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Synthesis and biological evaluation of a novel 99mTc-cyclopentadienyltricarbonyl technetium complex as a new potential brain perfusion imaging agent.

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Full Paper
Received: 8 March 2011
Revised: 23 May 2011
Accepted: 19 June 2011
Published online in Wiley Online Library: 10 August 2011
(wileyonlinelibrary.com) DOI 10.1002/aoc.1827
Synthesis and biological evaluation of a novel
99m Tc-cyclopentadienyltricarbonyl technetium
complex as a new potential brain perfusion
imaging agent
Nadia Malek-Saieda∗ , Radhia El Aissia – c , Sonia Ladeirad and Eric Benoistb,c∗
A new cytectrene prototype of general formula RCpTc(CO)3 (R = C6 H5 NHCO, Cp = cyclopentadienyl moiety) has been synthesized
from N-phenylferrocenecarboxamide 2, characterized and evaluated as a potential brain perfusion imaging agent. An improved
procedure has been developed to obtain both the ligand 2, characterized by its solid-state structure (orthorhombic, Pccn,
a = 10.4443(2) Å, b = 26.1467(6) Å, c = 9.9977(3) Å), and the corresponding metallic Tc- and Re-complexes in good yield.
These latter complexes possessed similar HPLC retention times, thereby indicating identity of their molecular structures. The
Tc-complex 99m Tc-2 is lipophilic enough to cross the blood-brain barrier. This complex exhibits good brain uptake (1.41%
injected dose per gram tissue at 5 min) combined with a fairly good retention of radioactivity in brain (0.48% injected dose
per gram tissue after 1 h). Then, the distribution of the activity at 5 min post-injection in various rat brain regions showed a
higher accumulation in the hippocampus area. The new 99m Tc-cyclopentadienyltricarbonyl technetium complex reported here
showed promising biological results, making it an interesting base for the development of a new generation of cytectrene as
c 2011 John Wiley & Sons, Ltd.
brain perfusion imaging agent. Copyright Keywords: radiopharmaceuticals; technetium(I); bioorganometallic chemistry; X-ray structure; biological applications
Introduction
680
Particular interest has been devoted to the development of
new 99m Tc-based radiopharmaceuticals, mainly owing to the
convenient availability in any hospital of the technetium-99m
radioisotope (through commercial 99 Mo/99m Tc generators) and
its ideal nuclear decay properties, e.g. a 140 keV γ -ray with 89%
abundance and a short half-life ca. 6.02 h, which minimizes the
radiation dose to the patient.[1 – 3] A plethora of chelating molecules
have been developed to efficiently coordinate diverse technetium
species like Tc(V) and Tc(I) cores, and to form stable 99m Tccomplexes under physiological conditions.[4 – 6] The structure of
these chelators is largely determined by the oxidation states of the
technetium-99m, but also by the nature of the biological target.[7]
In particular, in the case of 99m Tc-based brain imaging agents, the
technetium complexes require structural features summarized
as follows: a relative small size (MW < 600 Da), an appropriate
lipophilicity (logPo/w = 0.5–2.5) and a net charge of zero in
order to cross the blood–brain barrier by diffusion.[8,9] Among
those, 99m Tc-cyclopentadienyltricarbonyl technetium complexes
of general formula RCpTc(CO)3 (R = organic part or biomolecule, Cp
= cyclopentadienyl moiety) have been extensively studied.[10 – 15]
The neutral and lipophilic ‘piano stool’ organometallic core,
‘CpM(CO)3 ’ (M = 99m Tc or Re), is particularly attractive since
it has a low molecular weight, exhibits a high chemical and in vivo
stability resulting from the low-spin d6 electron configuration,
and unlike reported Tc(V)/Re(V) complexes, it does not possess
additional stereocenters, which often complicate the purification
process of radiopharmaceuticals.[16] Over the past decade, the
research of the radiopharmaceutical unit of the Centre National
des Sciences et Technologies Nucléaires, (Tunisia), have been
Appl. Organometal. Chem. 2011, 25, 680–686
focused on small Tc-cyclopentadienyl derivatives called cytectrene
I and II, as highlighted in Scheme 1.[17,18] Professor Saidi ‘s team
demonstrated that these complexes exhibited both high uptake
in the brain and fast blood clearance, these results making
the application of this kind of ‘CpTc(CO)3 ’ moiety-based small
complexes for brain imaging promising. Nevertheless, from a
chemical point of view, the structural modifications of these Tccomplexes are restricted and/or not easy, limiting the access to
novel brain imaging agents based on cytectrene I and II derivatives.
