Comparing hypoxia-targeting potential of 99mTc(CO) 3-labeled 2-nitro and 4-nitroimidazole

Article abstract of DOI:10.1002/jlcr.1525

Non-invasive determination of hypoxia is an important problem in clinical nuclear medicine. Although ¹⁸F-fluoromisonidazole is used clinically for hypoxia determination, the short half-life t_1/2 = 109.77 min), high cost and less availability of cyclotrone-produced ¹⁸F make ^99mTc (t_1/2 = 6 h)-based agents more desirable. With this aim, 99mTc(CO)₃-labeled iminodiacetic acid derivatives of 2- and 4-nitroimidazole are investigated for their ability to target hypoxic tumors. A bifunctional chelating agent, N, N-bis[(tert-butoxycarbonyl)methyl]-2- bromoethylamine, was synthesized in the first step, which was then conjugated to the nitroimidazoles in the second step. The tert-butyl ester derivatives formed were hydrolyzed to obtain the iminodiacetic acid derivatives of corresponding nitroimidazoles. The radiolabeling of the iminodiacetic acid derivatives with [""1Tc(CO)3(H2O)3I+ core was carried out following a reported protocol. Biodistribution of the prepared complexes was carried out in Swiss mice bearing fibrosarcoma tumor. The 2-nitroimidazole complex showed a steady retention in activity ( - 1.5% injected dose per gram [%ID/g]) in tumor throughout the period of study (3 h) indicating that it may be localized in the hypoxic cells. The 4-nitroimidazole counterpart, however, showed an initial uptake of ～4.6%ID/g at 30 min post injection, which was then observed to wash out rapidly. Copyright

Full text of DOI:10.1002/jlcr.1525

Research Article
Received 16 January 2008,
Revised 1 April 2008,
Accepted 11 May 2008
Published online 11 July 2008 in Wiley Interscience
(
www.interscience.wiley.com) DOI: 10.1002/jlcr.1525
Comparing hypoxia-targeting potential
3
-nitroimidazole
99m
of Tc(CO) -labeled 2-nitro and
4
Madhava B Mallia, Suresh Subramanian, Anupam Mathur, H.D. Sarma,
a
a
b
c
a
Meera Venkatesh, and Sharmila Banerjee
a
Ã
1
Non-invasive determination of hypoxia is an important problem in clinical nuclear medicine. Although F-fluoromisoni-
8
dazole is used clinically for hypoxia determination, the short half-life (t1/2 = 109.77 min), high cost and less availability of
cyclotrone-produced
1
8
99m
99m
F make
Tc (t1/2 = 6 h)-based agents more desirable. With this aim,
3
Tc(CO) -labeled
iminodiacetic acid derivatives of 2- and 4-nitroimidazole are investigated for their ability to target hypoxic tumors. A
bifunctional chelating agent, N, N-bis[(tert-butoxycarbonyl)methyl]-2-bromoethylamine, was synthesized in the first step,
which was then conjugated to the nitroimidazoles in the second step. The tert-butyl ester derivatives formed were
hydrolyzed to obtain the iminodiacetic acid derivatives of corresponding nitroimidazoles. The radiolabeling of the
9
9m
1
iminodiacetic acid derivatives with [ Tc(CO) (H O) ] core was carried out following a reported protocol. Biodistribution
3
2
3
of the prepared complexes was carried out in Swiss mice bearing fibrosarcoma tumor. The 2-nitroimidazole complex
showed a steady retention in activity (ꢀ1.5% injected dose per gram [%ID/g]) in tumor throughout the period of study (3 h)
indicating that it may be localized in the hypoxic cells. The 4-nitroimidazole counterpart, however, showed an initial uptake
of ꢀ4.6%ID/g at 30 min post injection, which was then observed to wash out rapidly.
