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

Article abstract of DOI:10.1007/s00706-016-1874-8

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: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1874-8

Monatsh Chem (2017) 148:83–90
DOI 10.1007/s00706-016-1874-8
ORIGINAL PAPER
Efficient preparation of 2-nitroimidazole nucleosides
as precursors for hypoxia PET tracers
1
Petra Krizkova Anna Wieczorek1 Friedrich Hammerschmidt1
•
•
ˇ
´
Received: 21 October 2016 / Accepted: 6 November 2016 / Published online: 7 December 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract 2-Deoxy-D-ribose was converted to a/b-mix-
tures of methyl 3-O-acetyl- and methyl 3-O-benzoyl-2-
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 moi-
ety 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 18F, 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 [18F]fluo-
romisonidazole (FMISO, 1) [7, 10]. A homologue thereof is
[18F]fluoroerythronitroimidazole (2) [11]. From the [18F]flu-
oro 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
precursorsderivedfroma-andb-2-deoxy-D-riboseanda-and
b-D-allofuranose. The b-anomers were radiolabeled and
deprotected to give tracers 7 [16] and 8 [17] so far and eval-
uated for imaging tumor hypoxia.
deoxy-5-(p-toluenesulfonyl)-D-ribofuranosides.
These
were reacted with boron trichloride to generate ribofura-
nosyl chlorides, which afforded precursors for tracers to
image tumor hypoxia on substitution with salts of 2-ni-
troimidazole. The anomeric ratio of the nucleosides was
delicately influenced by the reaction conditions.
Graphical abstract
Keywords Hypoxia Á 2-Deoxy-D-ribose nucleosides Á
2-Nitroimidazole Á Alkylation Á Halogenides
Introduction
Tumor hypoxia has a negative prognosis predictive value for
solid tumors, because it is associated with tumor aggressive-
ness, metastasis, and aberrant angiogenesis [1–3]. It reflects
increased resistance to anticancer treatment by radio- and
chemotherapy. Therefore, itisinthe interestof cancerpatients
toidentifyandtargethypoxicareasinsolidtumors[4, 5]. Non-
Results and discussion
& Friedrich Hammerschmidt
friedrich.hammerschmidt@univie.ac.at
The synthesis of the precursors for tracers a- and b-8 is
given in Scheme 1 [16]. In brief, it started from 2-deoxy-
D-ribose, which was converted via methyl glycosides 10 to
1
Department of Organic Chemistry, University of Vienna,
Vienna, Austria
123

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P. Krizkova et al.
84
Fig. 1 Known 2-nitroimidazole-based [18F]fluoro tracers
Scheme 1
fully protected methyl glycosides 11. Their mixture was
treated with 8 M HCl/Et2O/CH2Cl2 at 0 °C to form a
mixture of glycosyl chlorides which was reacted with the
tetrabutylammonium salt of 2-nitroimidazole. The two
nucleosides, a- and b-12, were separated by flash column
chromatography and individually desilylated and finally
tosylated to give the two desired precursors a- and 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 Et2O/CH2Cl2.
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 Et2O by a commercially
available and more reactive reagent, such as BCl3, 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 a- and 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 a- and b-15 with Ac2O in dry pyr-
idine delivered a mixture of acetates a- and b-17 in 92% (a/
b = 1.2/1) yield. This mixture could be separated by flash
123

Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers
85
Scheme 2
column chromatography and Zemplen saponification of
acetates (?)- and (-)-17 delivered homogenous samples of
a- and 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 a- and b-17 were less reactive with HCl/Et2O in
CH2Cl2, BCl3 in CH2Cl2 (1 M) was found to be an alterna-
tive 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-nitroimi-
dazole [21] was mixed with a solution of the 2-deoxy-D-
ribofuranosyl chloride at -30 °C in CH2Cl2. 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 a- and 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 a-
anomer was isolated in 53% yield. In the second case (gen-
eral procedure C), the mixture of glycosyl chlorides was
added to a mixture of 2-nitroimidazole/K2CO3/excess tris[2-
(2-methoxyethoxy)ethyl]amine (TDA-1) as phase transfer
catalyst [22] in CH3CN 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 a- or b-
anomer 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 a- and b-15 was benzoylated and gave again a
mixture of globally protected 2-deoxy-D-riboses a- and b-
18, which could not be separated by flash column chro-
matography to obtain homogeneous analytical samples.
Benzoylation of alcohols a- and b-15 with benzoyl chlo-
ride/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 BCl3
in CH2Cl2 according to general procedure A. Their isola-
tion without purification was immediately followed by
reaction with the tetrabutylammonium salt of 2-nitroimi-
dazole, starting the reaction at -50 °C and allowing it to
123

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P. Krizkova et al.
