Preparation and characterization of technetium and rhenium tricarbonyl complexes bearing the 4-nitrobenzyl moiety as potential bioreductive diagnostic radiopharmaceuticals. In vitro and in vivo studies

Article abstract of DOI:10.1016/j.ejmech.2007.05.015

The synthesis of a ligand containing a nitrobenzyl group as bioreductive pharmacophore and the preparation of the corresponding technetium and rhenium complexes are presented. ^99mTc labelling was performed in high yield (>90%) by ligand substitution using fac-[^99mTc(CO)₃(H₂O)₃]⁺ as precursor. The structure of the technetium complex was established by chromatographic comparison with the analogous rhenium compound which was fully characterized by elemental analysis, spectroscopic methods and X-ray crystallography. Reduction potential of the rhenium complex was in the characteristic range for bioreductive compounds. Biodistribution in normal mice was characterized by fast blood and soft tissue depuration and combined excretion via the hepatobiliary and urinary systems. Tumour uptake was low, probably due to low lipophilicity but tumour/muscle ratios were favourable as a consequence of high excretion.

Full text of DOI:10.1016/j.ejmech.2007.05.015

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European Journal of Medicinal Chemistry 43 (2008) 741e748
http://www.elsevier.com/locate/ejmech
Original article
Preparation and characterization of technetium and rhenium tricarbonyl
complexes bearing the 4-nitrobenzyl moiety as potential bioreductive
diagnostic radiopharmaceuticals. In vitro and in vivo studies
Javier Giglio a, Georgios Patsis b, Ioannis Pirmettis b, Minas Papadopoulos c,
b
b
Catherine Raptopoulou , Maria Pelecanou , Elsa Leon ,
a
´
c
Mercedes Gonzalez , Hugo Cerecetto , Ana Rey
c
a,
*
´
a Ca´tedra de Radioqu´ımica, Facultad de Qu´ımica, General Flores 2124, Montevideo 11800, Uruguay
b National Centre for Scientific Research ‘‘Demokritos’’, Athens, Greece
c Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica-Facultad de Ciencias, Montevideo, Uruguay
Received 30 March 2007; received in revised form 23 May 2007; accepted 25 May 2007
Available online 7 June 2007
Abstract
The synthesis of a ligand containing a nitrobenzyl group as bioreductive pharmacophore and the preparation of the corresponding technetium
and rhenium complexes are presented. 99mTc labelling was performed in high yield (>90%) by ligand substitution using fac-
[
99mTc(CO)3(H2O)3]þ as precursor. The structure of the technetium complex was established by chromatographic comparison with the analo-
gous rhenium compound which was fully characterized by elemental analysis, spectroscopic methods and X-ray crystallography. Reduction
potential of the rhenium complex was in the characteristic range for bioreductive compounds. Biodistribution in normal mice was characterized
by fast blood and soft tissue depuration and combined excretion via the hepatobiliary and urinary systems. Tumour uptake was low, probably due
to low lipophilicity but tumour/muscle ratios were favourable as a consequence of high excretion.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Bioreductive compounds; Technetium; Rhenium; Tricarbonyl complexes; Hypoxia imaging
1. Introduction
to a specific site in the body determined by the properties of
the transporter [2]. Current research is directed towards the de-
velopment of radiopharmaceuticals that can potentially serve
as ligands for bioactive substrates and can be applied in the
in vivo evaluation of biochemical functions in a non-invasive
way [3e5].
An area of special interest in radiopharmacy is the develop-
ment of suitable tracers for imaging hypoxic tissue. Oncology
would highly benefit from agents that effectively target hyp-
oxic cell populations of solid tumours that are characterized
by increased radioresistance and diffusion limitations that hin-
der the treatment [6,7]. Bioreductive compounds, which are
selectively reduced in hypoxic tissue to reactive intermediates
that bind to intracellular molecules, have been utilized for the
development of potential radiodiagnostic markers of tumour
Diagnostic radiopharmaceuticals are compounds containing
gamma-emitters, the radiation of which readily penetrates the
body, thus permitting external detection and measurement.
The pattern of biodistribution of radiation allows the physician
to make a diagnostic evaluation of both morphology and func-
tion [1]. 99mTc is the radionuclide of choice in nuclear medi-
cine due to its ideal nuclear properties for imaging (t1/2 ¼ 6 h,
Eg ¼ 140 keV). In technetium radiopharmaceuticals the metal
is bound to a transporting moiety that delivers the radioactivity
* Corresponding author. Tel.: þ598 2 924 85 71; fax: þ598 2 924 19 06.
