Synthesis and biological results of the technetium-99m-labeled 4-nitroimidazole for imaging tumor hypoxia

Article abstract of DOI:10.1016/j.bmcl.2003.11.017

1-(4-Nitroimidazole-1-yl)-propanhydroxyiminoamide (N4IPA) was synthesized. The biodistribution of ^99mTc-N4IPA in mice bearing S180 tumor demonstrated that the complex showed accumulation in tumor and slow clearance from it. The tumor-to-tissue uptake ratios increase with time. These results suggest that ^99mTc-N4IPA would be a marker for imaging tumor hypoxia.

Full text of DOI:10.1016/j.bmcl.2003.11.017

Bioorganic & Medicinal Chemistry Letters 14 (2004) 747–749
Synthesis and biological results of the technetium-99m-labeled
4-nitroimidazole for imaging tumor hypoxia
Taiwei Chu,a Shaowen Hu,a Bing Wei,b Yi Wang,a Xinqi Liua and Xiangyun Wanga,*
aDepartment of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
bCollege of Life Science, Peking University, Beijing 100871, China
Received 15 August2003; revised 11 November 2003; accepted 12 November 2003
Abstract—1-(4-Nitroimidazole-1-yl)-propanhydroxyiminoamide (N4IPA) was synthesized. The biodistribution of 99mTc-N4IPA in
mice bearing S180 tumor demonstrated that the complex showed accumulation in tumor and slow clearance from it. The tumor-to-
tissue uptake ratios increase with time. These results suggest that 99mTc-N4IPA would be a marker for imaging tumor hypoxia.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
useful new chelating moiety for design 99mTc radio-
pharmaceuticals.7 In this study, a hydroxyiminoamide
derivative with 4-nitroimidazole was synthesized, 99mTc
complex was prepared, and the behavior of the complex
as tumor hypoxia marker was evaluated in vivo (Scheme
1).
Tumor hypoxia is believed to be an important factor in
resistance of tumors to radiation and some other forms
of therapy. The identification and quantification of
tumor hypoxia may predict outcome and may identify
patients who might benefit from concomitant radio-
sensitizing therapy to overcome the hypoxia effect.
Current electrode measurement techniques are cumber-
some and not readily available to most investigators.
Nuclear medicine offers a non-invasive method for
demonstrating tumor hypoxia.1 Compounds that are
selectively accumulated in hypoxic tissue may be labeled
with either positron-emitting radionuclides or single-
photon gamma emitters such as 99mTc, thus allowing
wider availability. Although many types of nitroaro-
matic compounds have shown promise as probes for
hypoxic cells, almostall of htese have been based on
2. Synthesis and radiolabeling
Acrylonitrile (53.2 mL, 0.8 mol) was added to the sus-
pension of 4-nitroimidazole (22.6g, 0.2 mol) in triethyl-
amine (160 mL), and the mixture was refluxed over an
oil-bath for 12 h. After cooling, the white solid was fil-
tered off and washed with fresh water several times. 1-
(2-Cyanoethyl)-4-nitroimidazole was obtained as a
white solid by recrystallization from methanol, mp 109–
110 ꢀC (lit.8 mp 110–111 ꢀC). Yield: 21.73 g (65%). H
1
nitroimidazoles.2À6 Linker etal. found htat
99mTc–
NMR d (DMSO-d6): 3.17 (t, 2H, CH2), 4.39 (t, 2H,
CH2), 7.93 (s, 1H, imi-H), and 8.47 (s, 1H, imi-H).
BATO complex containing 4-nitroimidazole had the
potential for selective retention in hypoxic tissue.3 And
some 99mTc-labeled metronodazoles, 5-nitroimidazole
derivatives showed selectively accumulation in
tumors.4,5
The previous product(8.3 g, 0.05 mol) was refluxed for
17 h with a methanolic solution of hydroxylamine (0.25
mol). Standing at 4 ꢀC overnight gave the white crystal-
line powder, which was recrystallized from water to give
Recently, Nakayama et al. found that the bidentate
ligand hydroxyiminoamides could form highly in vivo
and in vitro stable complexes with 99mTc and mightbe a
7.18
g
of 1-(4-nitroimidazole-1-yl)-propanhydroxy-
iminoamide (N4IPA) as pale green crystals, mp 144.5–
146.0 ꢀC. Yield 72%. 1H NMR d (DMSO-d6): 2.50
(overlap between CH2 and solvent), 4.26 (t, 2H, CH2),
5.52 (s, 2H, NH2), 7.80 (s, 1H, imi-H), 8.37 (s, 1H, imi-
H), and 8.98 (s, 1H, OH). H NMR d (acetonitrile-d6):
2.62 (t, 2H, CH2), 4.36 (t, 2H, CH2), 5.21 (s, 2H, NH2),
7.63 (s, 1H, imi-H), 8.11 (s, 1H, imi-H), and 8.38 (s, 1H,
Keywords: 4-Nitroimidazole; Hypoxia imaging agent; Biodistribution;
Tumor.
* Corresponding author. Tel.: +86-10-627-65560; fax: +86-10-627-
55409; e-mail: xywang@pku.edu.cn
1
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.017

