Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Article abstract of DOI:10.1016/j.bmcl.2013.05.015

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.

Full text of DOI:10.1016/j.bmcl.2013.05.015

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3704–3708
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a novel 99mTc labeled
2-nitroimidazole derivative as a potential agent for imaging tumor
hypoxia
Yoann Joyard a,b, Vadim Le Joncour c, Hélène Castel c, Chérif Bounana Diouf a, Laurent Bischoff b,
Cyril Papamicaël b, , Vincent Levacher b, Pierre Vera a, Pierre Bohn a,
⇑
⇑
a Department of Nuclear Medicine, Henri Becquerel Center and Rouen University Hospital and QuantIF-LITIS (EA4108), 1 rue d’Amiens, 76038 Rouen Cedex, France
b UMR CNRS COBRA 6014, 1 rue Tesnière, 76130 Mont-Saint-Aignan, France
c Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation, University of Rouen, Mont-Saint-Aignan, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy
and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having
a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with
99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue.
The biodistribution and scintigraphic image of the corresponding 99mTc-complex showed accumulation
in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.
Published by Elsevier Ltd.
Received 5 April 2013
Revised 3 May 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
Technetium-99m
2-Nitroimidazole
Hypoxia imaging agent
Biodistribution
Tripeptide ligand
Scintigraphic image
Hypoxia is characterized by an imbalance between the rate of
cellular oxygen consumption and oxygen supply in cells. Hypoxic
tissues are a hallmark of advanced solid tumors. The distribution
of hypoxic tissue in tumors is heterogeneous and variable over
time. It has been shown at the beginning of the twentieth century
that hypoxia plays a role in increased radio resistance.1 Indeed,
oxygen has a crucial role in the response to radiotherapy. Being a
potent radiosensitizer, it enhances the effect of ionizing radiation
leading to a lethal effect on tumor cells by breaking the double-
stranded DNA. More recently it has been shown that hypoxia
was a predictor for drug resistance.2 The decrease in the response
to radiotherapy of hypoxic cells then leads to an increased risk of
metastasis and poor prognosis for the patient.3 It is therefore of
major interest to have a precise knowledge of the level of oxygen
in the tissues in order to understand the formation mechanism of
this hypoxic state, and develop strategies to address this imbal-
ance. This requires quantifying the level of oxygenation of tissues
with good spatial and temporal resolution.
We can distinguish two classes of markers of hypoxia: the nitro-
imidazole ligands and non-nitroimidazoles ligands. Nitroimidazole
derivatives are known for their ability to be selectively retained in
hypoxic tissues. Nakamura has first demonstrated in 1955 that, the
2-nitroimidazole showed activity against particular types of infec-
tions associated with a deficiency of oxygen.4 Therefore nitroimi-
dazole analogues were synthesized and proved to be effective
against bacteria and protozoa that proliferate in hypoxic cells.5
This particular behavior of nitroimidazole derivatives in oxygen-
deficient environment led to their study in the detection of
hypoxia.6,7
Most clinically used tracers are 18F-labeled tracers in order to
perform a PET imaging but this radiochemistry needs a cyclotron
and a GMP laboratory. Thus, owing to limited availability, high cost
of production and short half-life of cyclotron isotopes, a 99mTc-
based agent could be a better alternative, especially as all nuclear
medicine centers possess gamma cameras whereas only few has
one PET-CT camera. So, many radio pharmaceuticals8–10 (Fig. 1)
having a nitroimidazole moiety and radiolabeled with techne-
tium-99m for SPECT imaging of hypoxic tissues were developed.
However, due to their high background levels in normal tissues,
quantification of hypoxia may be difficult.11 So, there is a crucial
need to develop new technetium-99m candidates.
⇑
Corresponding authors. Tel.: +33 (0)2 355 224 84; fax: +33 (0)2 355 229 62
(C.P.); tel.: +33 (0)2 320 829 23; fax: +33 (0)2 320 825 50 (P.B.).
E-mail addresses: cyril.papamicael@insa-rouen.fr (C. Papamicaël), pierre.-
bohn@chb.unicancer.fr (P. Bohn).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.05.015

Y. Joyard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3704–3708
3705
with trifluoroacetic acid to lead to the expected derivative 1b.
