Influence of ligand denticity on the properties of novel 99mTc(I)-carbonyl complexes. Application to the development of radiopharmaceuticals for imaging hypoxic tissue

Article abstract of DOI:10.1016/j.bmc.2012.05.010

An important issue in the development of metal-based radiopharmaceuticals is the selection of the labelling strategy in order to couple the metal to the pharmacophore without losing the biological activity. With the aim to evaluate the correlation between ligand denticity and biological behaviour of the corresponding ^99mTc complexes, we designed a tridentate and a bidentate 5-nitroimidazole derivatives suitable for ^99mTc(I) tricarbonyl complexation and with potential use as radiopharmaceuticals towards hypoxic tissue diagnosis. Ligands were synthesized using metronidazol, a pharmaceutical containing the bioreductive pharmacophore as starting material. The chelating units were connected to the pharmacophore using the click reaction of Huisgen. Both ^99mTc complexes were obtained in high yield and were hydrophilic and stable in labelling milieu. The complex obtained from the tridentate ligand exhibited high stability in human plasma, low protein binding and a favourable biodistribution characterized by low blood and liver uptake, fast elimination and negligible uptake in other organs or tissues. Selective uptake and retention in tumour together with favourable tumour/muscle ratio makes this ^99mTc-complex a promising candidate for further evaluation as potential hypoxia imaging agent in tumours. The bidentate ligand, on the other hand, yielded a less stable ^99mTc-complex that experimented hydrolysis in vitro and decomposition in human plasma and showed high protein binding, high blood and liver uptake and moderate excretion. Although selective uptake and retention in tumour was also observed physicochemical and biological behaviour are inadequate for in vivo use, demonstrating that denticity of the ligand is particularly important and that tridentate ligands are preferable in order to prepare ^99mTc-tricarbonyl complexes for Nuclear Medicine imaging.

Full text of DOI:10.1016/j.bmc.2012.05.010

Bioorganic & Medicinal Chemistry 20 (2012) 4040–4048
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Influence of ligand denticity on the properties of novel 99mTc(I)–carbonyl
complexes. Application to the development of radiopharmaceuticals
for imaging hypoxic tissue
Soledad Fernández a, Javier Giglio a, Ana M. Rey a, , Hugo Cerecetto b,
⇑
⇑
a Cátedra de Radioquímica, Facultad de Química, Universidad de la República. Avda. Gral. Flores 2124, 11800 Montevideo, Uruguay
b Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Química—Facultad de Ciencias, Universidad de la República. Iguá 4225, 11400 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
An important issue in the development of metal-based radiopharmaceuticals is the selection of the
labelling strategy in order to couple the metal to the pharmacophore without losing the biological activ-
ity. With the aim to evaluate the correlation between ligand denticity and biological behaviour of the cor-
responding 99mTc complexes, we designed a tridentate and a bidentate 5-nitroimidazole derivatives
suitable for 99mTc(I) tricarbonyl complexation and with potential use as radiopharmaceuticals towards
hypoxic tissue diagnosis. Ligands were synthesized using metronidazol, a pharmaceutical containing
the bioreductive pharmacophore as starting material. The chelating units were connected to the
pharmacophore using the click reaction of Huisgen. Both 99mTc complexes were obtained in high yield
and were hydrophilic and stable in labelling milieu. The complex obtained from the tridentate ligand
exhibited high stability in human plasma, low protein binding and a favourable biodistribution charac-
terized by low blood and liver uptake, fast elimination and negligible uptake in other organs or tissues.
Selective uptake and retention in tumour together with favourable tumour/muscle ratio makes this
99mTc-complex a promising candidate for further evaluation as potential hypoxia imaging agent in
tumours. The bidentate ligand, on the other hand, yielded a less stable 99mTc-complex that experimented
hydrolysis in vitro and decomposition in human plasma and showed high protein binding, high blood and
liver uptake and moderate excretion. Although selective uptake and retention in tumour was also
observed physicochemical and biological behaviour are inadequate for in vivo use, demonstrating that
denticity of the ligand is particularly important and that tridentate ligands are preferable in order to pre-
pare 99mTc-tricarbonyl complexes for Nuclear Medicine imaging.
Received 17 February 2012
Revised 28 April 2012
Accepted 8 May 2012
Available online 14 May 2012
Keywords:
Tc(I)-carbonyl complexes
Denticity
Hypoxic tissue
Click reaction
Huisgen reaction
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ligand which stabilizes metals in low oxidation states. In aqueous
milieu, the well-defined aqua ion of technetium [Tc(CO)3(OH2)3]+
Nuclear Medicine techniques based on the administration of
small amounts of radioactive compounds, the so-called radiophar-
maceuticals, followed by detection of the radiation escaping from
the body, provide a non-invasive and accurate method for in vivo
imaging highly specific biochemical processes. An important issue
in the development of metal-based radiopharmaceuticals is the
selection of the labelling strategy in order to couple the metal to
the pharmacophore without losing the biological activity. Recently,
new 99mTc-coordinated moieties have been introduced as poten-
tially adequate for labelling small molecules. Alberto et al.1 have
succeeded in the low pressure synthesis of Tc-tricarbonyl com-
plexes, which can be used as precursors to obtain a great diversity
of potential radiopharmaceuticals.2–4 The CO is a tightly bound
is formed. This novel type of complexes are at the same time small
and very stable, due to the d6 configuration of the metallic centre,
and give the possibility to substitute the three weakly bound water
molecules by a great variety of bi and tridentate ligands. Derivati-
zation of different pharmacophores to introduce adequate chelator
units (see examples in Fig. 1a)5–10 could produce a variety of
Tc-labelled small molecules. This approach has been applied re-
cently to the labelling of peptides, CNS receptor ligands, myocar-
dial imaging agents, DNA intercalators, among others.11–15 In
these cases different ligand denticity were employed with different
success.
