Synthesis and biological characterisation of novel dithiocarbamate containing 5-nitroimidazole 99mTc-complexes as potential agents for targeting hypoxia

Article abstract of DOI:10.1016/j.bmcl.2010.10.130

With the aim to develop new potential ^99mTc-radiopharmaceuticals for imaging hypoxia based on the formation of Tc-nitrido complexes, two novel dithiocarbamate containing metronidazole derivatives (L1 and L2) have been prepared and characterised. The synthesis of L1 and L2 was achieved in excellent yield and high purity. Labelling with ^99mTc was successfully performed using a low ligand concentration (approximately 2-3 mg) and the desired products were obtained with high radiochemical purity (>90%). Lipophilicity, plasma protein binding, and biodistribution in normal- and tumour-bearing-CD1 mice studies were performed to asses the potentiality for nuclear medicine oncology. According to the physicochemical and biological behaviour both in healthy animals and in animals bearing solid tumours complex dtcTc1 could be considered as a starting point for the development of new radiopharmaceuticals for imaging hypoxia.

Full text of DOI:10.1016/j.bmcl.2010.10.130

Bioorganic & Medicinal Chemistry Letters 21 (2011) 394–397
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological characterisation of novel dithiocarbamate
containing 5-nitroimidazole 99mTc-complexes as potential agents
for targeting hypoxia
Javier Giglio a, Soledad Fernández a, Ana Rey a, , Hugo Cerecetto b,
⇑
⇑
a Cátedra de Radioquímica, Facultad de Química, Gral Flores 2124, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11800 Montevideo, Uruguay
b Grupo de Química Medicinal, Laboratorio de Química Orgánica, Iguá 4225, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
With the aim to develop new potential 99mTc-radiopharmaceuticals for imaging hypoxia based on the for-
mation of Tc-nitrido complexes, two novel dithiocarbamate containing metronidazole derivatives (L1 and
L2) have been prepared and characterised. The synthesis of L1 and L2 was achieved in excellent yield and
high purity. Labelling with 99mTc was successfully performed using a low ligand concentration (approx-
imately 2–3 mg) and the desired products were obtained with high radiochemical purity (>90%). Lipo-
philicity, plasma protein binding, and biodistribution in normal- and tumour-bearing-CD1 mice studies
were performed to asses the potentiality for nuclear medicine oncology. According to the physicochem-
ical and biological behaviour both in healthy animals and in animals bearing solid tumours complex
dtcTc1 could be considered as a starting point for the development of new radiopharmaceuticals for
imaging hypoxia.
Article history:
Received 23 September 2010
Revised 24 October 2010
Accepted 26 October 2010
Available online 30 October 2010
Keywords:
5-Nitroimidazoles
Symmetrical Tc-nitride complexes
hypoxia imaging
Ó 2010 Elsevier Ltd. All rights reserved.
Hypoxic regions in tumours are formed when tumour growth
exceeds the capacity of accompanying blood vasculature to deliver
adequate quantities of oxygen to the growing mass of cells.1 The
resistance of hypoxic cells to conventional radiotherapy and
chemotherapy2,3 is a major obstacle for the complete remission
of tumours. Due to the inherent drawbacks of invasive techniques
to detect hypoxia4 nuclear medicine imaging, based on the admin-
istration of small amounts of radioactive compounds, the so-called
radiopharmaceuticals, followed by the detection of the radiation
escaping from the body, is considered an interesting alternative.
