Synthesis and characterization of thiol containing furoxan derivatives as coligands for the preparation of potential bioreductive radiopharmaceuticals

Article abstract of DOI:10.1002/ardp.200500172

The synthesis and characterization of thiol-containing 1,2,5-oxadiazole N-oxide (TONO) derivatives and their use as monodentate coligands for the preparation of ^99mTc complexes is presented. 3-Mercaptomethyl-4- phenyl-1,2,5-oxadiazol N₂

Full text of DOI:10.1002/ardp.200500172

Arch. Pharm. Chem. Life Sci. 2006, 339, 59–66
H. Cerecetto et al.
59
Full Paper
Synthesis and Characterization of Thiol Containing Furoxan
Derivatives as Coligands for the Preparation of Potential
Bioreductive Radiopharmaceuticals
Hugo Cerecetto1, Mercedes Gonzꢀlez1, Silvia Onetto1, Mariela Risso1, Ana Rey2, Javier Giglio2,
Elsa Leꢁn2, Alba Leꢁn2, Pierina Pilatti3, Marcelo Fernꢀndez4
1 Departamento de Quꢀmica Orgꢁnica, Facultad de Quꢀmica-Facultad de Ciencias, Universidad de la
Repfflblica, Montevideo, Uruguay
2 Cꢁtedra de Radioquꢀmica, Facultad de Quꢀmica, Universidad de la Repfflblica, Montevideo, Uruguay
3 Liceo Juan Lacaze, Colonia, Uruguay
4 Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
The synthesis and characterization of thiol-containing 1,2,5-oxadiazole N-oxide (TONO) deriv-
atives and their use as monodentate coligands for the preparation of 99mTc complexes is presen-
ted. 3-Mercaptomethyl-4-phenyl-1,2,5-oxadiazol N2-oxide and 3-(4-mercaptophenylmethylidenhy-
drazinocarbonyloxymethyl)-4-phenyl-1,2,5-oxadiazol N2-oxide were successfully synthesized and
combined with the tridentate ligand N,N-bis(2-mercaptoethyl)-N9,N9-diethylethylenediamine
(BMEDA) to prepare “3+1 mixed ligand” technetium complexes. The 99mTc complexes were obtai-
ned in high yield and radiochemical purity using low concentration of ligand and coligand. An
alternative procedure using a xantate and a disulphide precursor of 3-mercaptomethyl-4-phenyl-
1,2,5-oxadiazol N2-oxide yielded the same complex. Biological evaluation of the potentiality of
the 99mTc complexes as bioreductive radiopharmaceuticals was performed in normal CD1 mice
and in mice bearing induced sarcoma. Tumour uptake was moderate but tumour/soft tissue
ratio was favourable. Although these results are encouraging, further development is still neces-
sary in order to achieve higher tumour uptake and lower gastrointestinal activity.
Keywords: 1,2,5-Oxadiazole N-oxide derivatives / Bioreductive99mTc radiopharmaceuticals / Solid tumour imaging /
Received: July 20, 2005; Accepted: October 09, 2005
DOI 10.1002/ardp.200500172
field dealing with the application of coordination chem-
istry for medicinal purposes [2]. One important area
related to this new discipline is radiopharmacy, the
science devoted to the development of radioactive tracers
for human diagnosis and therapy. Most of these tracers
called radiopharmaceuticals are coordination com-
pounds containing radioactive isotopes of Tc, Re, Sm, Lu,
Ho, among others [3]. Diagnostic radiopharmaceuticals
are based on gamma-emitters whose radiation readily
escapes from the body, permitting external detection
and measurement. The pattern of distribution of radia-
tion in the body allows the physician to make a diagnos-
tic evaluation of both morphology and function [4]. This
is a unique feature, since other imaging modalities such
as computed tomography or magnetic resonance give
only anatomical information.99mTc is the preferred radio-
Introduction
Many metallic elements play a crucial role in living sys-
tems. Electron-deficient metal ions interact with a vari-
ety of electron-rich molecules involved in essential biolo-
gical functions such as proteins, DNA, etc. [1]. A natural
consequence of this implication has been the develop-
ment of medicinal inorganic chemistry, a relatively new
Correspondence: Dr. Hugo Cerecetto, Facultad de Ciencias, Iguꢁ 4225,
11400 Montevideo, Uruguay.
E-mail: hcerecet@fq.edu.uy
Fax: +598 2 525-0749
or
Dr. Ana Rey, Facultad de Quꢀmica, General Flores 2124, 11800 Montevi-
deo, Uruguay.