Hypoxia in tumours is a pathophysiologic consequence of struc-
turally and functionally disturbed microcirculation and deteriora-
tion of diffusion conditions. The evaluation of tumour perfusion
and oxygenation status is very important in oncology since tumour
hypoxia appears to be strongly associated with tumour propaga-
tion, malignant progression, and resistance to therapy and it has
⇑
Corresponding authors. Tel.: +598 29248571; fax: +598 29241906 (A.R.);
tel.: +598 252586181; fax: +598 25250749 (H.C.).
E-mail addresses: arey@fq.edu.uy (A.M. Rey), hcerecet@fq.edu.uy (H. Cerecetto).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.010

S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
4041
(a)
Bidentate ligands
R
N
COOH
CH3
HN
H
NH2
N
N
N
N
OH
OH
O
PPh2
Tridentate ligands
HOOC
HN
N
COOH
COOH
R
COOH
NH2
S
N
NH2
(b)
NO2
NO2
OH2
Tc
O
c
N
N
N
N
CO
CO
CO
CO
N
N
N
N
a
i
a
i
Tc
CO
CO
N : imidazolic nitrogen; N : aminic nitrogen; O : carboxylate oxygen
i
a
c
Figure 1. (a) Bidentate and tridentate chelator units used in the preparation of 99mTc(I) tricarbonyl complexes. (b) General structures of the herein designed 99mTc-complexes.
thus become a central issue in tumour physiology and cancer treat-
ment.16,17 Bioreductive compounds, which are selectively reduced
in hypoxic tissue to reactive intermediates that bind to intracellu-
lar molecules, have been used for the development of potential
radiopharmaceuticals for targeting hypoxic tumours being nitroa-
ryl moiety one of the preferred pharmacophore.18,19 In this sense,
5-nitroimidazol-1yl moiety20,21 has recognized bioreductive capac-
ity since in vivo reduction is irreversible in hypoxic tissue resulting
in entrapping of metabolites within the cells. Combination of this
bioreductive pharmacophore with Tc is useful in the design of po-
tential markers of hypoxic tissue.
the other hand, the second designed ligand has three coordinating
groups, an imidazolic nitrogen, an aliphatic amine nitrogen and a
carboxylate oxygen, from a carboxylic acid. These ligands were
used to prepare the corresponding 99mTc(I)-tricarbonyl complexes
whose structures were proposed from the preparation of the corre-
sponding rhenium complexes. The main physicochemical and bio-
logical properties were studied both in vitro and in vivo in order to
assess the influence of the denticity of the ligands and to establish
their potentiality as hypoxia targeting radiopharmaceuticals.
2. Results and discussion
With the aim to evaluate the correlation between ligand
denticity and biological behaviour of the corresponding 99mTc com-
plexes, herein we designed 5-nitroimidazole derivatives with
different denticity suitable for 99mTc(I) tricarbonyl complexation
(Fig. 1b) and with potential use as radiopharmaceuticals towards
hypoxic tissue diagnosis. One of the designed ligands contains an
imidazolic nitrogen, from a 1,2,3-triazole system, and an aliphatic
amine nitrogen as electron donor atoms for Tc coordination. On
2.1. Synthesis of the ligands
Two different ligands were designed and synthesized in order to
achieve the proposed objectives, that is N-methyl-1-[1-(2-(2-
methyl-5-nitro-1H-imidazole-1-yl)ethyl)-1H-1,2,3-triazole-4-yl]me
thylamine (Ntm-1) and 2-amine-3-{1-[2-(2-methyl-5-nitro-
1H-imidazole-1-yl)ethyl]-1H-1,2,3-triazole-4-yl}propanoic acid
NO2
(a)
N
N
N
N
N
H
1
e
NO2
NO2
n
N
N
Ntm-1
y
k
l
PPh3, (CH3)3SiN3
DIAD
A
CuSO4
N
N
N
N
sodium ascorbate
OH
N3
NO2
THF
O
Mtz
2
N
N
Azd
N
N
OH
NH2
CH3SO2Cl
Et3N / CH2Cl2
Ntm-2
NaN3
DMF
NO2
O
N
H
OH
N
N
Alkyne
OH2
1
Alkyne 2
OSO2CH3
NH2
(b)
Tc-Ntm-1
or
Tc-Ntm-2
Na2CO3 / NaBH4
CO (g)
Ntm-1 or Ntm-2
H2O
H2O
CO
CO
Tc
[
99mTcO4]-
MeOH / H2O
75 °C, 30 min
CO
Scheme 1. Synthesis of ligands Ntm-1 and Ntm-2 (a) and the corresponding 99mTc complexes Tc-Ntm-1 and Tc-Ntm-2 (b).

4042
S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
(Ntm-2) (Scheme 1a). For their preparation the key reagent was the
azide Azd (Scheme 1a) that was obtained from the commercially
available antiparasite drug metronizadole (Mtz, Scheme 1a). As
shown in Scheme 1, in a first stage, the Mtz OH-group was trans-
formed into the azide in a single step through a Mitsunobu reac-
tion,22 employing azidotrimethylsilane as nucleophylic reagent.
Alternatively the Azd intermediate was also prepared via sulfonate
intermediate with similar yield but without chromatographic
purification processes (Scheme 1a).23 Following this step, Azd was
reacted with the adequate terminal alkyne, N-methyl-propargyl-
for biomolecules in the preparation of potential radiopharmaceuti-
cals because it is efficient, selective and devoid of side reactions.
The one-pot procedure represents a remarkable improvement since
derivatization of the pharmacophore and labelling is achieved in a
very short time and without need of purification of intermediates.