86
Scheme 3
warm to ambient temperature. The mixture of the nucleo-
sides a- and 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 nucleo-
sides 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- and b-19 (1H
NMR; a-1-H0: d, J = 6.6 Hz; b-1-H0: dd, J = 7.5 and
5.6 Hz) was assigned in analogy to nucleosides a- and b-14
(1H NMR; a-1-H0: dd, J = 6.3, 0.8 Hz; b-1-H0: dd,
J = 6.6, 6.1 Hz) and the literature known analogue [19] of
b-14 with two 4-toluoyl protecting groups (1H NMR; b-1-
H0: t, J = 6.5 Hz) instead of the acetyl and benzoyl group.
The 1-H0 hydrogen atoms of the a-anomers resonate as
doublets or as doublets of doublets with one coupling
constant being very small. However, the 1-H0 hydrogen
atoms of the b-anomers resonate as doublets of doublets or
as triplet with two similar coupling constants.
respectively. Chemical shifts d (ppm) were referenced to
residual
CHCl3
(dH = 7.24 ppm)
and
CDCl3
(dC = 77.00 ppm). IR spectra were recorded on a Bruker
VERTEX 70 IR spectrometer as ATR spectra or of films on
a silicon disc [23] on a Perkin Elmer 1600 FT-IR spec-
trometer. Optical rotations were measured at 20 °C on a
Perkin Elmer 351 polarimeter in a 10 cm cell. Melting
points were determined on a Reichert Thermovar instru-
ment. Elemental analyses (C, H, N, S) were conducted
using the Euro EA 3000 Elemental Analyser (for oxygen in
combination with a high temperature pyrolysis furnace
(1480 °C) and reduction with carbon) from Eurovector.
Their results were found to be in good agreement ( 0.3%)
with the calculated values.
Flash (column) chromatography was performed with
Merck silica gel 60 (230–400 mesh). TLC was carried out on
0.25 mm-thick Merck plates, silica gel 60 F254. Spots were
visualized by UV and/or dipping the plate into a solution of
23.0 g (NH4)6Mo7O24Á4H2O and 1.0 g Ce(SO4)2Á4H2O in
500 cm3 10% aqueous H2SO4, followed by heating with a
heat gun. Pyridine was dried by refluxing over powdered
˚
CaH2, then distilled and stored over molecular sieves (4 A).
Conclusions
Dichloromethane was dried by storage over molecular sieves
˚
(3 A). All other chemicals and solvents were of the highest
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 BCl3 for generation
of 2-deoxy-D-ribofuranosyl chlorides and benzoyl instead
of acetyl group as protecting group for OH at C-3.
purity available and used as received.
Mixture of methyl 2-deoxy-5-O-(p-toluenesulfonyl)-a-
and methyl 2-deoxy-5-O-(p-toluenesulfonyl)-b-D-ribofura-
noside (a- and b-15, C13H18O6S) and mixture of methyl
3,5-bis(p-toluenesulfonyl)-a- and methyl 3,5-bis(p-tolue-
nesulfonyl)-b-D-ribofuranoside
(a- and b-16, C20H24O8S2)
Dry pyridine (2.20 cm3, 27.24 mmol) was added to a
mixture of 1.345 g methyl glycosides a- and b-10
(9.08 mmol) [18] in 17 cm3 dry CH2Cl2 under Ar. The
stirred reaction mixture was cooled to 0 °C and 1.868 g p-
toluenesulfonyl chloride (9.08 mmol) was added. The flask
was stored at -25 °C for 3 days and afterwards 1 cm3
water was added. After stirring for 15 min, the reaction
mixture was concentrated under reduced pressure and
10 cm3 EtOAc was added. The organic phase was washed
Experimental
1H, 13C (J-modulated; not J-modulated spectra were
recorded of 2-nitroimidazole derivatives) NMR spectra
were recorded in CDCl3 on a Bruker AV III 400 (1H:
400.27 MHz, 13C: 100.65 MHz), AV 400 (1H:
400.13 MHz, 13C: 100.61 MHz), and AV III 600 (1H:
600.13 MHz, 13C: 150.90 MHz) spectrometer at 25 °C,
123

Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers
87
with 10 cm3 2 M HCl, 10 cm3 water, and 10 cm3
NaHCO3, then dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by flash
chromatography (hexanes/EtOAc = 1/1; Rf = 0.34 for
monotosylates, Rf = 0.75 for ditosylates) giving 1.631 g
mixture of monotosylates a- and b-15 (59%; a/b = 1.22/1)
and 0.458 g mixture of ditosylates a- and b-16 (11%), both
as colorless oils. The data of the individual anomers are
given later. Mixture of ditosylates a- and b-16:
[a]2D0 = ? 24.3 cm2 g-1 (c = 1.55, acetone); IR (ATR,
isomer: Rf = 0.11 for hexanes/EtOAc = 3/1) as colorless
oils.
