Re(I) and 99mTc(I) tricarbonyl complexes with ether-containing pyrazolyl-based chelators: Chemistry, biodistribution and metabolism We dedicate this paper to Professor Maria José Calhorda on the occasion of her 65th birthday.

Article abstract of DOI:10.1016/j.jorganchem.2013.11.013

Tris(pyrazolyl)methane chelators, L1-L3, containing one or two ether groups at different positions of the azole rings, were synthesized and fully characterized. These chelators enabled the synthesis of fac-[ ^99mTc(CO)₃{HC[4-(ROCH₂)pz]₃}] ⁺ (R = Me (Tc1), Et (Tc2)) and fac-[^99mTc(CO) ₃{HC[3,5-(EtOCH₂)₂pz]₃}] ⁺ (Tc3) which were identified by HPLC in comparison with the rhenium counterparts. The evaluation of Tc1-Tc3 in CD-1 mice has shown that the number and/or nature of the ether groups greatly influence the biodistribution profile, pharmacokinetics and metabolic stability of these complexes. Tc1 and Tc2, bearing a unique ether substituent at the 4-position of the pyrazolyl ring, undergo metabolic transformation in vivo while Tc3 is not metabolized. The metabolization of Tc1 and Tc2 enhanced their rate of excretion but, most probably, also justify their negligible heart uptake in contrast with the high heart uptake of congener non-metabolizable complexes (^99mTc-DMEOP and ^99mTc-TMEOP), which have recently emerged as potential myocardial imaging agents. The attempts made to identify the metabolites of Tc1 and Tc2 have shown that the metabolization of these compounds must involve the ether functions with probable formation of carboxylic acid derivatives. A comparative study with the congener fac-[^99mTc(CO)₃{[4-(MeOCH ₂)pz](CH₂)₂NH(CH₂) ₂NH₂}]⁺ (Tc6) led us to confirm the formation of such type of metabolites. In fact, Tc6 is also metabolized in mice with formation of fac-[^99mTc(CO)₃{[4-(HOCH₂)pz] (CH₂)₂NH(CH₂)₂NH₂}] ⁺ (Tc7) and fac-[^99mTc(CO)₃{[4-(HOOC)pz] (CH₂)₂NH(CH₂)₂NH₂}] ⁺ (Tc8), which were chemically identified by HPLC in comparison with the Re congeners (Re7 and Re8).

Full text of DOI:10.1016/j.jorganchem.2013.11.013

Journal of Organometallic Chemistry 760 (2014) 138e148
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Re(I) and 99mTc(I) tricarbonyl complexes with ether-containing
pyrazolyl-based chelators: Chemistry, biodistribution and metabolism
*
Célia Fernandes, Leonor Maria, Lurdes Gano, Isabel C. Santos, Isabel Santos, António Paulo
Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, 2695-066 Bobadela LRS, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Tris(pyrazolyl)methane chelators, L1eL3, containing one or two ether groups at different positions of the
azole rings, were synthesized and fully characterized. These chelators enabled the synthesis of fac-
[
Received 2 October 2013
Received in revised form
8 November 2013
99mTc(CO)3{HC[4-(ROCH2)pz]3}]þ (R ¼ Me (Tc1), Et (Tc2)) and fac-[99mTc(CO)3{HC[3,5-(EtOCH2)2pz]3}]þ
(Tc3) which were identified by HPLC in comparison with the rhenium counterparts. The evaluation of Tc1
eTc3 in CD-1 mice has shown that the number and/or nature of the ether groups greatly influence the
biodistribution profile, pharmacokinetics and metabolic stability of these complexes. Tc1 and Tc2,
bearing a unique ether substituent at the 4-position of the pyrazolyl ring, undergo metabolic trans-
formation in vivo while Tc3 is not metabolized. The metabolization of Tc1 and Tc2 enhanced their rate of
excretion but, most probably, also justify their negligible heart uptake in contrast with the high heart
uptake of congener non-metabolizable complexes (99mTc-DMEOP and 99mTc-TMEOP), which have
recently emerged as potential myocardial imaging agents.
The attempts made to identify the metabolites of Tc1 and Tc2 have shown that the metabolization of
these compounds must involve the ether functions with probable formation of carboxylic acid derivatives.
A comparative study with the congener fac-[99mTc(CO)3{[4-(MeOCH2)pz](CH2)2NH(CH2)2NH2}]þ (Tc6) led
us to confirm the formation of such type of metabolites. In fact, Tc6 is also metabolized in mice with
formation of fac-[99mTc(CO)3{[4-(HOCH2)pz](CH2)2NH(CH2)2NH2}]þ (Tc7) and fac-[99mTc(CO)3{[4-(HOOC)
pz](CH2)2NH(CH2)2NH2}]þ (Tc8), which were chemically identified by HPLC in comparison with the Re
congeners (Re7 and Re8).
Accepted 12 November 2013
We dedicate this paper to Professor Maria
José Calhorda on the occasion of her 65th
birthday.
Keywords:
Rhenium
Technetium-99m
Tricarbonyl
Tris(pyrazolyl)methanes
Metabolism studies
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
We have introduced the complexes fac-[99mTc(CO)3{HC[3,5-
(MeOCH2)2pz]3}]þ
(CH3OCH2)3pz]3}]þ
(
99mTc-DMEOP) and fac-[99mTc(CO)3{HC[3,4,5-
Tris(pyrazolyl)methanes are tridentate and tripodal ligands that
have been used to stabilize coordination and organometallic com-
plexes with a large variety of d- and f-transition metals, aiming
mainly at exploring their reactivity in stoichiometric and/or cata-
lytic transformation of organic substrates [1]. The research studies
on tris(pyrazolyl)methane complexes for biomedical applications
are more scarce and are restricted to a few examples, such as CO
releasing molecules (CORM) based on Mn(I) tricarbonyl complexes
[2]. Searching for new metal-based compounds with relevance for
nuclear medicine applications, our group has investigated in the
past few years tris(pyrazolyl)methane 99mTc(I) organometallic
complexes as radioprobes for myocardial imaging by Single Photon
Emission Computerized Tomography (SPECT) [3e6].
(
99mTc-TMEOP) (Fig. 1), anchored by
tris(pyrazolyl)methane chelators that contain two and three
methoxymethyl substituents at each azolyl ring, respectively. The
biological evaluation of 99mTc-DMEOP and 99mTc-TMEOP indicated
that these complexes hold potential as myocardial imaging agents,
particularly 99mTc-TMEOP [4e6]. Biodistribution studies and SPECT
images have demonstrated that 99mTc-TMEOP exhibits a cardiac
uptake comparable to 99mTc-Sestamibi and 99mTc-Tetrofosmin
(Fig.1), which are the 99mTc-radiopharmaceuticals in clinical use for
perfusion cardiac imaging. Most importantly, 99mTc-TMEOP
showed a significantly faster liver clearance than these radiophar-
maceuticals, pointing out that our 99mTc(I) tricarbonyl complex
might improve the diagnostic accuracy of coronary artery disease
(CAD). In fact, 99mTc-TMEOP is among the best performing cationic
and lipophilic 99mTc complexes that have been evaluated in recent
years as potential myocardial imaging agents, which include
mixed-ligand 99mTc(V) nitrido complexes and a 99mTc(I) tricarbonyl
* Corresponding author.
E-mail address: apaulo@ctn.ist.utl.pt (A. Paulo).