E-mail: arey@fq.edu.uy
Fax: + 598 2 924-1906
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Arch. Pharm. Chem. Life Sci. 2006, 339, 59–66
nuclide for diagnosis, due to its ideal nuclear properties
for imaging (t1/2 = 6 h, Ec = 140 keV). As a transition ele-
ment, its chemistry is dominated by the formation of
coordination compounds. Technetium radiopharmaceu-
ticals have the metal bound to a transporting moiety
that delivers the radioactivity to a specific site in the
body determined by the properties of the transporter [5].
Current research is directed towards compounds that
imitate bioactive substrates and can be used to evaluate
biochemical functions in vivo in a non-invasive way [6, 7].
The design of a ligand for the preparation of novel 99mTc-
radiopharmaceuticals involves the combination of the
active part of a biomolecule (pharmacophore) with
appropriate chelating groups to bind the metal. Techne-
tium chelate should be separated from the pharmaco-
phore in order to prevent interference with the biologi-
cal activity [8]. Proposal of a synthetic strategy for such a
ligand is a crucial step in the development of new com-
pounds.
An area of special interest in radiopharmacy is the
development of suitable tracers for imaging hypoxic tis-
sue. Oncology would highly benefit fromagents thateffec-
tively target hypoxic cell populations of solid tumours,
due to their increased radioresistance and diffusion lim-
itations that hinder the treatment [9]. Bioreductive com-
pounds, which are selectively reduced in hypoxic tissue to
reactive intermediates that bind to intracellular mole-
cules, have been utilized for the development of potential
radiodiagnostic markers of tumour hypoxia. 2-Nitroimi-
dazole has been the preferred bioreductive pharmaco-
phore and propylene amine oxime (PnAO) the chelator for
technetium attachment. Figure 1 shows two examples:
BMS-181321 ([99mTc]oxo[[3,3,9,9-tetramethyl-1-(2-nitro-1H-
imidazol-1-yl)-4,8-diazaundecane-2,10-dionedioximato]-
(3)-N,N9,N99,N999] technetium(V)) and BRU59-21 ([99mTc]oxo-[
[3,3,9,9-tetramethyl-6-[(2-nitro-1H-imidazol-1-yl)methyl]-
Scheme 1. Synthesis of thiol-containing 1,2,5-oxadiazole N-
oxide (TONO) derivatives.
5-oxa-4,8-diazadioximato]-(3)-N,N9,N99,N999] technetium (V))
[10].
With the aim to develop new potential99mTc-radiophar-
maceuticals for imaging hypoxia, we have selected the N-
oxide functional group as the bioreductive pharmaco-
phore [11]. In the past years, we have described different
approaches in the development of new N-oxide contain-
ing heterocycles as hypoxic selective cytotoxic agents
[12], among them 1,2,5-oxadiazole N-oxide scaffold has
been extensively studied [13, 14, 15]. The “3+1” mixed
ligand approach, based on the simultaneous coordina-
tion of a tridentate ligand and a monodentate coligand
to the metal, was chosen to attach the pharmacophore to
the radionuclide [16]. The diaminodithiol N,N-bis(2-mer-
captoethyl)-N9,N9-diethylethylenediamine (BMEDA) binds
to the [Tc(V)O]+3 core leaving an open coordination posi-
tion that can be occupied by a monodentate thiol con-
taining the pharmacophore [17].
Herein, we describe the synthesis and characterisation
of thiol-containing 1,2,5-oxadiazole N-oxide (TONO)
derivatives (Scheme 1) and their use as monodentate coli-
gands for the preparation of 99mTc complexes. In order to
assess their potentiality as hypoxia imaging radiophar-
maceuticals a preliminary evaluation in normal and
tumour bearing mice is also presented.