Radiochemical purities of precursor fac-[99mTc(CO)3(OH2)3]+
and final complexes (Tc-Ntm-1 and Tc-Ntm-2) were evaluated
by reverse phase HPLC (RP-HPLC, Fig. 4). Analysis of the labelling
mixture for Ntm-2 revealed the presence of a main radio-product
with retention time of 18 min (Fig. 4b). On the other hand, for
Ntm-1 labelling the RP-HPLC analysis of the reaction milieu indi-
cated the formation of two Tc-complexes with retention times of
15.0 and 16.0 min (Fig. 4c), ratio 1.0:2.5. Isolation of the main
product from the reaction milieu of the Tc-Ntm-1 synthesis, tR=
16.0 min, was attempted by HPLC (Fig. 4d). However, HPLC analy-
sis of the isolated peak, during the time, also showed the minor
product, tR= 15.0 min, (Fig. 4e), suggesting that an equilibrium be-
tween two 99mTc-species was established. The chromatogram also
showed a small amount of the precursor fac-[99mTc(CO)3(OH2)3]+
indicating that technetium complex Tc-Ntm-1 (Fig. 2), might suf-
fers, in aqueous milieu, transformation to the di-aquo minor Tc-
complex, tR = 15.0 min (Fig. 4c), and finally to the precursor,
tR = 3.5 min (Fig. 5a). However another possibility to explain the
presence of two Tc-complexes for the bidentate ligand Ntm-1 is
the presence of two configurational isomers (i.e. A-Tc-Ntm-1 and
B-Tc-Ntm-1, Fig. 5b). This kind of isomers, diastereomers, could be
chromatographically differentiated and could be in equilibrium
depending on the pH, as it was recently described.26 No further stud-
ies to explain the presence of two labelled entities in the Tc-Ntm-1
synthesis were done.
amine (alkyne 1) or D,L-propargylglycine (alkyne 2), to obtain the fi-
nal ligands Ntm-1 and Ntm-2, respectively. According to the NMR
experiments results, 1H NMR24 and NOE-diff, the Huisgen [4+2]
cycloaddition, click reaction, occurred regioselectively in presence
of Cu(I), produced in situ by reduction of Cu(II) with sodium ascor-
bate, to yield the desired 1,4-disubstituted-1,2,3-triazoles.25 All
the compounds were characterized by 1H NMR, 13C NMR, IR and
MS (see Supplementary data). The purity of the ligands was estab-
lished by HPLC.
2.2. 99mTc-labelling
Synthesis of 99mTc-tricarbonyl complexes was achieved by a
two-step synthetic route (Scheme 1b). The first stage consisted in
the preparation of the precursor, fac-[99mTc(CO)3(OH2)3]+, using so-
dium pertechnetate eluted from a 99Mo/99mTc generator as starting
material and sodium borohydride as reducing agent. The second
stage involved the substitution of the precursor by incubation with
the desired ligand, Ntm-1 or Ntm-2, and heating at 75 °C during
30 min. Proposed structures for final complexes, Tc-Ntm-1 and
Tc-Ntm-2, are shown in Figure 2 (see Section 2.3.). For the most
promising radiopharmaceutical, Tc-Ntm-2, a one-pot preparation
procedure (Fig. 3) was also assayed. The combination of the chelat-
ing unit with the pharmacophore was achieved as a first step by the
click reaction and the reaction mixture without any purification
was incubated with the neutralized 99mTc-tricarbonyl precursor
to yield the desired technetium complex in high purity. Click chem-
istry has been proposed as an innovative functionalization strategy
Taking into consideration that during the preparation of Tc-
Ntm-1 the two generated species were in equilibrium (Figs. 4c
and 5) and that the main species was converted to the other com-
plex in aqueous solution (Fig. 4e), (and probably also in the biolog-
ical milieu), we decided to perform all the biological studies using
the mixture of both Tc-complex, ratio = 2.5, obtained in the label-
ling reaction.
2.3. Preparation of rhenium analogues
O
NO2
N
The structure of 99mTc-complexes was studied using stable rhe-
nium as a surrogate for technetium. Rhenium, as technetium third
row congener, exhibits many of the chemical properties of Tc. Fur-
thermore, Re and Tc complexes with the same ligand have essen-
tially identical coordination parameters since the ionic radii of
both metals are about the same, due to the lanthanide contrac-
tion.27 In our case, the analogue rhenium complexes were prepared
by substitution of ligands Ntm-1 or Ntm-2 on the rhenium precur-
sor fac-[NEt4]2[Re(CO)3Br3] (Scheme 2).28–30 The HPLC analyses, UV
detection, of the reaction mixture for both processes showed main
peaks with the same retention time of the corresponding techne-
tium complexes (see Supplementary data). The results of the anal-
ysis of the isolated main complexes, Re-Ntm-1 and Re-Ntm-2, were
compatible with the proposed structures for the Tc-complexes
(Fig. 2) confirming the presence of one ligand molecule per
NO2
N
NH
99mTc
N
N
N
H2N
99mTc
N
OH2
CO
N
O
N
N
N
OC
OC
CO
CO
CO
Tc-Ntm-1
Tc-Ntm-2
Figure 2. Proposed structures for complexes Tc-Ntm-1 and Tc-Ntm-2.
[
99mTc(CO)3]+ core and the presence of one molecule of coordinated
water in the case of Re-Ntm1.
2.4. Physicochemical studies
In order to study the potentiality of the technetium com-
plexes as radiopharmaceuticals some relevant physicochemical
We would like to thank Referee #1 for his helpful suggestions concerning to this
point.
Figure 3. One-pot procedure to prepare Tc-Ntm-2.