Nitroimidazoles are reduced selectively under low oxygen pressure
and consequently accumulate into the hypoxic regions of tumours,
providing the attractive possibility of employing them as ligands
for the preparation of potential radiopharmaceuticals targeting
hypoxic tissues.5–7 The 5-nitroimidazole metronidazole (Mtz,
Fig. 1), which radiosensitizes hypoxic tumour cells in vivo, has
been selected due to its affinity for hypoxic tumours as starting
material for the preparation of 99mTc radiopharmaceuticals.8–14
99mTc is the radionuclide of choice in diagnostic nuclear medi-
cine due to its ideal nuclear properties for imaging (t1/2 = 6 h,
means to combine the pharmacophore with appropriate chelating
groups to bind the metal, separating them through a spacer to pre-
vent interference with the biological activity. Selection of the che-
lating groups is essential to achieve adequate stability and
pharmacokinetics of the resulting compound. A variety of chelating
groups and co-ligands, as well as Tc oxidation states, have been
investigated for the radiolabelling of biologically active com-
pounds. The Tc-nitrido core, initially developed by Baldas and
Bonnyman,16 is an auspicious tool for the design of novel radio-
pharmaceuticals due to their intrinsic structural robustness.17
The co-ordination sphere of these complexes is either build up
by two molecules of bidentate
r
-donor ligands or a combination
-acceptors diphosphinoamines
-donor ligands (X–Y). Various bidentate
of ‘pseudotridentate’
(PNP) and bidentate
r-donors p
r
r
-donor ligands can be used but the bis(dithiocarbamate)99mTc-
nitrido complexes, which are square pyramidal, neutral and highly
lipophilic compounds are specially useful for the preparation of
radiopharmaceuticals with adequate cell penetration. A remark-
able example is 99mTcN-NOEt, a symmetric 99mTc-nitrido complex
with outstanding properties for myocardial imaging (see Fig. 2).18
E = 140 keV). In technetium radiopharmaceuticals the metal is
c
bound to the bioactive group by means of a coordination com-
NO2
pound using the so-called pendant approach,15 which literally
Mtz
OH
N
N
(metronizadole)
⇑
CH3
Corresponding authors. Tel.: +598 9248571; fax: +598 9241906 (A.R.);
2
tel.: +598
2
5258618; fax: +1 598 2 5250749 (H.C.).
E-mail addresses: arey@fq.edu.uy (A. Rey), hcerecet@fq.edu.uy (H. Cerecetto).
Figure 1. Chemical structure of the radiosensitizer metronidazole.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.130

J. Giglio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 394–397
395
N
Tc
3 was transformed, via nucleophilic substitution, into the pro-
tected amine 4 which was deprotected in presence of CF3CO2H
and transformed into the desired dithiocarbamate, L2, in the same
condition that it was used for L1. The intermediates, 1–4,21 and
products, L1 and L2, were characterised by NMR (1H and 13C)
and IR spectroscopies and MS spectrometry.22 The presence of
the –N–CS2 moiety was clearly evidenced in the 13C NMR and IR
of L1 and L2 by a signal near to 200 ppm and the signals near to
1120 and 1000 cmꢀ1, corresponding to the thiocarbonyl carbon
and the C@S and –N–C from the dithiocarbamate moiety, respec-
tively. The ligands L1 and L2 were used as such without further
purification. Preparation of the 99mTc nitrido complexes was
accomplished by a two step procedure as follows: first, the
S
S
S
S
N
O
N
O
99mTcN-NOEt
Figure 2. Example of bis(dithiocarbamate)99mTc„N complex.
With the aim to develop new potential 99mTc-radiopharmaceu-
ticals for imaging hypoxia, we have selected the 5-nitroimidazol-
1-yl moiety as the bioactive group and the dithiocarbamate group
as chelator for the technetium. The 5-nitroimidazole Mtz (Fig 1),
with a pendant –CH2CH2OH linker, acts as a suitable substrate
for facile derivatisation to a dithiocarbamate containing ligand.
Herein, we report the synthesis of two new dithiocarbamate
containing 5-nitroimidazoles, L1 and L2, and its 99mTc-complexes
bearing the 99mTc„N core, dtcTc1 and dtcTc2. To the best of our
knowledge, this report constitutes the first of its kind in using
the dithiocarbamate as donor moiety in combination with a nitro-
imidazolic pharmacophore for the preparation of 99mTc complexes
for targeting tumour hypoxia. A preliminary evaluation of the main
physicochemical and biological properties is also presented in or-
der to asses the potentiality of our approach.