E-mail address: arey@fq.edu.uy (A. Rey).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.05.015

742
J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
hypoxia. 2-Nitroimidazole has been the preferred bioreductive
pharmacophore but other functional groups as nitroaromatics
and N-oxides have also been studied. Attachment of these bio-
reductive moieties to technetium is achieved through the use
of a variety of chelators, namely propylene amine oxime
(PnAO), diaminodithiols (DADS) and nitrido complexes [8e12].
However, properties of the synthesized technetium complexes
are not yet ideal and new potentially active compounds are
still being developed.
derivative, which is an adequate chelator for the synthesis of
fac-tricarbonyl complexes of technetium and rhenium (Fig. 1).
The ligand LH was synthesized by reacting 4-nitrobenzyl
bromide with ethyl 2-[(pyridin-2-ylmethyl)amino]acetate, fol-
lowed by alkaline hydrolysis as shown in Fig. 2. Charac-
terization was performed by the usual spectroscopic methods
(1H NMR, 13C NMR and IR). The overall yield was high
(>70%) and all analytical data were consistent with the
proposed structure.
With the aim to develop new potential 99mTc-radiopharma-
ceuticals for imaging hypoxia with potential application in on-
cology and cardiology, we have selected the nitrobenzyl
functional group as the bioreductive pharmacophore [13],
while the fac-[99mTc(CO)3]þ synthon was chosen as the metal
core for the synthesis of the complexes. The widely employed
99mTc-tricarbonyl core is under intense investigation for the la-
belling of small biomolecules. Alberto et al. succeeded in
carrying out low pressure synthesis of the Tcecarbonyl com-
plexes, which can be used as precursors to obtain a great diver-
sity of potential radiopharmaceuticals. In these complexes CO
is tightly bound and stabilizes the low oxidation state of the
metal. In aqueous medium, which is the usual medium for
the preparation of technetium-99m radiopharmaceuticals, the
well characterized aqua ion of technetium [Tc(OH2)3(CO)3]þ
is formed. The final step of the synthetic procedure consists of
exchanging the weakly bound water molecules with an incom-
ing mono or multidentate ligand attached to a derivatized
biomolecule [14,15]. A wide variety of functional groups con-
taining donor atoms are present in these ligands: N-heterocy-
cles such as imidazoles, pyridines and pyrazoles, amides,
carboxylic acids, thioethers, thiols, etc. According to the prop-
erties of the employed ligands, labelled molecules of different
charge, lipophilicity and stability might be obtained. This ap-
proach has been applied recently for the labelling of peptides,
CNS receptor ligands, myocardial imaging agents, DNA
intercalators, etc [16e20].
2.2. Preparation of the [99mTc] technetium complex
99mTc labelling was performed by substitution using fac-
[
99mTc(OH2)3(CO)3]þ as precursor. The tricarbonyl precursor
was successfully prepared by two different methods. Method
1 involves the reduction of pertechnetate with sodium borohy-
dride in the presence of CO (g) at atmospheric pressure, while
method 2 consists in the addition of pertechnetate to a lyophi-
lized kit formulation containing sodium boranocarbonate,
which acts simultaneously as reductant and ‘‘in situ’’ source
of CO [21]. Both methods produced the desired compound
in high yield (>95%) as shown by HPLC analysis. The tricar-
bonyl precursor had a retention time of approx. 4 min. Com-
parison with chromatographic profile of the pertechnetate
ion that has a retention time of 12 min demonstrated that the
formation of the precursor was quantitative. Minor peaks of
longer retention times probably corresponding to polymeric
species were observed. Radiochemical purity was also
checked by paper chromatography. Retardation factors of
fac-[99mTc(OH2)3(CO)3]þ in butanone and methanol/HCl
95:5 are 0 and 0.9e1, respectively, and allow differentiation
of potential radiochemical impurities, namely pertechnetate
and reduced hydrolyzed technetium.