∗
Correspondence to: Nadia Malek-Saied Radiopharmaceutical Unit, Centre
National des Sciences et Technologies Nucléaires, Sidi Thabet 2020, Tunisia.
E-mail: nadia.malek@gmail.com;
Eric Benoist, Université de Toulouse, UPS, Laboratoire de Synthèse et PhysicoChimie de Molécules d’Intérêt Biologique, SPCMIB, UMR 5068, 118, route de
Narbonne, F-31062 Toulouse cedex 9, France.
E-mail: benoist@chimie.ups-tlse.fr
a Radiopharmaceutical Unit, Centre National des Sciences et Technologies
Nucléaires, Sidi Thabet 2020, Tunisia
b CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt
Biologique, SPCMIB, UMR 5068, 118, route de Narbonne, F-31062 Toulouse
cedex 9, France
c Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de
Molécules d’Intérêt Biologique, SPCMIB, UMR 5068, 118, route de Narbonne,
F-31062 Toulouse cedex 9, France
d Université de Toulouse, UPS and CNRS, Institut de Chimie de Toulouse, FR2599,
118, route de Narbonne, F-31062 Toulouse cedex 9, France
c 2011 John Wiley & Sons, Ltd.
Copyright 99m Tc-cyclopentadienyltricarbonyl
technetium complex
R
O
R = CH3, Cytectrene I
N
99mTc-2
O
R = CH(CH3)2, Cytectrene II
N
H
O
Tc
OC
Tc
CO
CO
OC
CO
CO
Scheme 1. Structural features of cytetrenes I and II and evaluated 99m Tc-2 complex.
With these criteria in mind, we anticipated that the substitution
of both the ester function and the piperidine derivatives of
cytectrene I and II by an amido group and an aryl ring, respectively,
should (i) lead, in a few steps, to a new cytectrene with suitable
lipophilicity to cross the blood–brain barrier, and (ii) offer us the
possibility to develop a multitude of other cytectrenes based
on this new model, the presence of an aryl group allowing
easy chemical substitutions. As a proof of example, Tc-complex
99m Tc-2, synthesized from N-phenylferrocenecarboxamide 2, was
evaluated as a new model of cytectrene (Scheme 1). If the synthesis
of the molecule 2 was previously reported by Jaouen’s team,[19]
to the best of our knowledge, no preparation and evaluation
of the corresponding 99m Tc-complex have been described. In
this contribution, an improved synthesis of the ligand 2 and its
characterization including the solid-state structure, as well as the
preparation of the corresponding CpM(CO)3 complexes on the
macroscopic scale (M = Re) and radiotracer scale (M = 99m Tc), are
presented. The biological evaluation of the Tc-complex 99m Tc-2,
comprising the assessment of its lipophilicity and a biodistribution
study on healthy male Wistar rats, was also investigated.
Results and Discussion
Synthesis of Ligand 2 and its Corresponding Rhenium Complex
Re-2
Ferrocene amides were prepared by amide formation between
amines and ferrocene carboxylic acid derivatives, generally in
the presence of a base to prevent protonation of the attacking
nucleophile.[20] In our case, N-phenylferrocenecarboxamide ligand
2 was first prepared according to a previously reported one-pot
procedure[19] by in situ activation of ferrocene carboxylic acid
by oxalyl chloride and then reaction of the formed ferrocenoyl
chloride with an excess of aniline at ambient temperature
(Scheme 2). With this method, the molecule 2 was prepared in
64% yield. On the other hand, by using the following two-step
procedure, 2 was afforded with a 93% overall yield: (i) synthesis and
isolation of the ferrocenoyl chloride 1 (by recrystallization); and
(ii) treatment of 1 with aniline in the presence of Hünig’s base, in
freshly distilled tetrahydrifuran (THF) at ambient temperature.