(-)-17: Rf = 0.20 (hexanes/EtOAc = 3/1); colorless
crystals, m.p.: 50–51 °C (i-Pr2O/hexanes); [a]2D0 = -40.7 -
cm2 g-1 (c = 1.01, acetone); 1H NMR (600.13 MHz,
CDCl3): d = 7.81–7.78 (m, 2H, Htos), 7.34–7.30 (m, 2H,
Htos), 5.08 (ddd, J = 7.3, 5.3, 2.3 Hz, 1H, 3-H), 5.04 (dd,
J = 5.4, 2.0 Hz, 1H, 1-H), 4.19–2.13 (m, 2H, 4-H and 5-H),
4.04 (dd, 9.7, 6.6 Hz, 1H, 5-H), 3.21 (s, 3H, OCH3), 2.42 (s,
3H, CHt3os), 2.31 (ddd, J = 14.0, 7.3, 2.0 Hz, 1H, 2-H), 2.09
ꢀ
NMR sample in CDCl3): m = 1359, 1190, 1174, 1096,
(d, J = 14.0, 5.3 Hz, 1H, 2-H), 2.01(s, 3H, CH3CO) ppm; 13
C
976 cm-1
.
NMR (150.90 MHz, CDCl3): d = 171.0 (C=O), 144.9
1H NMR (400.27 MHz, CDCl3): a/b = 1.4/1.0; con-
tained 5% by weight of toluene; a-16: d = 7.76–7.71 (m,
4H, HAr), 7.36–7.30 (m, 4H, HAr), 4.92 (bd, J = 5.2 Hz,
1H, 1-H), 4.77 (ddd, J = 8.3, 3.4, 2.0 Hz, 1H, 3-H), 4.29
(q, J = 3.4 Hz, 1H, 4-H), 4.09 (d, J = 3.4 Hz, 2H, 5-H),
3.27 (s, 3H, OCH3), 2.43 (s, 6H, CHt3ol), 2.13 (ddd,
J = 14.8, 8.3, 5.2 Hz, 1H, 2-H), 1.96 (ddd, J = 14.8, 2.0.
0.8 Hz, 1H, 2-H) ppm; b-16: d = 7.76–7.71 (m, 4H, HAr),
7.36–7.30 (m, 4H, HAr), 5.00 (dd, J = 4.9, 2.6 Hz, 1H,
1-H), 4.88 (ddd, J = 9.0, 5.9 Hz, 1H, 3-H), 4.18 (td,
J = 5.9, 3.5 Hz, 1H, 4-H), 3.92 (AB part of ABX system,
JAB = 10.4 Hz, JAX = JBX = 5.9 Hz, 2H, 5-H), 3.18 (s,
3H, OCH3), 2.43 (s, 6H, CHt3ol), 2.20–2.15 (m, 2H, 2-H)
ppm; 13C NMR (100.65 MHz, CDCl3) of mixture:
d = 145.4 (Cq, b), 145.2(Cq, a), 145.0 (Cq, a), 145.0 (Cq,
b), 133.0 (Cq, a), 132.8 (Cq, b), 132.5 (Cq, b), 132.5 (Cq,
a), 130.1 (2 CH, b), 130.0 (2 CH, a), 129.8 (4 CH, a and b),
127.9 (4 CH), 127.8 (2 CH), 127.8 (2 CH), 105.1 (C-1, b),
104.7 (C-1, a), 80.5 (C-4, b), 80.4 (C-4, a), 80.1 (C-3, b),
79.1 (C-3, a), 68.8 (C-5, b), 68.4 (C-5, a), 55.1 (CH3O, b),
64.99 (OCH3, a), 39.0 (C-2, b), 38.9 (C-2, a), 21.6 (2 CH3),
21.55 (2 CH3) ppm.
(Cqtos), 132.8 (Cqtos), 129.8 (2 CH), 127.9 (2 CH), 105.2 (C-
1), 80.9 (C-4), 74.0 (C-3), 69.5 (C-5), 55.2 (OCH3), 38.8 (C-
tos
ꢀ
2), 21.6 (CH3 ), 21.0 (CH3) ppm; and IR (ATR): m = 2925,
1737, 1360, 1235, 1175, 1047, 973, 955 cm-1
.