Keywords: hypoxia; nitroimidazole; technetium-99m tricarbonyl core; fibrosarcoma; iminodiacetic acid
Introduction
behavior of the molecule may depend on other factors such as
the structure of the molecule. W. Peter et al. had earlier reported
In connection with our current interest of developing suitable
radiolabeled agents for targeting hypoxic tumors, it was felt derivatives in aqueous solution at pH 7. The estimated values
one electron reduction potentials of different nitroimidazole
3
pertinent to explore the potential of two differently substituted are in the range À243 millivolt (mV) to À398 mV for differently
nitroimidazoles complexes. Hypoxia plays a major role in
substituted 2-nitroimidazole derivatives. The values are between
contributing toward the resistance of tumors to both radiation À400 and À500 mV for substituted 5-nitroimidazoles and for 4-
1
,2
therapy and chemotherapy.
invasive methods available today for measuring hypoxia have
Inherent drawbacks in the nitroimidazoles the values are even less. The above data suggest
that 2-nitroimidazoles are the most promising candidates as
hypoxia-targeting agents. However, Linder et al. showed that
provided the impetus for the development of non-invasive
1
8
9
9m
modalities for the purpose. Currently, F-labeled fluoromisoni-
dazole is the standard used for the detection of hypoxia. potential for selective retention in hypoxic tissue, and Chu et
However, limited availability and high cost of the cyclotron-
Tc-based agents could serve as more desirable 4-nitroimidazole labeled with [ Tc = O] core, which showed
alternatives. A majority of nitroaromatics studied for targeting accumulation and slow clearance from tumor. These observa-
Tc–BATO complex containing 4-nitroimidazole has the
4
al. have reported a bidentate hydroxyiminoamide derivative of
1
8
99m
9
9m
31
produced F,
1
0
3
–10
hypoxia are based on nitroimidazoles.
Among the nitroimi-
tions highlight the importance of synthesis and in vivo testing of
dazole derivatives, the 2-nitroimidazole derivative is by far the different derivatives of nitroimidazoles irrespective of the
most studied agent. However, 4-nitro and 5-nitro derivatives are
also reported. Lipophilicity and the redox potential are the two
a
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai
major factors governing the uptake and retention of the
4
00085, India
molecule in hypoxic cells. Lipophilicity is primarily responsible
for the uptake in tumor, whereas the redox potential determines
the efficiency with which the agent gets reduced and is retained
in the hypoxic cells. A fine balance between the two factors is
sought, which will eventually lead to an ideal hypoxia-targeting
agent. Although broad guidelines are available regarding the
lipophilicity and the redox potential that a molecule should
b
Medical and Biological Products Program, Board of Radiation and Isotope
Technology, Mumbai 400705, India
c
Radiation Biology and Health Science Division, Bhabha Atomic Research
Centre, Mumbai 400085, India
*Correspondence to: Sharmila Banerjee, Radiopharmaceuticals Division, Bhabha
Atomic Research Centre, Mumbai 400085, India.
have to show hypoxia-targeting property, the actual in vivo E-mail: sharmila@apsara.barc.ernet.in
J. Label Compd. Radiopharm 2008, 51 308–313
Copyright r 2008 John Wiley & Sons, Ltd.

M. B. Mallia et al.
O
O
O
1
) Anhyd. KHCO3, DMF, RT
0
NH2
HO
+
Br
N
O
O
OC4H9
Br
2
) PPh3, NBS, CH2Cl2, 0 C
1
Scheme 1. Synthesis of N,N-bis[(tert-butoxycarbonyl)methyl]-2-bromoethylamine.
position of the nitro substituent. Generally, labeling of in near-quantitative yield to obtain the desired products 2 and
molecules via technetium oxo core requires high ligand 3, respectively (Scheme 2).
concentration to achieve satisfactory labeling yield. The
A reported procedure was followed for the preparation of
1
9
introduction of [ Tc(CO) (H O) ] core opened up the
9m
1
11
99m
[
11
Tc(CO) (H O) ] core, as mentioned in the Experimental
3 2 3
3
2
3
possibility of labeling molecules using low ligand concentration section. For the preparation of the complex, about 1–2 mg of the
to yield complexes with high stability. The three substitutionally corresponding ligand in 0.5 mL of phosphate buffer was mixed
9
labile water molecules in [ Tc(CO) (H O) ] core facilitate the with 0.5 mL of freshly prepared [ Tc(CO) (H O) ] core. The
9m
1
99m
1
3
2
3
3
2
3
formation of stable complexes with tridentate ligands such as mixture was vortexed for 1 min and then incubated at 801C for
1
2
iminodiacetic acid, diethylene triamine, histidine, etc. and 30min. The three labile water molecules are replaced by the
hence target-specific molecules of interest are often modified to iminodiacetic acid group resulting in a complex with overall unit
possess one of these ligating groups. In the present study, 2- negative charge. The probable structure of the complex is shown
1
4
nitro and 4-nitroimidazoles were synthetically modified to in Scheme 3. Allali et al.