0022-328X/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2013.11.013

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
139
Fig. 1. Molecular structures of 99mTc-Sestamibi, 99mTc-Tetrofosmin, 99mTc-DMEOP and 99mTc-TMEOP.
complex stabilized by a tridentate PNP ligand bearing a pendant
crown ether [7e16].
derivatives, 1-trityl-4-hydroxymethylpyrazole (3) and 1-trityl-3,5-
bis(hydroxymethyl)pyrazole (4), having the NeH function pro-
tected with a trityl group. Compound 3 was obtained using the
same approach that we have previously described for the synthesis
of 4, by reaction of ethyl pyrazole-4-carboxylate (1) with trityl
chloride [4]. The resulting tritylated pyrazole, compound 2, was
reduced almost quantitatively with LiAlH4 to the corresponding
alcohol 3 (Scheme 1). Compounds 3 and 4 were used to prepare the
ether-containing N-tritylated pyrazoles 5e7 by a Williamson
reaction [17]. This comprised the in situ preparation of the
respective alcoholates followed by reaction with excess of the
adequate alkyl halide. Removal of the trityl protecting group under
acidic conditions gave the final ether-containing pyrazoles 8e10
(Scheme 1). Compounds 8e10 are colourless to pale yellow oils that
have been characterized by 1H and 13C NMR spectroscopy.
As depicted in Scheme 2, the syntheses of the ether-containing
tris(pyrazolyl)methane chelators, HC[4-(ROCH2)pz]3 (R ¼ Me (L1),
Et (L2)) and HC[3,5-(EtOCH2)2pz]3 (L3) have been performed by
CHCl3/H2O phase transfer reaction with the pyrazoles 8e10 under
alkaline conditions, which is the most common synthetic approach
to obtain this type of chelators [18]. L1eL3 are colourless or pale
yellow compounds that have been isolated with yields ranging
from 16 to 73%, after adequate purification. L2 was purified by
extraction with ether followed by washing with distilled water. The
purification of L1 and L3 was achieved, respectively, by HPLC or
silica gel column chromatography. L1eL3 were characterized by 1H
and 13C NMR spectroscopy and by high resolution FT/ICR mass
spectrometry. The analytical data collected for L1eL3 were
consistent with their formulation as chelators of the tris(pyrazolyl)
methane type. In particular, the FT/ICR mass spectra obtained in the
The encouraging pre-clinical results that we have obtained with
99mTc-TMEOP prompted us to study related tris(pyrazolyl)methane
Re(I) and 99mTc(I) tricarbonyl complexes having different ether
functions at different positions of the azole rings, expecting to
understand the influence of the ether substitution pattern on the
physico-chemical (i.e. lipophilicity) and biological properties (i.e.
heart uptake, heart/liver and heart/lung ratios, metabolic stability)
of this family of complexes. Herein, we describe the new tris(pyr-
azolyl)methane chelators HC[4-(ROCH2)pz]3 (R ¼ Me (L1), Et (L2))
and HC[3,5-(EtOCH2)2pz]3 (L3) and report on the synthesis and
characterization of the respective tricarbonyl complexes fac-
[M(CO)3{HC[4-(ROCH2)pz]3}]þ (M ¼ Re, R ¼ Me (Re1), Et (Re2);
M ¼ 99mTc, R ¼ Me (Re1), Et (Re2)) and fac-[M(CO)3{HC[3,5-
(EtOCH2)2pz]3}]þ (M ¼ Re (Re3), 99mTc (Tc3)). The biological eval-
uation of Tc1eTc3, comprising biodistribution and metabolism
studies in mice, is also presented. Looking for a better under-
standing on the biological fate of 99mTc(I) tricarbonyl complexes
with ether-containing pyrazolyl-based chelators, we have also
studied the in vivo biological behaviour of fac-[99mTc(CO)3{[4-
(MeOCH2)pz](CH2)2NH(CH2)2NH2}]þ (Tc6), containing a N3-donor
ligand with a single ether-containing azolyl ring.
2. Results and discussion
2.1. Synthesis of ether-containing tris(pyrazolyl)methane chelators
To obtain pyrazoles functionalized with ether groups at different
positions of the azolyl ring we have started from alcohol

140
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
Scheme 1. Synthesis of ether-containing pyrazoles. (a) (i) NaH, DMF, 30 min, rt, (ii) triphenylmethylchloride, overnight (o.n.), rt; (b) LiAlH4, THF, o.n., rt; (c) (i) NaH, THF, 4 h, rt, (ii)
CH3I (5) or CH3CH2I (6, 7), 2 h, rt. (d) CF3COOH, CH2Cl2/MeOH (1:1), 75e80 ꢂC, 24 h (8) or HCl, EtOH/acetone, 80 ꢂC, 2 h (9 and 10).
positive mode showed the presence of prominent peaks (Mþ or
MHþ) which were assigned to the expected molecular-ions.
of these two bands, associated respectively with the asymmetrical
E mode and symmetrical A1 mode, are very similar for all the
complexes. The resonances present on the 1H and 13C NMR spectra
of Re1eRe3 display a splitting pattern consistent with the magnetic
equivalence of the three pyrazolyl rings. Some of these resonances
are downfield shifted compared with those in the respective free
tris(pyrazolyl)methane ligands, which corroborates the coordina-
tion of the azolyl rings to the fac-[Re(CO)3]þ unit. Mass spectrom-
etry analysis of Re1eRe3 led to the expected molecular-ions (Mþ)
in the respective positive ESI mass spectra. In summary, the spec-
troscopic data obtained for Re1eRe3 support their formulation as
cationic tricarbonyl complexes anchored by tridentate and facially
coordinated tris(pyrazolyl)methanes, a coordination mode that has
been confirmed in several instances by X-ray diffraction analysis for
other complexes of the type fac-[Re(CO)3(RCpz3*)]þ [3,20e23].
At the no-carrier added (n.c.a) level, the syntheses of fac-
2.2. Synthesis and characterization of the ether-containing
organometallic Re(I) and 99mTc(I) complexes
The synthesis of the compounds fac-[Re(CO)3{HC[4-(ROCH2)
pz]3}]Br (R
(EtOCH2)2pz]3}]Br (Re3) was achieved in moderate to high yields
(46e92%), by treatment of methanolic solution of fac-
¼
Me (Re1), Et (Re2)) and fac-[Re(CO)3{HC[3,5-
a
[Re(CO)3(H2O)3]Br with a stoichiometric amount of the respective
tris(pyrazolyl)methane ligand (Scheme 3).
Complexes Re1eRe3 are air- and water-stable microcrystalline
solids which were fully characterized by the common analytical
techniques, and were used to identify the chemical nature of the
99mTc congeners by HPLC, as described below. Due to the similar-
ities between the physico-chemical properties of Re and Tc com-
[
[
99mTc(CO)3{HC[4-(ROCH2)pz]3}]þ (R ¼ Me (Tc1), Et (Tc2)) and fac-
99mTc(CO)3{HC[3,5-(EtOCH2)2pz]3}]þ (Tc3) were done in aqueous
pounds, this is
a common and well accepted practice in
medium by reaction of the organometallic precursor fac-
[
radiopharmaceutical chemistry that avoids the manipulation of
99Tc, the long lived and soft bꢀ emitter used to isolate technetium
compounds at the macroscopic level, i.e. at the millimolar range.
The IR spectra of Re1eRe3 displayed two intense bands in the
1923e2038 cmꢀ1 range with the typical pattern of tricarbonyl
complexes with C3v symmetry [19]. The frequencies found for each
99mTc(CO)3(H2O)3]þ with L1eL3 (Scheme 3), using the experimental
conditions summarized in Table 1.