The syntheses of the ligands L1 and L2 (Scheme 1) were
achieved by two synthetic routes. In the first route Mtz was trans-
formed into its amino-analogue 2 using a previously described
Mitsunobu methodology19 and then transformed into the corre-
sponding dithiocarbamate L1 by reaction with CS2 in presence of
NaH in aprotic milieu. This procedure affords the desired product
in excellent yield and high purity by evaporation of the organic
solvent. However, when the process was assayed in aqueous milieu
(CS2/aqueous NaOH (50%)) the isolation and purification of L1 was
unsuccessful. In the second route, ligand L2 was prepared in a four-
step procedure from Mtz with good global yield. In the first step
Mtz was converted, via phosphonium ion (Ph3P/imidazole/I2),20
into the corresponding iodide derivative, 3, in excellent yield. Then
[
99mTc„N]2+ intermediate was obtained using an optimised proto-
col (Scheme 2). One millilitre of freshly eluted 99mTcVIIO4 (37–
50 MBq) was added to a commercial kit (Cis-Bio int.) containing
succinic acid dihydrazide as nitrido donor (5.0 mg), Na2EDTA
(5.0 mg), and SnCl2ꢁH2O (0.1 mg) as reducing agent; the mixture
was vortexed for one minute and kept at room temperature for
5 min. Formation of the [99mTc„N]2+ intermediate species was
ꢀ
controlled by paper chromatography using acetone as solvent (Rf
ꢀ
[
99mTc„N]2+-intermediate = 0, Rf 99mTcO4 ¼ 1);
a minimum
radiochemical purity of 90% was required to proceed to the second
step.
Substitution was achieved by addition of ligands L1 or L2 to the
precursor. The following reaction parameters: ligand concentration
(studied: 0.0035–0.030 mmol/0.1 mL), temperature (studied: 50–
90 °C), and time (studied: 20–40 min) were assayed to obtain max-
imum complexation yield. The desired complexes dtcTc1 and
dtcTc2 were finally obtained by substitution of L1 (0.011 mmol/
0.1 mL) or L2 (0.012 mmol/0.1 mL for L2) on the precursor fol-
lowed by incubation at 75 °C during 30 min. The radiochemical
purity for both complexes was determined by RP-HPLC (Fig. S1,
Supplementary data, Table 1) and found to be above 90%.
NO2
NO2
NO2
S
Ph3P (1 equiv)
DIAD (1 equiv)
O
1) HBr (48 %)
reflux
CS2 / NaH
THF, r.t.
S-Na+
N
NH2
N
H
N
N
N
N
N
N
2) Neutralization
phtalimide (1 equiv)
THF, r.t.
CH3
CH3
CH3
O
1
2
L1
NO2
OH
N
N
CH3
NO2
NO2
NO2
Ph3P (1 equiv)
1) CF3CO2H
CH2Cl2, r.t.
HN
N-Boc
(2 equiv)
N
imidazole (2.5 equiv)
S-Na+
I
N
N
N
N
N
N
N
N
N
S
THF reflux
I2 (1 equiv)
CH2Cl2, r.t.
Boc 2) CS2 / NaH
THF, r.t.
CH3
CH3
CH3
3
4
L2
Scheme 1. Synthesis of metronidazole dithiocarbamates.
NO2
NO2
S
O2N
N
N
N
Tc
linker
S-Na+
N
N
N
N
S
S
S
S
linker
linker
NH2NH2
CH3
-
99mTcO4
[
99mTcN]2+
CH3
CH3
dtcTc1, dtcTc2
Scheme 2. Synthesis of 99mTc-complexes.

396
J. Giglio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 394–397
Table 1
Table 2
Physicochemical parameters for 99mTc-complexes
Tumour/blood and tumour/muscle ratios of 99mTc-complex dtcTc1 at various time
points
a
Complex
tR (min)
Stability in plasmab (%)
PBPPc
log Pd
Time post-injection (min)
Tumour/blood
Tumour/muscle
dtcTc1
dtcTc2
7.5
8.5
>90.0
>87.0
31 3%
75 4%
0.63 0.04
0.7 0.1
30
60
120
240
0.28 0.03
0.30 0.17
0.65 0.21
1.15 0.32
1.63 0.26
1.29 0.10
1.80 0.17
2.43 0.75
a
HPLC conditions: reverse-phase Waters LC-18 column, 250 mm ꢂ 4.6 mm ID,
particle size 5 M, (Waters Corporation, Massachusetts, USA); mobile phase A: TFA/
water, 0.1:99.9 (v/v), mobile phase B: TFA/acetonitrile, 0.1:99.9 (v/v), 0–3 min:
l
100% A (0% B), 3–5 min: 100% A?0% A (0% B?100% B), 5–20% A (100% B); flow rate
1.0 mL minꢀ1
;
c
-detection.
b
Radioactivity after 4.0 h of incubation with human plasma at 37 °C.