S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
4043
Figure 4. Typical chromatograms for 99mTc(I)-tricarbonyl complexes (RP-HPLC eluted with phosphate buffer (pH 2.5):MeOH (0 to 100%), coupled to
c
-detector). (a) fac-
[
99mTc(CO)3(OH2)3]+. (b) Labelling mixture of Tc-Ntm-2. (c) Labelling mixture of Tc-Ntm-1. (d) Isolated main product from the reaction milieu of Tc-Ntm-1. (e) Example of
stability study by RP-HPLC of radio-product with tR = 16.0 min. The arrows show the presence of fac-[99mTc(CO)3(OH2)3]+ (dotted) and the minor 99mTc complex with
tR = 15.0 min at 4 h of incubation.
(a)
HN
NO2
N
NO2
N
NH
99mTc
OH2
99mTc
OH2
99mTc
H2O
H2O
N
N
OH2
CO
OH2
CO
OH2
CO
N
N
H2O
N
N
N
N
OC
OC
OC
CO
CO
CO
Tc-Ntm-1 (mono-acuo
)
di-acuo
tR= 15 min
fac-[99mTc(CO)3(OH2)3]+
tR= 3.5 min
tR= 16 min
(b)
H
NO2
NO2
H
OH2
CO
N
99mTc
N
99mTc
N
N
OH2
CO
N
N
N
N
N
N
N
N
OC
OC
CO
CO
A-Tc-Ntm-1
B-Tc-Ntm-1
Figure 5. Proposed processes that explain the presence of multiple labelled species in the Tc-Ntm-1 preparation.
2-
O
NO2
N
NO2
N
Br
Re
CO
NH
Re
Ntm-1 or Ntm-2
N
Br
Br
CO
CO
Br
CO
N
N+(Et)4
N
N
2
H2N
N
or
O
N
N
MeOH / H2O
70 °C, 3 h
N
OC
Re
OC
OC
CO
OC
Re-Ntm-1
Re-Ntm-2
Scheme 2. Synthesis of the Re-complexes analogues, Re-Ntm-1 and Re-Ntm-2.
properties were studied (i.e. stability, lipophilicity, and binding
to plasmatic proteins).
analysis for up to 4 h after labelling. The complexes, Tc-Ntm-1 as
the mixture of the two forms and Tc-Ntm-2, were stable for at least
4 h in the labelling milieu. On the other hand, stability after incuba-
tion in human plasma for 2 h, at 37 °C, was also studied (Table 1).
Tc-Ntm-1 showed a lower stability than Tc-Ntm-2 in agreement
with the bidentate-monodentate behaviour of the ligand, Ntm-1,
2.4.1. Stability
99mTc-complexes, in the labelling milieu, were incubated at
room temperature and the radiochemical purity checked by HPLC

4044
S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
Table 1
Some physicochemical and biological properties of the developed Tc-complexes
Complex
Physicochemical properties
Activity ind
Liver
Stability in plasmaa
Log Pb
Protein bindingc
Blood
Kidneys
Tc-Ntm-1e
Tc-Ntm-2
85%
92%
ꢀ0.82 0.02
ꢀ0.44 0.04
51.5 0.9%
13.0 3.0%
4.7 1.3
0.4 0.2
17.0 0.6
1.8 0.6
4.9 0.9
0.22 0.04
a
b
c
Percentage of radioactivity remaining in the main peak after 2 h of incubation in human plasma at 37 °C.
For experimental details see Text.
Percentage of radioactivity bound to plasma proteins after 2 h of incubation.
Percentage of injected dose in each organ after 2 h of complex injection.
Studied as the mixture of complexes.
d
e
that entails to
nucleophiles.
a more reactive complex towards plasma
Table 2
Uptake of 99mTc-Ntm-2 both in oxia and in hypoxia
2.4.2. Lipophilicity
The partition coefficient between 1-octanol and phosphate buf-
fer (0.1 M, pH 7.4) of the technetium complexes was measured in
order to assess their lipophilicity (Table 1). The values are in accor-
dance to the proposed structures of the complexes since Tc-Ntm-1
has
a formal positive charge and consequently the highest
hydrophilicity.
2.4.3. Protein binding
Binding to plasma protein was studied using size exclusion
chromatography (Table 1). Ideally, low protein binding is required
in order to ensure adequate pharmacokinetics of the potential
radiopharmaceuticals. Additionally, only the unbound fraction of
the radiotracer will penetrate cells and other biological mem-
branes.31 A relatively low protein binding was obtained for Tc-
Ntm-2, correlating with the highest lipophilicity of this complex.
On the other hand, protein binding of Tc-Ntm-1 was considerably
high. Once more the substitution of the labile-bound water mole-
cule in the coordination sphere of this complex by other potential
electron donor groups from the proteins, that is, imidazole, thio-
late, and carboxylate groups, could be considered as a possible
cause of this result.
Uptake hypoxia/oxia at 60 min (n = 3)
2.2 0.3
Value is shown as mean S.D.
(⁄) Not different (p <0.05).
by low blood and liver activity (Table 1), rapid depuration and
quantitative excretion through the urinary tract (58% of injected
activity in urine at 2 h after administration) and intestines (30%
at the same time). Activities in other organs and tissues were neg-
ligible. Uptake in tumour was low but high and statistical signifi-
cant (p <0.05) tumour/muscle ratios were achieved at all time
points (Table 3). Another favourable characteristic was fast clear-
ance from blood that lead to tumour/blood ratios that increased
significantly with time (Table 3). On the other hand, biodistribu-
2.5. Biological evaluation
2.5.1. Cell uptake studies
For the most promising radiopharmaceutical, Tc-Ntm-2, in vitro
uptake both in oxia and hypoxia was evaluated using human colon
adenocarcinoma HCT-15 cells in culture. Cells were incubated at
37 °C under an atmosphere of 95% air plus 5% carbon dioxide (aer-
obic exposure) or 95% nitrogen plus 5% carbon dioxide according to
literature oxygen concentration <1000 ppm is considered hypoxic
condition.32,33 After 60 min equilibration period, the radiolabeled
compound was added and incubated with the cells for other 60,
90, or 120 min. It has been shown by means of propidium iodide
viability test34 that HCT-15 cells maintain >90% of viability for at
least 90 min under the hypoxic conditions of this assay (data not
shown). Finally, cells were separated from supernatant and radio-
activity measured in order to compare the percentage taken up in
oxic and hypoxic conditions.21 Tc-Ntm-2 showed preferential up-
take in hypoxia, as shown in Table 2.