Substitution was performed by the addition of ligand LH to
an aqueous solution of fac-[99mTc(OH2)3(CO)3]þ at neutral
pH. HPLC analysis of the labelling mixture revealed the for-
mation of mainly one complex (retention time of 20 min)
with a high radiochemical purity (>90%). The radioactivity
recovery from the column was monitored by means of an
on-line solid scintillation detector coupled to the HPLC sys-
tem and found to be quantitative. The complex was stable
for at least 6 h. The HPLC purified complex is obtained in car-
rier free form, since the ligand LH has a retention time of ap-
prox. 15 min. The partition coefficient of the technetium
complex between octanol and phosphate buffer pH 7.4 was
measured in order to assess its lipophilicity. A log P of 0.02
Herein, we describe the synthesis and characterization of
the potentially bioreductive ligand 2-[(4-nitrobenzyl)(pyri-
din-2-ylmethyl)amino]acetic acid. The corresponding 99mTc
and Re complexes were also prepared. The rhenium complex
was structurally characterized by elemental analysis, IR,
UVevis and NMR spectra and X-ray crystallography. Electro-
chemical studies were carried out in order to determine the re-
duction potential of the nitroaromatic group. The potentiality
of the 99mTc complex as hypoxia imaging agent was evaluated
by biodistribution experiments in normal and tumour bearing
mice.
NO2
2. Chemistry
N
2.1. Synthesis of 2-[(4-nitrobenzyl)(pyridin-2-ylmethyl)-
amino]acetic acid (L)
N
OC
Tc
OC
O
The ligand 2-[(4-nitrobenzyl)(pyridin-2-ylmethyl)amino]-
acetic acid (LH) was designed by combining the 4-nitrobenzyl
bioreductive moiety with a pyridinmethylaminoacetic acid
CO
O
Fig. 1. Proposed structure of 99mTc complex.

J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
743
O2N
O
MEK/Et3N
H
N
+
NO2
O
Br
OC2H5
N
N
N
OC2H5
NaOH/H2O
O2N
O
N
N
OH
Fig. 2. Synthesis of ligand LH.
was obtained indicating that about half of the activity was ex-
tracted by octanol while the other half remained in the aqueous
layer.
Corroboration of the structure of the 99mTc complex was
achieved by comparing its HPLC profile with that of the cor-
responding Re complex upon coinjection [22] (see Section
2.3). Radioactivity and UV detectors showed identical chro-
matographic profiles, suggesting that the same chemical
structure was formed under both chelating conditions.
showed the expected symmetric and asymmetric stretching
bands of the C^O bond at 1883 (broad), 1923 and
2022 cmꢁ1, demonstrating the presence of the tricarbonyl
core in the molecule. The band at 1664 cmꢁ1 can be assigned
to the coordinated carboxylate anion of ligand LH and the ab-
sorptions at 1523 and 1338 cmꢁ1, which are characteristic of
the nitro group, corroborate the presence of the intact biore-
ductive pharmacophore [26,27]. In agreement with the pro-
posed structure, the NMR spectra revealed the presence of
the pyridinyl and nitrobenzyl moieties in the aromatic region,
as well as the presence of three pairs of diastereotopic methy-
lene protons in the aliphatic region. X-ray crystallographic
studies confirmed the proposed structure. The molecular struc-
ture of the rhenium complex is shown in Fig. 4 and selected
bond lengths and angles are listed in Table 1. The ligand LH
acts as an NNO monoanionic tridentate chelate towards the
fac-[Re(CO)3Br3]2ꢁ precursor and replaces the three bromine
atoms. The nitro group remains free to interact with the bio-
logical system. Bond distances and angles are consistent
with the octahedral geometry around the rhenium atom. There
are two 5-membered rings in the coordination sphere, defined
by the ReeNeCeCeN and the ReeNeCeCeO atoms,
which adopt the envelope configuration with C(6) and C(7)
2.3. Preparation of the rhenium compound
The structural characterization of 99mTc complexes pres-
ents some peculiarities derived from the nuclear properties
of the metal. 99mTc is used in vivo in very low concentration
(ꢀ10ꢁ9 M). Radioprotection of patients and health care per-
sonnel is the cause of this unusually low concentration. As
a consequence, the structural characterization of radioactive
99mTc complexes by the traditional methods (elemental analy-
sis, spectroscopy, X-ray crystallography) is not feasible and
usually their chemical evaluation is carried out by using the
stable rhenium as a surrogate for technetium [23,24]. Rhe-
nium, as technetium third row congener, exhibits many of
the chemical properties of Tc. Furthermore, Re and Tc com-
plexes with the same ligand have essentially identical coordi-
nation parameters since the ionic radii of both metals are about
the same, due to the lanthanide contraction. In addition, the
studies of 99mTc complexes may be further expanded to the
preparation of analogous radioactive rhenium complexes
with potential application in radiation therapy.