had earlier shown, through
H-NMR and IR, the tridentate coordination of the metal-
complexation with [ Tc(CO) (H O) ] core. Toward this, a tricarbonyl core (metal= Tc, Re) via iminodiacetic acid-functiona-
1
iminodiacetic acid derivatives to render them suitable for
9
9m
1
3
2
3
bifunctional chelating agent (BFCA) with a pendant bromo lized molecule. The two complexes reported herein were
substituent was synthesized and used for alkylation of the –NH– therefore presumed to have a similar structure. It is pertinent to
of the imidazole ring. The complexes were prepared, character- note that although the overall negative charge on the complexes
ized and their in vivo behaviour as markers for tumor hypoxia reported herein may not be a desirable feature as it may hinder/
were evaluated in the animal model.
reduce its diffusion across the cell membrane, it may facilitate
faster clearance of activity from the body, which is also an
important attribute of an imaging agent. Characterization of the
9
9m
1
[
Tc(CO)
using a dual pump high-performance liquid chromatography
HPLC) unit coupled with a radioactivity detector. The elution
3 2 3
(H O) ] core and the final complex was carried out
Results and discussions
(
Synthesis
9
9m
1
profiles of [ Tc(CO)
3 2 3
(H O) ]
core, 2-nitroimidazole complex
The iminodiacetic acid derivatives of both 2- and 4-nitroimida- and 4-nitroimidazole complex are shown in Figure 1(a), (b) and
9
zole were synthesized in two steps. In the first step, N, N- (c), respectively. It can be seen that [ Tc(CO) (H O) ] core
9m
1
3
2
3
bis[(tert-butoxycarbonyl)methyl]-2-bromoethylamine (1) was appeared as a broad peak at 17.4270.2 min. The 2-nitroimidazole
1
3
synthesized, following a reported procedure, by reacting complex was eluted out at 11.3270.2 min and the 4-nitroimida-
ethanolamine and tert-butylbromoacetate in the presence of zole complex at 15.2770.1 min. In vitro serum stability studies
potassium bicarbonate (KHCO
Scheme 1). The crude product, N, N-bis[(tert-butoxycarbonyl)- from an earlier reported protocol. About 50mL of the labeled
methyl]-2-hydroxyethylamine, was then treated with trisphenyl compound was added to 0.5 mL of human serum and this
phosphine (PPh ) and N-bromosuccinamide (NBS) in dichlor- mixture was incubated at 371C for 3 h. Aliquots were drawn at 1, 2
3
) in dimethyl formamide (DMF) were performed for the labeled complex using a method adapted
1
5
(
3
omethane to yield the target compound in 80% yield. This and 3 h and analyzed by HPLC to assess stability of the complex in
bifunctional chelator was used in the subsequent step for N- the serum. The elution profiles of the complexes were unaltered
alkylation of the ring –NH– of the 2- and 4-nitroimidazole to indicating that the complexes are not degraded in serum during
yield N, N-bis[(tert-butoxycarbonyl)methyl]-2-(2-nitroimidazoly- the period of the study. Lipophilicity of the complex was assessed
l)ethylamine and N, N-bis[(tert-butoxycarbonyl)methyl]-2-(4-ni- by determining Log Po/w following a multiple extraction proce-
1
6
troimidazolyl)ethylamine, respectively. The N-alkylation was dure reported by Troutner et al. The Log Po/w value of the
9
9m
3
carried out using potassium carbonate as the base in acetonitrile 4-nitroimidazole– Tc(CO) complex was found to be À0.86 and
99m
3
solvent under reflux conditions. During N-alkylation of 4- that of the 2-nitroimidazole– Tc(CO) complex was found to be
nitroimidazole, two products were observed by thin layer À0.87. Despite showing nearly identical LogPo/w values, it can be
chromatography (TLC) carried out in diethyl ether. Separation seen that there is significant difference in the retention times of
1
of the two products and their analysis by H-NMR showed that the two complexes in the HPLC profile. However, this apparent
one was the expected 4-nitroimidazole derivative and the other anomaly that could not be explained has been observed earlier
1
2
was the isomeric 5-nitroimidazole derivative. The former was by us as well as reported by Schibli et al.