Table 3
Tumour uptakes for developed complexes on C57 mice bearing Lewis carcinoma
% Injected dose/g
30 min
60 min
120 min
Tc-Ntm-1
Blood
Muscle
Tumour
4.83 0.43
0.96 0.15
1.10 0.20
5.50 1.10
0.82 0.02
0.85 0.22
3.11 0.74
0.69 0.10
1.33 0.32
Ratios
Tumour/Blood
Tumour/Muscle
0.23 0.05
1.17 0.27
0.16 0.07
1.04 0.30
0.44 0.15
1.93 0.34
Tc-Ntm-2
Blood
Muscle
Tumour
2.5.2. Biodistribution studies in animals bearing induced
tumours
1.20 0.60
0.19 0.05
0.63 0.06(*)
0.29 0.04
0.09 0.03
0.22 0.07(*)
0.15 0.04
0.08 0.02
0.28 0.07(*)
Biodistribution studies were carried out in C57 mice bearing in-
duced Lewis carcinoma. This animal model was selected because
histopathologic studies demonstrated high degree of hypoxia
within the tumours.21 Complete results of biodistribution for
Tc-Ntm-1 and Tc-Ntm-2 complexes are available as Supplemen-
tary Material. Biological behaviour of Tc-Ntm-2 was characterized
Ratios
Tumour/Blood
Tumour/Muscle
0.50 0.10
3.30 0.10
0.76 0.10
2.40 0.11
1.86 0.09
3.11 0.09
*
Significant differences between tumour and muscle uptake. p <0.05 (Student’s
test).

S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
4045
Figure 6. Biodistribution pattern of Tc-Ntm-1 and Tc-Ntm-2 complexes on C57 mice bearing Lewis carcinoma at 60 min post-injection.
Tc
Complex oxidation
state
t/ba ratio
t/mb ratio
O
NO2
N
Tc-Ntm-2
Tc-dtc-1c
Tc-iso-1d
I
V
III
1.86 0.09
0.65 0.21
1.0 0.4
3.11 0.09
1.80 0.17
N
N
H2N
99mTc
N
O
N
2.4 0.5
OC
CO
a
b
t/b: tumour/blood ratio at 2 h after injection;
t/m:
CO
c
tumour/muscle ratio at
2
h
after injection;
From
reference [20]; d From reference [21]
Tc-Ntm-2
S
S
99mTc
S
N
N
99mTc
-
N+
C
O2N
N
NO2
N
NO2
S
S
S
N
H
N
H
S
N
N
H
N
N
N
O
Tc-dtc-1
Tc-iso-1
Figure 7. Comparison of tumour uptakes, expressed as tumour/blood and tumour/muscle ratios, of different bioreducible-pharmacophore containing Tc-complexes
developed by our group.
tion of Tc-Ntm-1 complex was less favourable, as expected from its
high protein binding. Activity in blood and liver was high and up-
take in kidneys (Table 1) and muscle was also higher compared
with Tc-Ntm-2 complex. Excretion occurs through hepatobiliary
and renal systems. Although tumour uptake was considerably
higher (Table 3) than the observed for Tc-Ntm-2, tumour/muscle
ratios are lower and statistical analysis revealed that differences
in uptake between tumour and muscle were not significant
(p <0.05). Besides, tumour/blood ratios were unfavourable for all
time points because of the high level of activity retained in blood.
Figure 6 shows a comparison between biodistribution patterns of
both complexes at 60 min post-injection and clearly demonstrates
that Tc-Ntm-1 and Tc-Ntm-2 have completely different biological
behaviour. While the complex formed by tridentate coordination
of Ntm-2 to technetium has a low uptake in all organs and tissues
with rapid depuration, the complex formed by bidentate coordina-
tion of Ntm-1 shows low depuration from blood, liver, kidneys and
muscle, leading to an unfavourable biodistribution pattern for a
potential radiopharmaceutical. This behaviour correlates with the
high protein binding of the Tc-Ntm-1 (Table 1) and could be asso-
ciated with the bidentate coordination. Studies by Schibli et. al. on
the influence of ligand denticity in 99mTc(I) tricarbonyl complexes
indicate that clearance of bidentate coordinated complexes from
blood is slower and activity is generally retained in all organs,
especially in liver and kidneys.31,35 A lower thermodynamic stabil-
ity of this type of complexes is suggested as the reason for the high
retention in vivo, where adequate functional groups of proteins can
substitute the labile-bound water molecule in the coordination
sphere.36,37 Comparison between Tc-Ntm-1 and Tc-Ntm-2 support
this idea. In fact, the addition of a third electron donor group to ob-
tain a tridentate ligand had a great impact not only in vitro but also
in vivo behaviour of the final complex.
3. Conclusion
This work presents the development of two novel 5-nitroimi-
dazole derivatives and the corresponding 99mTc(I)-tricarbonyl com-
plexes as potential radiopharmaceuticals for hypoxia imaging.
Derivatization of the pharmacophore unit was made in order to
introduce a bidentate or a tridentate chelator unit, obtaining final
ligands Ntm-1 and Ntm-2, respectively. Final 99mTc(I)-tricarbonyl
complexes, Tc-Ntm-1 and Tc-Ntm-2, were characterized in vitro
and in vivo and results showed clearly that the complex with the
tridentate ligand, Tc-Ntm-2, had a good and fast clearance from
all organs and tissues, which correlates with a low protein binding.