(?)-17: Rf = 0.11 (hexanes/EtOAc = 3/1), oil;
[a]2D0 = ?93.6 1H
(c = 1.05, acetone); NMR
(600.13 MHz, CDCl3): d = 7.79–7.75 (m, 2H, Htos),
7.35–7.30 (m, 2H, Htos), 4.98 (dd, J = 5.3, 0.7 Hz, 1H,
1-H), 4.94 (ddd, J = 8.3, 3.5, 1.9 Hz, 1H, 3-H), 4.21 (AB
part of ABX system, JAB = 10.6 Hz, J4,5 = 3.5 and
3.2 Hz, 2H, 5-H), 4.15 (*q, J = *3.5 Hz, 1H, 4-H), 3.31
(s, 3H, OCH3), 2.42 (s, 3H, CH3 tol), 2.28 (ddd, J = 14.5,
8.3, 5.3 Hz, 1H, 2-H), 2.02 (s, 3H, CH3CO), 1.95 (ddd,
J = 14.5, 1.9, 0.7 Hz, 1H, 2-H) ppm; 13C NMR
(150.90 MHz, CDCl3): d = 171.0 (C=O), 144.9 (Cqtos),
132.8 (Cqtos), 129.8 (2 CH), 127.9 (2 CH), 105.2 (C-1),
80.9 (C-4), 74.0 (C-3), 69.5 (C-5), 55.2 (OCH3), 38.8 (C-
2), 21.6 (CHt3os), 21.0 (CH3) ppm; and IR (ATR):
-1
ꢀ
m = 2836, 1736, 1364, 1240, 1177, 1070, 1020, 978 cm
Methyl
noside (a-15, C13H18O6S)
solution of 0.291 g acetate (?)-17 (0.84 mmol,
.
2-deoxy-5-O-(p-toluenesulfonyl)-a-D-ribofura-
A
Mixture of (?)- and (-)-methyl 3-O-acetyl-2-deoxy-5-O-
(p-toluenesulfonyl)-D-ribofuranoside ((?)- and (-)-17,
C15H20O7S)
[a]2D0 = ?93.6 (c = 1.05, acetone)), 4.25 cm3 dry MeOH,
and 0.43 cm3 NaOMe/MeOH (0.425 mmol, 1 M) was
stirred for 30 min at 0 °C (TLC). Dry ice was added to
neutralize base. The reaction mixture was concentrated
under reduced pressure. Water (10 cm3) and 5 cm3 CH2Cl2
were added. The organic phase was separated and the
aqueous one extracted with CH2Cl2 (2 9 5 cm3). The
combined organic layers were dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by
flash chromatography (hexanes/EtOAc = 1/1, Rf = 0.47) to
yield 0.199 g alcohol a-15 (77%) as colorless oil.
[a]2D0 = ? 95.09° g cm2 (c = 1.12, acetone); 1H NMR
(400.13 MHz, CDCl3): d = 7.78–7.73 (m, 2H, Htos),
7.35–7.30 (m, 2H, Htos), 5.01 (d, J = 4.4 Hz, 1H, 1-H),
4.18 (&td, J = 4.1, 1.8 Hz, 1H, 4-H), 4.10 (bd, J = 5.9 Hz,
1H, 3H), 4.04 (AB part of ABX system, JAB = 10.7 Hz,
J = 4.3, 3.8 Hz, 2H, 5-H), 3.32 (s, 3H, CH3O), 2.75 (bs, 1H,
OH), 2.43 (s, 3H, CH3), 2.05 (ddd, J = 13.9, 5.9, 4.4 Hz, 1H,
To 1.631 g mixture of monotosylates a- and b-5
(5.39 mmol), 1.02 cm3 Ac2O (10.78 mmol) and 1.67 cm3
dry pyridine (21.56 mmol) in 13 cm3 dry CH2Cl2 were
added under Ar. The reaction mixture was heated at 40 °C
until the starting material was consumed (about 4 h). After
addition of 4 cm3 water, stirring was continued for 15 min.
The organic phase was separated and washed with 15 cm3
2 M HCl and 15 cm3 saturated aqueous solution of
NaHCO3, dried (MgSO4), and concentrated under reduced
pressure. The residue was purified by flash chromatography
(hexanes/EtOAc = 1/1, Rf = 0.82, 0.75 for anomers) to
yield 1.704 g mixture of anomers (92%, a/b = 1.2/1.0) as
colorless oil. Part of the mixture was flash chro-
matographed to get analytical samples of anomers (?)-
and (-)-17 (less polar isomer: Rf = 0.20, more polar
123

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´
P. Krizkova et al.
88
2-H), 1.96 (dd, J = 13.9, 0.8 Hz, 1H, 2-H) ppm; 13C NMR
(100.61 MHz, CDCl3): d = 145.0 (Cq, CSO3), 132.7
(Cqtos), 129.90 (2 HCtos), 127.9 (2 HCtos), 105.7 (C-1),
84.6 (C-4), 72.8 (C-3), 69.4 (C-5), 55.0 (OCH3), 41.0 (C-2),
overlapping with 1-H of a-chloride, integration was refer-
enced to resonance at 4.46 ppm (q, J = 2.9 Hz, 4-H).