then separated by column chromatography over silica gel using
diethyl ether as the solvent. Although the overall yield of
4-nitroimidazole tert-butyl ester derivative was 65%, the 2-nitro
Biodistribution
imidazole ester derivative could be synthesized in 85% yield. The Swiss mice (ꢀ25 g body weight) were used to develop in vivo
tert-butyl ester derivatives were then hydrolyzed using 6 N HCl tumor models for the biodistribution studies. Solid tumors were
J. Label Compd. Radiopharm 2008, 51 308–313
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

M. B. Mallia et al.
O
R2
O
NH
+
N
N
O
O
Br
R1
Anhyd. K2CO3, CH3CN, Reflux
O
O
O
O
N
N
R2
N
R1
6
N HCl, 2h, 50 ˚C
O
O
OH
OH
N
N
R2
N
R1
(2)
(3)
R1=NO2; R2=H - 2-nitroimidazole
R =H; R =NO - 4-nitroimidazole
1
2
2
Scheme 2. Synthesis of nitroimidazole derivative.
R2
N
R2
R1
N
N
+
OH2
CO
R1
N
H2O
99mTc
OH2
-
pH = 8
N
O
+
O
3
0 min at 80 0
C
OC
CO
O
O
N
O
O
99mTc
OH HO
OC
CO
CO
R1 = NO2, R2 = H --2-nitroimidazole
R1 = H, R2 = NO2 --- 4-nitroimidazole
99m
3
Scheme 3. Preparation of nitroimidazole- Tc(CO) complex.
propagated in the animals by subcutaneous administration of a specific organ/tissue per gram. The results of the biodistribution
6
fibrosarcoma cell line (ꢀ10 cells per animal) into the dorsal study were shown in Tables 1 and 2. Both the complexes under
region. The tumor size was allowed to reach approximately investigation showed uptake in tumor despite the presence of a
10 mm diameter after which the animals were used for the negative charge on them. The proposed mechanism of
experiment. For the in vivo distribution studies, about 0.1 mL of retention of nitroimidazoles in hypoxic cells is attributed to
the labeled product (ꢀ100 mCi) per animal was administered their stepwise reductions, catalyzed by various one-electron
intravenously. The animals were sacrificed at various time points reductases, followed by ring fragmentation forming reactive
1
7
(
30, 60 and 180 min p.i.) after which the relevant organs and radicals, which then bind to the cellular components. The first
tissue were excised for measurement of associated activity. reduction step is reversible under well-oxygenated conditions
Radioactivity measurements were carried out in a flat-bed-type and, hence, nitroimidazoles do not accumulate in normoxic cells.
NaI(Tl) scintillation counter with an optimal energy window for In the present study, the 2-nitroimidazole complex showed a
9
9m
Tc. The accumulated activity was expressed in terms of the steady retention of activity in the tumor throughout the period
percentage of the total injected dose associated with the of study. This can be attributed to efficient reduction of the
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 308–313

M. B. Mallia et al.
9
9
99m
saline from a Mo– Tc column generator. Silica gel plates
silica gel 60 F254) were obtained from M/s. Merck, U.S.A. The
(
HPLC analyses were carried out on a JASCO PU 2080 Plus dual
pump HPLC system, Japan, with a JASCO 2075 Plus tunable
absorption detector and a radiometric detector system, using a
1
C18 reversed phase HiQ Sil (5 mm, 4 Â 250 mm) column. H-NMR
spectra were recorded on a 300 MHz Varian VXR 300S spectro-
photometer, U.S.A. IR was recorded on a JASCO FT/IR-420
spectrophotometer, Japan. IR of the liquid samples are recorded
neat as a thin film over the NaCl/KBr window. For solid samples
KBr pellets are used.