On the other hand, the bidentate-coordinated complex, Tc-Ntm-1,
showed a high protein binding and revealed high retention of activ-
ity in blood, liver, kidneys, and muscle. Although several promising

4046
S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
99mTc(I)-tricarbonyl complexes have been reported using bidentate
ligands,5–7 literature also describes that in some cases a bidentate
ligand could lead to low thermodynamic stability of the coordina-
tion compounds due to the substitution of the labile-bound water
molecule in the coordination sphere. In our case, conclusion is that
denticity of the ligand has a deep impact on physicochemical and
biological behaviour of the final complex and tridentate chelating
systems are preferable for pharmacophore derivatization.
4.1. Synthesis of [2-(2-methyl-5-nitro-1H-imidazol-1-yl)]
ethylazide, Azd. Mtz
(0.5 g, 3.57 mmoles) and triphenylphosphine (1.4 g, 5.34 mmo-
les) in dried THF (25 mL) were stirred under ice bath. Azidotri-
methylsilane (1.40 mL, 5.38 mmoles) was added to the solution
and then diisopropyl azodicarboxylate (DIAD, 1.1 g, 5.4 mmoles)
in dried THF (10 mL) was added maintaining the reaction mixture
at 0 °C. After 12 h at room temperature the solvent was evaporated
in vacuo and the residue was purified by column chromatography
(SiO2, CH2Cl2:MeOH, 95:5). Beige solid. 1H NMR (CDCl3) d (ppm):
2.56 (s, 3H), 3.79 (t, 2H, J = 5.4), 4.46 (t, 2H, J = 5.4), 8.00 (s, 1H).
13C NMR (CDCl3) d (ppm): 14.9, 46.0, 51.4, 129.0, 133.9, 151.7. IR
Previously, we have studied the Tc(V)-nitride,20 and
Tc(III)-[4+1]21 cores as centres for coordination to the 5-nitroimidaz-
olyl-pharmacophore where the dithiocarbamate and isonitrile con-
taining complexes Tc-dtc-1 and Tc-iso-1 ( Fig. 7), respectively,
showed the best differential tumoural uptake in each group of com-
pounds. Clearly, the Tc(I)-tricarbonyl complex, Tc-Ntm-2, displayed
approximately 3-times and 1.5-times higher tumour/blood and tu-
mour/muscle ratios, respectively, than the Tc(V)-nitride complex,
Tc-dtc-1, at 2 h post-injection (Fig. 7). On the other hand, at the same
experimental time the complex with Tc in the intermediate
oxidation state, that is III, Tc-iso-1, displayed lower tumour/blood
and tumour/muscle ratios than Tc-Ntm-2 (Fig. 7). In conclusion,
the new 5-nitroimidazolyl bioreducible pharmacophore containing
Tc-complex Tc-Ntm-2, described herein, appears as the best radio-
pharmaceutical for hypoxic tumour diagnosis developed by our
group.
(KBr)
m
(cmꢀ1): 1386, 1528, 2064–2128. MS (EI, 70 eV) m/z (%):
196 (M+Å), 151 ([M–NO2–OH]+), 122 ([M–NO2–OH–NO]+).
4.2. Synthesis of N-methyl-1-[1-(2-(2-methyl-5-nitro-1H-
imidazole-1-yl)ethyl)-1H-1,2,3-triazole-4-yl]methylamine,
Ntm-1
A solution of sodium ascorbate (0.30 g, 1.49 mmoles) and cop-
per (II) sulphate (0.47 g, 2.97 mmoles) in distilled H2O (15 mL)
was added to a solution of Azd (1.50 g, 7.42 mmoles) and N-meth-
ylpropargylamine (0.63 mL, 7.42 mmoles) in t-butanol (15 mL) un-
der stirring at room temperature. The mixture was stirred for 24 h
and more sodium ascorbate (0.15 g, 0.75 mmoles) was added. After
extra 24 h at room temperature the t-butanol was evaporated in
vacuo, the residue was treated with aqueous solution of NaOH
(33%, 60 mL) and the aqueous phase was extracted with EtOAc
(4 ꢁ 60 mL). The organic phase was dried with Na2SO4 anhydrous
and evaporated in vacuo. The brown-beige solid corresponds to
the desired product (47%); mp: 86.5–88.9 °C. 1H NMR (CD3OD:-
D2O) d (ppm): 2.00 (s, 3H), 2.37 (s, 3H), 3.80 (s, 2H), 4.87 (t,
J = 3.0, 2H), 4.91 (m, 2H), 7.79 (s, 1H), 7.98 (s, 1H). 13C NMR
(CD3OD:D2O) d (ppm): 11.8, 33.9, 44.9, 46.1, 49.1, 123.9, 131.6,
4. Experimental
The precursors fac-[99mTc(CO)3(OH2)3]+ and fac-[NEt4]2[Re(-
CO)3Br3] were prepared according to literature.1,28–30 Distilled
and dried solvents were employed for synthesis. All laboratory
chemicals were reagent grade and were used without further puri-
fication. Solvents for chromatographic analysis were HPLC grade.
Thin-layer chromatography was carried out on alumina or silica
gel plates (Merck 60 F254). Column chromatography was carried
out on silica gel (Merck, 60–230 mesh) or neutral alumina (Merck
70–230 mesh).
138.7, 145.7, 151.0. IR (KBr)
m
(cmꢀ1): 1366, 1527, 3402. MS (EI,
70 eV) m/z (%): 264 ([M–H]+, 20), 248 ([M–OH]+, 22), 218 ([M–
99mTc]NaTcO4 was obtained from a commercial generator
HNO2]+, 9), 44 ([CH3NHCH2]+, 100).