The 99mTc complexes, Tc1eTc3, were obtained with high
radiochemical yield (>95%), using ligand (L1eL3) concentrations in
the 10ꢀ3e1.5 ꢁ 10ꢀ3 M range, and running the reactions at 100 ꢂC
Scheme 2. Synthesis of tris(pyrazolyl)methane chelators. (a) [NBu4]Br, Na2CO3, H2O/CHCl3, reflux, 3 days.

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
141
Scheme 3. Synthesis of 99mTc and Re complexes with tris(pyrazolyl)methane chelators.
for 30e60 min. The chemical identity of Tc1eTc3 was established
by comparing their HPLC radiochromatograms with the UV/vis
trace of the Re congeners, Re1eRe3 (Fig. 2).
there is no clear-cut “optimal” log P value [7]. The introduction of a
slightly longer ether, like ethoxymethyl groups, into the framework
of tris(pyrazolyl)methane chelators increases the lipophilicity of
the 99mTc(I) complexes to log P values higher than 1.5, which are
expected to induce a slow liver clearance.
The in vitro evaluation of Tc1eTc3 comprised the study of their
in vitro stability in PBS (pH 7.4, 37 ꢂC) and the determination of the
respective lipophilicity by measurement of the log Po/w values (n-
octanol/0.1 M PBS, pH 7.4). HPLC analysis of Tc1eTc3 has shown
that the compounds did not undergo any transformation, including
reoxidation to pertechnetate, even after 24 h of incubation under
physiologic conditions. The log P values measured for Tc1eTc3
spanned between 0.45 and 2.64. The log P value of 0.45 found for
Tc1 is rather similar to the values that we have previously found for
2.3. Biodistribution and metabolism studies in mice
The evaluation of the biodistribution and pharmacokinetics of
Tc1eTc3 has been performed in female CD-1 mice. The organ dis-
tribution (% ID/g) of Tc1eTc3 in mice as a function of time is
summarized in Table 2.
All the evaluated complexes presented a relatively fast blood
clearance in mice displaying blood activities between
0.36 ꢃ 0.03% ID/g for Tc2 and 1.2 ꢃ 0.2% ID/g for Tc3 at 1 h p.i. The
dominant excretory pathway is hepatobiliary as expected for lipo-
philic complexes but the overall rate of excretion depends very
much on the degree of substitution and/or on the nature of the
ether substituents. Compared with Tc3 that presents an overall
excretion of 4.9 ꢃ 0.8% ID at 2 h p.i., the compounds containing
fac-[99mTc(CO)3{HC[3,5-(MeOCH2)2pz]3}]þ
(
99mTc-DMEOP)
(log P ¼ 0.58) and fac-[99mTc(CO)3{HC[3,4,5-(CH3OCH2)3pz]3}]þ
(
99mTc-TMEOP) (log P ¼ 0.64) [5]. This result suggests that the
number of methoxymethyl substituents at the azolyl rings has little
influence on the lipophilicity of tris(pyrazolyl)methane 99mTc(I)
tricarbonyl complexes. By contrast, the replacement of methoxy by
an ethoxy group leads to an important increase of lipophilicity,
independently of the number of substituents per ring, as can be
seen by comparing the log P values of Tc1 and Tc2 (0.45 vs 1.80) and
99mTc-DMEOP and Tc3 (0.58 vs 2.64).
The lipophilicity is a physico-chemical parameter having a quite
determinant influence on the biological performance of 99mTc
compounds for myocardial imaging. It is generally considered that
cationic 99mTc radiotracers must have a log P value of 0.5e1.2 in
order to obtain high heart uptake and fast liver clearance, although
Table 1
Experimental conditions for the synthesis of complexes Tc1eTc3 and Tc6 and their
radio-HPLC retention times and log P values.
Complex Yield [L]/M
(%)
Time T/ꢂC pH tR (min)a
(min)
Log Po/w
Tc1
Tc2
Tc3
Tc6
ꢄ98
ꢄ98
ꢄ96
ꢄ97
1.5 ꢁ 10ꢀ3 30
100 4.0 17.9 (17.5)b 0.45 ꢃ 0.022
100 4.0 18.5 (18.2)b 1.80 ꢃ 0.019
100 4.0 21.9 (21.4)b 2.64 ꢃ 0.002
100 7.4 15.9 (15.5)c n. d.
1.5 ꢁ 10ꢀ3 30
10ꢀ3
10ꢀ4
60
30
a
The values in parentheses are for the corresponding Re complexes.
Using a gradient of acetonitrile and aqueous 0.1% CF3COOH as the solvent.
Using a gradient of acetonitrile and 0.01 M sodium acetate buffer (pH ¼ 5).
b
c
Fig. 2. HPLC chromatograms of co-injected rhenium complex (Re3) (UV detection) and
(n. d. ¼ not determined).
99mTc complex (Tc3) (
g detection).

142
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
Table 2
Biodistribution results of complexes Tc1eTc3 in CD-1 mice at 1 and 2 h after i.v. administration, expressed in % ID/g tissue ꢃ SD (n ¼ 3e5).
Organ
Tc1
Tc2
Tc3
1 h
2 h
1 h
2 h
0.6 ꢃ 0.4
1 h
2 h
Blood
Liver
Intestines
Spleen
Heart
0.4 ꢃ 0.2
0.08 ꢃ 0.02
1.4 ꢃ 0.9
0.36 ꢃ 0.03
6.0 ꢃ 1.4
1.2 ꢃ 0.2
0.75 ꢃ 0.01
8.9 ꢃ 1.7
18.9 ꢃ 6.4
2.8 ꢃ 0.5
2.5 ꢃ 0.7
1.9 ꢃ 0.6
27.1 ꢃ 9.1
0.7 ꢃ 0.1
1.3 ꢃ 0.1
10.0 ꢃ 2.9
4.9 ꢃ 0.8
4.9 ꢃ 1.5
17.9 ꢃ 3.5
0.4 ꢃ 0.2
4.9 ꢃ 2.0
17.6 ꢃ 3.9
1.7 ꢃ 0.4
0.5 ꢃ 0.1
0.49 ꢃ 0.07
6.2 ꢃ 0.2
0.4 ꢃ 0.1
0.30 ꢃ 0.06
1.1 ꢃ 0.5
28.6 ꢃ 2.8
16.3 ꢃ 2.6
11.9 ꢃ 3.3
3.8 ꢃ 0.6
3.7 ꢃ 0.6
3.2 ꢃ 0.6
29.1 ꢃ 5.8
1.0 ꢃ 0.1
1.7 ꢃ 0.1
7.3 ꢃ 2.2
3.7 ꢃ 1.1
18.9 ꢃ 2.9
0.16 ꢃ 0.06
0.07 ꢃ 0.01
0.31 ꢃ 0.05
4.4 ꢃ 1.0
14.1 ꢃ 0.8
1.1 ꢃ 0.3
0.3 ꢃ 0.1
1.0 ꢃ 0.2
Lung
0.27 ꢃ 0.06
11.8 ꢃ 2.7
0.21 ꢃ 0.04
0.27 ꢃ 0.06
2.4 ꢃ 0.9
0.69 ꢃ 0.06
9.2 ꢃ 2.9
Kidney
Muscle
Bone
Stomach
Excretion (% ID)
0.2 ꢃ 0.1
0.6 ꢃ 0.1
0.12 ꢃ 0.05
2.9 ꢃ 1.6
0.45 ꢃ 0.03
0.8 ꢃ 0.2
29.1 ꢃ 7.9
39.4 ꢃ 5.5
25.8 ꢃ 1.1
ether groups uniquely at the 4-position of the pyrazolyl rings, Tc1
and Tc2, display a fastest rate of excretion, respectively 39.4 ꢃ 5.5
and 28.6 ꢃ 2.8% ID at 2 h p.i.