A similar convenient two-steps synthesis of 2 was reported
by Rotello’s team using fluorocarbonylferrocene as versatile
intermediate and DMAP as a catalyst for the reaction of the
fluorocarbonylferrocene with the poorly nucleophilic aniline.[21] In
our hands, no difference in yield was observed using DMAP instead
of Hünig’s base in the second step. As expected, compound 2
exhibits a typical strong νC O band (amide I band) at 1643 cm−1
in IR and characteristic ferrocenyl moiety peaks in 13 C NMR
(68–77 ppm).[22] The structure of 2 was then confirmed by an
X-ray study. An ORTEP diagram of this molecule with its atomic
numbering scheme and significant crystallographic data are given
in Fig. 1 and Table 1, respectively.
The solid-state structure shows no unexpected features. The
bond lengths and angles about the cyclopentadienyl rings as well
as the amide bond length are unexceptional and close to those
reported for analogous N-substituted ferrocenecarboxamides.[23]
The crystal cohesion is provided by a network of inter-layer
hydrogen bonds involving the amide NH group (which acts
as a hydrogen bond donor) of one molecule and the carbonyl
oxygen (which acts as an acceptor) of a neighboring molecule.
These N–H· · ·O hydrogen bonds lead to an infinitive chain of
ligand 2 along the crystallographic a-axis. It is noteworthy that
the N–H· · ·O hydrogen bond distance is in the upper range of
previously reported values for this kind of N-monosubstituted
iii
O
COOH
COCl
N
H
Fe
i
Fe
1
ii
Fe
2
◦
Appl. Organometal. Chem. 2011, 25, 680–686
c 2011 John Wiley & Sons, Ltd.
Copyright wileyonlinelibrary.com/journal/aoc
681
Scheme 2. Synthesis of 2: (i) oxalyl chloride, CH2 Cl2 , 0 C then 4 h at ambient temperature, 97%; (ii) aniline, Hünig’s base, THF, ambient temperature, 4 h,
96%; (iii) Top et al.,[19] 64%.
N. Malek-Saied et al.
of ‘CpM(CO)3 ’-type complexes (M = 99m Tc or Re) from ferrocene
derivatives. Jaouen’s team reported recently the preparation of
complex Re-2 in 43% yield via a single-ligand transfer using the
water-soluble cation Re(CO)6 + as ReI (CO)3 source.[19] Nevertheless,
the [Re(CO)6 ][BF4 ] salt is not commercially available and requires
toxic sodium amalgam for its synthesis.[28] To circumvent this
problem, we investigated another convenient synthetic route
using classical tricarbonylrhenium precursors such as Re(CO)5 Cl
and [Re(CO)3 Cl3 ][NEt4 ]2 , as reported previously.[26] Briefly, 2 and
the rhenium precursor were refluxed for 2 h at 160 ◦ C in a 0.1
M HCl/DMSO mixture. Surprisingly, first attempts using Re(CO)5 Cl
as rhenium(I) precursor failed, the desired complex Re-2 being
observed only at trace level (less than 5%). On the other hand,
with [Re(CO)3 Cl3 ][NEt4 ]2 as starting material, the desired complex
Re-2 was obtained in good yield. It is noteworthy that this latter
procedure allowed the preparation of Re-2 in 75% yield vs 43%
with Jaouen’s method. Spectroscopic data were consistent with
the structure of the Re-complex, e.g. the facial arrangement of the
carbonyl groups is evidenced by the CO-stretching absorptions in
the IR spectrum (2027–1922 cm−1 ).
Figure 1. Molecular structure of 2; hydrogen atoms have been omitted for
clarity, excepted for the amide group nitrogen. Selected bond lengths (Å)
and angles (deg): C(10)–Fe(1), 2.033(2); C(10)–C(11), 1.483(3); C(11)–O(1),
1.235(3); C(11)–N(1), 1.360(3); C(12)–N(1), 1.426(3); Fe(1)–C(10)–C(11),
125.49(17); C(10)–C(11)–O(1), 121.4(2); C(10)–C(11)–N(1), 115.2(2);
O(1)–C(11)–N(1), 123.3(2).