Reaction of anomeric 2-deoxy-D-ribofuranosyl chlorides
with tetrabutylammonium salt of 2-nitroimidazole (general
procedure B)
21.6 (CHt3os) ppm; and IR (Si): m = 3445, 2923, 1354, 1173,
ꢀ
1081, 961, 908 cm-1
.
A solution of the above 2-deoxy-D-ribofuranosyl chlorides
derived from a- and b-17 in 3.5 cm3 dry CH2Cl2 (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 cm3 CH2Cl2 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 cm3 EtOAc and washed with water
(2 9 5 cm3). The organic phase was dried (MgSO4) and
concentrated under reduced pressure. The residue (a/b = 2/
1 by 1H NMR) was flash chromatographed (hexanes/
EtOAc = 1/1, a: Rf = 0.29; b: Rf = 0.49) to yield 0.060 g
b-14 (11%) and 0.231 g a-14 (41%), both spectroscopically
(1H, 13C NMR) identical to the ones described in Ref. [14].
Methyl
2-deoxy-5-O-(p-toluenesulfonyl)-b-D-ribofura-
noside (b-15, C13H18O6S)
A mixture of 0.066 g acetate (-)-17 (0.19 mmol, less polar
acetate, [a]2D0 = -40.7 cm2 g-1 (c = 1.01, acetone)), 2 cm3
dry MeOH, and 0.064 cm3 MeONa/MeOH (0.064 mmol,
0.33 equiv, 1 M) was stirred at -30 °C. The ester was
consumed after 5 h (TLC). Work up as for a-15 yielded
0.047 g alcohol b-15 (82%) as colorless oil. [a]2D0 = -40.4 -
g cm2 (c = 1.08, acetone); 1H NMR (400.13 MHz, CDCl3):
d = 7.81–7.74 (m, 2H, Htos), 7.36–7.31 (m, 2H, Htos), 5.01
(dd, J = 5.2, 1.8 Hz, 1H, 1-H), 4.41 (bs, 1H, 3-H), 4.07–3.99
(m, 3H, 5-H, 4-H), 3.20 (s, 3H, OCH3), 2.43 (s, 3H, CHt3os),
2.19 (ddd, J = 13.4, 6.9, 1.8 Hz, 1H, 2-H), 2.10 (bs, 1H, OH),
2.03 (ddd, J = 13.4, 6.2, 5.2 Hz, 1H, 2-H) ppm; 13C NMR
(100.61 MHz, CDCl3): d = 145.1 (Cq, CSO3), 132.7 (Cq,
Ctos), 129.9 (2C, HCtos), 128.0 (2C, HCtos), 105.3 (C-1), 82.9
(C-4), 72.65 (C-3), 70.14 (C-5), 50.02 (CH3O), 41.32 (C-2),
21.64 (CH3 tol) ppm.
Similarly, 0.536 g mixture of anomeric methyl glyco-
sides (1.56 mmol) were converted via chlorides to
nucleosides (reaction was started at -50 °C); ratio of a/
1
b = 5/1 by H NMR in crude product. Flash chromatog-
Preparation of anomeric 3-O-acetyl-2-deoxy-5-O-(p-
toluenesulfonyl)-D-ribofuranosyl chlorides (general pro-
cedure A) and their conversion to 1-(30-O-acetyl-20-deoxy-
50-O-(p-toluenesulfonyl)-a- and 1-(30-O-acetyl-20-deoxy-50-
O-(p-toluenesulfonyl)-b-D-ribofuranosyl)-2-nitroimidazole
(a- and b-14)
raphy furnished 0.318 g a-14 (53%).
Reaction of 3-O-acetyl-glycosyl chlorides with 2-nitroimi-
dazole/K2CO3/tris[2-(2-methoxyethoxy)ethyl]amine (TDA-
1) (general procedure C)
A mixture of 0.118 g 2-nitroimidazole (1.04 mmol, 0.8
equiv.), 0.225 g K2CO3 (1.63 mmol), 10 mm3 TDA-1 [23],
and 20 cm3 dry CH3CN was stirred for 10 min at RT under
Ar and then cooled to 0 °C. The chlorides prepared from
0.449 g methyl glycosides a- and b-17 (1.30 mmol) by the
above given general procedure A were dissolved in dry
CH3CN at 0 °C and added. Stirring was continued for 2 h at
0 °C and then the reaction mixture was filtered through
Celite (washing with CH2Cl2). The filtrate was concentrated
under reduced pressure and 20 cm3 EtOAc was added to the
residue. The mixture was washed with water (2 9 10 cm3),
dried (MgSO4), and concentrated under reduced pressure.