2.4. Chemical identification of metabolites
A common feature of all 99mTc complexes with adequate bio-
logical profile for cardiac imaging is the presence of several ether
groups on their structure. The role of the ether groups is not fully
understood, but it is generally accepted that they allow a fine-
tuning of the lipophilicity of the complexes, without changing their
overall charge and enhancing the liver washout kinetics. It has been
also invoked that the metabolic transformation of the ether func-
tions is another possibility to enhance the liver washout kinetics of
myocardial imaging radiotracers. Unlike Tc1 and Tc2, almost all of
the ether-containing 99mTc complexes are excreted as non-
metabolized species. The mechanisms involved in the metabolic
fate of complexes Tc1 and Tc2 have not been investigated in detail,
but we have considered that the formation of the three metabolites
could result from successive O-dealkylation reactions of the ether
groups, occurring most probably in the rodent liver microsomes
and involving catalysis by an isoform of cytochrome P-450 [24]. O-
Dealkylation reactions of methyl or ethyl ethers of the type R1eOe
R2 (R1 ¼ Me, Et) are usually accompanied by the release of these
alkyl groups as formaldehyde or acetaldehyde and by the formation
of R2eOH alcohol derivatives. As proposed in Scheme 4, in the case
of Tc1 and Tc2 this process should end-up with the formation of a
tri-alcohol derivative of these organometallic complexes, corre-
sponding to the common metabolite appearing at tR ¼ 13.0 min.
Searching to confirm this possibility, we have synthesized the
congener tri-alcohol Re complex (Re4), aiming at its use in the
chemical identification of this metabolite by means of HPLC
analysis.
Tc1 and Tc2 have a negligible or very low heart uptake in mice
(<1% ID/g). On the contrary, complex Tc4 has higher heart uptake
(3.7 ꢃ 0.03% ID/g, 1 h p.i.) but display a quite unfavourable heart/
liver ratio of 0.3 ꢃ 0.6% at 2 h p.i., which certainly reflects its slow
rate of excretion.
The metabolic stability of Tc1eTc3 was assessed by HPLC
analysis of the urine and blood serum of mice injected with these
complexes. Likewise the previously reported 99mTc-DMEOP and
99mTc-TMEOP with two or three ether substituents in the respec-
tive azolyl rings, Tc3 does not undergo any metabolic trans-
formation in mice [4,5]. Contrastingly, Tc1 and Tc2 are
metabolized in vivo. A considerable fraction of the urine activity at
1 h p.i. still corresponds to intact Tc1 (57%) and Tc2 (35%) but the
presence of three prominent metabolites was also detected
(Fig. 3). The same metabolites were also detected in the serum,
although in a smaller proportion than in urine. The three metab-
olites have lower retention times than the original compounds,
being eluted with retention times between 13 and 18 min. Co-
injection of the urine of mice injected independently with Tc1
and Tc2 indicated that the metabolite with the lowest retention
time (tR ¼ 13.0 min) corresponds to the same compound, as
revealed by the coincident retention time in both analysis. At 2 h
p.i., this metabolite is already the major radiochemical species in
the urine, corresponding to 46% and 51% of the excreted activity
for Tc1 and Tc2, respectively (Fig. 3).
As depicted in Scheme 5, complex Re4 has been obtained by
demethylation of the ether functions of Re1 with BBr3 [25]. Re4 has
been isolated as a white solid, after HPLC purification, and has been
fully characterized by mass spectrometry, spectroscopic techniques
(IR and 1H/13C NMR) and HPLC. The characterization of Re4
confirmed the formation of a tri-alcohol derivative as a conse-
quence of the full demethylation of the three ether functions. By
HPLC analysis Re4 has shown a tR ¼ 19.8 min, which is considerably
longer than the one (tR ¼ 13.0 min) exhibited by the common
metabolite, under the same analytical conditions. This result indi-
cated that the metabolites detected for Tc1 and Tc2 do not corre-
spond to alcohol derivatives, as hypothesized initially. If formed,
such alcohol derivatives undergo themselves a fast metabolic
transformation, eventually oxidation to aldehydes and subse-
quently to carboxylic acids [26]. To check for this possibility, we
have studied the synthesis of fac-[Re(CO)3{HC[4-(HOOC)pz]3}]þ.
However, we could not synthesize this complex as we were unable
to obtain the HC[4-(HOOC)pz]3 chelator. Alternatively, we have
prepared the Re complex of HC[4-(EtOOC)pz]3 (L4), fac-[Re(CO)3{HC
Tc1
Urine; 2h p.i.
Urine; 1h p.i.
0
5
10
15
20
25
Time (min)
Fig. 3. HPLC analysis (radiometric detection) of urine from mice administered with Tc1
at 1 h and 2 h p.i.

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
143
Scheme 4. Possible O-dealkylation reactions of complexes Tc1 and Tc2.
[4-(EtOOC)pz]3}]þ (Re5), expecting that the hydrolysis of its ester
functions could afford “fac-[Re(CO)3{HC[4-(HOOC)pz]3}]þ”. Com-
plex Re5 was obtained as described above for the congeners Re1e
Re3, and its characterization by 1H/13C NMR and ESI-MS confirmed
the proposed formulation. The hydrolysis of Re5 was also followed
by decomposition processes, and we were unable to isolate the
desired “fac-[Re(CO)3{HC[4-(HOOC)pz]3}]þ”.
synthesized by reacting fac-[Re(CO)3(H2O)3]Br with 4-(HOCH2)
pz(CH2)2NH(CH2)2NH2 (L6). In its turn, L6 was obtained by
treatment of L5 with BBr3 (Scheme 6). Complex Re7 has been
characterized by ESI-MS, by the common spectroscopic tech-
niques and X-ray diffraction analysis. The solid state structural
analysis confirmed the tridentate coordination of L6 and the
presence of the alcohol function at the 4-position of the pyr-
azolyl ring, as can be seen in the molecular structure of the
cation of Re7 presented in Fig. 5. The rhenium atom is six-
coordinate with a distorted octahedral coordination geometry
The angles and bond distances found for Re7 are comparable
to the values that we have previously reported for fac-
[Re(CO)3{(3,5-Me2pz)(CH2)2NH(CH2)2NH2}]Br [27].
The co-injection of Re7 and Re8 with the urine from a mouse
injected with Tc6 showed that the Tc6 metabolites, with retention
times of 9.8 and 10.8 min, correspond to the acid and alcohol de-
rivatives, respectively. These findings point out that in the case of
the tris(pyrazolyl)methane Tc(I) complexes, Tc1 and Tc2, their
metabolization also takes place at the methoxymethyl substituent
of the azolyl ring, and proceeds most probably with its oxidation to
a carboxylic acid function.
