Synthesis, radiofluorination, and hypoxia-selective studies of FRAZ: A configurational and positional analogue of the clinical hypoxia marker, [18F]-FAZA

Article abstract of DOI:10.1016/j.bmc.2010.01.064

The current work evaluates 1-α-d-(2-deoxy-2-fluororibofuranosyl)-2-nitroimidazole (FRAZ), a novel azomycin nucleoside that is a potential radiosensitizer of tumor hypoxia. FRAZ is a ribose analogue of 1-α-d-(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole ([¹⁸F]-FAZA), a clinically used hypoxia marker. Preliminary assessment of the cytotoxicity and hypoxia-specific in vitro binding in HCT-110 colorectal cancer cells indicate that the radiosensitization properties of FRAZ are similar to that of FAZA, with a sensitizer enhancement ratio (SER) of ～1.8. An automated radiosynthesis of [¹⁸F]-FRAZ using a commercial automated synthesis unit (ASU) was established (synthesis time ～32 min; radiochemical yield (decay uncorrecetd) ～22%) to facilitate its application in PET-based diagnosis of hypoxic tumors.

Full text of DOI:10.1016/j.bmc.2010.01.064

Bioorganic & Medicinal Chemistry 18 (2010) 2255–2264
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, radiofluorination, and hypoxia-selective studies of FRAZ:
A configurational and positional analogue of the clinical hypoxia marker,
[18F]-FAZA
*
Piyush Kumar , Ebrahim Naimi, Alexander J. McEwan, Leonard I. Wiebe
Division of Oncologic Imaging, Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Ave., Edmonton, Canada T6G1Z2
a r t i c l e i n f o
a b s t r a c t
Article history:
The current work evaluates 1-a-D-(2-deoxy-2-fluororibofuranosyl)-2-nitroimidazole (FRAZ), a novel azo-
mycin nucleoside that is a potential radiosensitizer of tumor hypoxia. FRAZ is a ribose analogue of 1-a-D-
Received 18 November 2009
Revised 20 January 2010
Accepted 22 January 2010
Available online 6 February 2010
(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole ([18F]-FAZA), a clinically used hypoxia marker. Pre-
liminary assessment of the cytotoxicity and hypoxia-specific in vitro binding in HCT-110 colorectal can-
cer cells indicate that the radiosensitization properties of FRAZ are similar to that of FAZA, with a
sensitizer enhancement ratio (SER) of ꢀ1.8. An automated radiosynthesis of [18F]-FRAZ using a commer-
cial automated synthesis unit (ASU) was established (synthesis time ꢀ32 min; radiochemical yield (decay
uncorrecetd) ꢀ22%) to facilitate its application in PET-based diagnosis of hypoxic tumors.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Tumor hypoxia
FRAZ
Azomycin nucleoside-based radiosensitizer
Positron emission tomography (PET)
1. Introduction
izing radiosensitivity) through an oxygen-mimicking process (radi-
osensitizer effect) that results from their selective bio-reductive
Hypoxic cells are viable, oxygen deficient, cells that are gener-
ally 2.5 to 3-fold more radioresistant than well-oxygenated cells
to the cytotoxic effects of ionizing radiation (oxygen effect). Hy-
poxia develops in many solid tumors, leading to the induction of
activation and consequent adduct formation to hypoxic tissue
components.15,16 The first-electron reduction (1E7) step is crucial,
and is reversible in the presence of oxygen. Therefore, the selectiv-
ity and the degree of binding of a radiosensitizer is a function of its
electron affinity and the concentration of oxygen in cells. Reducing
equivalents (electrons) are metabolically derived,17 and therefore
the adduct-based accumulation of azomycins is restricted to viable
but oxygen-deficient tissues, with no accumulation in necrotic
cells, and little accumulation and low toxicity in most normally-
oxygenated cells. If the single-electron reduction potential at pH
7 (1E7) approaches that of O2 (ꢁ155 mV), then selectivity for hy-
poxia will be diminished; if the drug is not sufficiently electron-
affinic (1E7 <ꢁ450 mV), then sensitivity will be lost. The 1E7’s of
most 2-nitroimidazoles, for example, misonidazole (MISO), lie
around ꢁ390 mV, the region for optimal selectivity18 and sensitiv-
ity.19,15,20 Nitroimidazole-based radiosensitizers, when radiohalo-
genated11,12,21,22 with radioiodine or radiofluorine, are of interest
as radiodiagnostics for imaging focal hypoxia, providing in vivo
pre- and post-therapeutic evaluation of cancer patients.12 Hypoxia
imaging is also of interest in determining the role of hypoxia-
adaptive
genomic
transcriptional
and
post-translational
changes1–4 that promote the establishment of an aggressive phe-
notype which induces metastatic potential, promotes angiogenesis
and supports local disease progression.5–8 Inadequate angiogenesis
and compression of blood and lymphatic vessels in rapidly prolif-
erating tumor cells change the composition and structure of the
extracellular matrix (ECM), which effectively reduces perfusion of
the tumor9 and, consequently, decreases the delivery of the drugs
to the hypoxic area. Since hypoxic tumor cells have an aggressive
and treatment-resistant phenotype, they respond poorly to radio-
therapy (external beam radiotherapy; XRT) and are major determi-
nants of XRT failure.10 This necessitates early identification of
hypoxic tumor cells11,12 and aggressive, innovative treatment to
combat their rapid progression and the resultant poor
prognosis.8,13
2-Nitroimidazoles (azomycins) have proven radiosensitization
and chemosensitization properties in viable but hypoxic cells14
(Nagasawa et al., 2006), restoring the ‘oxygen effect’ (i.e., normal-
upregulated transcription factors such as hypoxia-inducible fac-
23
tor-1a
(HIF1a)
and glucose transporter proteins (GLUTs)24 to
the therapeutic sensitivity of hypoxic tissues at cellular and molec-
ular levels,16,25 for PET mapping of heterogeneity in hypoxic tis-
sues,24 and to obtain valuable information on therapeutic efficacy
and the development of new treatment modalities.
* Corresponding author. Tel.: +1 780 989 4313; fax: +1 780 432 8483.
E-mail addresses: pkumar@ualberta.ca (P. Kumar), ebinaimi@hotmail.com (E.
Naimi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.064

2256
P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
Hypoxic cancer cells can bio-reductively activate radiosensitiz-
radiolabeling process, resulting in poor fluorination yields.47 In
addition, the fluorine atom in FAZA is located at C-50; as a primary
alkyl fluoride it is also prone to in vivo hydrolysis (Fig. 1). Certainly,
deiodination of [123I]-IAZA, the corresponding iodinated analogue,
occurs in vivo.48 It is postulated that placement of fluorine on a
secondary carbon, that is, C-20, as in FRAZ, may provide additional
chemical stability to the molecule49,50 thereby offering fewer side
products during the synthesis of FRAZ (and the corresponding
ers via reductive enzymes that are upregulated in hypoxia.26 Prop-
erties of hypoxia-selective radiosensitizers that will maximize
their accumulation in hypoxic cells include minimal in vivo metab-
olism other than reductive bio-activation, short residence time in
non-target tissues, rapid renal excretion and low hepatobiliary
clearance. 2-Nitroimidazole-based molecules enter cells ubiqui-
tously via diffusion, and are selectively trapped by bio-reductive
activation in hypoxic cells. This means that increasing the lipophil-
icity of a radiosensitizer27 will increase its non-specific accumula-
tion in lipoidal tissues and contribute to selective toxicities (e.g.,
neuropathies), whereas lowering lipophilicity will increase renal
clearance and reduce accumulation in the liver. 18F-labeled 1-(2-
[
18F]-labeled radiopharmaceutical) while still maintaining the clin-
ically desirable properties (e.g., optimal lipophilicity and hypoxia-
selective reductive activation) of FAZA. The current work describes
the synthesis of 1-
idazole (FRAZ, 7), the automated F-18 radiolabeling to manufacture
18F]-FRAZ ([18F]-7), its preliminary hypoxia-selective radiosensiti-
a-D-(2-deoxy-2-fluororibofuranosyl)-2-nitroim-
nitroimidazole)-3-fluoro-2-propanol
([18F]-fluoromisonidazole,
[
[
18F]-FMISO) was one of the first 2-nitroimidazole-based tracers
zation potency in the HCT-110 colorectal cancer cells in vitro, and
assessment of its in vitro cytotoxicity using a bank of cancer cell
lines.
