness-of-fit on F2: 1.078 Final R indices [I ⬎ 4␴(I)]: R1 ⫽ 0.0389, wR2 ⫽ 0.0951 R indices (all data) R1 ⫽ 0.0509, wR2 ⫽ 0.0990

Weighting scheme: calc. w ⫽ 1/[s2(F2o) ⫹ (0.0650P)2 ⫹ 0.0000P], where P ⫽ (F2o ⫹ 2F2c )/3 Absolute structure parameter: ⫺0.6 (6) Largest difference between peak and hole: 0.192 and ⫺0.259 electrons/Å3

RESULTS AND DISCUSSION In developing our no carrier added radiosynthesis of [18F]FLT we screened several labeling precursors and found that: 1) labeling precursors with masked pyrimidine-NH such as N-2,4-dimethoxybenzyl were essential to obtain reasonable yields of FLT, 2) precursors with N-alkyl groups performed better than N-acyl compounds, and 3) the nosylate leaving group gave the best compromise between stability and reactivity (13–15). Figure 13 illustrates our current three-step synthesis of [18F]FLT. The standard preparation of [18F]fluoride in acetonitrile with Kryptofix [2.2.2] and potassium carbonate in anhydrous MeCN was preferred for the labeling reaction, because we anticipate that most systems devoted to preparing 18F-labeled compounds can be adapted for the synthesis of [18F]FLT. The high volatility and water solubility of MeCN has also proven useful to us. CAN was chosen to deprotect pro-[18F]FLT based on several factors: 1) CAN is an easily handled, potent oxidant toward electron rich aromatic and benzylic systems, yet it is largely unreactive toward primary alcohols (17), 2) the mild acidic nature of aqueous CAN solutions facilitates removal of the dimethoxytrityl group (DMTr), and 3) based on the low solubility of cerium salts in basic solutions, these toxic salts can be removed after use by an efficient precipitation (6). Figure 2 shows the synthesis of the labeling precursor compound (8) prepared in seven steps from thymidine in 17% overall yield. Introduction of the 2,4-dimethoxybenzyl group on compound (5) proved difficult. Although a Mitsunobu reaction between the corresponding 3⬘,5⬘-isopropylidene-NH-nucleoside (4) and the

TABLE 1. Scan Data for Diffraction Data Collection Scan set 1 2 3

Scan type

Scan range (deg)

␪ (deg)

␾/␻ (deg)

␬ (deg)

␾ ␻ ␻

⫺208.5 to 93.5 ⫺176.6 to 145.0 ⫺210.0 to 152.8

4.303 ⫺1.500 ⫺1.479

180 ⫺126.789 59.633

0 ⫺100 ⫺82.891

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FIG. 12. ORTEP diagram for compound (5) benzyl alcohol can be used for the alkylation (15), the yield was unattractive (6 –16%) and a more practical method was sought. Our solution was to alkylate the nucleoside with 2,4-dimethoxybenzyl chloride, which can be done in moderate yield (63%) under phase transfer catalysis. However, this alkylating agent is too unstable to be isolated, because of its rapid and unpredictable polymerization. Nevertheless, the chloride can be conveniently prepared in situ by the phase transfer catalyzed thermal decarboxylation of 2,4-dimethoxybenzyl chloroformate (K2CO3/MEK, benzyltributylammonium chloride catalyst). Although the decarboxylation reaction has precedent (8) we believe this is the first time it has been used for synthetic purposes. The needed chloroformate is prepared in situ from 2,4-dimethoxybenzyl alcohol with phosgene in toluene/dichloromethane, but must be used quickly (see Materials and Methods). Compound (5) was the only crystalline material with all the necessary stereocenters and nontrivial protecting groups in place. Therefore, we confirmed its structure by X-ray crystallography. An ORTEP diagram for compound (5) is shown in Figure 12 and correctly shows an N-alkylated pyrimidine. This X-ray structure ruled out the possibility that we were dealing with the isomeric O-alkylated compound that could be formed by alternative O-

alkylation of the pyrimidine enol with highly reactive 2,4-dimethoxybenzyl chloride. The best method for introducing the nosylate group in compound (8) was to use a combination of 4-nitrobenzenesulfonyl chloride and silver triflate in pyridine. This reagent mixture greatly accelerated the otherwise slow nosylate formation and, under these conditions, 4-nitrobenzenesulfonyl triflate is the likely sulfonating agent. Although compound (8) is noncrystalline, it can be isolated pure as an amorphous powder and is easily handled and stored. As shown in Figure 13, reaction of (8) (25 mg, 1.4 equivalents vs. K2CO3) with [18F]fluoride for 10 min at 100°C gave pro-[18F]FLT in 38% (EOB) from several hundred millicuries of [18F]fluoride. The consistent yields found for this labeling reaction have markedly improved our ability to prepare doses of [18F]FLT large enough for routine imaging (13). Figure 6 shows a typical radiochromatogram for the labeling reaction product. This product and unreacted fluoride accounts for the activity used. The identity of labeled pro-FLT (9) was confirmed by comparison with a set of unlabeled authentic materials seen in Figure 6 (top). The same result was found by comparisons using silica gel based TLC or HPLC (data not shown). Labeling yields are not limited due to synthesis and then decomposition of pro-[18F]FLT during reaction, because this labeled