[
(Tecno Nuclear S.A., Argentine). The NMR spectra were recorded
on a Bruker DRX-400 spectrometer in the indicated deuterated sol-
vent. Chemical shifts are reported as d values (parts per million)
with respect to TMS. Coupling constants are reported in Hertz
(Hz). The multiplicity is defined by s (singlet), t (triplet), or m (mul-
tiplet). The mass spectra (MS) were conducted in a mass spectrom-
eter Hewlett Packard 5973 MSD or MICROMASS (Triple Quattro)
using electron impact (EI) or electrospray (ESI), respectively. IR
spectra were obtained in the range 4000–200 cmꢀ1 in KBr pellets
at 1% in a Bomen MB–102 FT-IR spectrometer. Melting points were
determined with an electrothermal melting point apparatus
(Electrothermal 9100) and were uncorrected. Microanalyses were
performed on a Fisons EA 1108 CHNS-O instrument and were with-
in 0.4% of the calculated compositions. HPLC analysis was devel-
oped on a LC-10 AS Shimadzu Liquid Chromatography System
using a reverse phase column Phenomenex Luna 5m, C18 column
(4.6 ꢁ 150 mm). Elution was performed with a binary gradient sys-
tem at 1.0 mL/min flow rate using triehtylamine-phosphate buffer
pH 2.5 as mobile phase A and methanol as mobile phase B; the elu-
tion profile was as follows: 0–3 min 100% A; 3–6 min linear gradi-
ent to 25% B; 6–9 min linear gradient to 34% B; 9–20 min linear
gradient to 100% B; 20–27 min 100% B; 27–30 min linear gradient
0% B. Detection was accomplished either with a photodiode array
detector (SPD-M10A, Shimadzu) that recorded UV-vis spectra on
flux or with a 300 ꢁ 300 NaI (Tl) crystal scintillation detector. Activity
4.3. Synthesis of 2-amine-3-{1-[2-(2-methyl-5-nitro-1H-
imidazole-1-yl)ethyl]-1H-triazole-4-yl}propanoic acid, Ntm-2
A solution of Azd (0.26 g, 1.32 mmoles), D,L-propargylglycine
(0.15 mL, 1.32 mmoles), sodium ascorbate (0.53 g, 2.6 mmoles)
and copper (II) sulphate (0.85 g, 5.3 mmoles) in t-butanol:distilled
H2O (60:15 mL) was stirring at room temperature for 24 h. Then
cationic exchange resin (Chelex 100-Biorad, 0.5 g) was added and
the mixture was stirred until disappearance of the blue colour
(2 h). The resin was filtered and the volume of the solution was re-
duced in vacuo. Then ethyl ether:acetone (1:1, 10 mL) was added
and the mixture was cooled at 4 °C for 12 h. The generated solid,
corresponding to the desired product, was filtered and washed
with cold ethanol. White solid (57%); mp: 134.0–137.0 °C. 1H
NMR (D2O) d (ppm): 1.83 (s, 3H), 3.16 (s, 2H), 3.89 (s, H), 4.77
(br s, 4H), 7.65 (s, H), 8.01 (s, 1H). 13C NMR (D2O) d (ppm): 12.5,
26.1, 33.9, 46.3, 49.45, 62.2, 125.2, 129.0, 146.6, 151.7, 177.1. IR
(KBr)
m
(cmꢀ1): 1366, 1527, 1603, 3149. MS (EI, 70 eV) m/z (%):
309 (M+Å, 3), 278 ([M–HNO]+, 15), 265 (18).
4.4. 99mTc-labelling
4.4.1. Synthesis of 99mTc-Ntm-1 complex
Substitution of fac-[99mTc(CO)3(OH2)3]+ was achieved by incu-
measurements were performed either in
a Dose Calibrator,
bation of Ntm-1 ligand (3 mg) dissolved in MeOH (100 lL) with
the neutralized precursor solution (1.2 mL) at 70 °C for 30 min.
Radiochemical purity was assessed by HPLC using the HPLC system
Capintec CRC- 5R or in a scintillation counter, 300 ꢁ 300 NaI (Tl) crys-
tal detector associated to an ORTEC monochanel analyzer.

S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
4047
described previously. tR = 15 and 16 min. Yield >90% (mixture of
two species).
were counted in a gamma counter. The partition coefficient was cal-
culated as the mean value of each cpm/mL of 1-octanol layer di-
vided by that of the buffer. Lipophilicity was expressed as logP.
4.4.2. Synthesis of 99mTc-Ntm-2 complex
Substitution of fac-[99mTc(CO)3(OH2)3]+ was achieved by incu-
4.6.3. Protein binding
bation of Ntm-2 ligand (4 mg) dissolved in MeOH (100
lL) with
99mTc-complexes (Tc-Ntm-1 or Tc-Ntm-2, 25
l
L) were incu-
L) at 37 °C for up to 120 min. At
L) were added to MicroSpin G-50
the neutralized precursor solution (1.5 mL) at 70 °C for 30 min.
Radiochemical purity was assessed by HPLC using the HPLC system
described previously. tR = 18 min. Yield >90%.
bated with human plasma (475
30 and 120 min aliquots (50
l
l
columns (GE Healthare), which have been pre-spun at 2000g for
1 min. Columns were centrifuged again at 2000g for 2 min and
the collected elute and the column were counted in a NaI(Tl)-scin-
tillation counter. Protein bound tracer was calculated as the per-
centage of activity eluted from the column.
4.4.3. One pot synthesis of 99mTc-Ntm-2 complex
A solution of propargylglycine in H2O (50
mixed with a solution of Azd in MeOH (65
l
l
L, 1.131 mg/mL) was
L, 1.286 mg/mL). A
L, 1.996 mg/mL) and a solu-
L, 1.980 mg/mL) were added
cooper acetate solution in H2O (7.5
tion of sodium ascorbate in H2O (15
l
l
4.7. Cell uptake studies
and the mixture was heated for 1 h at 75 °C. After that the labelling
process was done by addition of the neutralized precursor complex
(100–200 lL) and heating for 1 h at 100 °C. Radiochemical purity is
assessed by HPLC using the HPLC system described previously.
tR = 18 min. Yield >90%.