used in positron emission tomography (PET) to diagnose oncolog-
ical hypoxia (Fig. 1). It is currently an important clinical tool in
diagnostic oncology.28 Extensive pharmacokinetic (PK) and hypox-
ia-selective uptake studies have been used to validate this tra-
cer.21,22,29,30 However, its major disadvantage lies in its relatively
slow clearance from normal tissues.31 Other nitroimidazole-based
PET tracers that have found limited clinical utility include EFI32 and
FETNIM.33
2. Results and discussion
2.1. Chemistry
The syntheses of 7 and [18F]-7 were derived from 1-
a-D-(3,5-di-
The optimal hypoxia-selective reductive binding potential and
faster clearance properties of several radioiodinated sugar-coupled
O-benzoylarabinofuranosyl)-2-nitroimidazole, 2. This molecule
features a 20-OH group for reaction with DAST to afford 6
(Scheme 1), or for elaboration with an appropriate leaving group,
for example, trifluoromethanesulfonyl (triflate), 4-nitro-
benzenesulfonyl (nosylate), or toluenesulfonyl (tosylate), to afford
the precursors, 11a–11c, for making [18F]-7 (Scheme 2). Thus, the
2-nitroimidazoles (e.g., 1-a-D-(5-deoxy-5-iodoarabinofuranosyl)-
2-nitroimidazole ([123I]-IAZA), in comparison to non-sugared
azomycins, led to their clinical advancement as imaging radiodiag-
nostics using single-photon-emission computed tomography
(SPECT).34–36 Exploitation of the shorter half-life of F-18
(ꢀ110 min compared to 13 h for I-123) and the superior image res-
treatment of 1-a-D-(arabinofuranosyl)-2-nitroimidazole (AZA,
8)51 with benzoyl chloride in anhydrous pyridine afforded 1 in
quantitative yield (100%; Scheme 1). Selective 20-O-debenzoylation
of 1 was attempted following a literature method,52 however in
our case, treatment of 1 with potassium tert-butoxide (tert-BuOK;
olution of PET formed the basis to develop 18F-labeled 1-
deoxy-5-fluoroarabinofuranosyl)-2-nitroimidazole
a
-
D
-(5-
([18F]-
FAZA).37–41 In a recent study, the in vitro cell uptake and in vivo
imaging parameters of [18F]-FAZA were compared with those of
the established tracer [18F]-FMISO.42,43 The advantage of [18F]-
FAZA appears to lie in its faster clearance from the body, resulting
in shorter waiting times after tracer application and perhaps a low-
3 equiv) in tetrahydrofuran (THF) at ꢁ56 °C for 5 min gave 1-
a-D-
(3-O-benzoylarabinofuranosyl)-2-nitroimidazole, 4, a mono ben-
zoylated derivative as a major product (42% yield). 1H NMR spec-
troscopy confirmed the formation of this compound by
demonstrating two doublet (d) signals for H-20 and H-30 at d 4.31
and 4.05 ppm, respectively, that moved upfield in comparison to
corresponding resonances in 1. In addition, this reaction also affor-
er radiation exposure.42,44 18F]-FAZA is currently being used in
[
several tumor hypoxia based clinical diagnostic studies.12,45,46
Radiodosimetric calculations with [123/131I]-IAZA, a radioiodinated
azomycin nucleoside, indicate possible use of these compounds
in molecular radiotherapy (MRT).34 Surprisingly, evaluations of
these azomycin halonucleosides as radiosensitizers for therapeutic
XRT have not been reported.
Radiolabeled FAZA and IAZA, radiodiagnostics used extensively
in the nuclear medicine community, are C-50 substituted azomycin
nucleosides; they have free hydroxyl groups at C-20 and C-30, and a
strongly electron-withdrawing nitro group at the C-5 imidazole
position which may consequently become involved in secondary
intramolecular reactions with neighboring nucleophilic substitu-
ents (Fig. 1, structure of the C-20-C-5 cyclic product). The latter
reaction competes with fluorination of the precursor during the
ded 1-
1- -(2,5-di-O-benzoylarabinofuranosyl)-2-nitroimidazole,
and a 50-O-monobenzoylated analogue, 1-
-(5-O-benzoylarabi-
a
-D
-(3,5-di-O-benzoylarabinofuranosyl)-2-nitroimidazole, 2,
a-D
3,
a
-D
nofuranosyl)-2-nitroimidazole, 5, albeit in smaller quantities
(totaling ꢀ40%). It was observed that 1, when reacted with an equi-
molar quantity of tert-BuOK in THF (1 equiv) at a relatively low
temperature (ꢁ78 °C), afforded the 20-debenzoylated product, 2,
in excellent yield (ꢀ81%) in 30 min (Scheme 1). A strong upfield
movement of the chemical shifts of H-20 (by d 1.21 ppm) and C-
20 (by d 7.02 ppm) in the 1H NMR spectrum of 2 in comparison to
the tribenzoylated product 1 confirmed this selective debenzoyla-
tion at C-20-OH.
'F' at primary carbon
'F' at C-2' secondary carbon
F
N
HO
O
HO
O
O
HO
NO2
O2N
NO2
N
N
N
N
OH
F
HO
N
HO
O
HO
F
N
N
FMISO
FAZA
FRAZ
C-2'-C-5 cyclic product
The location of fluorine at a secondary carbon C-2' in FRAZ will minimize possible intramolecular
side reactions e.g. the formation of C-2'-C-5' cyclic produc4t7 as shown here.
Figure 1. Structures of FMISO, FAZA, FRAZ and C-20-C-5 cyclic product.

P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
2257
HO
NO2
BzO
2 +
BzO
NO2
BzO
O
OH
O
OBz
O
OH
O
OBz
ii
NO2
NO2
+
N
+
N
N
N
BzO
HO
HO
BzO
N
N
N
N
4
1
3
5
i
BzO
RO
O
HO
O
O
7
OH
NO2
NO2
NO2
iii
iv
N
N
N
BzO
RO
N
N
F
HO
N
F
2
6
Where, R and Bz = Benzoyl
Scheme 1. Synthetic route to FRAZ, 7. Reagents and conditions: (i) tert-BuOK (1 equiv)/THF, ꢁ78 °C; (ii) tert-BuOK (3 equiv)/THF, ꢁ56 °C; (iii) DAST (5 equiv)/CH2Cl2/pyridine
(4:1, v/v)/ 4 h at 0 °C then 14 h at 22 °C; (iv) 2 M NH3/MeOH, 0–15 °C, 15 h.
O
RO
O
TIPDS
O
OL
NO2
OH
NO2
N
N
O
N
RO
N
2
10
viii
RO
v
O
OL
Route A
NO2
N
vii
RO
N
11
11a: R = Bz, L = Tf ;
11b: R = Bz, L = p-Ns;
11c: R = Bz, L = Ts;
11d: R = Ac, L = Ts
HO
O
O
OH
O
TIPDS
OH
NO2
NO2
N
N
O
HO
N
N
vi
Route B
8: AZA
9
Where, Tf = Trifluoromethanesulfonyl; Ns = p-Nitrobenzenesulfonyl, Ts =
Toluenesulfonyl; Bz = Benzoyl; Ac = Acetyl
Scheme 2. Synthetic route to the precursors, 11a–11d, for preparing [18F]-7. Reagents and conditions: (v) For 11a, triflyl anhydride/DMAP/anhyd CH2Cl2/0 (1 h)?22 °C (3 h);
for 11b, nosyl chloride/AgOTf/0 (1 h)?22 °C (3 h); for 11c, tosyl chloride/DMAP/anhyd pyridine/45–50 °C (16 h); (vi) TIPDS-Cl/anhyd pyridine/22 °C (16 h); (vii) tosyl
chloride/DMAP/anhyd pyridine/22 °C (24 h); (viii) TBAF (THF)/Ac2O/acetonitrile/22 °C (20 h).