In our study the rhenium complex was prepared by reacting
ligand LH with the tricarbonyl rhenium precursor [Ne
(CH3CH2)4]2[Re(CO)3Br3] [25], as shown in Fig. 3.
HPLC analysis revealed the presence of only one species
with a retention time of 20 min and a maximum absorption
at 264 nm in agreement with the presence of aromatic systems
in the molecule. The rhenium complex was obtained as yellow
crystals in high yield (80%). It is very stable in solid state as
well as in solution. Elemental analysis performed for C, H and
N was consistent with the proposed structure. IR spectra
˚
lying 0.60 and 0.21 A, respectively out of the mean plane of
the remaining four atoms.
2.4. Electrochemical studies
The redox behaviour of ligand LH and the rhenium complex
was studied by cyclic voltammetry in anhydrous DMF. Ca-
thodic region was studied in order to assess the reduction poten-
tials of the ligand and the complex and compare these values
with the ones corresponding to enzymes responsible for biore-
duction in anaerobic organisms (approx. E1/2 ꢁ450 mV vs stan-
dard hydrogen electrode or ꢁ700 mV vs saturated calomel
electrode) [28e30]. Fig. 5 shows typical voltammograms for
ligand LH and the rhenium complex and Table 2 lists the values
of the first and second cathodic peaks. Voltammetric data of the
complex indicated that the reduction processes follow a simple
pattern that involves two to three reduction waves. All couples

744
J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
-2
CO
Re
+1
CO
OC
Br
Br
Br
Br
CO
CO
OC
OC
Br2
[NEt4]Br
Diglime
110 ºC
N2
CO
CO
OC
OC
Re
CO
N
Re
CO
2.
Hexane
25 °C
N2
Re
CO
OC
OC
CO
CO
NO2
O2N
Acetonitrile
Reflux 3 h
O
Yelow crystals
Yield = 80%
N
O
O
N
N
N
OH
Re
CO
OC
CO
Fig. 3. Preparation of rhenium complex.
correspond to the nitro redox process, while the reduction of the
metallic core does not fall within the studied range according to
the literature [31,32]. The first cathodic peak could correspond
to the nitro-anion radical generation while the second could be
attributed to the hydroxylamine reduction [29]. The ligand LH
presents the same voltammetric behaviour in the studied region.
The reduction of the nitro group in the complex occurs at
ꢁ805 mV while in the ligand the processes occur approx.
100 mV lower (at ꢁ895 mV).
significantly reduced in 2 h (11.2 ꢂ 1.4%), indicating rapid
clearance. Excretion occurs mainly through the hepatobiliary
system as demonstrated by high intestinal activity with urinary
excretion being also significant (66.0 ꢂ 9.1 and 15.3 ꢂ 2.4, re-
spectively at 2 h post-injection). Stomach and thyroid values
were within acceptable levels (0.1 ꢂ 0.01% and 0.2 ꢂ 0.06%,
respectively, at 30 min), indicating minimal in vivo
reoxidation.
3.2. Biodistribution studies in mice bearing induced
sarcoma
3. Biology
3.1. Biodistribution studies in normal mice
In order to assess the potentiality of our approach for the
design of potential radiopharmaceuticals for nuclear oncology,
evaluation in mice bearing induced sarcoma was performed.
This animal model was selected because histopathological
studies demonstrated a high degree of hypoxia within the
tumours.
Tumour uptake (% dose/g) is shown in Table 3. Although
uptake was low (0.19 ꢂ 0.03% at 0.5 min post-injection) prob-
ably due to poor cell penetration caused by low pipophilicity,
only minor depuration from the tumor was observed from 0.5
to 12 h suggesting selective retention. Tumour/muscle ratios
are favourable from 2 h post-injection due to rapid clearance
from blood and muscle (2.2e6.5).