Finally, we decided to synthesize and evaluate a pyrazolyl-
diamine 99mTc(I) tricarbonyl complex containing
a single
ether-containing pyrazolyl ring: fac-[99mTc(CO)3{[4-(MeOCH2)
pz](CH2)2NH(CH2)2NH2}]þ (Tc6). By studying this compound, we
have considered that it would also undergo metabolization in
mice and we have anticipated a more straightforward identifi-
cation of the involved transformations, i.e. conversion of the
ether function into an alcohol or an acid. Tc6 was synthesized in
high yield by reaction of fac-[99mTc(CO)3(H2O)3]þ with the
respective pyrazole-diamine ligand (L5), which has been ob-
tained using the synthetic approach that we have previously
reported for other pyrazolyl-diamine derivatives (Scheme 6)
[27]. The chemical identification of Tc6 has been achieved by
HPLC comparison with the congener fac-[Re(CO)3{[4-(MeOCH2)
pz](CH2)2NH(CH2)2NH2}]þ (Re6) (Scheme 6), isolated at macro-
scopic level and fully characterized by the common analytical
techniques. Tc6 has been administered to female CD-1 mice, and
the urine and serum of the injected mice was analyzed by HPLC.
The HPLC analysis of urine revealed the formation of two me-
tabolites with retention times of 9.8 and 11.8 min, respectively,
as well as the presence of Tc6 (Fig. 4). These findings confirmed
that Tc6 also underwent metabolic transformations, like Tc1 and
Tc2 bearing the same ether-containing pyrazolyl ring. Moreover,
Tc6 has shown a biodistribution profile (Table S1) similar to the
one of Tc1 and Tc2, although exhibiting a faster clearance from
main organs and an increased overall excretion.
3. Conclusions
The functionalization of tris(pyrazolyl)methane chelators with
methoxymethyl or ethoxymethyl groups at different position of the
azole rings has a marked influence on the biodistribution profile,
pharmacokinetics and metabolic stability of the respective 99mTc(I)
tricarbonyl complexes. The complexes containing ether groups
uniquely at the 4-position of the azolyl ring, fac-[99mTc(CO)3{HC[4-
(ROCH2)pz]3}]þ (R ¼ Me (Tc1), Et (Tc2)), undergo metabolic trans-
formation to more polar compounds. Interestingly, the presence of
additional ether groups at the 3- and 5-positions prevents the
metabolization of the ethers at the 4-position, as the previously
We have synthesized the alcohol and acid derivatives fac-
[Re(CO)3{[4-(HOCH2)pz](CH2)2NH(CH2)2NH2}]Br (Re7) and fac-
[Re(CO)3{[4-(HOOC)pz](CH2)2NH(CH2)2NH2}]Br (Re8), aiming
the identification of the Tc6 metabolites by HPLC. Complex Re8
has been reported previously by our research group [28]. The
alcohol derivative, Re7, is a new compound that has been
reported fac-[99mTc(CO)3{HC[3,4,5-(CH3OCH2)3pz]3}]þ
(
99mTc-
TMEOP) is not metabolized in mice. Despite being cationic and
lipophilic, Tc1 and Tc2 did not accumulate in the mice heart, on
contrary to 99mTc-TMEOP that holds potential as a SPECT radiop-
robe for cardiac imaging. The ether metabolization observed for Tc1
Scheme 5. Synthesis of complex Re4.

144
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
Scheme 6. Synthesis of pyrazolyl-diamine chelators and respective 99mTc and Re complexes. (a) [NBu4]Br, NaOH, reflux, 1.5 h; (b) EtOH, reflux, 5 h; (c) CH2Cl2, ꢀ20 ꢂC, 3 h.
and Tc2 enhanced their rate of excretion and certainly justifies their
negligible heart uptake.
pz](CH2)2NH(CH2)2NH2}]þ (Tc6), containing
a single ether-
containing pyrazolyl ring, is metabolized to the corresponding
alcohol and acid derivatives. These findings corroborate the
possible oxidation of the ether functions of Tc1 and Tc2 to car-
boxylic acid groups.
In summary, we have proved for the first time that ether-
containing 99mTc complexes can be metabolized in vivo with for-
mation of alcohol and carboxylic acid derivatives. Such possibility
was invoked previously by other authors but was not demon-
strated before [7]. The metabolization observed for the ether-
The metabolic transformation of Tc1 and Tc2 is most probably
due to O-dealkylation processes. However, the final metabolites
are not of the alcohol type, as confirmed by HPLC in comparison
with the fully characterized fac-[Re(CO)3{HC[3,5-(HOCH2)2pz]3}]
Br (Re4). Most probably, such metabolites are carboxylic acid
derivatives which, despite our efforts, could not be synthesized.
Nevertheless, we have proved that fac-[99mTc(CO)3{[4-(MeOCH2)
Re7
Re8
Urine 1h p.i.
Tc6
5
10
15
Time (min)
Fig. 5. ORTEP view of complex Re7 with ellipsoids drawn at the 40% probability level.
Selected bond distances (A): Re(1)eC(1) 1.919(6), Re(1)eC(2) 1.923(6), Re(1)eC(3)
1.923(7), Re(1)eN(1) 2.169(5), Re(1)eN(3) 2.227(4), Re(1)eN(4) 2.217(5).
ꢀ
Fig. 4. HPLC analysis (radiometric detection) of urine from mice administered with Tc6
at 1 h p.i. and comparison with the chromatograms of complexes Re7 and Re8.

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
145
containing tris(pyrazolyl)methane (Tc1 and Tc2) and pyrazolyl-
4.2.2. HC[4-(EtOCH2)pz]3 (L2)
diamine (Tc6) 99mTc(I) tricarbonyl complexes involves the ether
function, did not affect their coordination sphere and enhanced
their overall excretion. These features indicate that the introduc-
tion of ether functions at the 4-position of the azolyl ring of
tris(pyrazolyl)methane and pyrazolyl-diamine ligands might allow
the tuning of the pharmacokinetics of the corresponding 99mTc(I)
tricarbonyl complexes, which is a key issue in radiopharmaceutical
applications.
Compound L2 was synthesized as above described for L1,
starting from 4-ethoxymethylpyrazole (9) (234 mg, 1.86 mmol) and
using the same proportions of reagents and solvents. After 3 days of
reaction, the solvent was removed under vacuum and the residue
was redissolved in diethyl ether, and washed with saturated brine
and water. The ethereal phase was treated with decolorizing
charcoal, filtered and the solvent removed under vacuum to afford
compound L2 as a white microcrystalline solid. Yield: 176 mg
(0.453 mmol), 73%.
1H NMR (CDCl3,
7.55 (3H, s, H-3/5 (pz)), 4.32 (6H, s, 6.9 Hz, CH2), 3.45 (6H, q, OCH2),
1.15 (9H, t, CH3). 13C NMR (CDCl3,
ppm): 141.6 (C-3/5 (pz)), 128.6
d ppm): 8.25 (1H, s, CH), 7.60 (3H, s, H-3/5 (pz)),
4. Experimental section
d
4.1. General procedures
(C-3/5 (pz)), 120.2 (C-4 (pz)), 83.15 (CH), 65.6 (CH2), 63.0 (CH2), 15.0
(CH3). FTICR/MS m/z: 388.2214 (Mþ, calcd for C19H28N6O3,
388.2217).
All chemicals were of reagent grade and were used without
purification. 1H and 11C NMR spectra were recorded on a Varian
Unity 300 MHz spectrometer; 1H and 11C chemical shifts were
referenced with the residual solvent resonances relative to tetra-
methylsilane. IR spectra were recorded as KBr pellets on a Bruker 27
Tensor spectrometer. C, H and N analyses were performed on an EA
110 CE Instruments automatic analyser. The compounds ethyl 1H-
pyrazole-4-carboxylate (1) [29], 1-trityl-3,5-bis(hydroxymethyl)
pyrazole (4) [4], [Re(H2O)3(CO)3]Br [30] and fac-[Re(CO)3{[4-
(HOOC)pz](CH2)2NH(CH2)2NH2}]Br (Re8) [28] were prepared
according to published methods. The description of the synthesis of
compounds 2, 3, 5 and 6e10 is presented as supplementary data.