Table 1. Selected structural parameters for 2
Formula
M
Crystal system, space group
Crystal size (mm)
Unit cell dimensions
V (Å 3 )
Z, d (g cm−3 )
T (K)
F(000)
Reflections collected/unique
Data/restraints/parameters
Final R indices [I > 2σ (I)]
R indices (all data)
Goodness-of-fit on F 2
Largest difference peak and hole
Radiolabeling of 2 with 99m Tc
C17 H15 FeNO
305.15
orthorhombic, Pccn
0.28 × 0.08 × 0.02
a = 10.4443(2) Å, α = 90◦
b = 26.1467(6) Å, β = 90◦
c = 9.9977(3) Å, γ = 90 deg.
2730.21(12)
8, 1.485
193(2)
1264
38 614/4159 [R(int) = 0.1024]
4159/1/184
R1 = 0.0447, wR2 = 0.0835
R1 = 0.1074, wR2 = 0.1032
0.991
0.425 and −0.314 e Å −3
The double-ligand transfer reaction was privileged for the Tcradiolabeling of 2. We demonstrated previously that microwave
irradiation could significantly improve the 99m Tc-labeling reaction
kinetics of ferrocenecarboxamide derivatives compared with
classical heating.[29] We were delighted to see that, under
microwave irradiation for ca. 5 min (5 irradiation periods of
40 s plus a 30 s period between two irradiations), the expected
radiocomplex 99m Tc-2 could be prepared in 85% yield. After HPLC
purification, the radiochemical purity was more than 98%. The
structure of the 99m Tc-complex was established by comparison of
its HPLC retention time with that of the well-characterized rhenium
complex, as illustrated in Fig. 2. The stability of the purified complex
was assessed in at least solution and in rat serum. When 99m Tc-2
was incubated with serum at 37 ◦ C, ITLC analysis of the samples
after 30 min and 1 h of incubation showed broadly no free 99m Tc
release. After 24 h, less than 15% complex had decomposed to
pertechnetate (Fig. 3). It is clear from this study that the complex
99m Tc-2 was stable enough in solution and in rat serum, thus
indicating its high stability.
It is generally accepted that only the lipid pathway provides
access to the brain for 99m Tc-complexes.[9] Consequently, radiocomplexes have to be relatively lipophilic. The lipophilic character
of our radiocomplex was assessed by determination of the partition
coefficient (P) in physiological conditions (0.05 M Tris–HCl buffer,
pH 7.4/n-octanol) and was expressed as logPo/w . A value of 1.60
was found, indicating that 99m Tc-2 is moderately lipophilic. Interestingly, this value is within the range for related radiocomplexes
ferrocenecarboxamides [N(1)–H(100)· · ·O(1) = 3.043(3) Å, with
N(1)–H(100) = 0.853(10) Å and N(1)–H(100)–O(1) = 154(2)◦ ].[23]
In order to characterize the technetium-99m complex, we
prepared first the corresponding rhenium analog (Scheme 3).
Depending of both the nature of the ferrocene derivatives and
the oxidation state of the metallic precursor, different synthetic
strategies (via double-ligand transfer[13,24,25] or single-ligand
transfer reactions[18,25 – 27] ) were described for the preparation
O
N
H
OC
CO
CO
Re-2
N
H
N
H
(i)
Re
O
O
Fe
(ii)
Tc
OC
CO
CO
99mTc-2
2
682
Scheme 3. Preparation of Re/Tc-complexes: (i) [Re(CO)3 Cl3 ][NEt4 ]2 , DMF, 160 ◦ C, 2 h, 75%; (ii) Mn(CO)5 , [Na][99m TcO4 ], DMSO, MW, 5 × 40 s, 85%.
wileyonlinelibrary.com/journal/aoc
c 2011 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2011, 25, 680–686
99m Tc-cyclopentadienyltricarbonyl
technetium complex
Arbitrary unit
Re-2
Tc-2
0
1
2
3
4
5
6
7
8
time (min.)
Figure 2. HLPC comparison of rhenium complex Re-2 (gray line) and
radiocomplex 99m Tc-2 (black line).
Figure 4. Summary of the biodistribution data (percentage of injected
dose per gram of tissue) of the radiocomplex 99m Tc-2 in healthy Wistar
rats at 5 and 60 min post-injection (n = 3).