The in vivo behaviour of the 99mTc complex was evaluated
by biodistribution studies in normal mice between 0.5 and 24 h
post-injection. Fig. 6 shows the results expressed as % dose/
organ in the most significant organs and fluids as a function
of time. The complex demonstrated low initial blood and
lung activity (1.5 ꢂ 0.4% and 0.11 ꢂ 0.4%, respectively at
30 min post-injection) and quantitative clearance after 24 h
(0.06 ꢂ 0.05% and 0.01 ꢂ 0.01%, respectively). Initial liver
activity was relatively high (21.0 ꢂ 1.1%) but was
Table 1
Selected bond lengths (A) and angles ( ) for the rhenium complex
ꢃ
˚
Lengths
ReeC(23)
1.902(5)
1.908(6)
1.928(6)
ReeO(1)
ReeN(1)
ReeN(2)
2.108(3)
2.191(4)
2.239(4)
ReeC(22)
ReeC(21)
Angles
C(21)eReeN(1)
C(22)eReeN(2)
C(23)eReeO(1)
C(23)eReeN(2)
C(22)eReeN(1)
N(1)eReeN(2)
C(21)eReeN(2)
C(22)eReeC(21)
172.4(2)
170.8(2)
176.0(2)
97.6(2)
96.9(2)
76.0(1)
97.4(2)
89.3(3)
C(23)eReeC(22)
C(22)eReeO(1)
O(1)eReeN(2)
C(23)eReeN(1)
O(1)eReeN(1)
C(21)eReeO(1)
C(23)eReeC(21)
89.1(3)
93.7(2)
79.4(1)
98.2(2)
78.5(1)
96.8(2)
86.1(2)
Fig. 4. Labelled ORTEP plot of the rhenium complex showing 30% thermal
probability ellipsoids (hydrogen atoms have been omitted for clarity).

J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
745
Fig. 5. Voltammogram of rhenium complex and ligand LH (inset).
4. Conclusions
probably due to poor cell penetration. However, the observed
tumour/muscle ratios were favourable due to slow clearance
from the tumour combined with fast clearance from blood
and soft tissues. These ratios are higher than the ones obtained
for potentially bioreductive mixed ligand oxotechnetium com-
plexes bearing the same pharmacophore [12], demonstrating
the potentiality of our approach. However tumour uptake is in-
sufficient for imaging purposes.
Overall, the technetium-99m complex synthesized may be
considered as a starting point for the development of new
radiopharmaceuticals for imaging hypoxia. We are currently
investigating the application of other pharmacophores and la-
belling methods in the search for compounds with improved
properties.
The ligand 2-[(4-nitrobenzyl)(pyridin-2-ylmethyl)amino]-
acetic acid was designed combining the 4-nitrobenzyl
bioreductive moiety with the chelator (pyridin-2-ylmethyl)-
aminiacetate. The ligand synthesized was successfully utilized
in the preparation of a new 99mTc complex with potentiality
for hypoxia imaging. 99mTc complex was obtained with high
radiochemical purity (>90%). It is very stable and has me-
dium lipophilicity. The proposed structure was corroborated
by means of the comparison with that of the analogous rhe-
nium complex. Reduction potentials of the nitroaromatic
group in both the ligand and the rhenium complex were found
to be similar to the values of enzymes responsible for
the bioreduction in anaerobic organisms. Different types of
bioreductive compounds (N-oxides, nitrofurans, nitrophenyl
mustards, nitroquinolines, etc.) have similar values of reduc-
tion potentials [33e36].
80.00
0.5 hs
70.00
2 hs
12 hs
60.00
Overall biodistribution in normal animals was the expected
for medium lipophilicity compounds. Tumour uptake was low,
24 hs
50.00
40.00
30.00
20.00
10.00
0.00
Table 2
Cyclic voltammetric parameters of ligand LH and the rhenium complex vs
saturated Ag/AgCl electrode (scan rate 500 mV sꢁ1
)
Compounda
b
Epc (mV)
c
Epc (mV)
L
ꢁ895
ꢁ805
ꢁ1140
ꢁ974
Blood
Liver
Stomach Intestine Blader + Faeces
Urine
ORGANS OR FLUIDS
fac-[Re(CO)3L]
a
Concentration 1 mM in DMF.
Epc ¼ potential of the first cathodic peak.
Epc ¼ potential of the second cathodic peak.
b
c
Fig. 6. Biodistribution of 99mTc complex in normal CD1 mice from 0.5 to 24 h.