Synthesis of N, N-bis[(tert-butoxycarbonyl)methyl]-2-bromoethyla-
mine (1)
To tert-butylbromoacetate (3.3 g, 17.3 mmol) dissolved in 15 mL
of DMF, powdered KHCO
suspension was cooled to 01C, and ethanolamine (0.46 mL,
.5 mmol) was added dropwise over a period of 5 min. The
3
(1.92 g, 19.2 mmol) was added. The
7
9
9m
1
reaction mixture was stirred at 01C for 30 min and then at room
temperature for 24 h. The semisolid residue obtained after
removing the solvent was dissolved in NaHCO3 solution and
extracted with ether. The ether layer was then washed with
brine, dried and evaporated to give the crude product (ꢀ1.8 g,
Figure 1. HPLC elution profile of (a) [ Tc(CO) (H O) ]
3
2
3
core, (b) 2-nitroimida-
complex and (c) 4-nitroimidazole– Tc(CO) complex.
99m
99m
zole– Tc(CO)
3
3
complex in hypoxic cells. On the contrary, a steady retention of
activity was not observed with 4-nitroimidazole complex,
despite its high initial uptake. This indicates that the
8
of dichloromethane, Ph P (2.2 g, 8.45 mmol) was added. The
3%) as oil. To the crude dialkylated product dissolved in 20 mL
3
solution was cooled to 01C, and solid NBS (1.5 g, 8.45 mmol) was
added over a period of 5 min. The reaction was continued for a
period of 2 h after which, the solvent was removed to yield a
semisolid residue. The semisolid residue was triturated with
ether and the precipitate was filtered off. The ether layer was
concentrated and passed through a small column of silica
eluting with ether. Evaporation of ether yielded a yellow oil,
which was then purified by silica gel chromatography using 5:1
4
-nitroimidazole derivative may be less efficient in being
reduced within the time frame that it spends inside the tumor,
before being washed out. It is also interesting to observe that
after 1 h p.i., the retention of the 4-nitroimidazole complex is
similar to that of the 2-nitroimidazole complex, though not
identical. However, on careful observation it is evident that the
rate of washout of the two complexes from the tumor is not
similar, the 4-nitroimidazole complex being cleared at a faster
rate than the 2-nitroimidazole complex. Although the present
studies carried out for a limited time (3 h max.) do not
adequately indicate the clear superiority of one complex over
the other, there are possible indications to demonstrate the
favorable features of the 2-nitroimidazole complex as a hypoxia-
imaging agent in comparison with that of the 4-nitroimidazole
complex. The biodistribution results also showed that major
clearance of the injected activity was through the hepatobiliary
route. The clearance of activity from blood was slow with both
the complexes. This affected the tumor to blood ratio, which
hexane/ether mixture as an eluent to get the target compound
1
(
1
(
1.7 g, 80%); R
.46 (s, 18H, tert-butyl CH
t, 2H, J = 7.5 Hz, BrCH CH N); 3.5 [s, 4H, (tert-butoxycarbo-
f
0.28 (5:1 hexane/ether); H-NMR (CDCl
3
, ppm)
3
); 3.1 (t, 2H, J = 7.5 Hz, BrCH
2
CH
2
N); 3.4
2
2
À1
nyl–CH ) N]. IR (Neat, cm ) 2985 (m); 2943 (m); 1747 (vs); 1451
2
2
(
(
m); 1379 (m); 1254 (s); 1190 (vs); 1145 (w). 1030 (s); 993 (w); 918
w); 864 (w); 741 (w); 659 (w).
Synthesis of iminodiacetic acid derivative of 2-nitroimidazole (2)
was found to be lower than one throughout the period of study. To 2-nitroimidazole (100 mg, 0.88 mmol) in 15 mL of acetonitrile,
Although the tumor to muscle ratio of 2-nitroimidazole complex anhydrous K CO (2 eq) was added and the suspension was
showed improvement with time, that in case of the 4- stirred for 15 min. To this mixture N, N-bis[(tert-butoxycarbo-
2
3
nitroimidazole was found to decrease with time.