Compound 2 was the main synthon for synthesizing 7 and the
precursors that would be used to develop [18F]-7). 1-
-(3,5-Di-
O-benzoyl-2-deoxy-2-fluororibofuranosyl)-2-nitroimidazole, 6,
was obtained in excellent yield (>72% isolated yield) by fluorinat-
ing 2 with diethylaminosulfurtrifluoride (DAST) under anhydrous
conditions. Strong downfield movement in the chemical shifts for
H-20 (by d 1.08 ppm), H-30 (by d 0.15 ppm), H-10 (by d 0.32 ppm)
protons, and the split in these resonances were attributed to their
coupling with fluorine (JF–H) which confirmed the incorporation of
fluorine at C-20 in 6 (Table 1).
a-D
The incorporation of fluorine at C-20 in 6 impacted the NMR sig-
nals of H-10, H-20, and H-30 (neighboring protons), as evident by the
appearance of
a doublet of doublet (dd) at d
6.94 (H-10;
0
JF,H1 = 12 Hz), a doublet of doublet of doublet (ddd) at d 5.77 (H-
20; JF,H2 = 54 Hz) and a ddd at d 5.64 for (H-30; JF,H3 = 18 Hz) in its
0
0
1H NMR spectrum. 19F NMR and 13C NMR spectroscopic data pro-
Table 1
1H, 19F and 13C NMR chemical shifts and the coupling constants (J) of specific compounds 2–11 and related coupling constants
Compounds
Chemical shifts (d ppm) and the splitting patterns (in italic)
Coupling constant ‘J0 (Hz)
H-10
H-20
H-30
H-40
19F
C-10
C-20
C-30
C-40
JF,1
JF,2
0
0
0
JF,3
2
3
6
6.62 s
5.34 s
6.94 dd
4.69 s
5.34 s
5.77 ddd
5.49 s
4.58 s
5.64 ddd
5.03 dd
4.90 dd
4.99 dd
96.51
94.65
79.05
89.88
86.23
81.32
82.07
80.11
79.83
ꢁ47.85 ddd
ꢁ46.65 ddd
88.43 d
JF,C = 17
90.11 d
JF,C = 15
92.91
88.04 d
JF,C = 202
91.94 d
JF,C = 195
84.75
71.60 d
JF,C = 15
71.17 d
JF,C = 18
80.82
12
13
54
54
18
20
7
6.77 dd
5.34 ddd
4.70
4.34 dd
86.01
11b
11c
11d
6.58 s
6.74 s
6.58 s
5.44 s
5.70 s
5.40 s
5.11 d
5.34 d
5.04 s
4.65 ddd
4.94 dt
4.69 ddd
76.21
76.67
76.32
92.53
92.53
85.88
85.50
85.88
85.50

2258
P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
vided additional support to the site-specific fluorination at C-20 in
formed as shown below (Scheme 3). The –OH groups at the C-50
and C-30 positions in the tosylate precursors were protected by
the base labile protective groups (acetyl/benzoyl) to render selec-
tive fluorination at the C-20 position possible, to ensure facile
post-fluorination deprotection. An automated radiofluorination
process for [18F]-7 at 110 °C (5 min labeling time) using a commer-
cially available automated synthesis unit (ASU), Tracerlab-FX (GE,
USA) provided complete synthesis in ꢀ32 min.
6. The 19F NMR spectrum included a ddd signal at d ꢁ47.85
(JF,2 = 54 Hz, JF,1 = 12 Hz, and JF,H3 = 18 Hz) while 13C NMR spec-
trum revealed a strong downfield shift of the signal for C-20 (by d
8.99 ppm) in comparison to the corresponding non-fluorinated
compound 2, and exhibited fluorine-specific interaction
0
0
0
0
(JF,C2 = 202 Hz). The incorporation of fluorine also created a shield-
ing effect on neighboring carbons, which was reflected by the sig-
nals for C-10 (d 88.43 ppm, JF,C1 = 17 ppm) and C-30 (d 71.60 ppm,
The incorporation of F-18, due to its short half-life, requires
optimal labeling conditions to avoid unnecessary losses. It has
been reported that the base-catalyzed radiofluorination of
azomycin nucleoside-based precursors containing vicinal –OH
groups competes with the formation of several secondary fluori-
nated and non-fluorinated products (due to the intramolecular
elimination).47 The leaving groups that create a stronger polari-
zation of ‘C-sulfonate’ bond, for example, nosylate or triflate55
may also promote the rate of formation of undesired side prod-
ucts even though they undergo higher rates of substitution.
Nucleophilic substitution of the tosyl group is slow relative to
triflyl or nosyl groups, and this may translate into lower radio-
fluorination yields, however, the slower reaction kinetics may
also reduce decomposition and/or side product formation during
the labeling process. The F-18 labeling of 7 was attempted from
its tosylated precursor 11d. HPLC analysis of the labeled reaction
mixture revealed the formation of [18F]-7 along with unreacted
fluoride and several unidentified labeled species which were
not isolated (Fig. 2A and B). Purified [18F]-7 (195 MBq) was col-
lected after HPLC in ꢀ22% radiochemical yield (uncorrected
yield; specific activity = 63 GBq/nmol); TLC co-spotting with
standard FRAZ indicated a radiochemical purity (Fig. 2C) >97%
using a AR 2000 radio-scanner (Bioscan Inc., USA) and UV visu-
0
JF,C1 = 15 ppm) in the 13C NMR spectrum of 6 (Table 1). The conver-
0
sion of 6 into 7 was facilitated by NH3/MeOH-assisted debenzoyla-
tion, which proceeded smoothly (74% yield). Removal of benzoyl
groups shielded H-10 and H-20 in 7 (chemical shifts moved upfield
by d 0.17 ppm and 0.45 ppm, respectively) in comparison to the
corresponding dibenzoyl product 6. In addition, 19F and 13C NMR
spectra also demonstrated relevant impact on the carbon chemical
shifts (C-10, C-20, and C-30) and exhibited typical fluorine–carbon
(F–C) coupling patterns reconfirming the presence of fluorine in
the structure of 7 as shown by the relevant chemical shifts and cor-
responding coupling constants in Table 1.
F-18 radionuclide is short-lived (t1/2 = ꢀ110 min), so its facile
incorporation requires both an easily substitutable leaving group
and easily hydrolyzed protective groups in the precursor molecule
to minimize total reaction time. Scheme 2 depicts the syntheses of
four precursors that possess these features. They were prepared via
two synthetic routes, A and B. Route A followed simple substitution
of C-20-OH group in 2 with an appropriately substituted alkyl/aryl
sulfonylchloride.
Thus, the syntheses of 1-
binofuranosyl)-2-nitroimidazole, 11a, and 1-
a
-
D
-(3,5-di-O-benzoyl-2-O-triflyl-ara-
-(3,5-di-O-ben-
a
-D
zoyl-20-O-tosyl-arabinofuranosyl)-2-nitroimidazole, 11c, were
catalyzed by dimethylaminopyridine (DMAP), while silver triflate
was used to promote the reaction of nosyl chloride with 2. The
chemical yields of these derivatives were satisfactory, ranging from
62% to 82%. Compound 11a, the triflyl analogue, was not very sta-
ble even under cold storage conditions. It is reported that the re-
moval of benzoyl groups takes longer and requires stronger alkali
which, in the case of azomycin nucleosides, results in the forma-
tion of secondary products.53 Therefore, compound 11d, an acetyl
substituted analogue of 11c, was also synthesized following route
B. This route followed the protection of 30- and 50-OH groups in
AZA, 8, with the tetraisopropyldisilanyloxy (TIPDS) group to afford
alization of standard FRAZ (UV at 254–400 nm; Rf[18
RfSTD. UV).