746
J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
Table 3
Tumour uptake as a function of time
% Dose/organ or fluid
0.5 h
2 h
12 h
24 h
% Tumour/g (T )
% Blood/g (B)
% Muscle/g (M )
T/M
0.19 ꢂ 0.03
0.85 ꢂ 0.06
0.26 ꢂ 0.04
0.73 ꢂ 0.05
0.22 ꢂ 0.09
0.14 ꢂ 0.09
0.74 ꢂ 0.08
0.04 ꢂ 0.01
2.2 ꢂ 0.2
0.13 ꢂ 0.06
0.34 ꢂ 0.02
0.02 ꢂ 0.01
6.5 ꢂ 0.9
0.07 ꢂ 0.01
0.03 ꢂ 0.01
0.01 ꢂ 0.01
6.0 ꢂ 1.0
T/B
0.18 ꢂ 0.5
0.38 ꢂ 0.07
2.3 ꢂ 0.9
1
5. Experimental
(78%). H NMR dH (ppm, CDCl3): 1.20 (t, 3H, CH3), 3.32
(s, 2H, CH2), 3.89 (s, 2H, CH2), 3.87 (s, 2H, CH2), 4.09 (q,
2H, COOCH2), 7.10 (m, 1H, PyrH), 7.44 (m, 1H, PyrH),
7.57 (m, 2H, ArH), 7.61 (m, 1H, PyrH), 8.10 (m, 2H, ArH),
8.48 (m, 1H, PyrH). IR (KBr, cmꢁ1): 1735 (s) (C]O ester),
1518 (s), 1345 (s) (NO2).
5.1. General
All laboratory chemicals were reagent grade and were used
without further purification. Solvents for chromatographic
analysis were HPLC grade. [99mTc]NaTcO4 was obtained
from a commercial generator (Tecnonuclear SA, Argentina).
Carbonyl labelling agent Isolink was provided by Mallinck-
rodt Medical B.V, The Netherlands. IR spectra were recorded
as KBr pellets in the range 4000e200 cmꢁ1 with a Bomen
MB-102 FT-IR spectrophotometer. Elemental analysis was
performed on a Carlo Erba EA 1108 analyzer. The NMR spec-
tra were recorded in deuterium chloroform on a Bruker AC
250E spectrometer. Chemical shifts are reported with respect
to TMS. HPLC analysis was developed on an LC-10 AS Shi-
madzu Liquid Chromatography System using a reverse phase
column Phenomenex Luna 5 m, C18 column (4.69 ꢄ 300 mm).
Elution was performed with a binary gradient system at
1.0 mL/min flow rate using phosphate buffer pH 2.5 with
2% triethylamine as mobile phase A and methanol as mobile
phase B; the elution profile was as follows: 0e3 min 100%
A; 3e6 min linear gradient to 75% A; 6e9 min linear gradient
to 66% A; 9e20 min linear gradient to 0% A; this composition
was held for another 10 min. Detection was accomplished ei-
ther with a photodiode array detector (SPD-M10A, Shimadzu)
that recorded UVevis spectra on flux or with a 300ꢄ300 NaI (Tl)
crystal scintillation detector. Activity measurements were per-
formed either in a Dose Calibrator, Capintec CRC-5R or in
a scintillation counter, 300ꢄ300 NaI (Tl) crystal detector associ-
ated to an ORTEC monochannel analyzer.
Ethyl [(4-nitrobenzyl)(pyridin-2-ylmethyl)amino]acetate
(3.08 g, 9.7 mmol) was treated with 0.1 M NaOH (15 mL)
for 18 h at room temperature under stirring. The mixture
was neutralized and the product crystallized from the reaction
mixture. Filtration afforded 2-[(4-nitrobenzyl)(pyridin-2-ylme-
1
thyl)amino]acetic acid (LH). Yield: 2.6 g (93%). H NMR dH
(ppm, D2O): 3.38 (s, 2H, CH2), 3.93 (s, 2H, CH2), 3.97 (s,
2H, CH2), 7.18 (m, 1H, PyrH), 7.39 (m, 1H, PyrH), 7.52 (m,
2H, ArH), 7.67 (m, 1H, PyrH), 8.10 (m, 2H, ArH), 8.50 (m,
1H, PyrH). 13C NMR dH (ppm, DMSO): 172.12, 158.72,
148.74, 147.51, 146.58, 136.65, 129.48, 122.71, 122.25,
104.37, 58.84, 56.65, 53.95. IR (KBr, cmꢁ1): 1709 (s)
(C]O), 1517 (s), 1344 (s) (NO2), 1217 (s).