Results
Chemistry
Using the approach shown in Scheme 1, we designed two
different series of TONO derivatives. For series-I deriv-
atives, the procedures depicted in Scheme 2 were assayed
in order to prepare compounds 7 and 11. However, nitra-
tion of compound 5, in different conditions, did not
afford the desired product 6, precursor of TONO 7. On the
Figure 1. Proposed 99mTc bioreductive radiopharmaceuticals
bearing 2-nitroimidazoles as pharmacophore.
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Furoxans as Coligands for Radiopharmaceuticals
61
Scheme 2. Procedures tested to pre-
pare compounds 7 and 11.
Scheme 3. Synthetic routes of thiol
14.
other hand, we assayed the preparation of series-I deriv- gen atmosphere and at –208C in order to avoid decompo-
atives from phenylalkene bearing a thiol moiety. Thus, sition. For the synthesis of “3+1” Tc complexes (see below)
we prepared via Wittig procedure compound 9 which freshly prepared derivative 14 was used.
was unable to react with NaNO2 in acid medium to yield
the furoxan heterocycle [18].
For series-II derivatives with different linker moieties
(Scheme 1), two different approaches were assayed
For series-II derivatives, we designed TONO with and (Scheme 4). Starting from chloride 12, achievement of
without linker moiety (see Scheme 1). In the first case, derivative 22 was intended via a nucleophilic substitu-
derivative 14 synthesis was assayed using different meth- tion process involving the reactant 21, prepared from
odologies (Scheme 3) [19]. The pathway from alcohol 5, aldehyde 17. Because the sulphur is the best nucleophile
via the chloride 12 [14] and the salt 13, produced the in the reaction media, derivative 24 was the main prod-
desired product in very low yield because the last step uct of reaction. However, this product is not adequate for
(hydrolysis in basic medium) produced a complex mix- coordination with Tc in our “3+1” approach. Derivative
ture of products. The direct transformation of alcohol 5 22 and dimmer 23 were generated as marginal products
to thiol 14 using Lawesson’s reagent yielded the desired in the assayed conditions. So, we decided to use another
product (in low yield remaining 5). Finally, thiol 14 was approach in order to generate a TONO with phenyl lin-
prepared via the xantate 15, followed by the reduction in ker. Consequently, the synthetic procedures shown in
mild conditions, as shown in Scheme 3. In these condi- Scheme 4 were chosen to prepare derivative 28 [15]. This
tions 14 was isolated chromatographically together with compound was obtained in good yields from alcohol 5,
the dimmer 16. Stability of derivative 14 was not exten- via the carbonate 25 and the carbazate 26, which reacted
sively studied, but it was necessary to store it under nitro- with aldehyde 27, obtained from aldehyde 8 [20]. All new
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H. Cerecetto et al.
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Scheme 4. Synthetic procedures for deriv-
atives 22 and 28 [12c].
1,2,5-oxadiazole N-oxide derivatives were characterised cal purity (>90%, determined by HPLC), using low amount
1
by IR, MS, and one- and two-dimensional H-NMR, 13C- (2610–5 mol) of the coligand that carries the pharmaco-
NMR experiments, and their purity was established by phore group. The radioactivity recovery from the column
TLC and microanalysis.
after injection of complexes was monitored by means of
The TONO derivatives 14 and 28 were used as mono- an on-line solid scintillation detector coupled to the
dentate coligands together with the tridentate ligand HPLC system and found to be quantitative.
BMEDA in the preparation of “3+1” 99mTc mixed ligand
An alternative route for the preparation of 99m Tc com-
complexes. Both compounds were prepared by ligand- plexes is shown in Scheme 5b. Xantate 15 and dimmer 16
exchange reaction using 99mTc-glucoheptonate as precur- were used as precursors of compound 14 and reacted
sor together with ligand and coligand in a ratio of 1:1, as with 99mTc-glucoheptonate in the presence of BMEDA to
shown in Scheme 5. According to previous experience achieve complex 29. Although the thiol groups are neces-
and literature data about “3+1 mixed ligand” technetium sary for the complexation, the presence of an excess of
complexes bearing BMEDA as tridentate ligand, we the strong reducing agent SnCl2 produced enough
expect the formation of neutral and lipophilic complexes amount of compound 14 to achieve the desired final
due to the ionisation of the sulphur groups of ligand and product as shown by HPLC analysis.
coligand [16, 21]. Consequently, isolation of the mixed
ligand complexes from the reaction mixture was Biodistribution studies
achieved upon extraction by dichloromethane. Com- In order to assess the potentiality of our approach for the
plexes 29 and 30 were obtained in high yield (>85%, deter- design of radiopharmaceuticals, a preliminary evalua-
mined by CH2Cl2 extraction) and with high radiochemi- tion in normal and tumour-bearing mice was performed.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 59–66
Furoxans as Coligands for Radiopharmaceuticals
63
Scheme 5. Preparation of 99m Tc complexes
29 and 30.