= 0.05
F]-FRAZ
Chemical stability is essential for in vivo and in vitro evalua-
tions (e.g., PET imaging and radiosensitization measurements). It
is well known that halogens substituted on a primary carbon are
relatively unstable chemically in comparison to the halogens
substituted at a secondary carbon. In the case of IAZA, which
has an iodine at the primary C-50 position, and FMISO, which
has fluorine on a primary carbon, in vivo deiodination48 and
metabolic degradation,56 respectively, occur. On the other hand,
[
18F]-FAZA, a fluorinated analogue of IAZA, is stable over a 24 h
1-a
-D
-(3,5-O,O-TIPDS-arabinofuranosyl)-2-nitroimidazole, 954 in
storage period when formulated in 8% ethanol solution in sterile
water.57 Although there is no literature report available on its
in vivo stability, other fluorinated nucleosides have been re-
ported to be stable.49 The reason for the stability of the C–F
bond (length 1.35 Å) relative to the C–H bond (length 1.43 Å)
may be due to stabilization of the ‘endo’ conformation in a
two carbon unit bonded vicinally to electronegative elements
as in 2-deoxy-2-fluoro ribonucleosides.49,50 It has been proposed
that this effect results from the high electronegativity of the
fluorine atom acting in conjunction with the ‘gauche effect’.58
The placement of fluorine at C-20 position in 7 therefore may
provide it a superior in vivo stability in comparison to non-fluo-
rinated azomycin nucleosides.
86% yield. Compound 9, on reaction with tosyl chloride in the pres-
ence of DMAP, gave 1-a-D
-(3,5-O,O-TIPDS-20-O-tosylarabinofur-
anosyl)-2-nitroimidazole, 10. 1H and 13C NMR spectra of 10
confirmed the incorporation of tosyl group at C-20 OH group. Acet-
ylation of 10 replaced the TIPDS group from 30-O and 50-O positions
to provide the corresponding 30,50-di-O-acetyl product 11d in high
yield.
2.2. 18F-labeling
The radiofluorination chemistry of [18F]-FRAZ was based on the
nucleophilic substitution of its tosylate precursor, and was per-
RO
O
HO
O
OL
NO2
NO2
1. Kryptofix 2.2.2/18F
2. 0.1N.NaOH
N
N
RO
HO 18F
N
N
18F]-FRAZ, [18F]-7
[
11a
Scheme 3. Radiofluorination process to synthesize [18F]-7.

P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
2259
Figure 2. Radio (2A) and UV (2B; 320 nm) HPLC chromatograms of post-labeling reaction mixture of [18F]-7 from its tosylate precursor, and TLC chromatogram of purified
18F]-7 (2C). [18F]-FRAZ is seen at a retention time of 21 min in the radiochromatogram (2A). A major peak in the UV chromatogram (2B) at a retention time of 23.5 min relates
[
to the formation of an unknown side product.
3.2. Radiosensitization
3. Biological assessment
3.1. Cytotoxicity
These studies with 7 were performed in HCT-116 colorectal car-
cinoma cells (Fig. 4, top frame) at a concentration of 100 lM and
were compared with standard hypoxia radiosensitizer FAZA
(Fig. 4, lower frame). The toxicity to normoxic cells at this concen-
tration is expected to be minimal. The data showed that the radio-
FRAZ was evaluated for its cytotoxic effects in vitro in EMT-6
(murine mammary tumor), KBALB, KBALB-STK (Kirsten virus-
transformed murine sarcoma cell lines), 143B (human osteosar-
coma), and 143B-LTK (human leukemia) cancer cell lines at the
concentrations ranging from 10ꢁ7 to 10ꢁ3 M (Fig. 3). These data
indicated that 7 has only low toxicity in these cell lines at concen-
trations below 10ꢁ4 M, and that all cells except the EMT-6 line
showed marked reductions in survival at mM (10ꢁ3 M)
concentrations.
sensitization by FRAZ, 7, at 100 lM concentration is comparable to
FAZA, since its oxygen enhancement ratio was ꢀ2.0, although 7
demonstrated a slightly superior sensitization enhancement ratio
(SERFRAZ ꢀ1.4 vs. SERFAZA ꢀ1.25) for HCT-116 cancer cell lines. This
indicates that the sensitizer enhancement ratio of 7 falls within the
range of 1.20–2.14 at 1 mM, and is therefore a potentially useful
nitroimidazole-based hypoxia radiosensitizer.36
KBALB
143B
EMT-6
KBALB-STK
143B-LTK
3.3. Partition coefficient (P)
The retention times of FRAZ, FAZA and selected
a-azomycin
nucleosides were compared on reverse phase HPLC. It was ob-
served that the retention times for FRAZ and FAZA were almost
identical. Interpolation from a plot of partition coefficients (P) of
known azomycin nucleosides as a function of their retention times
showed that FRAZ is essentially equi-lipophilic (P = 1.08) to FAZA
(P = 1.10).38 The positional difference in fluorine placements (C-50
in FAZA and C-20 in FRAZ), as expected, resulted in very little vari-
ation in the lipophilicity (Fig. 5). This falls within the range
(P = 0.1–10) of log partition coefficients thought to be compatible
with good tissue penetration by diffusion.18,59
80.00
40.00
0.00
4. Conclusion
0
1x10-7 1x10-6 1x10-5 1x10-4 1x10-3
Concentration (M)
The synthesis of FRAZ, 7, a positional ribose analogue of FAZA,
was achieved by DAST-assisted fluorination of 2 in ꢀ74% yield. Pre-
liminary in vitro radiosensitization studies with 7 against HCT-
Figure 3. In vitro cytotoxicity of FRAZ, 7, in KBALB, KBALB-STK, 143B, 143B-LTK and
EMT-6 cancer cell lines as assessed by MTT assay.

2260
P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
Figure 4. In vitro radiosensitization of HCT-116 colorectal cancer cells (top) by 7 (indicated as
a-2-FAZR in the top frame; 100 lM), and by FAZA (100 lM) (bottom), as
demonstrated by their survival curves.
116/100 colorectal carcinoma cells indicated that 7 is equipotent to
FAZA as a radiosensitizer. The toxicity of FRAZ to oxygenated cells
was low, with minimal effect on cell survival at concentrations less
that 10ꢁ3 M. The precursors required for the radiochemical devel-
opment of [18F]-7 were based on simple, readily removable pro-
tecting groups and leaving groups. Initial F-18 labeling
experiments afforded [18F]-7 in ꢀ22% radiochemical yield (decay
uncorrected). FRAZ, 7, may be more stable in vivo than FAZA due
to the influence of fluorine substitution at C-20 (in comparison to
C-50 in FAZA). It is expected that this positional isomer will possess
similar hypoxia-selective accumulation in hypoxic tumors as FAZA
since the placement of fluorine at a C-20 (in comparison to C-50 in
FAZA) in the sugar moiety does not impact its lipophilicity, and
therefore, its diffusion properties. It is known that the clinical use-
fulness of the radiosensitizers with substantially lower partition
coefficients is greatly enhanced due to their faster pharmacokinetic
renal clearance, as in the case of FAZA,44 in comparison to more
lipophilic compounds like MISO and FMISO that have poor egres-
sion resulting in undesirable neurotoxicities60,61 and slower hepa-
tobiliary clearance.44 PET imaging and hypoxia-selective in vivo
biodistribution and pharmacokinetic studies of FRAZ are
warranted.