O
N
The so-obtained amine was reacted with commercially available
NO2
N
N
H
O
Fmoc-L
-Cys(Trt)-OH using propylphosphonic anhydride T3PÒ to af-
O
H
N
N
O
N
ford the desired protected nitroimidazole derivative 2a. The subse-
quent Fmoc-deprotection was achieved by classical reaction with
piperidine, leading to amine 2b. On the other hand, a peptidic cou-
pling reaction with available glycine methyl ester hydrochloride
N
Tc
HO
N
O
B
O
O
Tc
N
O
N
Cl
O
and Boc-L-Lys(Boc)-OH, DCHA gave the protected dipeptide 3a.
The ester group of 3a was also converted into an acid moiety to
give 3b. Then, the required protected tripeptide 4a was prepared
from ester 3a following a classical hydrolysis with LiOH and cou-
pling with 2b in the presence of the cationic coupling agent HBTU.
Moreover, it should be noted that the protected tripeptide 4a was
also synthesized starting from 3b and T3PÒ. Finally, the cleavage of
both NH-Boc and S-Trityl protecting groups was conducted by
means of TFA in the presence of triethylsilane and phenol as scav-
engers to give the desired tripeptide 4b. Optimization of the radio-
labeling procedure of 4b was then conducted (Scheme 2, Table 1).
N
N
O2N
nitroimidazole-BATO
Schiff Base nitroimidazole
O
Tc
N
O
N
N
N
N
N
O
NO2
H
BMS181321
So, peptide 4b (10
(20 mg in 0.5 mL, pH 7), 99mTcOÀ4 (740 MBq) and tin(II) chloride
(40 g in 0.1 mL). The resulting solution was analyzed by radio-
lg) was radiolabeled with sodium tartrate
Figure 1. Structures of selected technetium-containing nitroimidazoles tested for
imaging.
l
HPLC. The reaction was first studied at 25 °C and a 95% radiochem-
ical yield was obtained when the reaction time was changed from
10 to 60 min (Table 1, entries 2 and 3). We then decided to perform
the reaction at 100 °C (Table 1, entry 4). These conditions have
enabled us to considerably reduce the reaction time to 10 min
(Table 1, entry 4). Using this labeling procedure, the complex
was obtained with a radiochemical purity of 95% as a syn and
anti-diastereoisomers mixture 5a,b (Fig. 2). It has been demon-
strated that the presence of two radiometric peaks is due to the
resolution of diasteroisomers resulting from the chiral centers on
the peptide backbone and the chiral technetium.12b,16 It is difficult
to determine directly the formation of the 99mTc-complex because
of the extremely small amount of the compound present. However,
the formation of 99mTc-complex 5a,b could be indirectly deter-
mined by comparison with 6a,b which can be easily prepared. Re
and Tc belongs to the same group of the periodic table and are sim-
ilar in size. In our case, the analogue rhenium complex 6a,b was
There are many examples in the literature12 reporting the use of
peptides acting as chelating agent of 99mTc (NxS4-x). But to date
there are only few examples of hypoxia radiotracers using such li-
gands to complex 99mTc. Indeed, only Ballinger and co-workers.13
report the use of sequences glycine–serine–cysteine and glycine–
cysteine–glycine to chelate 99mTc.
In this study, a cysteine–glycine–lysine sequence with a bio-
reducible moiety, 2-nitroimidazole, was synthesized and labeled
with 99mTc. We chose to use a 2-nitroimidazole moiety as a vector
since it has a better response to hypoxia than 4- and 5-nitroimi-
dazoles isomers.14 The latter was then evaluated as a tumor hypox-
ia marker.
The chelating agent 4b was synthesized through a multi-step
reaction using 2-nitroimidazole as a starting material. The synthe-
sis procedure is outlined in Scheme 1. On the one hand, according
to a procedure reported in the literature by Hay et al.,15 2-nitroim-
idazole was reacted with tert-butyl 2-bromoethylcarbamate to
give compound 1a. Then, the Boc protecting group was cleaved
O
R
O
N
N
O
O
M
NH2
5a,b
6a,b
N
N
S
N
H
NO2
N
Trt
S
NO2
4b
NO2
N
N
N
H
N
5a
a for
b for 5b
H2N
a
STrt
OH
NO2
HN
RHN
N
: syn and anti, M = 99mTc
: syn and anti, M = Re
FmocHN
O
1a
: R = NHBoc
1b : R = NH2.TFA
O
b
2a
: R = Fmoc
d
c
2b : R = H
Scheme 2. Reagents and conditions. (a) SnCl2, 99mTcO4À, sodium tartrate pH 7.0,
100 °C, 10 min (radiochemical yield 95%); (b) ReOCl3(PPh3)2, NaOAc, MeOH, 70 °C,
NHBoc
NHBoc
4 h.