5.2.2. 99mTc labelling
5.2.2.1. Preparation of fac-[99mTc(OH2)3(CO)3]þ precursor
5.2.2.1.1. Preparation using a kit formulation. 99mTc-
sodium pertechnetate (185e1850 MBq, 1 mL) was added to
the IsolinkÔ kit formulation (Mallinckrodt Medical B.V.) con-
taining sodium tetraborate (2.85 mg), sodium carbonate
(7.15 mg), sodium boranocarbonate decahydrate (2.85 mg)
and sodium tartrate dihydrate (8.5 mg) and the mixture was in-
cubated at 75 ꢃC for 30 min. After cooling the pH was adjusted
to 7 with 1 N HCl. Complex formation was checked by HPLC
as described in Section 5.1. Radiochemical purity was also
checked by ascending chromatography on Whatman 1 paper/
butanone and Whatman 1 paper/methanol/HCl conc. 95:5.
5.2.2.1.2. Preparation using CO (g). The 99mTc precursor
was prepared according to a previously described method
[18] as follows. Sodium and potassium tartrate (20 mg), so-
dium carbonate (4 mg) and sodium borohydride (5.5 mg)
were placed in a stoppered vial and flushed with CO (g) for
30 min. 99mTc-sodium pertechnetate (185e1850 MBq) was
added and the mixture incubated at 75 ꢃC for 30 min. After
cooling, the pH was adjusted to 7 with 1 N HCl. The complex
formation was checked as in Section 5.2.2.1.1.
5.2. Chemistry
5.2.1. Synthesis of 2-[(4-nitrobenzyl)(pyridin-2-ylmethyl)-
amino]acetic acid (LH)
Ethyl [(pyridin-2-ylmethyl)amino]acetate (3.00 g, 15.46
mmol) was dissolved in methylethylketone (50 mL) and 4-ni-
trobenzyl bromide (3.30 g, 15.46 mmol) and triethylamine
(1.56 g, 15.46 mmol) were added. The mixture was heated at
reflux for 2 h and the solvent was removed under vacuum.
The residue was extracted with dichloromethane and con-
centrated under vacuum. The crude product was purified by
silica gel flash column chromatography using 2:1 mixture of
ether/petroleum ether to give ethyl [(4-nitrobenzyl)(pyridin-
2-ylmethyl)amino]acetate as viscous oil. Yield: 3.80 g
5.2.2.2. Preparation of technetium-99m complex. A solution of
ligand LH, 2-[(4-nitrobenzyl)(pyridin-2-ylmethyl)amino]acetic

J. Giglio et al. / European Journal of Medicinal Chemistry 43 (2008) 741e748
747
Table 4
Summary of crystal, intensity collection and refinement data for the rhenium
complex
acid, in methanol (12.5 mg/mL, 0.1 mL) was added to an aque-
ous solution of the fac-[99mTc(OH2)3(CO)3]þ precursor. After
incubation at 50 ꢃC for 30 min an aliquot of the reaction mixture
was analyzed by HPLC to verify complex formation.
Empirical formula
Formula weight
Temperature
C18H14N3O7Re
570.52
298
Mo Ka 0.71073
5.2.3. Preparation of the rhenium complex
Wavelength
Space group
A solution of [NEt4]2[Re(CO)3Br3] (0.077 g, 0.1 mmol)
and ligand LH (30 mg, 0.1 mmol) in acetonitrile (20 mL)
was heated at reflux for 3 h. The solution was cooled to
room temperature. Light yellow plates were obtained upon
standing and purified by recrystallization from acetonitrile.
[Re(CO)3[(C5H4N)CH2N(CH2-p-NO2C6H4)CH2COO]: yel-
low crystals, yield: 80.0%. Analysis for C18H14N3O7Re, theo-
retical: C: 37.89; H, 2.47; N, 7.37. Experimental: C, 37.80; H,
2.35; N, 7.42. IR (KBr, cmꢁ1): 1883 (broad), 1923 and 2022
P21/n
13.174(5)
˚
a (A)
˚
b (A)
10.315(4)
15.210(5)
˚
c (A)
b (ꢃ)
112.09(1)
1915.2(12)
3
˚
V (A )
Z
4
1.979
Dcalcd (Mg mꢁ3
)
Absorption coefficient m (mmꢁ1
F(000)
Goodness-of-fit on F2
R indices
)
6.392
1096
1.071
R1 ¼ 0.0248,a wR2 ¼ 0.0622a
1
(C^O), 1664 (COOeRe), 1523 and 1338 (NO2). H NMR
dH (ppm, DMSO-d6): 8.78 (m, 1H, PyrH), 8.33 (m, 2H,
ArH), 8.11 (m, 1H, PyrH), 8.07 (m, 2H, ArH), 7.64 (m, 1H,
PyrH), 7.57 (m, 1H, PyrH), 4.91 (d, 1H, CH2Pyr), 4.90 (d,
1H, CH2ArH), 4.69 (d, 1H, CH2ArH), 4.28 (d, 1H, CH2Pyr),
4.26 (d, 1H, CH2CO), 2.92 (d, 1H, CH2CO). 13C NMR dC
(ppm, DMSO-d6): 198.40, 197.84, 179.07, 159.90, 152.87,
148.78, 141.41, 140.64, 134.32, 126.77, 124.86, 124.25,
70.95, 68.12, 61.33.
a
For 2977 reflections with I > 2s(I ).