The residue (a/b = 1/3, by 1H NMR) was purified by flash
chromatography (hexanes/EtOAc = 1/1) to yield 54 mg a-
14 (12%) and 160 mg b-14 (36%).
General procedure A: To a solution of 0.507 g methyl
glycosides, a- and b-17 (1.47 mmol) in 4.5 cm3 dry Et2O at
0 °C under Ar 3.68 cm3 BCl3 (3.68 mmol, 2.5 equiv, 1 M
in CH2Cl2) 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 Rf = 0.34) at
0 °C. CH2Cl2 (12 cm3, 0 °C) was added and the mixture
was washed with 4 cm3 cold brine (-18 °C), which was
then extracted with 5 cm3 cold CH2Cl2 (0 °C). The
combined organic phases were washed with 5 cm3 cold
aqueous solution of NaHCO3 (0 °C), dried (Na2SO4) at
0 °C, and concentrated first to 5–10 cm3 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 1H NMR
spectroscopy; ratio of chlorides: a/b = 3.6/1.0.
Mixture of methyl 3-O-benzoyl-2-deoxy-5-O-(p-toluene-
sulfonyl)-a- and methyl 3-O-benzoyl-2-deoxy-5-O-(p-
toluenesulfonyl)-b-D-ribofuranoside
(a- and b-18, C20H22O7S)
To 0.800 g, mixture of anomeric monotosylates 15
(2.65 mmol) and 0.64 cm3 dry pyridine (7.95 mmol) in
dry CH2Cl2 (6.3 cm3) under Ar was added 0.64 cm3
benzoyl chloride. The reaction mixture was stirred at RT
1H NMR of anomeric 2-deoxy-D-ribofuranosyl chlo-
rides
(400.27 MHz, CDCl3):
d = 6.21 ppm
(d,
J = 5.3 Hz, 1-H of a-chloride), 1-H of b-chloride
123

Efficient preparation of 2-nitroimidazole nucleosides as precursors for hypoxia PET tracers
for 18 h. After addition of 0.5 cm3 water, stirring was
89
1H, 5-H), 3.26 (s, 3H, OCH3), 2.43 (ddd, J = 14.2, 7.3,
2.0 Hz, 1H, 2-H), 2.39 (s, 3H, CHt3os), 2.25 (td, J = 14.2,
5.4 Hz, 1H, 2-H) ppm; 13C NMR (100.61 MHz, CDCl3):
d = 165.95 (CO), 144.90 (CSO3), 133.38 (HCPh), 132.84
(CH3Ctos), 129.86 (2C, HCtos), 129.64 (2C, HCPh), 129.35
(CCO), 128.44 (2C, HCPh), 128.02 (2C, HCtos), 105.70 (C-
1), 81.39 (C-4), 75.10 (C-3), 70.35 (C-5), 55.19 (CH3O),
continued for 15 min. The mixture was concentrated under
reduced pressure and 15 cm3 EtOAc and 5 cm3 water were
added. The organic phase was separated and washed with
5 cm3 2 M HCl, 5 cm3 water, and 5 cm3 saturated aqueous
solution of NaHCO3, dried (MgSO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography (hexanes/EtOAc = 2/1, Rf = 0.76) to
yield 0.972 g mixture of anomeric benzoates 18 (90%; a/
b = 1.2/1.0 by 1H NMR) as a colorless oil possibly
containing some benzoic acid; [a]2D0 = ? 41.0 g cm2
(c = 1.35, acetone). The individual anomers of 18 for
characterization were prepared by esterification of homo-
geneous anomers a- and b-15 with PhC(O)Cl/pyridine.
39.07 (C-2), 21.60 (CHt3os) ppm; and IR (Si): m = 2924,
ꢀ
1721, 1365, 1274, 1178, 1110 cm-1
.
Preparation of mixture of anomeric 3-O-benzoyl-2-deoxy-
5-O-(p-toluenesulfonyl)-D-ribofuranosyl chlorides and
their conversion to 1-(30-O-benzoyl-20-deoxy-50-O-(p-
toluenesulfonyl)-a- and 30-O-benzoyl-20-deoxy-50-O-(p-
toluenesulfonyl)-b-D-ribofuranosyl)-2-nitroimidazole
(a- and b-19, C22H21N3O8S)
Methyl
3-O-benzoyl-2-deoxy-5-O-(p-toluenesulfonyl)-a-
and methyl 3-O-benzoyl-2-deoxy-5-O-(p-toluenesulfonyl)-
b-D-ribofuranoside (a- and b-18, C20H22O7S)
A mixture of 0.609 g methyl glycosides a- and b-19
(1.50 mmol) was converted to 3-O-benzoyl-glycosyl chlo-
rides (TLC: hexanes/EtOAc = 1/1, Rf = 0.58) by the
procedure used for methyl glycosides a- and b-17 (general
procedure A). The crude product was used immediately for
the next step. 1H NMR spectrum of crude 3-benzoyl
2-deoxy-D-ribofuranosyl chlorides (400.27 MHz, CDCl3):
d = 6.31 (d, J = 5.0 Hz, 1-H of a-chloride), 6.28 (dd,
J = 5.5, 1.6 Hz, 1-H of b-chloride), integration referenced
to resonance at 4.60 ppm (q, J = 2.6 Hz, 4-H); a/b = 1/
0.13; fairly pure.