P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
2261
5.2.2. 1-
nitroimidazole (2)
a-D-(3,5-Di-O-benzoylarabinofuranosyl)-2-
6.00
5.00
4.00
3.00
2.00
1.00
0.00
-1.00
-2.00
y = 0.2241x - 2.6303
R2 = 0.9697
tert-BuOK (1.8 mL of 1 M solution in THF, 1.8 mmol, 1 equiv)
was added to a solution of 1 (1 g, 1.8 mmol) in THF (15 mL), pre-
chilled at ꢁ78 °C (dry-ice/acetone bath), with vigorous stirring un-
der an inert (argon) atmosphere. After stirring for 30 min, another
portion of 1 M solution of tert-BuOK in THF (0.45 mL, 0.45 mmol)
was added to the reaction mixture and the resulting solution was
stirred for an additional 10 min. A TLC check (solvent system A)
at this time indicated complete disappearance of the starting mate-
rial 2. The reaction was quenched by the addition of Dowex-50 W
resin that neutralized the base (by monitoring with colorpHast™
Indicator strips; pH 0–14). The resin was filtered off, further
washed with CH2Cl2 (30 mL) and CH3OH (10 mL) and then the fil-
trate and the washings were combined to evaporate the solvents
under reduced pressure. The residue was purified via flash silica
gel column chromatography using hexanes/EtOAc (2:1?1:1, v/v)
as eluent to give 2 (0.66 g, 81%) as a white solid; mp 70–71 °C; Rf
5-α-IAZA
RT=33.25 min
2-α-FRAZ;
RT=17.11 min
5-α-FAZA
RT=18.57 min
α−AZA
RT=12.28 min
3
8
13
18
23
28
33
38
Retention time(min)
Figure 5. Correlation between partition coefficient (P values) and reverse phase
HPLC retention times for FRAZ, FAZA and selected -azomycin nucleosides. The
linear regression line was drawn using Microsoft Excel™.
a
1
0.44 (solvent system A); H NMR (CDCl3): d 4.69 (s, 1H, H-20),
4.74 (dd, J4 ,5 = 6 Hz, Jgem = 12 Hz, 1H, H-50), 4.82 (dd, J4 ,5 = 7 Hz,
0
0
0
00
5. Experimental
5.1. General
Jgem = 12 Hz, 1H, H-500), 5.03 (dd, J5 4 = 6 Hz, J5 ,4 = 7 Hz, 1H, H-40),
5.49 (s, 1H, H-30), 6.62 (s, 1H, H-10), 7.22 (s, 1H, H-4), 7.39 (s, 1H,
H-5), 7.34–8.14 (m, 10H, two phenyls); 13C NMR (CDCl3): d 63.94
(C-50), 79.05 (C-20), 82.07 (C-40), 86.23 (C-30), 96.51 (C-10),
129.46–130.10 (phenyl carbons), 133.30 (imidazole C-4), 133.87
(imidazole C-5), 143.77 (C-2, imidazole), 165.13 and 166.25 (2
C@O); HRMS for C22H19N3O8Na, Calcd: 453.07514. Found:
453.07519 (M+Na, 100%).
0
0
00
0
All chemicals used were of reagent grade. Solvents were dried
over appropriate drying agents and freshly distilled before use.
The progress of synthetic reactions was monitored by thin layer
chromatography (TLC) in a suitable solvent system (system A—
5:95, v/v, CH3OH/CH2Cl2; system B—10:90, v/v, CH3OH/CH2Cl2;
system C—hexanes/EtOAc, 2:1, v/v; system D—hexanes/EtOAc,
1:2, v/v; and system E—hexanes/EtOAc 1:1, v/v). Column chroma-
tography was performed on Merck Silica Gel 60 (70–200 and
230–400 mesh ASTM). Melting points were determined on a Büchi
capillary melting point apparatus and are uncorrected. 1H, 19F, and
13C NMR spectra were recorded on a Bruker AM-300 spectrometer
in deuterated chloroform (CDCl3), dimethylsulfoxide (DMSO-d6) or
methanol (CD3OD), depending on the solubility of the product.
Chemical shifts are reported in d ppm downfield with respect to
tetramethylsilane as an internal standard in 1H and 13C NMR spec-
tra, and trifluoroacetic acid as an external standard for 19F NMR
spectra. The protons and carbons of the sugar moiety and nitroim-
idazole are represented by a single prime (0) and no prime, respec-
tively. HRMS were recorded using an AEI-MS-12 mass
spectrometer. High performance liquid chromatography (HPLC)
of 18F-labeled FRAZ was performed on a Beckman HPLC system
equipped with an isocratic solvent single flow pump, a reverse
phase C-18 guard column (Phenomenex 50 ꢂ 10 mm) and column
(Phenomenex 250 ꢂ 9 mm), dual detectors (UV and radioactive),
and Gold software to analyze and quantify the eluted components.
The purification was performed using EtOH/sterile H2O (12:88; v/
v) as an eluent at a flow rate of 1.8 mL/min.
5.2.3. 1-a-D-(2,5-Di-O-benzoylarabinofuranosyl)-2-
nitroimidazole (3)
This product was formed during the synthesis of 2, and was iso-
lated as a white solid (0.16 g, 20%) during the chromatographic
purification of the debenzoylation reaction mixture. Mp 69–
70 °C; Rf 0.48 (solvent system A); 1H NMR (DMSO-d6 + CD3OD): d
3.89 (dd, J4 ,5 = 6 Hz, Jgem = 12 Hz, 1H, H-50), 3.94 (dd, J4 ,5 = 5 Hz,
0
0
0
00
Jgem = 12 Hz, 1H, H-500), 4.58 (s, 1H, H-20), 4.90 (dd, J5 4 = 6 Hz,
0
0
J5 ,4 = 5 Hz, 1H, H-40), 5.34 (s, 1H, H-30), 6.56 (s, 1H, H-10), 7.24 (s,
1H, H-4), 7.43–7.74 (5H, phenyl), 7.85 (s, 1H, H-5); 13C NMR
(DMSO-d6 + CD3OD): d 62.85 (C-50), 80.11 (C-40), 81.32 (C-20),
89.88 (C-30), 94.65 (C-10), 124.79 (imidazole C-4), 129.24–134.35
(phenyl carbons), 128.30 (imidazole C-5), 144.92 (C-2, imidazole),
166.89 (C@O); HRMS for C22H19N3O8Na, Calcd: 453.07514. Found:
453.07521 (M+Na, 100%).
00
0
5.2.4. 1-
a
-D
-(3-O-Benzoylarabinofuranosyl)-2-nitroimidazole
(4) and 1-
(5)
a-D-(5-O-benzoylarabinofuranosyl)-2-nitroimidazole
These compounds were formed when the synthesis of 2 from 1
was attempted at a higher temperature (ꢁ56 °C), and using three
chemical equivalents of tert-BuOK solution (1 M in anhydrous
THF). The reaction was complete in 5 min, and afforded 4 as a ma-
jor product along with small quantities of 2 (ꢀ3%), 3 (5%), and 5
(35%). Flash column chromatography of the impure mixture using
gradients of hexanes/EtOAc (1:1?1:2?1:3; v/v; 200 mL of each
composition) separated these products and gave pure 4 as a solid.
Recrystallization from hexanes/EtOAc (3:1; v/v; 6 mL) afforded 4 as
white crystals, and 5 as a white solid. The chemical data for these
compounds are given below.
5.2. Chemistry
5.2.1. 1-a-D-(2,3,5-Tri-O-benzoylarabinofuranosyl)-2-
nitroimidazole (1)
Benzoyl chloride (2.30 g, 16.32 mmol) was added drop-wise to a
stirred solution of 8 (1 g, 4.08 mmol) in dry pyridine (16 mL) at 0 °C
(ice/water bath). The reaction mixture was stirred at 0 °C for 2 h
and at 22 °C for 20 h under argon. At this time, the solvent was re-
moved under vacuum (rotary evaporator and water pump) and co-
evaporated with toluene (2 ꢂ 5 mL). Purification of the residue via
flash column chromatography on silica gel using hexanes/EtOAc
(2:1?1:1, v/v) as eluent afforded a light yellowish oil, which was
crystallized from EtOAc/hexanes (2:1, v/v, 15 mL) to yield 1
(2.26 g, 100%) as white crystals; mp 105–106 °C (observed);
106 °C (reported); Rf 0.68 (system E).51
Compound 4: Yield 300 mg (42%); Mp 194–196 °C; Rf 0.34 (sol-
vent system A); 1H NMR (CD3OD + DMSO-d6):
d 3.91 (dd,
J4 ,5 = 5 Hz, Jgem = 12 Hz, 1H, H-50), 3.94 (dd, J4 ,5 = 6 Hz,
0
0
0
00
Jgem = 12 Hz, 1H, H-500), 4.58 (s, 1H, H-20), 4.90 (dd, J5 4 = 5 Hz,
0
0
J5 ,4 = 6 Hz, 1H, H-40), 5.34 (s, 1H, H-30), 6.56 (s, 1H, H-10), 7.24 (s,
1H, H-4), 7.43–7.75 (m, 5H, phenyl), 7.85 (s, 1H, H-5); 13C NMR
(CDCl3): d 62.85 (C-50), 80.11 (C-40), 81.33 (C-20), 89.88 (C-30),
00
0

2262
P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
97.42 (C-10), 129.03 and 130.12 (phenyl carbons), 130.26 (imidaz-
ole C-4), 134.35 (imidazole C-5), 144.92 (C-2, imidazole), 165.89
(C@O); HRMS (EI+) for C15H15N3O7Na, Calcd: 352.0754. Found:
352.0752 (M+Na, 100%).