Figure 2. Biodistribution of complex
29 in normal CD1 mice.
Normal biodistribution of 99m Tc complex 29 as a func- respectively at 2 h post-injection) demonstrating no in
tion of time is shown in Figure 2. The compound showed vivo reoxidation.
high initial blood, lung, and liver uptake (13.3%, 6.8%,
Behaviour of 99mTc complex 29 in mice bearing induced
and 24.4%, respectively). This behaviour is similar to the sarcoma as a function of time is shown in Figure 3. Initial
previously reported “3+1” complexes obtained with the tumour uptake was moderate (1.2%/g at 30 min post-
same ligand [16, 21]. Blood and lungs clearance was quite injection) but clearance was rather slow (0.7%/g and
fast (% injection dose in blood and lungs 0.36% and 0.35%/g after 2 and 24 h, respectively). Tumour to muscle
0.18%, respectively at 2 h post-injection), while liver activ- ratio was favourable at all time points and increased
ity remained high for a longer period (% injection dose with time (1.3, 1.8, and 1.8 at 30 min, 2 h, and 24 h post-
20.7% at 2 h post-injection). The radioactivity from the injection, respectively) due to soft tissue depuration.
novel technetium complex was excreted mainly through
Overall biodistribution and tumour uptake of complex
the hepatobiliary system (56% at 2 h post-injection). On 30 was studied at one time point (12 h) for comparison. In
the other hand, urinary excretion was low (8.2% in urine vivo behaviour was analogous to complex 29, with low
at 2 h post-injection). Depuration from blood and soft tis- blood, lung, and liver activity (0.2%, 0.7%, and 3.6%,
sues, after 24 h, was almost complete and excretion after respectively) and very high hepatobiliary elimination
this period above 98%. Stomach and thyroid values were (above 80%). Tumour uptake was also moderate (0.4%/g)
within acceptable levels (approximately 0.4% and 0.1%, and tumour/muscle ratio was about 2. Due to these simi-
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The xantate 15 and disulphide 16 were able to produce,
in the reductive medium of the reaction, complex 29
using 99mTc-glucoheptonate as precursor.
In vivo behaviour for complexes 29 and 30 was the
expected for this kind of compounds. Tumour uptake
was moderate but tumour/soft tissue ratio was favour-
able. Although these results are encouraging, further
development is still necessary in order to achieve higher
tumour uptake and lower gastrointestinal activity.
This research has been supported by Comisiꢀn Honoraria de
Lucha Contra el Cꢁncer (Uruguay). We thank for a scholarship
grant for P.P. from Programa de Desarrollo de Ciencias Bꢁsicas
(PEDECIBA-Uruguay).
Experimental
Chemistry
All starting materials were commercially available research-
grade chemicals and used without further purification. All sol-
vents were dried and distilled prior to use. All the TONO synth-
eses were carried out in nitrogen atmosphere. The typical work-
up included washing with brine and drying the organic layer
with sodium sulphate before concentration. Melting points were
determined using a Leitz Microscope Heating Stage Model 350
(Leitz, Wetzlar, Germany) apparatus and are uncorrected. Ele-
mental analyses were obtained from vacuum-dried samples (over
phosphorous pentoxide at 3–4 mm Hg, 24 h at room tempera-
ture), performed on a Fisons EA 1108 CHNS-O analyzer (Fisons,
Valencia, CA, USA), and were within (€0.4% of theoretical values.