Benzoyl chloride (0.143 g, 1.02 mmol, 0.118 cm3) and
0.120 g dry pyridine (1.52 mmol, 0.122 cm3) were added
to 0.153 g alcohol a-15 (0.51 mmol) dissolved in 1.5 cm3
dry CH2Cl2 and the solution was stirred for 20 h at RT.
Water (0.5 cm3) was added and the reaction mixture was
stirred for 15 min. The mixture was concentrated under
reduced pressure, 10 cm3 water was added, and it was
extracted with ethyl acetate (3 9 5 cm3), dried with
Na2SO4, and concentrated under reduced pressure. The
crude product was purified by flash chromatography
(hexanes/ethyl acetate = 1/1, Rf = 0.55) to yield 0.183 g
benzoate a-18 (88%) as colorless oil. Similarly, 0.096 g
alcohol b-15 (0.32 mmol) was converted to 0.104 g
benzoate b-18 (81%).
The above mixture of chlorides was converted to a
mixture of a- and b-19 using the procedure (general pro-
cedure B) given for the corresponding 3-O-acetyl-glycosyl
chlorides. Tetrabutylammonium salt of 2-nitroimidazole
(0.481 g, 1.36 mmol) was used; the reaction was started at
-50 °C; and the reaction mixture was allowed to warm to
1
a-18: [a]2D0 = ? 98.23 (c = 1.015, acetone); H NMR
(400.13 MHz, CDCl3): d = 8.01–7.96 (m, 2H, HPh),
7.81–7.76 (m, 2H, Htos), 7.58–7.52 (m, 1H, HPh), 7.45–7.38
(m, 2H, HPh), 7.34–7.28 (m, 2H, Htos), 5.22–5.16 (m, 1H,
3-H), 5.06 (d, J = 5.2 Hz, 1H, 1-H), 4.35–4.27 (m, 3H,
4-H, 5-H), 3.34 (s, 3H, CH3O), 2.41 (s, 3H, CHt3os), 2.40
(ddd, J = 14.5, 8.1, 5.2 Hz, 1H, 2-H), 2.11 (dd, J = 14.5,
1.5 Hz, 1H, 2-H) ppm; 13C NMR (100.61 MHz, CDCl3):
d = 166.41 (CO), 144.92 (CSO3), 133.27 (HCPh), 132.86
(CH3Ctos), 129.86 (2C, HCtos), 129.73 (2C, HCPh), 129.58
(CCO), 128.39 (2C, HCar), 127.99 (2C, HCar), 105.26 (C-
1), 80.945 (C-4), 74.49 (C-3), 69.56 (C-5), 55.14 (CH3O),
1
RT in 18 h. The crude product (a/b = 2/1, by H NMR)
was flash chromatographed (hexanes/EtOAc = 2/1, a-19:
Rf = 0.25; b-19: Rf = 0.21) to yield 0.536 g mixture
(81%, a/b = 2/1) of a- and b-19. The anomers were sep-
arated by flash chromatography (CH2Cl2/EtOAc = 20/1; a:
Rf = 0.42; b: Rf = 0.36) using a long column to yield
homogenous anomers and mixture of anomers.
a-19: Oil, which decomposed at room temperature
within a few days, but it was more stable at 4 °C. When a-
19 was crystallized from C2H4Cl2/i-Pr2O by slowly cooling
from RT to -18 °C, a white powder was obtained, which
contained after drying at 0.5 mbar/RT for 10 h 0.05 mol%
of i-Pr2O; m.p.: 63–65 °C (powder became glassy); this
powder was ideal for storage at 4 °C and handling.