The radioactive precursor fac-[99mTc(OH2)3(CO)3]þ was prepared
using a IsoLinkÒ kit (Covidien) following described procedures [31].
Na[99mTcO4] was eluted from an Elumatic 99Mo/99mTc generator
(CisBio) with 0.9% saline. HPLC analysis of the Re and 99mTc com-
plexes was performed on a PerkineElmer LC pump 200 coupled to
a LC 290 tunable UV/Vis detector and to a Berthold LB-507A
radiometric detector. Separations were achieved on a Nucleosil
4.2.3. HC[3,5-(EtOCH2)2pz]3 (L3)
Compound L3 was synthesized as above described for L1,
starting from 3,5-bis(ethoxymethyl)pyrazole (10) (485 mg,
2.63 mmol). After 3 days of reflux, the organic phase was separated,
washed with water and dried over MgSO4. The brown residue oil
was applied on a silica gel column which was eluted with a gradient
from 100% CHCl3 to 98% CHCl3/2% MeOH. Removal of the solvent
from the collected fractions gave L3 as a pale yellow oil. Yield:
230 mg (0.41 mmol), 47%.
1H NMR (CDCl3,
d ppm): 8.68 (1H, s, CH), 6.32 (3H, s, H-4 (pz)),
4.41 (6H, s, CH2), 4.32 (6H, s, CH2), 3.46 (6H, q, 6.9 Hz, CH2), 3.34
(6H, q, 6.9 Hz, CH2), 1.16 (9H, t, 6.9 Hz, CH3), 1.06 (9H, t, 6.9 Hz, CH3).
13C NMR (CDCl3,
d ppm): 149.9 (C-3/5 (pz)), 141.7 (C-3/5 (pz)), 107.2
(C-4 (pz)), 79.0 (CeH), 66.3 (CH2), 65.7 (CH2), 65.5 (CH2), 62.9 (CH2),
15.1, (CH3), 14.8 (CH3). FTICR/MS m/z: 563.3552 ([M þ H]þ, calcd for
C28H46N6O6, 563.3553).
column (10
mm, 250 mm ꢁ 4 mm), using a flow rate of 1 mL/min;
4.2.4. HC[4-(EtO(O)C)pz]3 (L4)
UV detection, 254 nm; eluents, A e aqueous 0.1% CF3COOH solution,
B e acetonitrile; method, t ¼ 0e3 min, 0% B; 3e3.1 min, 0e25% B;
3.1e9 min, 25% B; 9e9.1 min, 25e34% B; 9.1e20 min, 34e100% B;
20e22 min, 100% B; 22e22.1 min, 100-0% B; 22.1e30 min, 0% B. The
HPLC analysis of the Tc6 metabolites and complexes Re7 and Re8
was performed using the following eluent: The HPLC analysis of the
metabolites of Tc6 and complexes Re7 and Re8 was performed
under the same experimental conditions but using the following
eluent: A e 0.01 M sodium acetate buffer (pH ¼ 5), B e acetonitrile.
Compound L4 was synthesized as above described for L1,
starting from ethyl 1H-pyrazole-4-carboxylate (1) (602 mg,
4.27 mmol). After 3 days of reflux, the organic phase was separated,
washed with water and dried over MgSO4. The brown residue oil
was applied on a silica gel column which was eluted with a gradient
from 100% CHCl3 to 95% CHCl3/5% MeOH. Removal of the solvent
from the collected fractions gave L4 as a pale yellow oil. Yield:
94 mg (0.22 mmol), 15%.
1H NMR (CDCl3,
d ppm): 8.35 (1H, s, CH), 8.13 (3H, s, H-3/5 (pz)),
8.04 (3H, s, H-3/5 (pz)), 4.29 (6H, q, OCH2), 1.31 (9H, t, CH3). ESI/MS
m/z: 429.3 ([M ꢀ H]ꢀ, calcd for C19H21N6O6, 429.41).
4.2. Synthesis of the ligands
4.2.5. {[4-(MeOCH2)pz](CH2)2NH(CH2)2NH2} (L5)
4.2.1. HC[4-(MeOCH2)pz]3 (L1)
A solution of 10 (0.25 g, 1.1 mmol) and 1,2-ethylenediamine
(1.5 mL, 22 mmol) in ethanol (2 mL) was refluxed for 5 h. The
solvent was removed under reduced pressure and the residue
extracted with CHCl3. After filtration and removal of the solvent
under vacuum L5 was obtained as a yellow oil. Yield: 190 mg
(0.96 mmol), 87%.
To a suspension of 4-methoxymethylpyrazole (8) (60 mg,
0.54 mmol) and [NBu4]Br (9 mg) in distilled water (2 mL) was
slowly added Na2CO3 (500 mg). Then, 0.5 mL of CHCl3 was added
and the mixture was gently refluxed for 3 days. The organic phase
was separated, washed with water and dried over MgSO4. Evapo-
ration of the solvent under vacuum gave a brown oily residue
which was purified by gradient HPLC (100% aqueous 0.1% CF3COOH
1H NMR (CDCl3,
d ppm): 7.48 (1H, s, H-3/5 (pz)), 7.43 (1H, s, H-3/
5 (pz)), 4.31 (2H, s, CH2), 4.20 (2H, t, CH2), 3.33 (3H, s, CH3), 3.05
solution / 100%$CH3CN) using a Nucleosil column (10
mm,
(2H, t, CH2), 2.88 (2H, m, CH2), 2.68 (2H, m, CH2). 13C NMR (CDCl3,
250 mm ꢁ 4 mm) and a flow rate of 1 mL/min. Compound L1 was
recovered as a colourless oil by removal of the solvent from the
collected fractions. Yield: 10 mg (0.029 mmol), 16%.
d ppm): 139.6 (C-3/5 (pz)), 129.3 (C-3/5 (pz)), 118.1 (C-4 (pz)), 65.3
(OCH2), 57.7 (CH2), 52.2 (CH2), 51.9 (CH2), 49.2 (CH2), 41.5 (CH3).
1H NMR (CDCl3,
d
ppm): 8.24 (1H, s, CH), 7.62 (3H, s, H-3/5 (pz)),
7.54 (3H, s, H-3/5 (pz)), 4.31 (6H, s, 6.9 Hz, CH2), 3.32 (9H, s, OCH3).
13C NMR (CDCl3,
ppm): 141.7 (C-3/5 (pz)), 128.7 (C-3/5 (pz)), 120.1
4.2.6. {[4-(HOCH2)pz](CH2)2NH(CH2)2NH2} (L6)
To a solution of L5 (289 mg, 1.46 mmol) in CH2Cl2 (5 mL) were
added dropwise 4 mL of a 1 M solution of BBr3 in CH2Cl2, while
keeping the reaction vial at ꢀ20 ꢂC. At this temperature, the reac-
tion mixture was stirred for 3 h. After this time, 5 mL of distilled
d
(C-4 (pz)), 83.3 (CH), 65.1 (CH2), 57.9 (OCH3). FTICR-MS m/z:
346.1747 (Mþ, calcd for C16H22N6O3, 346.1748).

146
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
water were added to the mixture. The aqueous phase was separated
and freeze-dried to afford a yellowish residue, which was extracted
with CHCl3. Compound L6 was recovered as a yellow oil, after
filtration and removal of the solvent under vacuum. Yield: 109 mg
(0.59 mmol), 40%.
Re4 was recovered as a white solid after removal of the solvent
from the collected fractions. Yield: 33% (8 mg, 0.014 mmol).