The cell culture studies were performed using the adherent cell
line HCT-15 (CCL-255TM ATCC) corresponding to human
adenocarcinoma. Cells were cultured in RPMI-1640 (R6504
Sigma–Aldrich) supplemented with 10% fetal bovine serum
(Gibco), penicillin 100 U/mL (Sigma) and 100 lg/mL streptomicine
4.5. Preparation of rhenium analogues
(Sigma) in T75 tissue culture flasks (Nunc, Denmark) at 37 °C and
5% CO2 until approximately 7.5 ꢁ 106 cells were obtained. Then,
the flasks were preincubated in a chamber gassed with N2 for 1 h
to remove oxygen from the milieu and afterwards radiopharma-
ceutical was added and incubated for additional predefined incu-
bation times (60–120 min). The same procedure was repeated in
normal culture conditions (37 °C and 5% CO2) to use as control.
After incubation time elapsed culture milieu was removed, cells
were washed with PBS and treated with Trypsin-EDTA (Sigma). Fi-
nally, activity in the supernatant and the cells was measured in a
solid scintillation counter and results were expressed as the ratio
between the percentages of activity taken up by cells incubated
in nitrogen (hypoxia) and incubated in normal conditions (oxia).
4.5.1. Synthesis of Re(I) tricarbonyl complex with Ntm-1 ligand,
Re-Ntm-1
A solution of Ntm-1 ligand (64 nmoles) in MeOH:H2O (1:1,
10 mL) was added to a solution of fac-[NEt4]2[Re(CO)3Br3] precur-
sor (64 nmoles) in methanol:H2O (1:1, 10 mL). The mixture was
stirred at 70 °C for 3 h and the reaction progress was monitored
by the HPLC system described previously, coupled to a UV detector.
The solid product obtained was filtrated, washed, dried and ana-
lyzed by HPLC and elemental microanalysis. tR (HPLC) = 16 min.
Calculated analysis for C13H18BrN7O5Re: C: 25.3; H: 2.9; N, 15.9.
Found: C: 26.3; H: 3.1; N: 15.5.
4.5.2. Synthesis of Re(I) tricarbonyl complex with Ntm-2 ligand,
Re-Ntm-2
4.8. Biodistribution studies in animals bearing induced tumours
A solution of Ntm-2 ligand (64 nmoles) in methanol:H2O (1:1,
10 mL) was added to a solution of fac-[NEt4]2[Re(CO)3Br3] precur-
sor (64 nmoles) in methanol:H2O (1:1, 10 mL). The mixture was
stirred at 70 °C for 3 h and the reaction progress was monitored
by the HPLC system described previously, coupled to a UV detector.
The desired product was purified by HPLC under conditions men-
tioned previously and analyzed by mass spectrometry. tR
(HPLC) = 18 min. MS (ESI) m/z: 971 ([2M–2 CO–2 CO2–2 H–NO2]+).
All animal studies were approved by the Ethics Committee of
the Faculty of Chemistry, Universidad de la República (Uruguay).
A culture of 3LL Lewis murine lung carcinoma cells was expanded
and treated with trypsine previous to inoculation. A cell suspen-
sion in PBS containing 3 ꢁ 106 cells was prepared and injected
subcutaneously in the right limb of C57B16 mice (8–10 weeks
old). 20–30 days later the animals developed palpable tumour nod-
ules (1.5 ꢁ 0.5 ꢁ 0.5 cm, approximately) and were used for
biodistribution.
Three animals per group and per intervals were injected via a
lateral vain in the tail with 99mTc-complexes (Tc-Ntm-1 or
Tc-Ntm-2, 0.1 mL, 0.037–0.37 MBq). At different intervals, 30, 60,
and 120 min, after injection the animals were sacrificed by neck
dislocation. Whole tumour and samples of blood and muscle were
collected, weighed and assayed for radioactivity. Results were ex-
4.6. Physicochemical studies
4.6.1. Stability
99mTc-complexes (Tc-Ntm-1 or Tc-Ntm-2), in the labelling mili-
eu, were incubated at room temperature and the radiochemical
purity checked by HPLC analysis as described previously during
30 min for up to 4 h after labelling.
pressed as %injected dose/g of tissue (x
rn-1, n = 3).
99mTc-complexes (Tc-Ntm-1 or Tc-Ntm-2, 200
plasma (1000
different incubation times (30, 60 and 120 min), samples (200
were precipitated with ethanol (200 L), centrifuged (12000 rpm,
l
L), in human
L), were incubated at 37 °C for up to 120 min. After
L)
l
Acknowledgements
l
l
We thank PEDECIBA-Química and ANII for scholarships for
J. Giglio and S. Fernández, Gramón-Bagó S.A. for providing metro-
nidazole, Dr. María Moreno for providing the animals bearing tu-
mours and IAEA and Dr. Roger Schibli for providing some reagents.
5 min) and analyzed by HPLC as described previously.
4.6.2. Lipophilicity
Lipophilicity was studied through the apparent partition coeffi-
cient between 1-octanol and phosphate buffer (0.125 M, pH 7.4). In
a centrifuge tube, containing 2 mL of each phase, 0.1 mL of the
99mTc-complex (Tc-Ntm-1 or Tc-Ntm-2) solution was added, and
the mixture was shacked on a Vortex mixer and finally centrifuged
at 5000 rpm for 5 min. Three samples (0.2 mL each) from each layer
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.010.

4048
S. Fernández et al. / Bioorg. Med. Chem. 20 (2012) 4040–4048
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