F2 using SHELXL-97 [38]. Further crystallographic details:
2qmax ¼ 50ꢃ, reflections collected/unique/used 3486/3354
[Rint ¼ 0.0354]/3354, 318 parameters refined, [Dr]max
/
3
˚
[Dr]min ¼ 0.747/ꢁ1.089 e/A , [D/s]max ¼ 0.003, R1/wR2 (for
all data) ¼ 0.0308/0.0651. All hydrogen atoms were located
by difference maps and were refined isotropically, all non-H
atoms were refined anisotropically.
5.2.4. Electrochemical studies
Electrochemical behaviour of the rhenium complex and the
ligand (LH) was studied by cyclic voltammetry (Table 2).
Anhydrous DMF (spectroscopic grade) was obtained from
Aldrich. Tetrabutylammonium perchlorate (0.1 M), used as
supporting electrolyte, was obtained from Fluka (electrochem-
ical grade). Cyclic voltammetry was carried out with computer
controlled BAS-Epsilon EC equipment. A standard electro-
chemical three-electrode cell was used with a platinum disc
as the working electrode, a platinum wire as the counter elec-
trode and saturated Ag/AgCl as the reference electrode. Cyclic
voltammograms were obtained at different scan rates, between
50 and 2000 mV/s in 1 mM DMF solutions of the compounds.
Oxygen was removed by purging the solution with extra-pure
nitrogen and a continuous stream of this gas was passed over
the solutions during the measurements.
5.3. Biological studies
All animal studies were approved by the Ethics Committee
´
of the Faculty of Chemistry from Universidad de la Republica,
Uruguay.
5.3.1. Biodistribution studies in normal animals
In vivo evaluation of 99mTc complexes was performed by
biodistribution using normal CD1 mice. Four animals per
group (female, 8e10 weeks old and 25e30 g) were injected
via a lateral tail vein with the HPLC purified 99mTc complex
reconstituted with saline (0.1 mL, 37e370 kBq [1e10 mCi]).
At different intervals after injection (0.5e24 h) the animals
were sacrificed by neck dislocation. Whole organs and sam-
ples of blood and muscle were collected, weighed and assayed
for radioactivity. Total urine volume was collected during the
biodistribution period and also removed from bladder after
sacrifice. The bladder, urine and intestines were not weighed.
Corrections by different sample geometry were applied when
necessary. Results were expressed as % dose/organ or fluid.
5.2.5. X-ray crystal structure determination
A crystal with approximate dimensions 0.50 ꢄ 0.35 ꢄ
0.24 mm was mounted in air. Diffraction measurements were
made on a Crystal Logic Dual Goniometer diffractometer using
graphite monochromated Mo radiation. Unit cell dimensions
were determined and refined by using the angular settings of
25 automatically centered reflections in the range 11 <
2q < 23ꢃ and they appear in Table 4. Intensity data were re-
corded using a qe2q scan to 2qmax ¼ 50ꢃ, with scan speed
4.0/min and scan range 2.3 plus a1a2 separation. Three standard
reflections monitored every 97 reflections showed less than 3%
variation and no decay. Lorentz polarization and psi-scan ab-
sorption corrections were applied using Crystal Logic software.
The structures were solved by direct methods using SHELXS-
86 [37] and refined by full-matrix least squares techniques on
5.3.2. Biodistribution studies in mice bearing induced
sarcoma
A suspension of sarcoma cells CCRF-180II in PBS, contain-
ing 2.5 ꢄ 106 cells/mL was prepared and 200 mL was injected
subcutaneously in the right limb of normal CD1 mice (female,
8e10 weeks old and 25e30 g). After 10e15 days the animals
developed palpable tumour nodules and were used for the bio-
distribution studies according to the procedure described in
Section 5.3.1. Results were expressed as % dose/gram for

748
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culated from the corresponding % dose/gram values.
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