5.2.7. 1-a-D-(3,5-Di-O-benzoyl-2-O-
trifluoromethanesulfonylarabinofuranosyl)-2-nitroimidazole
(11a)
Triflyl anhydride (0.0.06 g, 0.35 mmol) was added to a solution
of 2 (0.08 g, 0.176 mmol) and DMAP (0.065 g, 0.53 mmol) in anhy-
drous CH2Cl2 (5 mL), and stirred for 2 h at 0 °C under argon. The
TLC at this stage showed no change. Another aliquot of triflyl anhy-
dride was added to the reaction mixture, the temperature was then
raised to 22 °C, and the stirring was continued for an additional 3 h
under argon. The progress of the reaction was quenched by adding
a small piece of ice, the solution diluted by adding CH2Cl2, and ex-
tracted. The organic phase was separated, dried over anhydrous
Na2SO4, filtered, and the solvent was evaporated under reduced
pressure to afford a viscous residue which was purified by flash sil-
ica gel column chromatography using hexanes/EtOAc (1:1, v/v) as
an eluent to give pure 11a (0.064 g, 62%) as a colorless viscous
oil that was not very stable under normal storage conditions, and
could be characterized only by its proton spectrum. Rf 0.6 (system
Compound 5: Yield 250 mg; Mp 198–199 °C; Rf 0.29 (solvent
system A); 1H NMR (CDCl3): d 4.05 (d, J2 ,3 = 3 Hz, 1H, H-30),
0
0
4.31 (d, J3 ,2 = 3 Hz, 1H, H-20), 4.55 (d, J4 ,5 = 4 Hz, Jgem = 12 Hz,
0
0
0
0
1H, H-50), 4.84 (dd, J4 ,5 = 3 Hz, Jgem = 12 Hz, 1H, H-500), 4.83
0
00
(dd, J5 4 = 4 Hz, J5 ,4 = 3 Hz, 1H, H-40), 6.87 (s, 1H, H-10), 7.19 (s,
1H, H-4), 7.48 (s, 1H, H-5), 7.49–8.05 (m, 5H, phenyl); HRMS
(EI+) for C15H15N3O7Na, Calcd: 352.0754. Found: 352.0749
(M+Na, 100%).
0
0
00
0
5.2.5. 1-a-D-(3,5-Di-O-benzoyl-2-deoxy-2-fluororibofuranosyl)-
2-nitroimidazole (6)
Compound 2 (0.60 g, 1.32 mmol) was dissolved in CH2Cl2/pyri-
dine (10 mL, 98:2, v/v). The solution was cooled to 0 °C, diethyla-
minosulfurtrifluoride (DAST, 1.06 g, 6.6 mmol) was added, and
the mixture was stirred for 4 h at 0 °C, and then for 14 h at 22 °C,
under argon. Once the starting material was completely exhausted
(TLC), the reaction progress was quenched by adding a small quan-
tity of methanol (5 mL). The solvents were removed under vacuum
by toluene co-evaporation (2 ꢂ 5 mL) under reduced pressure and
the residue was purified by flash silica gel column chromatography
using hexanes/EtOAc (1:1, v/v) as eluent which gave 6 (0.435 g,
>72%) as a white foam. Mp 56–57 °C; Rf 0.59 (solvent system E);
E); 1H NMR (CDCl3): d 4.68 (dd, J5 ,4 = 6 Hz, Jgem = 13 Hz, 1H, H-50),
0
0
4.76 (dd, J5 ,4 = 6 Hz, Jgem = 12 Hz, 1H, H-500), 5.09 (dd, J5 ,4 = 5 Hz,
00
0
0
0
J5 ,4 = 6 Hz, 1H, H-40), 5.73 (s, 1H, H-30), 5.81 (s, 1H, H-20), 6.96 (s,
1H, H-10), 7.31 (s, 1H, H-4), 7.39 (s, 1H, H-5), 7.41–8.11 (m, 10H,
aromatic).
00
0
5.2.8. 1-a-D-(3,5-Di-O-benzoyl-2-O-(4-
nitrobenzenesulfonyl)arabinofuranosyl)-2-nitroimidazole
(11b)
1H NMR (CDCl3): d 4.58 (dd, J4 ,5 = 4 Hz, Jgem = 13 Hz, 1H, H-50),
0
0
4.63 (dd, J4 ,5 = 3 Hz, Jgem = 13 Hz, 1H, H-500), 4.99 (dd, J3 4 = 4 Hz,
Compound 2 (0.15 g, 0.33 mmol) was dissolved in anhydrous
pyridine (10 mL) at 0 °C, and nosyl chloride (0.22 g, 0.99 mmol)
and silver triflate (AgOTf; 0.255 g, 0.99 mmol) were added to this
solution under argon. The reaction mixture was stirred at 0 °C for
1 h and then the temperature was raised to 22 °C. The stirring
was continued at this temperature for an additional 3 h, and then
the reaction was quenched by adding a small piece of ice. The sol-
vents were evaporated and co-evaporated with toluene
(2 ꢂ 10 mL) under reduced pressure and the viscous residue was
purified by flash silica gel column chromatography using 3%
CH3OH in CH2Cl2 as eluent to afford 11b as a solid. Recrystalliza-
tion of this material from hexanes/EtOAc (1:1, v/v, 5 mL) produced
white crystals of 11b. Yield 0.20 g (95%); Mp 53–54 °C; Rf 0.6 (sys-
tem A); 1H NMR (CDCl3): d 1.99 and 2.20 (2 s, each for 3H of CH3),
0
00
0
0
J5 ,4 = 3 Hz, 1H, H-40), 5.64 (ddd, J2 ,3 = 4 Hz, J4 ,3 = 4 Hz, JF,3 = 18 Hz,
0
0
0
0
0
0
0
1H, H-30), 5.77 (ddd, J1 ,2 = 4 Hz, J3 ,2 = 4 Hz, JF,2 = 54 Hz, 1H, H-20),
0
0
0
0
0
6.94 (dd, J2 ,1 = 4 Hz, JF,1 = 13 Hz, 1H, H-10), 7.26 (s, 1H, H-4), 7.41
0
0
0
(s, 1H, H-5), 7.44–8.05 (m, 10H, two phenyls); 13C NMR (CDCl3):
d 62.98 (C-50), 71.60 (d, JF,4 = 15 Hz, C-30), 79.83 (C-40), 88.04 (d,
0
JF,2 = 202 Hz, C-20), 88.43 (d, JF,1 = 17 Hz, C-20), 128.06–129.85
(phenyl carbons), 133.55 (imidazole C-4), 133.94 (imidazole C-5),
145.00 (C-2, imidazole), 165.1 and 165.8 (2 C@O); 19F NMR
0
0
0
0
0
(CD3OD): d ꢁ47.85 (ddd, JF,1 = 12 Hz, JF,3 = 18 Hz, JF,2 = 54 Hz);
HRMS (EI+) for C22H18N3O7FNa, Calcd: 478.10210. Found: 478.
10231.