1H-NMR, 13C-NMR spectra and HETCOR experiments were
recorded on a Bruker DPX-400 (at 400 MHz and 100 MHz (Bruker,
Rheinstetten, Germany)) instrument, with tetramethylsilane as
the internal reference and in the indicated solvent. Mass spectra
were recorded on a Shimadzu GC-MS QP 1100 EX (Shimadzu,
Kyoto, Japan) instrument using electron impact ionization at 70
eV. The compounds 5, 12, 27, BMEDA and 99mTc-glucoheptonate
were prepared as previously reported [14, 20, 21, 22]. [99mTc]
NaTcO4 was obtained from an Elumatic III generator (Cis-Bio-
international). High performance liquid chromatography (HPLC)
analysis was performed on a LC-10 AS Shimadzu Liquid Chroma-
tography System coupled to both SPD-M10A, Shimadzu photo-
diode array detector (UV trace for ligands) and a Parken 3"63"
NaI (Tl) crystal scintillation detector (c trace for 99mTc). Separa-
tions were achieved on a reverse phase l Bondapack C18 column
(3.96300 mm), eluted with a binary gradient system at a 1.0 mL/
min flow rate. Mobile phase A was phosphate buffer pH 7.4 with
2% triethylamine while mobile phase B was methanol. The elu-
tion profile was: 0 min 0% B followed by a linear gradient to
100% B in 7 min; this composition was held for another 15 min.
Activity measurements were performed either in a Capintec
CRC-5R dose calibrator or in a scintillation counter, using a
3"63" NaI (Tl) crystal detector associated to an ORTEC monocha-
nel analyzer.
Figure 3. Tumour uptake and retention of complex 29 in CD1
mice bearing induced sarcoma.
larities in in vivo behaviour no other time points were
studied.
Discussion
A series of new furoxan derivatives bearing thiol moieties
were synthesized and assayed as coligands for the pre-
paration of technetium “3+1 mixed ligand” complexes
with potential application as bioreductive radiopharma-
ceuticals.
,
Preparation of phenylthio derivatives of series I’
(Scheme 1) was attempted by different procedures but
,
unfortunately without success. Derivatives of series II’
(Scheme 1) were successfully produced, in this sense two
different linkers were used to join the thiol moiety with
the furoxan system. Derivative 14 was developed from
chloride 12 (Scheme 3) and derivative 28 from alcohol 5
(Scheme 4).
These compounds were used together with equimole-
cular amounts of the well known ligand BMEDA to pre-
pare two novel 99mTc compounds, whose proposed struc-
ture based on the ligand system used and the existing lit-
erature data is shown in Scheme 5. The 99mTc complexes
were obtained in high yield and radiochemical purity
using low concentration of ligand and coligand.
O-Ethyl S-[(2-oxide-4-phenyl-1,2,5-oxadiazole-3-yl)-
methyl]xantate 15
A mixture of 3-chloromethyl-4-phenyl-1,2,5-oxadiazole N2-oxide,
12 (1 eq.), potassium ethylxantate (1 eq.) and THF as solvent was
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Arch. Pharm. Chem. Life Sci. 2006, 339, 59–66
Furoxans as Coligands for Radiopharmaceuticals
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stirred at reflux until the chloride reactant was not present
(Al2O3, 5% EtOAc in petroleum ether). The resulting precipitate,
KCl, was filtered and washed with THF. The resulting organic
layer was evaporated in vacuo and the residue was purified by
chromatography. Yellow-white needles (60%). 1H-NMR (CDCl3,
400 MHz) d (ppm): 1.39 (t, 3H, J = 7.1 Hz, –CH3), 4.45 (s, 2H, Ar-
CH2), 4.63 (q, 2H, J = 7.1 Hz, O–CH2), 7.58 (m, 3H, Ph), 7.69 (m, 2H,
Ph). 13C-NMR (HMQC, HMBC) (CDCl3, 100 MHz) d (ppm): 14.05
(–CH3), 28.62 (Ar-CH2), 71.36 (O–CH2), 112.11 (–C=N+–O–), 126.69,
128.15, 129.76, 131.68 (Ph–C), 156.84 (–C=N), 210.96 (–C=S). MS
m/z (rel. int.%): 296 (M+, 30.0%), 280 (8.0). mp (8C) 56.0–57.0. Anal.
Calcd. for C12H12N2O3S2: C, 48.6; H, 4.1; N, 9.5; S, 21.6. Found: C,
48.5; H, 4.0; N, 9.1; S, 21.2.
