Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers

Abstract 2-Deoxy-D-ribose was converted to α/β-mixtures of methyl 3-O-acetyl- and methyl 3-O-benzoyl-2-deoxy-5-(p-toluenesulfonyl)-D-ribofuranosides. These were reacted with boron trichloride to generate ribofuranosyl chlorides, which afforded precursors for tracers to image tumor hypoxia on substitution with salts of 2-nitroimidazole. The anomeric ratio of the nucleosides was delicately influenced by the reaction conditions. Graphical abstract


Introduction
Tumor hypoxia has a negative prognosis predictive value for solid tumors, because it is associated with tumor aggressiveness, metastasis, and aberrant angiogenesis [1][2][3]. It reflects increased resistance to anticancer treatment by radio-and chemotherapy. Therefore, it is in the interest of cancer patients to identify and target hypoxic areas in solid tumors [4,5]. Non-invasive in vivo quantification of hypoxic areas of solid tumors with radiolabeled tracers attracted much attention and was studied extensively in recent years [6,7]. Fluorine-18 containing tracers derived from 2-nitroimidazole (azomycin) are the most important ones used for positron emission tomography (PET) to image hypoxia for diagnostic purposes. Under hypoxic conditions in cells, the 2-nitroimidazole moiety of the tracer is reduced stepwise by electron transfers via reactive intermediates [8,9]. These attack low-molecular weight compounds, preferably glutathione, and to a lesser extent high molecular weight compounds, and the nitro group ends up as amino group. The modified compounds with the bound 18 F, which is detected by PET, stay in the cells and are accumulated. Figure 1 is a compilation of those tracers, nucleosides derived from carbohydrates, such as various D-pentoses and D-hexoses, except compounds 1 and 2. The first azomycin-based tracer and, at the same time, the gold standard up to now for imaging tumor hypoxia are [ 18 F]fluoromisonidazole (FMISO, 1) [7,10]. A homologue thereof is [ 18 F]fluoroerythronitroimidazole (2) [11]. From the [ 18 F]fluoro nucleosides 3-8 derived from a-arabinose, tracer 3 [12,13], from b-arabinose, tracer 4 [14], from b-xylose, tracer 5 [14], and from b-glucose, tracer 6 [15], only 3 gained prominence. Recently, we synthesized 2-nitroimadazole precursors derived from aand b-2-deoxy-D-ribose and aand b-D-allofuranose. The b-anomers were radiolabeled and deprotected to give tracers 7 [16] and 8 [17] so far and evaluated for imaging tumor hypoxia. fully protected methyl glycosides 11. Their mixture was treated with 8 M HCl/Et 2 O/CH 2 Cl 2 at 0°C to form a mixture of glycosyl chlorides which was reacted with the tetrabutylammonium salt of 2-nitroimidazole. The two nucleosides, aand b-12, were separated by flash column chromatography and individually desilylated and finally tosylated to give the two desired precursors aand b-14. This sequence was selected, because we thought that introduction of the tosyl group right from the beginning would not be tolerated by 8 M HCl in Et 2 O/CH 2 Cl 2 . However, if that worked, the synthesis of both precursors could be shortened. Furthermore, we wanted to replace the tedious preparation of 8 M HCl in Et 2 O by a commercially available and more reactive reagent, such as BCl 3 , for the conversion of the methyl glycosides into the glycosyl chlorides.
The improved synthesis is given in Scheme 2. Although the mixture of methyl glycosides aand b-10 [18] was tosylated [19] at -25°C for 3 days in 59% yield (a/b = 1.2/ 1), some ditosylate 16 was formed as well (11%, a/b = 1.4/ 1). Analytical samples of the anomers for characterization could not be obtained by column flash chromatography. However, they could be obtained in homogeneous form by deacetylation of (?)-and (-)-17 and allowed to assign the anomeric configuration as will be shown later. Acetylation of the mixture of tosylates aand b-15 with Ac 2 O in dry pyridine delivered a mixture of acetates aand b-17 in 92% (a/ b = 1.2/1) yield. This mixture could be separated by flash column chromatography and Zemplen saponification of acetates (?)-and (-)-17 delivered homogenous samples of aand b-15, respectively. The latter one is a literature known compound whose b-configuration has been determined by 2D NMR methods [20]. It allowed to assign a-configuration to (?)-15 and a and b to (?)-and (-)-17, respectively. As glycosides aand b-17 were less reactive with HCl/Et 2 O in CH 2 Cl 2 , BCl 3 in CH 2 Cl 2 (1 M) was found to be an alternative to generate the glycosyl chlorides at 0°C (general procedure A). Rapid aqueous work up at 0°C allowed to isolate the labile chlorides, which were immediately reacted in two ways with 2-nitroimidazole. In the first case (general procedure B), the tetrabutylammonium salt of 2-nitroimidazole [21] was mixed with a solution of the 2-deoxy-Dribofuranosyl chloride at -30°C in CH 2 Cl 2 . The reaction mixture was allowed to warm slowly to 0°C within 2 h and was then extractively worked up. Flash chromatography furnished known anomers aand b-14 over two steps in 41 and 11% yield, respectively. When the reaction was started at -50°C, the a/b-14 ratio was 5/1 (by NMR) and only the aanomer was isolated in 53% yield. In the second case (general procedure C), the mixture of glycosyl chlorides was added to a mixture of 2-nitroimidazole/K 2 CO 3 /excess tris[2-(2-methoxyethoxy)ethyl]amine (TDA-1) as phase transfer catalyst [22] in CH 3 CN at 0°C. Work up after 2 h and purification delivered 12% of nucleoside a-14 and 36% of b-14 starting from methyl glycosides. Satisfyingly, the two complementary procedures gave either preferably aor banomer 14 [16].
We aimed to increase the yields of the nucleosides by replacing the acetyl protecting group by the more stable benzoyl group (Scheme 3). Therefore, the mixture of tosylates aand b-15 was benzoylated and gave again a mixture of globally protected 2-deoxy-D-riboses aand b-18, which could not be separated by flash column chromatography to obtain homogeneous analytical samples. Benzoylation of alcohols aand b-15 with benzoyl chloride/pyridine affords the individual anomers of 18 for analytical purposes, although the mixture was used for the next step. It was converted to chlorides as before with BCl 3 in CH 2 Cl 2 according to general procedure A. Their isolation without purification was immediately followed by reaction with the tetrabutylammonium salt of 2-nitroimidazole, starting the reaction at -50°C and allowing it to