FIG. 13. Radiosynthesis of [18F]fluorothymidine (FLT). CAN, ceric ammonium nitrate; HPLC, high performance liquid chromatography; EOB,

154

product can be isolated (HPLC), then re-subjected to the labeling reaction and the activity recovered (⬎90%) as pro-FLT. In addition, we verified that the fluoride routinely prepared for this synthesis is highly reactive (97% radiochemical yield of [18F]-tertBuPh2SiF from BuPh2SiCl; 89% radiochemical yield for the triflate displacement in a [18F]FDG synthesis). Drying of [18F]fluoride and the labeling reaction are done in the same open borosilicate test tube fitted into an aluminum dry block at 100°C. The open test tube method was used to facilitate the processes under robotic control. From experience, this method is as good or better than the same operations done in vented or closed glass vials. Figure 3 shows two simple setups used to separately process the labeling and deprotection reaction mixtures. The purpose of the left unit is to strip unreacted fluoride, Kryptofix, and salts from the labeling reaction mixture onto the alumina SepPak. The whole reaction mixture, loaded into the top syringe, is pulled through the SepPak with suction applied by the larger syringe. This preliminary clean up is necessary for the success of the deprotection of pro-[18F]FLT with CAN, because CAN is deactivated in basic solution. We found that the alumina column removes ⬎90% of the Kryptofix and ⬎95% of [18F]fluoride. After the alumina column is used, it is removed, and the column eluate is concentrated in the collection tube, which is also used for the subsequent CAN deprotection reaction. The deprotection of pro-[18F]FLT with CAN is a quick, 3-min reaction in a small volume of hot MeCN/EtOH/water. Adding aqueous bicarbonate quenches this reaction and forms a sparingly soluble cerium precipitate. It was shown that formation and filtration of the precipitate reduces the soluble cerium content to 6 ppm (0.2 ␮g). However, to later aid in an efficient HPLC purification of [18F]FLT, it is first necessary to evaporate as much of the organic materials as possible before using the filtration unit on the right of Figure 3. This is done because it is possible to load as much as 4 mL of a dilute aqueous FLT solution onto a semi-preparative (250 ⫻ 10 mm) HPLC column, eluted with 10% EtOH-water, and obtain nearly the same retention and peak volume as injecting the same FLT mass in 40␮L. This is shown in Figure 5 and described in the experimental section dealing with HPLC optimization. Operation of the filtration unit in Figure 3 (right) accomplished two tasks: 1) It removed the cerium precipitate over the self-venting micron filter and 2) it removed [18F]fluoride from the crude [18F]FLT preparation by adsorption onto the alumina SepPak. The side-mounted syringes are for flushing the filter and SepPak with water and air. Incorporating the alumina SepPak in the process ensures collection of fluoride-free [18F]FLT. The alumina SepPak treatment is important for efficient purification of [18F]fluoride,

J. R. Grierson & A. F. Shields

TABLE 2. Relative Decay Corrected Distribution of the Synthesis [18F]Fluoride First alumina column Eluted pro-[18F]FLT Micron filter and second alumina column Crude [18F]FLT: HPLC injection Purified [18F]FLT

18

F in

100% 44 38 100% 8 21 22 58 100% 13 34 54

FLT, fluorothymidine; HPLC, high performance liquid chromatography.

because omitting it invariably leads to [18F]fluoride breakthrough during FLT’s HPLC purification. [18F]Fluoride breakthrough is hard to visualize in radiochromatograms, because the bulk of fluoride is seen to clear near the void, but tailing persists throughout column elution. Figure 4A illustrates a typical purification of [18F]FLT on a semi-preparative C-18 column. A simple separation of FLT for other labeled and unlabeled materials is seen. The largest UV active peak displayed is 2⬘,3⬘-didehydro-2⬘,3⬘-dideoxythymidine (d4T), which forms from deprotection of the elimination product seen in Figure 6. Figure 4B shows that there is little if any nucleoside compounds (UV at 267 nm) contaminating purified [18F]FLT. Figure 4C shows that the specific activity of [18F]FLT can be assayed over a wide range. The deprotection reaction with CAN has not been optimized, but currently we use an excess of the reagent (4 equivalent vs. labeling precursor) to ensure extensive reaction in a short time. With this excess, we see evidence of byproduct formation and FLT degradation. For example, Table 2, column 2 (normalized percentages), shows that approximately 20% of the [18F]FLT degrades to [18F]fluoride during the deprotection reaction, and this fluoride can be captured by alumina. We are uncertain how this fluoride release occurs. Handling losses were also significant (approximately 20%) and substantially less [18F]FLT than expected was isolated after its HPLC purification. Because the material injected onto the HPLC column was fluoride-free, washing the column with 50% EtOHwater recovers most of the lipophilic, column-bound activity. The sensitivity of FLT to oxidation by CAN was studied. Figure 10 shows a series of chromatograms and the structures of some products identified by LC-MS (Fig. 11). It is evident that C-5 methyl group on FLT is prone to oxidation and that de-glycosylation is not significant. Possible oxidation of FLT’s primary alcohol is not supported by this data. Based on this study it is hard to reconcile significant degradation of [18F]FLT to [18F]fluoride by CAN.