Figure 5. Time course of brain uptake (percentage of injected dose per
gram of tissue) after injection of 99m Tc-2 in rats.
Figure 3. Stability study of 99m Tc-2 in serum at 37 ◦ C.
able to cross the blood–brain barrier (logPo/w =
It
is noteworthy that the synthesized cytectrene was slightly more
lipophilic than both cytectrenes I (logP = 0.93) and II (logP = 1.23),
as expected.[17,31]
0.5–2.5).[9,30]
In Vivo Studies
Appl. Organometal. Chem. 2011, 25, 680–686
Tissue
Medulla
Striatum
Hippocampus
Cortex
% ID g−1
0.60 ± 0.02
0.50 ± 0.04
0.78 ± 0.02
0.54 ± 0.02
(1.3% ID g−1 at 2 min post-injection).[32,33] Additionally, although
a relatively fast brain washout was observed for 99m Tc-2, the
retention of the radioactivity in brain was fairly good at 60 min
post-injection (0.48% ID g−1 ), as illustrated in Fig. 5. The ability of
99m
Tc-2 to penetrate the blood–brain barrier can be explained by
its good properties in terms of charge, small size, lipophilicity and
in vitro and in vivo stability. These good biological properties are
in agreement with the possibility that this kind of 99m Tc-complex
could be used to monitor regional cerebral blood perfusion.
The biodistribution of the activity in selected regions of the brain
was also investigated at 5 min (Table 2). The highest concentration
of radioactivity was found in the hippocampus area, where the
5-HT1A receptor density was important. For the cortex region,
where the 5-HT1A receptor density was less important than in
the hippocampus, the radioactivity value was lower. Nevertheless,
the repartition of the radioactivity in the different regions of the
brain seems too homogeneous to clearly establish a correlation
c 2011 John Wiley & Sons, Ltd.
Copyright wileyonlinelibrary.com/journal/aoc
683
The in vivo behavior of 99m Tc-2 was evaluated in healthy Wistar
rats at different time points, in order to check its ability to cross the
blood–brain barrier. Figure 4 shows the tissue distribution results
expressed as percentage of injected dose per gram tissue (% ID
g−1 ) at 5 and 60 min, in the most relevant organs. Interestingly,
the complex exhibits a fast blood clearance (0.33 and 0.22% at 5
and 60 min, respectively) combined with a good brain uptake at
earlier post-injection time (1.41% ID g−1 at 5 min). As expected
for lipophilic compounds, there is a high and rapid liver uptake
that decreases over the time. Excretion of the radioactivity occurs
mainly via the renal urinary pathway (1.66 and 1.97% at 5 and
60 min respectively) and through the hepatobiliary system, as
evidenced by the decreasing activity in liver.
These data were promising since the complex showed fast
blood clearance and excellent brain uptake at 5 min (1.41% ID
g−1 ). It is noteworthy that the brain to blood ratio was >2
at 60 min post-injection. More interestingly, the brain uptake
value was comparable to the value reported for the ECD, a
radiopharmaceutical used clinically for brain perfusion studies
Table 2. Regional distribution in rat brain at 5 min post injection
(percentage of injected dose per gram of tissue, % ID g−1 ; n = 3)
N. Malek-Saied et al.
between the repartition of the 5-HT1A receptors and the brain
distribution of the radioactivity. Further studies are ongoing to
clarify this point.
To conclude, we demonstrated that the Tc-complex is lipophilic
enough to cross the blood–brain barrier and exhibits good brain
retention. Surprisingly, if the complex 99m Tc-2 is a little more
lipophilic than cytectrenes I and II, its brain uptake is slightly
lower (0.51 vs 0.87% ID g−1 for cytectrene I and 0.75% ID
g−1 for cytectrene II at 20 min post-injection).[17] Nevertheless,
these encouraging biological results conjugated to the fact that
chemical and structural modifications could be easily achieved
make this model an interesting base for the development of a new
generation of cytectrene as a brain perfusion imaging agent.