[a]2D0 = -9.78 g cm2 (c = 1.15, acetone); 1H NMR
(400.13 MHz, CDCl3): d = 7.85–7.80 (m, 2H, Har),
7.66–7.62 (m, 2H, Har), 7.57–7.52 (m, 1H, Har), 7.41–7.33
(m, 4H, Har), 7.32 (d, J = 1.0 Hz, 1H, Him), 7.13 (d,
J = 1.0 Hz, 1H, Him), 6.62 (d, J = 6.6 Hz, 1H, 10-H), 5.43
(d, J = 6.6, 0.7 Hz, 1H, 30-H), 4.74 (td, J = 3.0, 1.0 Hz,
38.90 (C-2), 21.62 (CHt3os) ppm; and IR (Si): m = 3016,
ꢀ
2970, 2946, 1738, 1725, 1365, 1229, 1217 cm-1
.
b-18: [a]2D0 = -16.75 (c = 0.83, acetone); 1H NMR
(400.13 MHz, CDCl3): d = 7.98–7.93 (m, 2H, HPh),
7.83–7.77 (m, 2H, Htos), 7.59–7.53 (m, 1H, HPh), 7.46–7.38
(m, 2H, HPh), 7.33–7.27 (m, 2H, Htos), 5.32 (ddd, J = 7.5,
5.4, 3.3 Hz, 1H, 3-H), 5.13 (dd, J = 5.4, 2.0, Hz, 1H, 1-H),
4.32 (ddd, J = 7.1, 5.1, 3.3 Hz, 1H, 4-H), 4.26 (dd,
J = 10.1, 5.1 Hz, 1H, 5-H), 4.14 (dd, J = 10.1, 7.1 Hz,
123

ˇ
´
P. Krizkova et al.
90
1H, 40-H), 4.37 (AB part of ABX system, JAB = 11.4 Hz,
JAX = JBX = 3.0 Hz, 2H, 50-H), 3.05 (td, J = 15.5,
6.6 Hz, 1H, 20-H), 2.48 (d, J = 15.5 Hz, 1H, 20-H), 2.45 (s,
3H, CH3) ppm; 13C NMR (100.61 MHz, CDCl3):
d = 165.7 (CO), 145.6 (Cqtos), 143.7 (Cqim), 133.9 (HC),
132.43 (Cq), 130.1 (2 HCtos), 129.5 (2 HCar), 128.6 (2
HCar), 128.4 (Cqar), 128.2 (HCim), 127.9 (2 HCar), 122.2
(HCim), 91.4 (C-10), 86.1 (C-40), 74.6 (C-30), 69.0 (C-50),
41.1 (C-20), 21.7 (CHt3os) ppm; and IR (ATR, NMR sam-
use, distribution, and reproduction in any medium, provided you give
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ple): m = 2971, 1709, 1535, 1476, 1355, 1270, 1240, 1175,
1092, 1075 cm-1
.
b-19: [a]2D0 = -16.89 g cm2 (c = 1.06, acetone); m.p.:
90 °C (decomp., CH2ClCH2Cl/i-Pr2O, solution not heated
above 50 °C); 1H NMR (400.13 MHz, CDCl3):
d = 8.03–7.96 (m, 2H, Har), 7.82–7.75 (m, 2H, Har),
7.63–7.57 (m, 2H, Har), 7.60 (d, J = 1.0 Hz, 1H, Him),
7.50–7.43 (m, 2H, Har), 7.38–7.32 (m, 2H, Har), 7.17 (d,
J = 1.0 Hz, 1H, Him), 6.78 (dd, J = 7.6, 5.6 Hz, 1H, 10-
H), 5.42 (td, J = 6.6, 2.3 Hz, 1H, 30-H), 4.48–4.37 (m, 3H,
50-H and 40-H), 2.99 (ddd, J = 14.3, 5.6, 2.3 Hz, 1H, 20-H),
2.43 (s, 3H, CH3), 2.41 (ddd, J = 14.3, 7.6. 6.6 Hz, 1H, 20-
H) ppm; 13C NMR (100.61 MHz, CDCl3): d = 165.9
(CO), 145.7 (Cqtos), 144.0 (Cqim), 133.9 (HCPh), 132.2
(Cqtos), 130.2 (2C, HCtos), 129.7 (2C, HCtos), 128.96
(HCim), 128.7 (CqPh), 128.6 (2CPh), 127.9 (2CPh), 121.8
(Cim), 88.8 (C-10), 83.2 (C-40), 74.4 (C-30), 68.5 (C-50), 40.1
(C-20), 21.7 (CHt3os) ppm; and IR (ATR, NMR sample):
-1
ꢀ
m = 1713, 1544, 1352, 1279, 1174, 1096 cm
.
Preparation of 3-O-benzoyl-2-deoxy-5-O-tosyl-D-ribofu-
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general procedure C
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1
procedure C. The crude product (a/b = 1/5, by H NMR)
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a long column to yield 0.066 g nucleoside a-19 (14%) and
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Acknowledgements Open access funding provided by University of
Vienna. The authors thank S. Felsinger for recording NMR spectra
and J. Theiner for combustion analyses.
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Open Access This article is distributed under the terms of the
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123