nyl)methyl]-2-bromoethylamine (1) (311 mg, 0.88 mmol) was
added and the reaction mixture refluxed for 12 h. The progress
of the reaction was monitored by TLC. After removing the
solvent, the residue was dissolved in 20 mL water and extracted
with two 15 mL portions of chloroform. The combined chloro-
form layer was washed with brine and dried. The pure product,
N, N-bis[(tert-butoxycarbonyl)methyl]-2-(2-nitroimidazolyl)ethy-
Experimental
Synthesis
General
2-nitroimidazole, 4-nitroimidazole, 2-aminoethanol, tert-butyl- lamine, was then obtained by silica gel chromatography, using
bromoacetate, NBS, triphenylphosphine (PPh ) and carbon ethyl acetate as the eluant (287 mg, 85%). R 0.88 (ethyl acetate);
3
f
9
9m
monoxide gas, used for the preparation of the [ Tc(CO)
1
3
(-
H-NMR (CDCl , d ppm) 1.45 (s, 18H, tert-butyl CH ); 3.16 (t, 2H,
3
3
1
]
H
2
O)
3
core, were purchased from M/s. Aldrich, U.S.A. J = 6 Hz, 2-nitroimidazole–CH CH N); 3.37 [s, 4H, (tert-butoxycar-
2 2
Potassium carbonate and potassium bicarbonate were procured bonyl–CH ) N]; 4.55 (t, 2H, J = 6 Hz, 2-nitroimidazole–CH CH N);
2
2
2
2
from M/s. Fluka, Germany. All solvents used for syntheses were 7.12 (d, 1H, J = 1.2 Hz, nitroimidazole C2-H); 7.46 (d, 2H, J = 1.2 Hz,
À1
dried as per the general procedure. All reagents were of nitroimidazole C4-H). IR (Neat, cm ) 3126 (m); 2978 (m); 2930
analytical grade. Sodium pertechnetate was eluted using normal (m); 1740 (vs); 1540 (s); 1487 (m); 1343 (m); 1216 (m); 1150 (vs);
J. Label Compd. Radiopharm 2008, 51 308–313
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

M. B. Mallia et al.
99m
Table 1. Distribution of 2-nitroimidazole– Tc(CO) complex
Organ
3
% Injected dose per gram (7 SD) (n = 3)
3
0 min p.i.
60 min p.i.
180 min p.i.
Liver
Intestine1gall bladder
Kidney
Heart
Lungs
Muscle
Blood
8.22 (0.92)
7.88 (1.97)
4.57 (0.36)
1.25 (0.17)
3.72 (0.26)
0.61 (0.03)
3.71 (0.55)
11.45 (2.31)
17.71 (4.28)
5.73 (0.60)
1.42 (0.11)
4.98 (0.70)
0.80 (0.15)
3.61 (0.36)
15.14 (1.25)
24.13 (1.56)
5.05 (0.42)
1.04 (0.13)
3.37 (0.26)
0.44 (0.09)
2.89 (0.37)
Tumor
1.36 (0.09)
1.77 (0.15)
1.60 (0.32)
Tumor/blood
Tumor/muscle
0.37
2.23
0.49
2.38
0.55
3.64
9
9m
Table 2. Distribution of 4-nitroimidazole– Tc(CO) complex
3
%
Injected dose per gram (7SD) (n = 3)
3
0 min p.i.
60 min p.i.
180 min p.i.
Liver
Intestine1gall bladder
Kidney
Heart
Lungs
Muscle
Blood
13.32 (1.45)
20.45 (1.77)
6.80 (0.29)
2.07 (0.35)
3.94 (0.57)
0.44 (0.03)
3.87 (0.52)
11.76 (0.95)
11.43 (0.28)
6.62 (0.94)
1.47 (0.20)
3.42 (0.30)
0.55 (0.05)
3.33 (0.48)
9.24 (1.25)
11.89 (0.61)
3.90 (0.14)
0.59 (0.05)
2.41 (0.78)
0.51 (0.18)
1.66 (0.05)
Tumor
4.69 (0.27)
1.93 (0.31)
1.25 (0.03)
Tumor/blood
Tumor/muscle
1.23
0.58
3.50
0.76
2.61
10.77
1
077 (m); 982 (m); 938 (w); 850 (m); 823 (m). The purified ester gel column chromatography using ether as an eluent. The H-
1
was dissolved in 1 mL of methanol and to this solution 7 mL of NMR of the two compounds revealed that one was the expected
6
solvent was removed under vacuum to yield the iminodiacetic nitroimidazole ester derivative. The target compound N, N-