Conclusion
The design, preparation and biological evaluation of a new
lipophilic cytectrene as a 99m Tc-based brain perfusion imaging
agent were the main goals of this work. The synthesis and
characterization of the Tc-complex required the preparation of
the previously described N-phenylferrocenecarboxamide and its
corresponding rhenium complex. We developed an improved
procedure, allowing us to obtain the ligand and the Re-complex
in 96 and 75% yield, respectively. Using microwave irradiation,
the Tc-radiolabeling procedure was compatible with the period of
technetium-99m and gave 99m Tc-2 in good yield.
The preliminary biological studies are interesting. Firstly, the
Tc-complex is lipophilic enough to cross the blood–brain barrier,
as expected (logP = 1.60). Secondly, biodistribution studies in
healthy male rats indicated that this complex presented a very
good brain uptake combined with a fairly good retention of
radioactivity in brain (0.48% ID g−1 after 1 h). Then, the distribution
of the activity at 5 min post-injection in various rat brain regions
showed a higher accumulation in the hippocampus area, which
is rich in 5-HT1A receptors. The biological behaviour mentioned
above and the data presented in this paper suggest that our
Tc-complex probably passes through the blood–brain barrier
via a simple diffusion mechanism rather than via a specific
5-HT1A receptor targeting. Nevertheless, these first promising
results encourage us to pursue our efforts and to develop
other 99m Tc-complexes based on this new cytectrene prototype
system in order to improve the biological behavior, especially
the brain uptake and the 5-HT1A receptor affinity. In particular,
we are currently carrying out structural modifications on the
ferrocenecarboxamide backbone, such as the introduction of an
arylpiperazine pharmacophore, this pharmacophore being found
in numerous selective 5-HT1A imaging agents.[34]
Experimental
Materials and Equipment
684
All purchased chemicals were of the highest purity commercially
available and used without further purification. Analytical-grade
solvents were used and not further purified unless specified.
Reactions were monitored by TLC on a Kieselgel 60 F254 (Merck)
on aluminum support under UV light (254 nm). Chromatographic
purification was conducted using ‘gravity’ silica gel obtained
from Merck. Re(CO)5 Cl was purchased from Aldrich Chem. Co.
NMR spectra were recorded with a Bruker AC 300 (300 MHz)
spectrometer. Chemical shifts are indicated in δ values (ppm)
wileyonlinelibrary.com/journal/aoc
downfield from internal TMS, and coupling constants (J) are given
in Hertz. Infrared spectra were recorded on a Perkin Elmer FTIR 1725
spectrophotometer in the range 4000–400 cm−1 . Mass spectra
(chemical ionization) were obtained on a DSQ2 Thermofisher
mass spectrometer. Melting points were determined on a Wagner
& Mung Köfler bench and are uncorrected.
RP-HPLC were performed using a Waters 600E gradient
chromatography coupled to a UV–vis detector (ICS) and a
γ -detector (Raytest) for the radiolabeled compound. Analysis and
purification of the 99m Tc-complex were carried out on a Nucleodur
C18 endcapped RP column (Macherey-Nagel analytical column,
125 × 4 mm, 5 µm). The RP-HPLC conditions were as follows:
the flow rate was 1 ml/min, isocratic gradient mixture of 62%
methanol, 38% H2 O and 0.1% TFA. Then, radiochemical purity was
assessed by thin-layer chromatography on Alugram C18W plates
(eluent MeOH–H2 O–TFA, 62 : 38:0.1) using a Berthold LB 2832
detector coupled to a radiochromatogram scanner.
Synthesis of N-Phenylferrocenecarboxamide, 2
One-pot method
The desired compound 2 was prepared according to a literature
procedure.[19] A small improvement of yield (64 vs 57%) was
obtained.
Two-step synthesis
For ferrocenoyl chloride, 1, under a nitrogen atmosphere, to a
stirred solution of ferrocene carboxylic acid (1.20 g, 5.2 mmol) in
freshly distilled dichloromethane (10 ml), was added dropwise
oxalyl chloride (4 ml, 46.8 mmol), at 0 ◦ C. The resulting mixture
was stirred at ambient temperature for 4 h, then the solvent was
removed under reduce pressure. The solution was triturated with
hot pentane, then the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was crystallized from pentane to give a red crystalline solid (1.25 g, 97%).