3H, Ph), 7.70 (m, 2H, Ph), 8.00 (brs, 1H, NH). MS m/z (rel. int.%):
250 (M+, 2.0), 234 (6.0).
3-(4-Mercaptophenylmethylidenhydrazinocarbonyloxy-
methyl)-4-phenyl-1,2,5-oxadiazol N2-oxide 28
To a stirred mixture of aldehyde 27 (1 eq.) and p-toluenesulfonic
acid (catalytic amounts) in toluene, derivative 26 was added in
three portions. The suspension was stirred at room temperature
until the aldehyde was not present any more. The mixture of
reaction was evaporated in vacuo and the residue was purified by
chromatography (SiO2, CH2Cl2 : MeOH (0 to 5%)). Yellow oil (50%).
1H-NMR (CDCl3, 400 MHz) d (ppm): 5.35 (s, 2H, Ar-CH2), 7.20–7.80
(m, 10H, Ph+SH), 8.00 (s, 1H, CH=N), 9.00 (brs, 1H, NH). 13C-NMR
(HMQC, HMBC) (CDCl3, 100 MHz) d (ppm): 55.00 (Ar-CH2), 109.00
(–C=N+–O–), 125.00–133.00 (Ph–C), 144.00 (–C=N), 155.40 (–C=N),
156.00 (–C=O). MS m/z (rel. int.%): 370 (M+, 3.5), 354 (1.0). Anal.
Calcd. for C17H14N4O4S: C, 55.1; H, 3.8; N, 15.1; S, 8.7. Found: C,
54.9; H, 3.6; N, 14.7; S, 8.4.
3-Mercaptomethyl-4-phenyl-1,2,5-oxadiazol N2-oxide 14
Derivative 15 (1 eq.) dissolved in THF was cooled at 08C. Then,
three portions of NaBH4 (1 eq.) were added over the vigorously
stirred solution. The mixture was stirred at room temperature
until the xantate reactant was not present any more (SiO2, 5%
EtOAc in petroleum ether). The mixture of reaction was evapo-
rated in vacuo and the residue was purified by chromatography
(SiO2, petroleum ether : EtOAc (0 to 10%)). In the chromato-
graphic process, thiol 14 was mainly converted into derivative
16.
General procedure for the preparation of complexes
29 and 30
A vial containing a lyophilized mixture of 200 mg calcium glu-
coheptonate and 0.2 mg SnCl262H2O was reconstituted with
5 mL water, and 0.5 mL of this solution was mixed with 0.5 – 1.0
mL [99mTc]NaTcO4 with an activity of 185 – 1850 MBq (5 – 50 mCi).
The ligand BMEDA (0.02 mmol, 4.7 mg) and the coligand 14 or 28
(0.02 mmol) were added and the mixture was agitated in a vortex
mixer and left to react at room temperature for 10 minutes. The
lipophilic species were extracted with CH2Cl2 and the organic
layer dried with MgSO4, filtered, and analysed by HPLC. When
the same procedure was performed with 15 or 16 instead of coli-
gand 14, formation of complex 29 was evidenced by HPLC.
14: Brown oil (5%). 1H-NMR (CDCl3, 400 MHz) d (ppm): 2.50 (brs,
1H, SH), 4.15 (s, 2H, Ar-CH2), 7.55 (m, 3H, Ph), 7.70 (m, 2H, Ph).13C-
NMR (HMQC, HMBC) (CDCl3, 100 MHz) d (ppm): 22.00 (Ar-CH2),
110.00 (–C=N+–O–), 126.75, 128.50, 129.80, 132.00 (Ph–C), 155.90
(–C=N). MS m/z (rel. int.%): 208 (M.+, 5.0), 192 (1.5), 178 (0.5).
16: Yellow oil (75%). 1H-NMR (CDCl3, 400 MHz) d (ppm): 4.20 (s,
2H, Ar-CH2), 7.60 (m, 3H, Ph), 7.68 (m, 2H, Ph). 13C-NMR (HMQC,
HMBC) (CDCl3, 100 MHz) d (ppm): 30.00 (Ar-CH2), 111.00 (–C=N+–
O–), 126.60, 128.55, 129.70, 131.90 (Ph–C), 156.00 (–C=N). MS m/z
(rel. int.%): 414 (M+, 35.0), 398 (2.0), 382 (1.0), 358 (0.5). Anal.