H3CO
+
O
O
e
H
N
OH.DCHA
BocHN
BocHN
NH2.HCl
OR
O
O
Table 1
3a : R = CH3
Labeling conditions of 99mTc-complex 5a,b with sodium pertechnetate and tin(II)
f
NHR1
3b
: R = OH
chloride
g
h
SR2
Entry 4b
g)
Sodium tartrate Final
Temperature
(°C)
Time
(min)
RCY
(%)
O
NO2
N
(
l
(mg)
pH
: R1 = Boc, R2 = Trt
4b : R1 = R2 = H
H
H
4a
N
N
i
R1HN
N
H
N
1
2
3
4
5
6
100
10
10
10
10
10
—
5
25
25
25
100
100
100
10
10
60
10
10
10
76
89
95
95
38
38
O
O
20
20
20
20
20
5
5
Scheme 1. Reagents and conditions: (a) tert-Butyl 2-bromoethylcarbamate, K2CO3,
DMF, 110 °C, 5 h (57%); (b) TFA, 25 °C, 10 min (99%); (c) T3PÒ, DIEA, CH2Cl2, 25 °C,
12 h (86%); (d) piperidine (20% mol), DMF, 25 °C, 16 h (53%); (e) T3PÒ, DIEA, CH2Cl2,
25 °C, 12 h (86%); (f) LiOH, H2O, 25 °C, 12 h (85%); (g) LiOH, H2O, DMF, CH2Cl2 then
2b, HBTU, DIEA, DMF, 25 °C, 12 h (43%); (h) 2b, T3PÒ, DIEA, CH2Cl2, 25 °C, 12 h
(53%); (i) TFA, H2O, TES, phenol, 0 °C, 1 h (52%).
5
9a
4b
a
Phosphate buffer 0.2 M pH 9, 0.5 mL.
Citric acid buffer, 20 mM pH 4, 0.5 mL.
b

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Y. Joyard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3704–3708
Figure 2. Representative radio-HPLC pattern of syn and anti-diasteroisomers 5a,b. Peaks A and B are thought to be syn and anti-forms of the Tc@O group.
prepared by substitution of ligand 4b on the rhenium precursor
ReOCl3(PPh3)2 (Scheme 2).
The HPLC analysis, UV detection, of the reaction mixture
showed two main peaks with similar retention times of the corre-
sponding technetium-99m complex 5a,b.
cellular stress in T98 cells. However, at this time, no significant
uptake of tracer 5a,b occurred. (Fig. 4) After 48 h in the same
hypoxic conditions, expression of HIF1 increased by a factor of
2.5 (Fig. 3) and subsequently the tracer retention became highly
significant (Fig. 4). Nitroimidazole moiety is only reduced in cells
with long-lasting hypoxia, and reduction of this compound leads
in covalent bonding to macromolecules and subsequently in tracer
retention. Reduction of nitroimidazole only occurs in cells deprived
of oxygen with an acidic and reductive cytoplasm. After 24 h in
hypoxic conditions, T98 cells were stressed but their cytoplasms
were not sufficiently reductive to retain compound 5a,b. After
48 h, cell cytoplasm has reached optimal conditions to reduce the
nitroimidazole moiety. Compound 5a,b is thus a potential tracer
of severe hypoxia.
Besides, the LC-MS analysis of the reaction mixtureexhibited two
identical molecular ions at m/z 645.1 ([M+H]+), which confirm the
presence of syn and anti-diastereoisomers. In addition, the molecu-
lar ion value gives a further indication concerning the nature of the
bond with the metal and the amine function of the lysine. Indeed, in
this case, the metal undergoes a dative bond with amine function in-
stead of a bond with the corresponding amide ion12a
Complex 5a,b was in vitro evaluated. The human glioblastoma
cell line was cultured,17 and used as a model of brain cancer.