Compound 1 was characterized by comparing its IR spectrum
[particularly the ν(CO) stretching band at 1760 cm−1 ] with that
found in the literature.[20]
For N-phenylferrocenecarboxamide, 2, under a nitrogen atmosphere, to a stirred solution of aniline (274 µl, 3 mmol) and Hünig’s
base (536 µl, 3 mmol) in freshly distilled THF (4 ml), was added
dropwise a THF solution (6 ml) of ferrocenoyl chloride, 1 (250 mg,
1 mmol). The resulting mixture was stirred at ambient temperature for 4 h, then the solvent was removed under reduce pressure.
The resulting solution was diluted with dichloromethane (15 ml)
and washed successively with 1N HCl solution (2 × 20 ml) and
saturated NaHCO3 solution (1 × 30 ml). The organic extract was
dried over Na2 SO4 and evaporated. The crude product was purified by column chromatography on silica gel (eluent CH2 Cl2 then
CH2 Cl2 –ethyl acetate, 95 : 5) to afford the desired product as an
orange solid (293 mg, 96% yield). The crystals suitable for X-ray
structure determination were obtained by slow recrystallization
from a dichloromethane solution. Compound 2 was characterized
by comparing the NMR spectra, IR spectrum and mass spectrum
with those found in the literature.[19] Additional analytical data: IR
(KBr): νC O = 1643 cm−1 .
X-ray Data Collection and Refinement for 2
The selected crystals of 2 were mounted on a glass fibber
using perfluoropolyether oil and cooled rapidly to 193 K in a
c 2011 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2011, 25, 680–686
99m Tc-cyclopentadienyltricarbonyl
technetium complex
stream of cold N2 . For all the structures data were collected at
low temperature (193 K) on a Bruker-AXS APEX II diffractometer
equipped with the Bruker Kryo-Flex cooler device and using a
graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The
structure was solved by direct methods[35] and all nonhydrogen
atoms were refined anisotropically using the least-squares method
on F 2 .[36] ORTEP plot was drawn with the program ORTEP-3 for
Windows at a probability of 50%.[37] Crystallographic data for
the structural analysis have been deposited with the Cambridge
Crystallographic Data Centre, CCDC 804 953.
Synthesis of tricarbonyl(N-phenylcyclopentadienylcarboxamide)rhenium, Re-2
For synthesis of [Re(CO)3 Cl3 ][NEt4 ]2 , according to a slight modification of the literature,[38] a degassed suspension of Re(CO)5 Cl
(0.36 g, 1 mmol) and NEt4 Cl (0.86 g, 5.2 mmol) in freshly distilled
diglyme (130 ml) was stirred at 130 ◦ C for 3 h, under a nitrogen
atmosphere. The formed solid was then collected and washed
with absolute ethanol (10 ml). The residue was dissolved entirely
on shaking with an additional amount of absolute ethanol (10 ml),
then diethyl ether was layered on top to afford a precipitate. The
white solid was collected, washed with cold diethyl ether then
dried to yield a white solid (549 mg, 86%). The rhenium precursor
was characterized by comparing its elemental analysis with that
reported in the literature.[38] Additional analytical data: IR (KBr):
νCO = 1887, 2014 cm−1 ; m.p. > 260 ◦ C.
For synthesis of rhenium complex, Re-2, according to a slight
modification of the literature,[18] a solution of [Re(CO)3 Cl3 ][NEt4 ]2
(30 mg, 0.047 mmol) and 2 (30 mg, 0.098 mmol) in DMF (2.5 ml)
and a solution of 0.1N HCl (1.8 ml) were combined in a 5 ml
glass vial. The mixture was purged with argon and the vial was
sealed with a Teflon cap. The solution was stirred vigourously
for 2 h at 160 ◦ C. After cooling, the cap was removed and the
solution poured in a dichloromethane solution (10 ml). After three
washings with water (3×10 ml), the organic extract was dried over
Na2 SO4 and evaporated. The crude product was purified by column
chromatography on silica gel (eluent petroleum ether–ethyl
acetate, 80 : 20 then 70 : 30) to afford the desired complex Re2 as an orange powder (16 mg, 75% yield). The rhenium complex
Re-2 was characterized by comparing both the elemental analysis
and the mass spectrum with those reported in the literature.[19]
The proton NMR spectrum having been made in another solvent
than that used in Top et al.,[19] we added it.