N HCl was added. The reaction was stirred at 501C for 2 h. The 4-nitroimidazole ester derivative and the other was the 5-
1
acid derivative 2 as a white powder (193 mg, 95%). H-NMR bis[(tert-butoxycarbonyl)methyl]-2-(4-nitroimidazolyl)ethylamine
(
D
s, 4H, [(HO C–CH N–]); 4.43 (t, 2H, J = 5.1 Hz, nitroimidazo- acetate); H-NMR (CDCl
2
O, ppm) 3.51 (t, 2H, J = 5.1 Hz, nitroimidazole–CH
2
CH
2
N); 3.81 was obtained in 65% (219 mg) overall yield. R
1
f
0.85 (ethyl
, ppm) 1.46 (s, 18H, tert-butyl CH ); 3.13
(
le–CH CH N); 7.34 (d, 2H, J = 1.2 Hz, nitroimidazole C5-H); 7.50 (d, (t, 2H, J = 6 Hz, 2-nitroimidazole–CH CH N); 3.35 [s, 4H, (tert-
2
2
2
)
2
3
3
2
2
2
2
H, J = 1.2 Hz, nitroimidazole C4-H). IR (KBr, cmÀ1) 3136 (m); 2970 butoxycarbonyl–CH ) N]; 4.11 (t, 2H, J = 6 Hz, 2-nitroimidazo-
2 2
(
1
7
m); 2847 (m); 1760 (vs); 1724 (s); 1559 (m); 1499 (m); 1392 (m); le–CH
2
CH
350 (m); 1230 (m); 1198 (m); 1138 (s); 1006 (w); 860 (m); 827 (m); 2H, J = 1.5 Hz, nitroimidazole C4-H). IR (Neat, cm ) 3133 (m);
51 (w); 653 (m). 2983 (m); 2931 (m); 2954 (m); 1738 (vs); 1537 (s); 1497 (m); 1336
2
N); 7.62 (d, 1H, J = 1.5 Hz, nitroimidazole C2-H); 8.05 (d,
À1
(
(
m); 1229 (m); 1147 (vs); 1070 (m); 987 (m); 938 (w); 857 (m); 825
m); 767 (m). The purified ester was dissolved in 1 mL of
Synthesis of iminodiacetic acid derivative of 4-nitroimidazole (3)
methanol and to this 7 mL of 6 N HCl was added. The reaction
To 4-nitroimidazole (100 mg, 0.88 mmol) in 15 mL of acetonitrile, was kept stirring at 501C for 2 h. The solvent was removed under
anhydrous K CO3 (2 eq) was added and the suspension was vacuum to yield the iminodiacetic acid derivative 3 as a white
2
1
stirred for 15 min. To this, N, N-bis[(tert-butoxycarbonyl)methyl]- powder (147 mg, 95%). H-NMR (D O, ppm) 3.71 (t, 2H, J = 6.6 Hz,
2
2
-bromoethylamine] (311 mg, 0.88 mmol) was added and the nitroimidazole–CH CH N); 3.99 (s, 4H, [(HO C–CH ) N–]); 4.48
2 2 2 2 2
reaction mixture was refluxed for 12 h. Upon completion of (t, 2H, J = 6.6 Hz, nitroimidazole–CH CH N); 7.72 (d, 2H, J = 1.2 Hz,
2
2
the reaction, the target compound was recovered by following nitroimidazole C5-H); 8.16 (d, 2H, J = 1.2 Hz, nitroimidazole C4-H).
À1
the same work up procedure as described above. The TLC of the IR (KBr, cm ) 3130 (m); 2974 (m); 2847 (m); 1768 (vs); 1724 (s);
reaction mixture in ether showed two spots, corresponding to 1559 (m); 1499 (m); 1392 (m); 1350 (m); 1230 (m); 1185 (m); 1143
the formation of two products, which were separated by silica (s); 1017 (w); 863 (m); 825 (m); 760 (w).
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 308–313

M. B. Mallia et al.
subcutaneous administration of murine fibrosarcoma cell line
6
Radiolabeling
(
ꢀ10 cells/animal) on the dorsal region. The tumors were
99m
Preparation of [ Tc(CO)
1
3
(H
2
O)
3
]
core
allowed to grow till they reached a size of approximately 1 cm in
diameter and then the animals were used for the biodistribution
study. The radioactive preparation (ꢀ37 MBq per animal) was
injected intravenously into the tumor-bearing mice via the tail
vein. Individual sets of animals (n = 3) were utilized for studying
the biodistribution at different time points (30, 60 and 180 min).