PBPP: percentage of binding to plasmatic proteins.
Table 3
c
Tumour/blood and tumour/muscle ratios of 99mTc-complex dtcTc2 at 240 min post-
d
log P = Soctanol/Sbuffer
.
(pH 7.4)
injection
Tumour/blood
0.28 0.07
Tumour/muscle
1.50 0.37
Stability of dtcTc1 and dtcTc2 complexes in human plasma at
37 °C was studied using RP-HPLC (Table 1). Both complexes exhibit
significant stability since the radiochemical purity remained near
to 90% for at least 4 h. Protein binding (PBPP) of the studied com-
plexes, dtcTc1 and dtcTc2, was determined using blood plasma by
size exclusion chromatography (Table 1). Low protein binding val-
ues are associated with fast blood clearance. High values, on the
other hand, indicate normally poor in vivo stability, high blood
activity and high hepatobiliary excretion. Lipophilicity is also a rel-
evant indication of pharmacokinetic behaviour and determines the
excretion pattern of the radiopharmaceutical. It was determined as
the partition coefficient between 1-octanol and saline (Table 1).
According to PBPP dtcTc1 complex could probably have a more
favourable biodistribution pattern than dtcTc2.
The biodistribution studies were carried out using healthy CD1
mice between 30 min and 6 hours post-injection for dtcTc1 and
120 and 240 min for dtcTc2 complex. Biological behaviour of
dtcTc1 was characterised by high initial blood and muscle activity,
rapid depuration and quantitative excretion through the urinary
tract (70% of injected activity in urine at 6 h after administration,
Fig. S2a). Activity from other organs and tissues was also washed
out with time. On the other hand, biodistribution of complex
dtcTc2 was less favourable as expected from high protein binding.
Activity uptake in liver was very high, above 50% of injected dose
and urinary excretion very low (9% at 4 h post-injection) (Fig. S2b).
The results of the biodistribution of complexes dtcTc1 and
dtcTc2, in C57 mice bearing induced Lewis carcinoma, are repre-
sented in Fig. 3. Overall in vivo behaviour was very similar to the
observed in healthy animals. The tumour retains about 1.0% of in-
jected dose per gram for dtcTc1 (Fig. S3) and this remains nearly
constant over the period of study up to 240 min post-injection.
The striking observation in the present study was the desirable fea-
ture of steady retention of the tumour activity throughout the per-
iod of study. Another favourable characteristic was fast clearance
from blood and soft tissues that led to tumour/blood and
tumour/muscle ratios that increased significantly with time
(Table 2) reaching values of 1.15 and 2.43, respectively, at
240 min post-injection. Student’s test was applied to asses the sig-
nificance of the difference in uptake between tumour and muscle
and results demonstrate that uptake in tumour of complex dtcTc1
is significantly higher in comparison to muscle (p = 0.05) at 120
and 240 min post-injection.
On the other hand, the results of biodistribution for dtcTc2, in
C57 mice bearing Lewis carcinoma, showed a very different biolog-
ical behaviour. Uptake in tumour was significantly lower than the
observed for dtcTc1, that is, 0.40 0.10% of injected dose per gram
of tumour at 4 h after administration. As shown in Fig. 3, the
70
60
50
40
30
20
10
0
dtcTc1
dtcTc2
B
Li
H
Lu
Sp
K
T
M
G
St
I
B+U
Figure 3. Biodistribution pattern of 99mTcN–metronidazole complexes dtcTc1 and dtcTc2 on C57 mice bearing Lewis carcinoma, n = 3, at 240 min post-injection. B: blood; Li:
liver; H: heart; Lu: lung; Sp: spleen; K: kidney; T: thyroid; M: muscle; G: gallbladder; St: stomach; I: intestine; B + U: bladder + urine.