FIG. 14. Reaction scheme for the divergent reaction of 2,4-DMBn-fluorothymidine (FLT) (10) with ceric ammonium nitrate (CAN). Tentative structural assignment of (G) in Figure 15.

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FIG. 15. Following the oxidation of no carrier added 2,4-DMBn-[18F]fluorothymidine (FLT) with excess ceric ammonium nitrate (CAN). Column B: 45% MeCN-water, 1 mL/min.

It was possible that nucleoside isomers of [18F]FLT, which are more lipophilic than FLT, might be formed during the deprotection reaction and were responsible for the late eluted, column-bound radioactivity (removed with 50% EtOH-water). To test this, a mixture of FLT nucleoside isomers was prepared by treating FLT with iodotrimethylsilane in dry MeCN. Figure 8 shows a chromatogram of these isomers using the same HPLC conditions needed to purify FLT. Although the structural assignments are tentative, all these compounds have the same molecular weights, give thymine as a mass fragment by LC-MS, and exhibit UV maxima at 267 nm. From Figures 4B and 8, it is evident that FLT isomers are not formed during the synthesis of [18F]FLT. Based on the known chemistry of CAN oxidations, it is believed that CAN removes the pyrimidine N-dimethoxybenzyl group on pro-[18F]FLT (9) by benzylic oxidation, forming an intermediate hemiaminal that spontaneously hydrolyzes to give the free nucleoside (17, 38). Because benzylic alcohols are susceptible to oxidation with CAN, we also suspected that the excess of CAN intercepts the hemiaminal before it hydrolyzes, producing 2,4-dimethoxybenzoyl[18F]FLT (DMBz-FLT, Fig. 14). In that case, the product DMBzFLT would be lipophilic and resistant to further deprotection under the reaction conditions. To test this, the DMTr group on pro[18F]FLT was selectively removed with PPTS in hot ethanol (as for the synthesis of (10)), and then the released N-2,4-DMBn-[18F]FLT was purified by HPLC (data not shown). Subsequently, this compound was subjected to the oxidation reaction with a large excess of CAN. Figure 15 shows the progressive conversion of N-2,4-DMBn[18F]FLT into [18F]FLT and another labeled product (Fig. 14). Although the structural assignment for this new labeled compound (G) is tentative, we believe it is DMBz-[18F]FLT, because its C18 retention time is intermediate to FLT and N-2,4-DMBn-FLT (10) and we have ruled out its identity with a wide range of alternative candidates shown in Figures 6, 8, and 10. The distribution of Kryptofix [2.2.2] and cerium salt through the overall [18F]FLT synthesis process was determined to assure that these toxic materials were low in concentration or absent from purified [18F]FLT solutions. The LD50 (following intravenous injection in rats) for Kryptofix is 32 mg/kg and severe toxic effects (headache, sweating, muscle cramps, nausea, vomiting) have been noted for cerium (III) acetate in humans at a dose of 1.6 mg/kg (3, 4). The amounts of Kryptofix and cerium found in [18F]FLT were

estimated to be 1 ␮g and 0.2 ␮g, respectively. Although alumina was capable of removing significant amounts of Kryptofix (⬎90%) during the preliminary clean-up of the labeling reaction product, the balance was later removed during HPLC purification of [18F]FLT, in a fraction eluted after FLT. Cerium salts were efficiently removed (⬎99.999%) by precipitation in basic solution and micron filtration (see Materials and Methods).

CONCLUSIONS Although the overall synthesis of [18F]FLT has not been optimized, the simple procedure we describe will be useful for producing radiochemically pure doses of [18F]FLT (⬎10 mCi, specific activity ⬎1Ci/␮mol EOB, within 100 min) for PET imaging. We present our synthesis in its simplest format, with the aim that it will be easy to identify how this synthesis can be adapted to dedicated systems that produce other 18F-labeled radiopharmaceuticals. At least three chemical correlations were used as structure proof for [18F]FLT. Authentic standards for all protecting group combinations on FLT were prepared and at least three of these compounds (pro-FLT (9), N-2,4-dimethoxybenzyl-FLT (10), and FLT), were matched with predominant radioactive components in chromatograms. The radiosynthesis of [18F]FLT presented here is not exclusive, and simpler syntheses have been accomplished (20, 40). However, the approach we present has built into it the greatest amount of flexibility for improving the synthesis by application of different protection groups and deprotection protocols and the potential for employing more reactive leaving groups.

This work is dedicated to Professor Edward Piers in honor of his 60th birthday. Financial support was provided by the National Institutes of Health (CA42045 and CA39566) and the Department of Energy (DE-FG06-93ER61653). We thank Ross Lawrence and Jeanne Link for assistance with LC-MS measurements, Jeanne Link for use of an automated system for processing and drying [18F]fluoride, Scott Lovell for obtaining the crystal structure of compound (5), and Ken Krohn and Tim Tewson for helpful discussions.

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