Glioblastoma multiforme is the most common and most aggressive
malignant primary brain tumor in humans. Treatments are
surgery, chemotherapy with temozolomide or bevacizumab and
radiation therapy.18 Glioblastoma is well-known to form tumors
with hypoxic zones, which are resistant to standard radiation ther-
apy.19 The use of hypoxic tracers could help to customize treat-
ments20 with a real-time adjustment of the therapeutic beams
(Image-guided radiation therapy).21
Four nude mice25 were xenografted with T98 cells in their left
hind legs. 3 weeks after xenografting, the tumors have reached
an average diameter of 10 mm. Compound 5a,b was injected in tail
vein (10 MBq). Two hours later, animals were sacrificed and the
percent of injected dose per gram of tissue was calculated (Table 2).
The tumor-to-muscle ratio was 2.70 0.23 in these conditions.
This result is better than the reported uptake of [18F]-FMISO in
CaNT tumor, but lower than
[ [
99mTc]-HL91 and 64Cu]-ATSM
In hypoxic conditions, after 24 h, an increased expression of
hypoxia-inducible factor (HIF1) was observed (Fig. 3) as a sign of
(Table 3).26 This comparison should be interpreted carefully since
this is not the same cell line used. To the best of our knowledge,
Figure 3. Effect of hypoxia on HIF1 production and luciferase activity. T98 glioblastoma cell line transfected22 with no cDNA (Mock, A) and with cDNA encoding the HIF1
luciferase reporter vector (HIF1-Luc, B) were cultured in normoxic (20% O2) or hypoxic (1% O2) conditions, 24 h or 48 h post-transfection. The luciferase activity was expressed
as arbitrary bioluminescence units (au). In T98 not transfected with the HIF1-Luc cDNA vector, both normoxic and hypoxic conditions failed to evoke luciferase production. In
T98-expressing luciferase under the HIF1-responsive element, 24 h and 48 h of hypoxic conditions drastically stimulate luciferase activity, suggesting enhanced production of
HIF1.23 Bars represent means SEM. of a representative experiment with 10 replicates in each group. Statistical significance ⁄⁄⁄P <0.001; hypoxia versus normoxia in B (one-
way Anova followed by a Bonferroni post-test).

Y. Joyard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3704–3708
3707
xenografted in the right hind limb of nude mouse and after four
weeks the tumor diameter was 10 mm. Scintigraphic image was
obtained with 10 MBq of compound 5a,b 60 min after injection
into the tail vein. An important uptake was observed in kidneys
and intestinal tract as a sign of tracer excretion. A heterogeneous
tracer uptake was observed in the tumor (an interesting tumor-
to-muscle ratio of 10 was determined) as a potential uptake in
hypoxic areas (Fig. 5). Besides, scintigraphic images were also ob-
tained after 2 and 3 h and we observed a constant tumor-to-mus-
cle ratio.
In conclusion, the tripeptide 4b can be labeled with techne-
tium-99m to give the complex 5a,b with a radiochemical purity
of 95%. A specific uptake of the latter compound 5a,b in hypoxic tu-
mor cells was observed in vitro. A first biodistribution assay
showed a tumor-to-muscle ratio of 2.70 0.23 in vivo. To complete
this study, a scintigraphic image showed a specific tracer uptake in
hypoxic areas. These results suggest that the complex 5a,b might
be an useful tracer for hypoxic tumor imaging.
Figure 4. Effect of chronic hypoxia on tracer 5a,b uptake.T98 glioblastoma cell lines
were cultured in normoxic (20% O2) or hypoxic (1% O2) conditions during 24 or 48 h.
After 24 h, no significant difference in uptake of tracer 5a,b was observed in cells
regardless of culture conditions. After 48 h, an uptake and retention of tracer 5a,b
was only observed in cells cultured in hypoxic conditions, suggesting that the
nitroimidazole could be reduced in the cytoplasm of cells deprived of oxygen.24
Bars represent means SEM of a representative experiment with 8 replicates in
each group. Statistical significance ⁄⁄⁄P <0.001; hypoxia versus normoxia after 48 h
(one-way Anova followed by a Bonferroni post-test).
Acknowledgements
The authors thank the Association Nationale de la Recherche et
de la Technologie (Grant No. #543/2010) and La ligue contre le
Cancer (CD76) for funding Yoann Joyard.