4.41 (dd, J5 ,4 = J4 ,5 = 5 Hz, Jgem = 17 Hz, 2H, H-50 and H-500), 4.65
5.2.6. 1-a-D-(2-Deoxy-2-fluororibofuranosyl)-2-nitroimidazole
0
0
0
00
(ddd, J3 ,4 = 3 Hz, J5 ,4 = 5 Hz, Jgem = 17 Hz, 1H, H-40), 5.11 (d,
(7, FRAZ)
0
0
0
0
J4 ,3 = 3 Hz, 1H, H-30), 5.44 (s, 1H, H-20), 6.58 (s, 1H, H-10), 7.19 (s,
1H, H-4), 7.31 (s, 1H, H-5), 8.15 and 8.43 (2 d, each for 2H of nosy-
late phenyl); 13C NMR (CDCl3): d 20.42 and 20.48 (2 CH3), 68.55 (C-
50), 76.21 (C-40), 80.82 (C-30), 84.75 (C-20), 92.91 (C-10), 124.52 (two
phenyl carbons), 121.77 (imidazole C-4), 128.54 (imidazole C-5),
129.30 (two phenyl carbons), 141.02 (C-4 phenyl), 146.74 (C-2,
imidazole), 150.95 (C-1 of phenyl), 168.68 and 169.28 (2 ꢂ C@O).
Anal. Calcd for C18H18N4O12S (514.43): C, 52.67; H, 3.47; N, 8.77.
Found: C, 52.22; H, 3.14; N, 8.56.
Compound 6 (0.050 g, 0.11 mmol) was treated with a solution
of NH3 in CH3OH (4 mL of 2 M solution) at 0 °C under argon and
the resulting solution was stirred at this temperature for 4 h, and
then for 12 h at 22 °C. Once the reaction was complete, the solvent
was removed under vacuum and the residue was purified by flash
column chromatography on silica gel using 10% CH3OH in CH2Cl2
that gave a white solid. Recrystallization of this material from
EtOAc/hexanes (1:1, v/v, 5 mL) yielded 7 (20 mg, 74%) as white
crystals. Mp 155–157 °C; Rf 0.46 (system B); 1H NMR (CD3OD): d
0
0
3.71 (dd, J4 ,5 = 3 Hz, Jgem = 13 Hz, 1H, H-50), 3.90 (dd, J2 ,5 = 2 Hz,
0
0
0
00
Jgem = 13 Hz, 1H, H-500), 4.34 (dd, J3 4 = 4 Hz, J5 ,4 = 3 Hz, 1H, H-40),
0
0
0
0
5.2.9. 1-
toluenesulfonylarabinofuranosyl)-2-nitroimidazole (11c)
Compound (0.20 g, 0.44 mmol), tosyl chloride (0.252 g,
a-D-(3,5-Di-O-benzoyl-2-O-
4.70 (ddd, J2 ,3 = 4 Hz, J4 ,3 = 4 Hz, JF,3 = 20 Hz, 1H, H-30), 5.34
0
0
0
0
0
(ddd, J1 ,2 = 4 Hz, J3 ,2 = 4 Hz, JF,2 = 54 Hz, 1H, H-20), 6.77 (dd,
0
0
0
0
0
2
J2 ,1 = 4 Hz, JF,1 = 13 Hz, 1H, H-10), 7.19 (s, 1H, H-4), 7.72 (s, 1H,
0
0
0
1.32 mmol) and DMAP (0.161 g, 1.32 mmol) were dissolved in
anhydrous pyridine (15 mL) and stirred at 45–50 °C for 16 h under
argon. The TLC examination of the reaction mixture at this time
showed no evidence of 2. The reaction progress was quenched by
adding a small piece of ice to this mixture and the solvent was re-
moved under reduced pressure using two co-evaporations with
toluene (2 ꢂ 20 mL). The viscous residue so obtained was purified
by flash silica gel column chromatography. An elution with hex-
anes/EtOAc (2:1?1:1, v/v, 5 mL) gave pure 11c (0.22 g, 82%) as a
white foam. Mp 60–61 °C; Rf 0.64 (system E); 1H NMR (CDCl3): d
13
H-5); C NMR (CD3OD): d 61.93 (C-50), 71.17 (d, JF,3 = 18 Hz, C-
0
30), 86.01 (C-40), 90.11 (d, JF,1 = 15 Hz, C-10), 91.94 (d, JF,2 = 195 Hz,
0
0
C-20), 124.97 (imidazole C-4), 128.44 (imidazole C-5), 139.00 (C-2,
imidazole); 19F NMR (CD3OD):
d
ꢁ46.65 (ddd, JF,1 = 13 Hz,
0
0
0
JF,3 = 20 Hz, JF,2 = 54 Hz). Anal. Calcd for C8H10FN3O5 (247.18): C,
38.87; H, 4.08; N, 17.00. Found: C, 38.80; H, 4.01; N, 16.89.
The syntheses of the compounds 11a–11c involved the reaction
of compound 2 with the appropriate sulfonyl chloride/anhydride
following route A, and are described below.

P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
2263
2.39 (s, 1H, tosyl CH3), 4.61 (dd, J5 ,4 = 6 Hz, Jgem = 12 Hz, 1H, H-50),
J5 ,4 = 3 Hz, Jgem = 12 Hz, 1H, H-500), 4.69 (ddd, J5 ,4 = 6 Hz,
0
0
00
0
0
0
4.72 (dd, J5 ,4 = 6 Hz, Jgem = 12 Hz, 1H, H-500), 4.94 (dt,
J5 ,4 = 3 Hz, J3 ,4 = 5 Hz, 1H, H-40), 5.04 (s,1H, H-30), 5.40 (s, 1H, H-
20), 6.58 (s, 1H, H-10), 7.18 (s, 1H, H-4), 7.27 (s, 1H, H-5), 7.38 and
7.83 (2 d, 4H, aromatic); 13C NMR (CDCl3): d 20.48 and 20.71 (acet-
yl carbons), 21.79 (tosyl CH3), 62.86 (C-50), 76.32 (C-40), 85.50 (C-20
and C-30), 92.53 (C-10), 122.16 (C-4), 128.18 (C-5), 130.14, 131.77
and 146.26 (phenyl carbons) and 151.16 (C-2 imidazole), 168.83
and 170.40 (2 C@O); HRMS (EI+) for C19H21N3O10NaS, Calcd:
506.08399. Found: 506.08409.
00
0
00
0
0
0
J5 ,4 = J5 ,4 = 6 Hz, J3 ,4 = 2 Hz, 1H, H-40), 5.34 (d, J4 ,3 = 2 Hz, 1H, H-
30), 5.70 (s, 1H, H-20), 6.74 (s, 1H, H-10), 7.21 (s, 1H, H-4), 7.39 (s,
1H, H-5), 7.30–8.13 (m, 14H, aromatic); 13C NMR (CDCl3): d
21.72 (CH3), 63.56 (C-50), 76.67 (C-40), 85.88 (C-20 and C-30),
92.94 (C-10), 127.86 (C-4), 128.12–129.73 (phenyl carbons),
131.96 (C-5), 133.37 and 134.09 (tosyl aromatic carbons), 146.21
(C-2, imidazole), 164.47 and 165.91 (2 ꢂ C@O). Anal. Calcd for
(607.59): C, 57.33; H, 4.15; N, 6.92. Found: C, 57.98; H, 4.07; N,
6.41.
0
0
00
0
0
0
0
0
5.3. Radiochemistry
Synthesis of compound 11d was developed from compound 9
via route B, and is described below.
A TR19 negative ion accelerating cyclotron capable of accelerat-
ing protons to 19 MeV and deuterons to 9.5 MeV (Advanced Cyclo-
tron Systems, Inc.) with an external high performance multicusp
5.2.10. 1-a-D-(3,5-O,O-
Tetraisopropyldisilanyloxyarabinofuranosyl)-2-nitroimidazole
(9)
This product was synthesized following a reported procedure;
mp 83–84 °C (observed); 83–84 °C (reported).54
ion source greater than 150 lA beam current with dual simulta-
neous beam extraction was used for 18F production. A radiofluoride
target consisting of a niobium body with HAVAR foil window was
used for production of 18F via 18O(p,n)18F reaction by irradiating
highly 18O enriched (98%) water (H218O). Tracerlab-FX automated
synthesis units (ASU) employed for the manufacture of [18F]-FRAZ
was purchased from G.E. Medical Technologies Inc., Canada which
operates on the basis of performing unit operations under com-
puter (CPU) control, involving the movement of liquids, movement
of gases (vacuum and pressure), valve actuation and heating sys-
tems on a time dependent basis through a well defined flow path.