Scheme 2
Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers 85 warm to ambient temperature. The mixture of the nucleosides aand b-19 was isolated in 81% yield (a/b = 2/1) by flash chromatography. The individual anomers were obtained by a second flash chromatography. When general procedure C was used for the preparation of the nucleosides from the chlorides, the yield of a-19 was 14% and that of b-19 was 69%. As envisioned, the yields with the benzoyl protecting group were higher than with the acetyl version. The anomeric configuration of a-

Conclusions
The synthesis of known 2-nitroimidazole nucleosides derived from 2-deoxy-D-ribose used as precursors for tracers was shortened if tosylation is performed at the beginning instead of at the end of the reaction sequence. The yield was further improved using BCl 3 for generation of 2-deoxy-D-ribofuranosyl chlorides and benzoyl instead of acetyl group as protecting group for OH at C-3.  SO 4 , followed by heating with a heat gun. Pyridine was dried by refluxing over powdered CaH 2 , then distilled and stored over molecular sieves (4 Å ). Dichloromethane was dried by storage over molecular sieves (3 Å ). All other chemicals and solvents were of the highest purity available and used as received.

Experimental
Mixture of methyl 2-deoxy-5-O-(p-toluenesulfonyl)-aand methyl 2-deoxy-5-O-(p-toluenesulfonyl)-b-D-ribofuranoside (a-and b-15, C 13 H 18 O 6 S) and mixture of methyl 3,5-bis(p-toluenesulfonyl)-a-and methyl 3,5-bis(p-toluenesulfonyl)-b-D-ribofuranoside (a-and b-16, C 20 H 24 O 8 S 2 ) Dry pyridine (2.20 cm 3 , 27.24 mmol) was added to a mixture of 1.345 g methyl glycosides aand b-10 (9.08 mmol) [18] in 17 cm 3 dry CH 2 Cl 2 under Ar. The stirred reaction mixture was cooled to 0°C and 1.868 g ptoluenesulfonyl chloride (9.08 mmol) was added. The flask was stored at -25°C for 3 days and afterwards 1 cm 3 water was added. After stirring for 15 min, the reaction mixture was concentrated under reduced pressure and 10 cm 3 EtOAc was added. The organic phase was washed  General procedure A: To a solution of 0.507 g methyl glycosides, aand b-17 (1.47 mmol) in 4.5 cm 3 dry Et 2 O at 0°C under Ar 3.68 cm 3 BCl 3 (3.68 mmol, 2.5 equiv, 1 M in CH 2 Cl 2 ) was added. The reaction mixture was stirred for 2 h (TLC: hexanes/EtOAc = 1/1; virtually no starting material was present; new strong spot with R f = 0.34) at 0°C. CH 2 Cl 2 (12 cm 3 , 0°C) was added and the mixture was washed with 4 cm 3 cold brine (-18°C), which was then extracted with 5 cm 3 cold CH 2 Cl 2 (0°C). The combined organic phases were washed with 5 cm 3 cold aqueous solution of NaHCO 3 (0°C), dried (Na 2 SO 4 ) at 0°C, and concentrated first to 5-10 cm 3 on a rotavapor without warming with the water bath and then the remaining solvent was removed on the vacuum pump (1 mbar) within a few min without warming. The clear somewhat coloured solution was used immediately for the next step after withdrawing a sample for 1 H NMR spectroscopy; ratio of chlorides: a/b = 3.6/1.0. Reaction of anomeric 2-deoxy-D-ribofuranosyl chlorides with tetrabutylammonium salt of 2-nitroimidazole (general procedure B) A solution of the above 2-deoxy-D-ribofuranosyl chlorides derived from aand b-17 in 3.5 cm 3 dry CH 2 Cl 2 (0°C) was added to a solution of the 0.450 g tetrabutylammonium salt of 2-nitroimidazole (1.32 mmol, 0.9 equiv. relative to methyl glycosides) [21] in dry 4 cm 3 CH 2 Cl 2 at -30°C under Ar. Stirring was continued for 2 h, while the cooling bath was allowed to reach 0°C. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in 15 cm 3 EtOAc and washed with water (2 9 5 cm 3 ). The organic phase was dried (MgSO 4 ) and concentrated under reduced pressure. The residue (a/b = 2/ 1 by 1 H NMR) was flash chromatographed (hexanes/ EtOAc = 1/1, a: R f = 0.29; b: R f = 0.49) to yield 0.060 g b-14 (11%) and 0.231 g a-14 (41%), both spectroscopically ( 1 H, 13 C NMR) identical to the ones described in Ref. [14].