At the end of the various time periods, the animals in the
respective sets were immediately sacrificed and the relevant
organs excised for measurement of retained activity. The activity
A typical procedure involves dissolution of NaBH
4
(ꢀ5.5 mg),
Na CO (ꢀ4 mg) and Na/K tartrate (ꢀ15 mg) in 0.5 mL double-
2
3
distilled water in a 10 mL glass vial. The vial was sealed and
carbon monoxide gas was purged through the solution for
10 min. After the addition of 1 mL of the generator eluate
containing 37–74 MBq of
9
9m
À
4
TcO , the vial was heated at 801C
for 20 min. After cooling the vial and re-equilibrating with
atmosphere, the pH of the reaction mixture was adjusted to 7
using a 1:3 mixture of 0.5 M phosphate buffer (pH 7.5):1 M HCl.
associated with each organ was then measured in a flat-bed-
99m
type NaI(Tl) counter with a suitable energy window for
Tc. All
9
9m
1
The [ Tc(CO) (H O) ] core thus prepared was characterized
2
3
3
procedures performed herein were in strict compliance with the
national laws of governing the conduct of animal experiments.
by HPLC.
9
9m
Preparation of
Tc(CO) -complex
3
Conclusions
Under optimized conditions, 0.5 mL of freshly prepared
9
9m
1
The potential of 2-nitroimidazole and 4-nitroimidazole in
targeting tumor hypoxia was explored in tumor-bearing animal
models. Towards this, the iminodiacetic acid derivatives were
synthesized in excellent yield using the BFCA approach for
[
Tc(CO)
3
(H
2
O)
3
]
ligand in 0.5 mL of the phosphate buffer at pH 7.4. The mixture
precursor was added to 1–2 mg of the
was vortexed for a minute and then incubated at 801C for
30 min. The complex thus formed was characterized by HPLC.
9
9m
1
subsequent radiolabeling with the [ Tc(CO) (H O) ] core in
3
3
2
over 95% yield, under mild conditions and low ligand
concentration. In vivo distribution studies in Swiss mice showed
a steady retention of activity in the case of 2-nitroimidazole
derivatives, whereas the 4-nitroimidazole derivative showed a
high initial uptake but rapid washout from the tumor. The study,
carried out for a limited time, demonstrated a better potential of
the 2-nitroimidazole derivative over the 4-nitroimidazole deri-
vative, as a tumor-targeting agent.
Quality control
HPLC
9
9m
1
3 2 3
The radiochemical purity of the prepared [ Tc(CO) (H O) ]
core as well as the complex was assessed by HPLC using a C18
reversed phase column. Water (A) and acetonitrile (B) mixtures
with 0.1% trifluoroacetic acid were used as the mobile phase
and the following gradient elution technique was adopted for
the separation (0 min 90% A, 28 min 10% A, 30 min 10% A). The
flow rate was maintained at 1 mL/min. About 25 mL of the test
solution was injected into the column and the elution was
monitored by observing the radioactivity profile.
REFERENCES
Partition coefficient (Log Po/w
)
[1] L. H. Gray, A. D. Conger, M. Elbert, Br. J. Radiol. 1953, 26, 638–648.
About 100 mL of the labeled compound was mixed with 0.9 mL
of water and 1 mL of octanol on a vortex mixer for about 1 min.
The two phases were then allowed to separate. About 0.8 mL of
the octanol layer was withdrawn and an equal volume of fresh
saline was added. The mixture was re-vortexed for a minute and
then the two layers were allowed to separate. Equal aliquots of
the two layers were withdrawn and measured for the radio-
activity. The readings thus obtained were used to calculate the
Log Po/w value of the complex.
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[
[
[
[
[
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[
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separate centrifuge tubes and an equal volume of ethanol was
added to precipitate the serum proteins. The mixture was
centrifuged and the supernatant was analyzed by HPLC to
assess the stability of the complex in the serum.
[
12] R. Schibli, R. La Bella, R. Alberto, E. Garcia-Garayoa, K. Ortner, U.
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[
[
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Biodistribution studies
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J. Label Compd. Radiopharm 2008, 51 308–313
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org