Feedback control, monitoring and diagnostic functions are built
into the operation using various sensor-based systems.
5.2.11. 1-a-D-(3,5-O,O-Tetraisopropyldisilyloxy-2-O-
toluenesulfonylarabinofuranosyl)-2-nitroimidazole (10)
A mixture of 955 (0.30 g, 0.615 mmol), tosyl chloride (0.35 g,
1.85 mmol), and DMAP (24 mg, 0.02 mmol) were taken in anhy-
drous pyridine (5 mL) and stirred at 22 °C under an atmosphere
of argon for 24 h. At this time, a TLC examination of the reaction
mixture showed no change in the status of starting material.
Therefore, additional quantities of tosyl chloride (1.85 mmol) and
DMAP (0.06 mmol) were added to this reaction mixture and the
reaction was subjected to stirring at 60 °C for an additional 21 h
that led to the completion of tosylation of 9. Excess tosyl chloride
was hydrolyzed by addition of a small piece of ice, and then the
solvents were removed under reduced pressure by evaporation
and co-evaporation using toluene (2 ꢂ 20 mL). The resulting vis-
cous mass was purified by flash silica gel column chromatography
using hexanes/EtOAc (3:1, v/v) as eluent to afford pure solid prod-
uct, 10. Recrystallization of this solid from hexanes (20 mL) gave
pure white crystals of the product. Yield 340 mg (86%); mp 113–
115 °C; Rf 0.5 (solvent system C); 1H NMR (CDCl3): d 0.98–1.1 (m,
[
18F]Fluoride (1.2 GBq) was transferred to the ASU reaction ves-
sel in a solution containing potassium carbonate (3.5 mg in 100
of sterile water for injection, SWFI) and Kryptofix 2.2.2 (15 mg in
900 L of CH3CN). The solvents from the fluoride complex were re-
lL
l
moved by two azeotropic evaporations and then 11a (5 mg pre-
dissolved in 1 mL of anhydrous DMSO) was added to it and heated
at 110 °C (5 min). The total reaction time was ꢀ32 min. The result-
ing product was then deprotected using 0.1 N NaOH (2 min at
30 °C) and the pH was adjusted to ꢀ5–7.5 by adding 0.4 M NaH2-
PO4 buffer (1 mL). HPLC purification of this crude mixture on a C-
18 reversed phase column (10 ODS3, 25 ꢂ 0.9 cm) using 12% EtOH
in SWFI as eluent (flow rate 1.8 mL/min) revealed the presence of
0
0
28H, TIPDS protons), 2.44 (s, 1H, tolyl CH3), 3.91 (dd, J5 ,4 = 6 Hz,
[
[
18F]-7 along with unreacted [18F]-fluoride (10–13 min); pure
18F]-7 (195 MBq; ꢀ22% radiochemical yield, decay uncorrected,
Jgem = 12 Hz, 1H, H-50), 4.03 (dd, J5 ,4 = 3 Hz, Jgem = 12 Hz, 1H, H-
00
0
500), 4.19 (ddd, J5 ,4 = 6 Hz, J5 ,4 = 3 Hz, J3 ,4 = 5 Hz, 1H, H-40), 4.67
0
0
00
0
0
0
radiochemical purity = >97%) was recovered at a retention time
of 21 min ( 5%) (specific activity = 63 GBq/nmol). The purity of
the HPLC-purified radiopharmaceutical was checked by TLC exam-
ination using a BIOSCAN TLC AR2000 scanner and the radiochem-
ical identity was confirmed by comparing its Rf with by co-spotting
the standard FRAZ, 7 (Fig. 2C).
(dd, J4 ,3 = 5 Hz, J2 ,3 = 5 Hz,1H, H-30), 5.21 (dd, J3 ,2 = 5 Hz,
0
0
0
0
0
0
J1 ,2 = 4 Hz, 1H, H-20), 6.55 (d, J1 ,2 = 4 Hz, 1H, H-10), 7.18 (s, 1H,
H-4), 7.36 (s, 1H, H-5), 7.27–7.65 (2 d, 4H, aromatic); 13C NMR
(CDCl3): d 12.28–17.42 (TIPDS carbons), 21.70 (CH3), 62.03 (C-50),
75.75 (C-40), 85.01 (C-30), 87.91 (C-20), 88.82 (C-10), 122.10 (C-4),
128.66 (C-5), 127.61, 130.01, 131.92 and 146.08 (phenyl carbons)
and 144.51 (C-2 imidazole); HRMS (EI+) for C27H43N3O9NaSi2S,
Calcd: 664.21508. Found: 664.21529.
0
0
0
0
5.4. Cytotoxicity
Briefly, exponentially-growing cells were trypsinized and col-
lected in the appropriate medium. The cell concentrations were ad-
justed to 8 ꢂ 103 cells/mL with the corresponding medium. The
5.2.12. 1-a-D-(3,5-Di-O-acetyl-2-O-
toluenesulfonylarabinofuranosyl)-2-nitroimidazole (11d)
TBAF (0.94 mL, 1 M solution in anhydrous THF) was added to a
solution of 10 (0.30 g, 0.47 mmol) and acetic anhydride (0.38 g,
3.76 mmol) in acetonitrile (6 mL), and the resulting mixture was
stirred at 22 °C for 20 h under argon. At this time, TLC of the mix-
ture showed complete disappearance of the silylated precursor and
appearance of a new product at a higher Rf. The solvents were re-
moved under reduced pressure, and the dry residue was purified
by flash silica gel column chromatography using hexanes/EtOAc
(1:2, v/v) to afford pure 11d as a white foam. Yield 203 mg
(90%); mp 50–51 °C; Rf 0.52 (solvent system D); 1H NMR (CDCl3):
d 1.96 and 2.14 (2 s, each for 3H of acetyl CH3), 2.44 (s, 1H, tosyl
cells (8 ꢂ 102 cells in 100
lL) were seeded into wells of a 96-well
plate and incubated for 24 h at 37 °C with 5% CO2 and 95% air.62
Compound 7 was dissolved at the desired concentrations in growth
medium and the resulting solution (100
added to each cell-containing well. In controls, 100
l
L) of test compound was
L of the med-
l
ium replaced the compound solution. After incubation for 72 h at
37 °C, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT, 50
lL of 1 mg/mL solution) was added to each well,
and after 4 h the supernatant was removed and DMSO (150
lL)
was added to each well to dissolve the formazan crystals. The
plates were then scanned at 540 nm using an ELISA reader. The re-
sults indicating cytotoxicity potential of 7 are shown in Figure 3.
CH3), 4.03 (dd, J5 ,4 = 6 Hz, Jgem = 12 Hz, 1H, H-50), 4.36 (dd,
0
0

2264
P. Kumar et al. / Bioorg. Med. Chem. 18 (2010) 2255–2264
17. Biaglow, J. E.; Varnes, M. E.; Roizen-Towle, L.; Clark, E. P.; Epp, E. R.; Astor, M.
5.5. Radiosensitization
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a reverse phase analytical column
(25 ꢂ ꢁ0.4 cm) HPLC, and interpolating from a plot (Fig. 5) of
retention time plotted as a function of 1-octanol/water partition
coefficients taken from the literature.37,63,64
Acknowledgments
The authors thank Alberta Cancer Research Institute and a
Proof-of-Principal grant from Canadian Institutes of Health Re-
search for financial support to this project. Automated equipment
support and the 18F-supply from the Edmonton PET Centre are
gratefully acknowledged.
46. clinicaltrials.gov/ct2/show/NCT00756691. Cross Cancer Institute, Edmonton,
Canada, 2007.
47. Kumar, P.; Emami, S.; McEwan, A. J. B.; Wiebe, L. I. Lett. Drug Design 2009, 6, 82.
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