748
T. Chu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 747–749
99mTc–N4IPA complex was determined by TLC in
which a polyamide strip was used as the fixed phase and
a 0.9% NaCl solution as the developer. The water-
soluble complex 99mTc–N4IPA migrates to the top half
of the strip (Rf=0.4–0.6) while the lipophilic and/or
insoluble radiochemical impurities remain near the ori-
gin (Rf=0–0.3). Radiochemical purity was >95%. The
complex was observed to be stable for a period of 6 h
with retention of radiochemical purity to the extent of
ꢁ90%.
Scheme 1. Synthesis of 1-(4-nitroimidazole-1-yl)-propanhydroxy-
iminoamide and preparation of 99mTc–N4IPA complex.
OH). 13C NMR d (DMSO-d6): 31.98, 44.51, 121.67,
137.50, 146.77, and 149.40. MS (EI) calculated for
C6H9N5O3 (M+): m/z 199. Found: 199.
3. Biodistribution results
In vivo biodistribution studies were performed in
Kunming mice bearing S180. Male mice were inoculated
subcutaneously in the left axilla with a suspension of
approximately 106 S180 cells. Over the course of 7–8
days, tumors reached 10–15 mm in diameter, corre-
sponding to tumor weights of 0.5–1.5 g, each mouse
received an intravenous dose of the 99mTc complex
(1Â105 Bq, 100 mL) via tail vein. The mice were sacri-
ficed by cervical dislocation in groups of five at various
time intervals after injection and the organs or tissues of
interest were removed, weighed and counted. The
counting tubes, including a standard equivalent to 1%
of the injected dose, were assayed in a well-type gamma
counter and the results were calculated as percentages of
injected dose per gram tissue. Tumor-to-organ ratios
were calculated from the percentages of the injected
dose per gram for tumor and relevant organs. The final
results are expressed as meanÆone standard deviation
(SD). All experiments were carried out following the
principles of laboratory animal care and the China Law
on the protection of animals.
Analytical calculated for 1-(4-nitroimidazole-1-yl)-pro-
panhydroxyiminoamide, C6H9N5O3: C, 36.18; H, 4.52;
N, 35.18; O, 24.12. Found: C, 36.25; H, 4.52; N, 35.41;
O, 23.92.
Sehgal and Agrawal reported that alkylation of 4(5)-
nitroimidazole with chloropropionitrile resulted in the
formation of 1-(2-cyanoethyl)-5-nitroimidazole and the
isomeric 1-(2-cyanoethyl)-4-nitroimidazole in poor
yields 3.34 and 1.63%, respectively.8 We found acrylo-
nitrile reacted easily with 4-nitroimidazole in triethyl-
amine, and the desired product, 1-(2-cyanoethyl)-4-
nitroimidazole, was obtained after recrystallization with
high yield 65%. And the isomeric 1-(2-cyanoethyl)-5-
nitroimidazole was not found in the reaction mixture.
Obviously, itis an ideal mehtod for producing 1-(2-
cyanoethyl)-4-nitroimidazole.
The action of hydroxylamine on nitriles is the most
widely used process for the preparation of hydroxy-