Table 2
Biodistribution (%ID/g, mean SD) in mice (n = 4) bearing a T98 tumor (glioblastoma
cell line) 2 h following the injection of 99mTc labeled 5a,b
Organ
5a,b uptake (%ID/g)
References and notes
Kidney
Liver
Large intestine
Muscle
T98 tumor
T/M ratio
3.73 0.59
2.50 0.50
0.20 0.03
0.11 0.02
0.29 0.06
2.70 0.23
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for imaging tumor hypoxia, T98 glioblastoma cell line was
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Table 3
Reported T/M ratio for hypoxia imaging agents in nude mice (isoflurane + room air;
adenocarcinoma NT; 2 h post-injection)26
Imaging agent
T/M ratio
[
[
[
99mTc]-HL91
18F]-FMISO
64Cu]-ATSM
4.6 0.7
2.2 0.4
4.0 0.3
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17. Cell culture. The human glioblastoma cell line T98 was obtained from the
American Type Culture Collection (ATCC, LGC Standards, Molsheim, France).
The T98 cells were maintained in HAM-F12 (Invitrogen, Cergy Pontoise,
France) supplemented with 10% fetal calf serum (FBS, Lonza, Bâle, Suisse),
2 mM
L-glutamine and 100 U/ml penicillin/streptomycin (Gibco, Fischer
Scientific, Illkirch, France) at 37 °C in a humidified 5% CO2 incubator. Culture
media were replaced every 3 days.
18. Wainwright, D. A.; Nigam, P.; Thaci, B.; Dey, M.; Lesniak, M. S. Expert Opin.
Emerg Drugs 2012, 17, 181.
Figure 5. Scintigraphic image obtained with 10 MBq of complex 5a,b 60 min after
injection into the tail vein.

3708
Y. Joyard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3704–3708
19. Yang, L.; Lin, C.; Wang, L.; Guo, H.; Wang, X. Exp. Cell. Res. 2012, 318, 2417.
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lysis buffer was then added to the cells. Samples were lysed and collected and
20 l aliquots were assayed using luciferase assay reagent. Luminescence was
measured using a Victor V plate reader (PerkinElmer, Courtaboeuf, France).
Background absorbance from samples not transfected was systematically
measured.
l
22. For transfection, T98 cells were plated in 24-well plates (Costar, 10 Â 103 cells/
well) for 24 h before transfection in HAM-F12 (for T98 cells), supplemented
with 10% FBS. The pHIF1-pTL-Luc (HIF1 Luciferase reporter vector, Panomics,
Milano, Italy) is designed to measure transcriptional activity of hypoxia
inducible factor-1 (HIF-1). HIF-1 binds to the hypoxia responsive element and
activates genes involved in angiogenesis, glucose metabolism, cell
proliferation, survival and invasion/metastasis. The transient transfection of
24. Tracer uptake assay. T98 cells were incubated with 5a,b (2–3 MBq) for 1 h then
washed twice with cold PBS. Cells radioactivity was measured by a Wallac
Wizard 1470 Gamma Counter. Results were expressed as decay-corrected
counts in 106 cells/MBq.
25. Animals were maintained in a facility approved by the French ministry of
agriculture in accordance with current regulations and standards and
registered under the number B76-450-05. ‘Principles of laboratory animal
care’ (NIH publication No. 86-23, revised 1985) were strictly followed. Animals
were housed four per cage and fed ad libitum. The experiments have been
approved by an ethical committee and registered under the number N/02-06-
12/12/06-15.
the Plasmid pHIF1-Luc (0.2
lg/well) or without vector (Mock) was performed
using FuGene 6 (1.8 l/well, Promega) following the manufacturer’s protocol.
l
The response of these cells to hypoxia was obtained after 24 or 48 h incubation
in the presence of a gas mixture containing 1% O2, 5% CO2 and balanced N2
(Incu Safe Incubator, VWR, Fontenay-sous-Bois, France).
26. Kersemans, V.; Cornelissen, B.; Hueting, R.; Tredwell, M.; Hussien, K.; Allen, P.
D.; Falzone, N.; Hill, S. A.; Dilworth, J. R.; Gouverneur, V.; Muschel, R. J.; Smart,
S. C. PLoS One 2011, 6, e25911.
23. Luciferase assay. All cell extracts were prepared and analyzed using the
Luciferase Assay System (Promega, Madison, WI). Briefly, 24-well plates were
washed twice with cold 1Â phosphate-buffered saline (PBS) and 100 ll of 1Â