Caution: the manipulation of sealed reaction vial during and
after the reaction was done in a fume hood with suitable protective
equipment.
1 H NMR (300 MHz, CDCl ): δ (ppm) = 5.45 (bs, 2H, CH ), 6.00
3
H
Cp
(ls, 2H, CHCp ), 7.20 (t, 1H, J = 7.8 Hz, CHAr ), 7.39 (t, 2H, J = 7.8 Hz,
CHAr ), 7.54 (d, 2H, J = 7.8 Hz, CHAr ). 9.10 (s, 1H, NH).
radiochemical purity (>98%). The retention time of the Tc-complex
was similar to that of the cold rhenium analog, −3.89 min for
99m Tc-2 vs 3.70 min for Re-2, confirming the chemical identity of
99m
Tc-2.
Octanol–Water Partition Coefficient (logP) for 99m Tc-2
A mixture of 1 ml of 0.05 M Tris–HCl buffer, pH 7.4 with
approximatively 5.9 MBq of HPLC purified complex 99m Tc-2 (10 µl)
and 1 ml of n-octanol was shaken at room temperature for 1 min
then centrifuged at 3500 rpm for 10 min. Aliquots of 10 µl of the
buffer solution and of the organic layer were counted. The partition
coefficient was calculated using the formula: logP = counts in
n-octanol/counts in buffer. The reported value represents the
average of triplicate measurements.
In Vitro Stability Studies
The in vitro stability of the purified complex 99m Tc-2 was evaluated
at different time points using the following procedure: in a
borosilicated vial, 99m Tc-2 (100 µl) was added to 900 µl of fresh rat
serum. Aliquots were withdrawn during the incubation at different
time intervals until 24 h and subjected to chromatography using
ITLC-GS paper and AcOEt–hexane 7 : 3 w/w as solvent. Any
increase in the free pertechnetate was considered to be the
degree of degradation. The complex was found to be stable.
Biodistribution of 99m Tc-2 in Healthy Wistar Rats
All animal experiments were performed in compliance with
Tunisian laws relating to the conduct of animal experimentation. A
300 µl aliquot of 99m Tc-2 complex (20 MBq, saline–10% absolute
ethanol) was injected into the tail vein. After the injection, the
healthy male Wistar rats (250–300 g), anesthetized with diethyl
ether, were sacrificed at 5, 10, 15, 20, 30 and 60 min postinjection (n = 3). The major organs (liver, spleen, heart, lungs,
kidneys and brain), as well as selected regions of the brain, were
dissected, weighed and their radioactivity measured in a Packard
autogamma counter. Results, expressed as percentage of injected
dose per gram of tissue, are summarized in Fig. 4 and Table 2. The
percentage of injected dose per gram was calculated by dividing
the % ID per organ by the weight of the organ or tissue.
Acknowledgments
We are grateful to Dr Y. Coulais (Purpan Hospital, Toulouse, France)
for the Tc-radiolabeling procedure and Professor M. Saidi (Centre
National des Sciences et Technologies Nucléaires, Tunis, Tunisie)
for helpful discussions.
References
Radiolabeling Procedure: Preparation of the
99m Tc-2
99m Tc
Complex
Appl. Organometal. Chem. 2011, 25, 680–686
c 2011 John Wiley & Sons, Ltd.
Copyright wileyonlinelibrary.com/journal/aoc
685
A 2.5 mg aliquot of 2 and 5.5 mg of Mn(CO)5 Br dissolved in DMSO
(300 µl) were added to 0.07 ml of sodium pertechnetate eluate
(∼300 MBq). The reaction mixture was purged with nitrogen,
then heated by microwave irradiation (800 W) for five periods
of 40 s (a 30 s period was observed between two irradiations).
Without optimizing the conditions, the resulting Tc-complex was
obtained in 85% yield. After RP-HPLC purification (see above
for elution conditions), complex 99m Tc-2 was obtained with high
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complex, evaluation, biological, 99mtc, brain, technetium, new, potential, synthesis, cyclopentadienyltricarbonyl, agenti, imagine, perfusion, novem
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