Calcd. for C18H14N4O4S2: C, 52.2; H, 3.4; N, 13.5; S, 15.5. Found: C,
51.8; H, 3.1; N, 13.1; S, 15.1.
[
99mTcO(BMEDA)(14)] 29
Yield: >85%. Radiochemical purity: >90% tR, HPLC = 13.5 min.
Phenyl (4-phenyl-2-oxide-1,2,5-oxadiazole-3-yl)methyl
carbonate 25
[
99mTcO(BMEDA)(28)] 30
Yield: >85%. Radiochemical purity:>90% tR, HPLC =14.1 min.
Phenyl chloroformate (1 eq.) was added to a stirred and cooled
(08C) solution of the alcohol 5 (1 eq.) and triethylamine (1 eq.),
using toluene as solvent. After addition, the mixture was stirred
at room temperature for 45 min. After the work-up the solvent
was evaporated to give product 25 which were pure enough to
be used in the next preparation without further purification.
Biodistribution studies
Animal studies were approved by the Ethics Committee of the
Faculty of Chemistry from Uruguay. Ex vivo evaluation of 99mTc
complex 29 was performed by biodistribution using either nor-
mal mice or animals bearing induced tumours.
1
Colourless oil (95%). H-NMR (CDCl3, 400 MHz) d (ppm): 5.40 (s,
2H, Ar-CH2), 7.00 (m, 3H, Ph), 7.20 (m, 2H, Ph), 7.60 (m, 3H, Ph),
Normal CD1 mice were purchased from the Animal Experi-
mental Laboratory, – Faculty of Chemistry, Universidad de la
Repfflblica, Uruguay – (female, 25–30 g, 4 animals per group)
were injected via a lateral tail vein with the HPLC purified 99mTc
complex reconstituted with 30% methanol (0.1 mL, 3.7 MBq
[100 lCi]). At different intervals after injection (5 min, 30 min,
2 h, and 24 h) the animals were sacrificed by neck dislocation.
Whole organs and samples of blood and muscle were collected,
weighed, and assayed for radioactivity. Animals were kept in a
metabolic cage during the biodistribution period in order to
collect total urine volume. Urine was also removed from blad-
der 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 and % Dose/g.
7.72 (m, 2H, Ph). MS m/z (rel. int.%): 312 (M+, 8.5), 296 (0.5).
O-(4-Phenyl-2-oxide-1,2,5-oxadiazole-3-yl)methyl
carbazate 26
A mixture of 25 (1 eq.) and hydrazine monohydrate (1 eq.) in THF
as solvent was stirred at room temperature until the carbonate
was not present any more. The solvent was evaporated and the
residue was treated with ethyl acetate and washed with aqueous
NaOH (5%). The organic layer was dried with sodium sulphate
and evaporated to give product 26 which were pure enough to
be used in the next preparation without further purification.
1
Yellow-brown oil (69%). H-NMR (DMSO-d6, 400 MHz) d (ppm):
5.25 (s, 2H, Ar-CH2), 6.00 (brs, 2H, –NH2), 7.00 (m, 3H, Ph), 7.55 (m,
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

66
H. Cerecetto et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 59–66
Animals bearing induced tumours were obtained by subcuta-
M. L. Lavaggi, A. Azqueta, A. López de Cerain, A. Monge,
U. Abram, A. J. Costa-Filho, Eur. J. Med. Chem. 2005, 40,
473–480.
neous inoculation of CCRFS-180 II murine sarcoma cells (5–
66106 cells/animal in 200 mL PBS) in the right limb of CD1 mice
(female, 8–10 wk old, 25–30 g). After 15–20 days post-inocula-
tion, animals developed palpable nodules and were used for bio-
distribution studies at 0.5 to 24 h post-injection using the pre-
viously described procedure. The tumour/muscle ratio was cal-
culated from the corresponding percent dose/g values.
[13] A. Monge, A. López de Cerµin, O. Ezpeleta, H. Cerecetto,
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Uptake of 99mTc complex 30 in induced tumors at 12 h post-
injection was also evaluated by the above described technique.
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