IR Data (KBr,
ppm): 11.58 (1H, s, CH), 8.72 (3H, s, H-3/5 (pz)), 8.08 (3H, s, H-3/5
(pz)), 4.32 (6H, s, CH2). 13C NMR (CDCl3,
ppm): 149.4 (C-3/5 (pz)),
n
/cmꢀ1): 2042s, 1921s (C^O). 1H NMR (CDCl3,
d
d
1H NMR (CDCl3,
d
ppm): 7.38 (1H, s, H-3/5 (pz)), 7.36 (1H, s, H-3/
5 (pz)), 4.42 (2H, s, CH2), 4.09 (2H, tr, CH2), 2.93 (2H, tr, CH2), 2.61
(2H, tr, CH2), 2.52 (2H, tr, CH2). 13C NMR (CDCl3,
ppm): 138.6 (C-3/
135.6 (C-3/5 (pz)), 124.2 (C-4 (pz)), 78.1 (CH), 55.1 (CH2). ESI-MS m/
z: 575.2 (Mþ, calcd for C16H16N6O6Re, 575.1). RP-HPLC, retention
time: tR ¼ 19.8 min.
d
5 (pz)),128.7 (C-3/5 (pz)),121.9 (C-4 (pz)), 83.3 (CH), 55.1 (CH2), 51.9
(CH2), 51.5 (CH2), 48.9 (CH2), 41.1 (CH2). FTICR-MS m/z: 207.1196
([M þ Na]þ, calcd for C8H16N4ONa, 207.1216).
4.3.5. fac-[Re(CO)3{HC[4-(EtO(O)C)pz]3}]Br (Re5)
A solution of [Re(CO)3(H2O)3]Br (18.6 mg, 46
mmol) and L4
(20 mg, 46 mol) in water was refluxed overnight. The solvent was
m
removed under vacuum and the residue was washed with diethyl
ether. The residue was purified by gradient HPLC (100% aqueous
0.1% CF3COOH solution / 100% CH3CN) using a Nucleosil column
4.3. Synthesis of the Re complexes
4.3.1. fac-[Re(CO)3{HC[4-(CH3OCH2)pz]3}]Br (Re1)
(10
Re5 was recovered as a beige solid after removal of the solvent from
the collected fractions. Yield: 21% (6.9 mg, 9.8 mol).
IR Data (KBr,
/cmꢀ1): 2036s, 1944s, 1923s (C^O). 1H NMR
(CDCl3, ppm): 12.54 (1H, s, CH), 9.10 (3H, s, H-3/5 (pz)), 8.46 (3H, s,
H-3/5 (pz)), 4.32 (6H, q, CH2), 1.32 (9H, t, CH3). 13C NMR (CDCl3,
ppm): 159.7 (COOEt), 149.1 (C-3/5 (pz)), 143.5 (C-3/5 (pz)), 119.2
(C-4 (pz)), 76.2 (CH), 62.3 (CH2), 14.5 (CH3). ESI-MS m/z: 701.0 (Mþ,
calcd for 22H22N6O9Re, 700.7). RP-HPLC, retention time:
tR ¼ 21.1 min.
m
m, 250 mm ꢁ 7 mm) and a flow rate of 2 mL/min. Compound
A solution of [Re(CO)3(H2O)3]Br (3.5 mg, 8.7 mmol) and L1 (3 mg,
8.7 m
mol) in CD3OD was heated at 60 ꢂC during 3 h. After this time,
1H NMR analysis of the reaction mixture has shown an almost
quantitative formation of Re1. The solvent was removed under
vacuum and the residue was washed with diethyl ether and dried
under vacuum to afford a beige solid which was formulated as
m
n
d
d
compound Re1. Yield: 4.9 mg (7.0
IR Data (KBr,
/cmꢀ1): 2035s, 1943s (C^O). 1H NMR (CDCl3,
ppm): 12.62 (1H, s, CH), 8.92 (3H, s, H-3/5 (pz)), 7.98 (3H, s, H-3/5
(pz)), 4.30 (6H, s, CH2), 3.36 (9H, s, CH3). 13C NMR (CDCl3,
ppm):
mmol), 81%.
n
C
d
d
146.6 (C-3/5 (pz)), 132.8 (C-3/5 (pz)), 122.1 (C-4 (pz)), 74.0, (CH),
4.3.6. fac-[Re(CO)3{[4-(MeOCH2)pz](CH2)2NH(CH2)2NH2}]þ (Re6)
A solution of [Re(CO)3(H2O)3]Br (25 mg, 62 mol) and L5 (13 mg,
mol) in methanol (5 mL) was refluxed overnight. The methanol
64.0 (CH2), 58.8 (OCH3). ESI-MS m/z: 616.9 (Mþ, calcd for
m
C
19H22N6O6Re, 617.1).
62
m
was removed under vacuum and the residue was washed with
hexane and chloroform. The recovered insoluble solid was dried
under vacuum and formulated as compound Re6. Yield: 23 mg
4.3.2. fac-[Re(CO)3{HC[4-(CH3CH2OCH2)pz]3}]Br (Re2)
A solution of [Re(CO)3(H2O)3]Br (25 mg, 62 mol) and L2 (25 mg,
mol) in methanol was refluxed overnight. The solvent was
removed under vacuum and the residue was washed with diethyl
ether. Compound Re2 was recovered as a beige solid after drying
m
64
m
(48
IR Data (KBr,
ppm): 7.99 (1H, s, H-3/5 (pz)), 7.96 (1H, s, H-3/5 (pz)), 6.88 (1H, m,
mmol), 79%.
n
/cmꢀ1): 2020s, 1916s (C^O). 1H NMR (CD3OD,
d
under vacuum. Yield: 42 mg (57
IR Data (KBr,
/cmꢀ1): 2036s, 1944s, 1923s (C^O). 1H NMR
(CDCl3, ppm): 12.54 (1H, s, CH), 8.85 (3H, s, H-3/5 (pz)), 7.98 (3H, s,
H-3/5 (pz)), 4.33 (6H, s, CH2), 3.52 (6H, q, 6.9 Hz, CH2), 1.20 (9H, t,
6.9 Hz, CH3). 13C NMR (CDCl3,
ppm): 146.7 (C-3/5 (pz)), 133.0 (C-3/
mmol), 92%.
NeH), 4.58 (2H, m, CH2), 4.30 (1H, m, CH2), 3.72 (3H, s, CH3), 3.65
(2 þ 1H, m, CH2), 3.07 (1H, m, CH2), 2.79e2.80 (1 þ1H, m, CH2), 2.64
n
d
(1H, m, CH2), 2.18 (1H, m, CH2). 13C NMR (CDCl3,
d ppm): 196.5 (CO),
195.9 (CO), 195.7 (CO), 146.2 (C-3/5 (pz)), 134.5 (C-3/5 (pz)), 121.7
(C-4 (pz)), 65.4 (OCH2), 58.3 (CH2), 56.5 (CH2), 52.5 (CH2), 49.4
(CH2), 41.6 (CH3). ESI-MS m/z: 469.4 (Mþ, calcd for C12H18N4O4Re,
469.5).
d
5 (pz)),122.2 (C-4 (pz)), 73.8 (CH), 66.6 (CH2), 62.0 (CH2),15.0 (CH3).
ESI-MS m/z: 658.9 (Mþ, calcd for C22H28N6O6Re, 659.2).