J. Giglio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 394–397
397
activity at 240 min was mainly uptaken by liver and intestinal tract
A. Supplementary data
indicating that dtcTc2 is excreted predominantly through the
hepatobiliary system, without tumour retention. A minor portion
of the injected dose is also excreted renally, as seen from the activ-
ity associated with the kidneys, urine, and bladder. Although
tumour/muscle ratio resulted a little favourable at 240 min post-
injection (Table 3) statistical analysis showed that this difference
in uptake was not significant (p = 0.05).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.130.
References and notes
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between the 5-nitroimidazole pharmacophore and the dithiocar-
bamate chelating group, altered significantly the physicochemical
properties and biological behaviour in comparison to dtcTc1 (Table
1). The differential tumour and -organs uptake could be partially
due to a higher dtcTc2-lipophilicity, which promotes hepatic up-
take but the main factor seems to be protein binding of dtcTc2
to plasmatic proteins that could endorse high hepatic uptake, pref-
erential hepatobiliary excretion and lower tumour accumulation.
To conclude, the ligands L1 and L2 were designed combining
the 5-nitroimidazole bioreductive moiety with the chelator dithio-
carbamate. These were successfully synthesised and utilised in the
preparation of two novel 99mTc complexes, dtcTc1 and dtcTc2,
with potentiality for hypoxia imaging. 99mTc complexes were ob-
tained with high radiochemical purity (>90%), but only dtcTc1
showed adequate chemical and biological stability together with
low protein binding and intermediate lipophilicity. Overall biodis-
tribution in normal animals was very favourable due to fast clear-
ance from blood and soft tissues via kidney. Finally, according to
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lid tumours, this system could be considered as a starting point for
the development of new radiopharmaceuticals for imaging hypox-
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other labelling methods in the search for compounds with im-
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21. See Supplementary data.
22. L1—Oil, that solidifies in vacuum. 1H NMR (DMSO-d6/D2O, 9:1) d (ppm): 2.57
(s, 3H, CH3), 3.80 (t, 2H, J = 5.4 Hz, CH2), 4.46 (t, 2H, J = 5.4 Hz, CH2), 8.00 (s, 1H,
CH); 13C NMR (DMSO-d6/D2O, 9:1) d (ppm): 14.9 (CH3), 45.9 (CH2), 51.4 (CH2),
130.0 (CH), 133.9 (CNO2), 151.7 (C), 190.4 (CS2); IR (KBr)
m
(cmꢀ1): 1001 (NH–
Acknowledgments
CS2), 1118 (–(C@S)–NH), 1373 (CH), 1374 (NO2), 1576 (NO2), 3520 (NH); MS
(EI, 70 eV), m/z: 246 (M+Å+H–Na); C7H9N4NaO2S2. L2—Oil, that solidifies in
vacuum. 1H NMR (CD3OD) d (ppm): 2.55 (br t, 4H, J = 6.2 Hz, CH2–N), 2.58 (s,
3H, CH3), 2.73(br t, 4H, J = 6.2 Hz, N–CH2), 3.33 (t, 2H, J = 6.0 Hz, CH2), 4.54 (t,
2H, J = 6.0 Hz, CH2), 7.93 (s, 1H, CH); 13C (MeOD) d (ppm): 15.3 (CH3), 39.9
(CH2), 51.4 (CH2), 54.3 (CH2–N), 58.5 (N–CH2), 133.0 (C), 142.9 (CNO2), 158.7
We thank PEDECIBA and ANII for scholarships to J.G. and S.F.,
Dr. A. Duatti and Cis Bio International for providing kits for the
preparation of nitrido precursor, Gramón-Bagó del Uruguay S.A.
for providing metronidazol, Dr. A Chabalogoity and M. Moreno
for providing tumour cells.
(C), 206.3 (CS2); IR (KBr)
(CH), 1380 (NO2), 1579 (NO2), 3528 (NH); MS (EI, 70 eV), m/z: 315 (M+Å+H–Na);
11H16N5NaO2S2.
m
(cmꢀ1): 1005 (NH–CS2), 1124 (–(C@S)–NH), 1370
C