iminoamides.9 In this study, 1-(4-nitroimidazole-1-yl)-
propanhydroxyiminoamide (N4IPA) was synthesized
easily in a similar manner.
In Table 1 are presented the tissue distribution data for
five mice bearing S180 tumor at each of five time inter-
vals after injection of 99mTc–N4IPA. Itcan be seen that
the initial distribution of 99mTc–N4IPA appears to
reflect blood perfusion in each organ or tissue, and there
is no indication of selective uptake or binding of radio-
activity in the studied normal tissues. The blood clear-
ance data is characterized by an initial very rapid
distribution phase and a much slower clearance during
The 99mTc complex was prepared by reconstituting a
lyophilization kit containing 200 mg of N4IPA, 25 mg of
sodium tartrate, 1.36 mg of KH2PO4, 14.39 mg of
.
.
Na2HPO4 12H2O, 10 mg of SnCl2 2H2OÀand 25 mg of
urea with 3.0 mL of generator 99mTcO4 effluent. The
solution was heated to 75 ꢀC for 10–15 min. The yield of
Table 1. Biodistribution of 99mTc–N4IPA in mice bearing S180 tumor (%/g)a
Tissue
30 min
1 h
2 h
4 h
8 h
Blood
0.70Æ0.05
Æ0.08
0.28Æ0.08
0.14Æ0.03
0.22Æ0.03
0.79Æ0.06
5.44Æ0.79
0.10Æ0.01
0.45Æ0.07
0.11Æ0.07
0.04Æ0.01
1.19Æ0.39
4.25Æ2.78
3.22Æ0.85
2.06Æ0.43
0.57Æ0.10
0.22Æ0.02
0.12Æ0.05
0.17Æ0.04
0.46Æ0.07
3.80Æ1.17
0.09Æ0.02
0.39Æ0.10
0.05Æ0.01
0.04Æ0.01
1.81Æ0.49
8.12Æ2.64
3.39Æ1.66
2.24Æ0.78
0.85Æ0.25
0.18Æ0.04
0.08Æ0.02
0.12Æ0.01
0.39Æ0.09
3.14Æ0.88
0.08Æ0.01
0.34Æ0.06
0.04Æ0.01
0.04Æ0.00
1.88Æ0.53
8.60Æ2.63
4.47Æ1.37
2.94Æ0.57
0.88Æ0.26
0.15Æ0.05
0.06Æ0.01
0.10Æ0.02
0.23Æ0.04
2.26Æ0.57
0.06Æ0.01
0.32Æ0.07
0.04Æ0.01
0.04Æ0.01
2.06Æ0.82
8.83Æ2.93
5.39Æ1.48
3.09Æ0.92
1.41Æ0.39
Heart0.24
Lung
Liver
Kidney
Spleen
Tumor
Muscle
Brain
Tumor/blood
Tumor/muscle
Tumor/heart2.31
Tumor/lung
Tumor/liver
0.39Æ0.15
0.98Æ0.21
5.63Æ1.22
0.13Æ0.02
0.55Æ0.08
0.15Æ0.05
0.04Æ0.01
0.78Æ0.13
3.66Æ1.33
Æ0.84
1.42Æ0.58
0.56Æ0.14
a Each value is meanÆSD.

T. Chu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 747–749
749
the 2–8 h period. Interestingly, the decline in tumor
Acknowledgements
activity can be neglected. This slow decline in radio-
activity is in contrast to most other tissues. And the
tumor-to-tissue ratios increase with time going. At 2 h
after injection, the tumor/muscle ratio is 8.1, tumor/
blood 1.8, tumor/heart 3.4, tumor/lung 2.2, tumor/liver
0.9. Moreover, the tumor/liver ratio attains 1.4 at 8 h.
The results demonstrate that S180 tumor contains
hypoxic cells, and the radiolabeled tracer may be selec-
tively accumulated; meanwhile the tracer may flow out
from other tissues.
Financial support for this research was provided by the
National Natural Science Foundation of China (No.
20301001). The assistance of A. Prof. Zhi Yang, Beijing
Institute for Cancer Research, is gratefully acknowledged.
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