4.3.3. fac-[Re(CO)3{HC[3,5-(CH3CH2OCH2)2pz]3}]Br (Re3)
Compound Re3 is a beige solid which was obtained according to
the procedure described for Re2, starting from [Re(CO)3(H2O)3]Br
4.3.7. fac-[Re(CO)3{[4-(HOCH2)pz](CH2)2NH(CH2)2NH2}]Br (Re7)
A solution of [Re(CO)3(H2O)3]Br (48 mg, 119
mmol) and L6
(22 mg,199 mol) in distilled water (10 mL) was refluxed overnight.
m
(34 mg, 84
38 mol).
IR Data (KBr,
ppm): 9.29 (1H, s, CH), 6.72 (3H, s, H-4 (pz)), 5.05 (6H, s, CH2), 4.67
(6H, s, CH2), 3.67 (6H, q, 6.9 Hz, CH2), 3.70 (6H, s, CH2), 1.25 (18H, t,
6.9 Hz, CH3). 13C NMR (CDCl3,
ppm): 157.5 (C-3/5 (pz)), 144.9 (C-3/
mmol) and L3 (47 mg, 84
mmol). Yield: 46% (35 mg,
The water was removed under vacuum and the residue was washed
with chloroform. The recovered insoluble beige solid was dried
under vacuum and formulated as compound Re7. Yield: 50 mg
m
n
/cmꢀ1): 2038s, 1949s (C^O). 1H NMR (CDCl3,
d
(94
IR Data (KBr,
ppm): 7.94 (1H, s, H-3/5 (pz)), 7.93 (1H, s, H-3/5 (pz)), 4.52
mmol), 78%.
n
/cmꢀ1): 2025s, 1906s (C^O). 1H NMR (CD3OD,
d
d
5 (pz)), 109.4 (C-4 (pz)), 71.9 (CH), 67.5 (CH2), 67.4 (CH2), 66.3 (CH2),
(2 þ 1H, m, CH2), 4.25 (1H, m, CH2), 3.58 (1H, m, CH2), 2.97 (1H, m,
62.6 (CH2), 62.6 (CH3). ESI-MS m/z: 833.1 (Mþ, calcd for
CH2), 2.79e2.83 (1 þ 1H, m, CH2), 2.54 (1H, m, CH2), 2.16 (1H, m,
CH2). 13C NMR (CDCl3,
d ppm): 198.1 (CO), 194.8 (CO), 194.5 (CO),
C
31H46N6O9Re, 833.3).
145.2 (C-3/5 (pz)), 134.0 (C-3/5 (pz)), 125.1 (C-4 (pz)), 55.8 (CH2),
55.2 (CH2), 53.2 (CH2), 49.4 (CH2), 41.6 (CH2). ESI-MS m/z: 455.1
(Mþ, calcd for C11H16N4O4Re, 455.1). RP-HPLC, retention time:
tR ¼ 11.3 min.
4.3.4. fac-[Re(CO)3{HC[4-(HOCH2)pz]3}]Br (Re4)
To a solution of Re1 (26 mg, 0.042 mmol) in CH2Cl2 (10 mL)
at ꢀ20 ꢂC was added 0.38 mL of 1.0 M boron tribromide in CH2Cl2.
The mixture was allowed to react for 3 h at ꢀ20 ꢂC and was sub-
sequently quenched with water. The organic phase was separated,
dried over MgSO4 and the solvent removed under vacuum. The
residue was purified by gradient HPLC (100% aqueous 0.1%
CF3COOH solution / 100% CH3CN) using a Nucleosil column
4.4. X-ray diffraction analysis
Single crystals of Re7 adequate for X-ray diffraction analysis
were obtained from a saturated solution of the complex in meth-
anol. The X-ray diffraction analysis has been performed on a Bruker
(10
mm, 250 mm ꢁ 7 mm) and a flow rate of 2 mL/min. Compound

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 138e148
147
AXS APEX CCD area detector diffractometer, using graphite mono-
4.7. Biodistribution studies
ꢀ
chromatic Mo Ka radiation (0.71073 A). The crystal data are sum-
marized in Table 3.
The biodistribution of complexes Tc1eTc3 and Tc6 was evalu-
ated in groups of 3e5 female CD-1 mice (randomly bred, Charles
River) weighing approximately 20e25 g each, at 1 h and 2 h after
Empirical absorption correction was carried out using SADABS
[32]. Data collection and data reduction for 1 were done with
SMART and SAINT programs [33]. The structure was solved by
direct methods with SIR97 [34] and refined by full-matrix least-
squares analysis with SHELXL97 [35] using the WINGX [36] suite of
programs. Non-hydrogen atoms were refined with anisotropic
thermal parameters whereas H-atoms were placed in idealized
positions and allowed to refine riding on the parent C atom. Mo-
lecular graphics were prepared using ORTEP3 [37]. Crystallographic
data for the structure of 1 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC-835282. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Fax:
int. code þ44-1223/336-033; e-mail: deposit@ccdc.cam.ac.uk].
intravenous administration with 100 mL (5e11 MBq) of each prep-
aration via the tail vein as previously described. Studies were car-
ried out according the EU guidelines for Animal Care and Ethic for
Animal Experiments. Biodistribution results were expressed as
percentage of the injected dose per gram tissue (% ID/g) and are
shown in Table 2 and Table S1.
4.8. Metabolism studies
Blood and urine samples of mice injected with Tc1eTc3 and Tc6,
collected at sacrifice time, were analyzed by HPLC to check the
in vivo stability of the complexes. Prior to HPLC analysis urine
samples were centrifuged and the serum from the blood samples
was separated and treated with ethanol to precipitate proteins. The
supernatant from these biological samples was analyzed using the
conditions referred above for the HPLC analysis of the Re and 99mTc
complexes.
4.5. General procedure for the synthesis of the 99mTc complexes
(Tc1eTc3 and Tc6)
In a nitrogen-purged glass vial, 900 mL of the organometallic pre-
cursor fac-[99mTc(OH2)3(CO)3]þ at pH ¼ 4 (Tc1eTc3) or at pH ¼ 7.4
(Tc6) were added to 100
m e
L of an ethanolic solution (10ꢀ2
Acknowledgements
6.2 ꢁ 10ꢀ2 M) of L1eL3 or to an aqueous solution of L5 (10ꢀ4 M). The
resulting solutionswere heated at100ꢂC for30e60min. Aftercooling
to room temperature, the pH of the solutions was adjusted to pH 7.4
with 1 N NaOH in the case of Tc1eTc3. Complexes Tc1eTc3 and Tc6
have been obtained typically with a radiochemical yield ꢄ95%, as
checked by gradient HPLC analysis, and were used in the bio-
distribution and metabolism studies without further purification.
The authors acknowledge Covidien (Mallinckrodt Med. BV) for
financial support. L. Maria is grateful to Fundação para a Ciência e a
Tecnologia (“Ciência 2008” Programme). J. Marçalo is acknowledged
for the ESI-MS analyses which were run on a QITMS instrument
acquired with the support of the Programa Nacional de Reequipa-
mento Científico (Contract REDE/1503/REM/2005 e ITN) of Funda-
ção para a Ciência e a Tecnologia and is part of RNEM e Rede
Nacional de Espectrometria de Massa.
4.6. Octanolewater partition coefficient
The log Po/w values of complexes Tc1eTc3 (Table 2) were
determined by the shake-flask method [38] under physiological
conditions (n-octanol/0.1 M PBS, pH 7.4).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2013.11.013.
Table 3
Crystallographic data for complex Re7.
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Empirical formula
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Crystal system
Crystal size/mm
Space group
C11H16BrN4O4Re$H2O
552.40
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