Preparation and biodistribution of 1-((2-methoxyphenyl) piperazine)ferrocenecarboxamide labeled with technetium-99m as a potential brain receptor imaging agent

Article abstract of DOI:10.1016/j.ejmech.2015.05.014

The goal of this study is to develop a novel brain receptor imaging agent. This study reports the synthesis, characterization and the biological evaluation of 1-((2-methoxyphenyl) piperazine)ferrocenecarboxamide labeled with technetium-99 m (^99mTc-MP). The ^99mTc-MP was obtained quickly (radiolabelling time < 5 min), in 90% yield. The ^99mTc-complex, characterized by HPLC (20-50% ACN of 0 at 5 min then 50% ACN of 5 at 17 min to finally with 50 at 20% ACN of 17 at 20 min), is stable, neutral and lipophilic enough to cross the blood-brain barrier which was confirmed by octanol/water partition coefficient (LogP = 1.82). In vivo biodistribution indicated that this complex had exceptional brain uptake (2.47% ID/g at 5 min and 0.75% ID/g at 60 min). The distribution of the activity at 15 min post-injection in various rat brain regions showed a higher accumulation in the hippocampus area. After blocking with 8-hydroxy-2-(dipropylamino) tetralin, the uptake of hippocampus was decreased significantly from 0.87% ID/g to 0.21% ID/g at 15 min p.i., while the cerebellum had no significant decrease. The new ^99mTc-cyclopentadienyltricarbonyl technetium complex reported here showed promising biological results, making it an interesting starting point for the development of a new ^99mTc-complex as brain receptor imaging agent.

Full text of DOI:10.1016/j.ejmech.2015.05.014

European Journal of Medicinal Chemistry 97 (2015) 280e288
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Review article
Preparation and biodistribution of 1-((2-methoxyphenyl) piperazine)
ferrocenecarboxamide labeled with technetium-99m as a potential
brain receptor imaging agent
Nadia Malek Saied , Najoua Mejri , Radhia El Aissi a c, Eric Benoist b c
Mouldi Saidi
a
a
,
*
a
,
b
,
, , **
,
a
Radiopharmaceutical Unit, National Centre of Sciences and Nuclear Technology, Sidi Thabet 2020, Tunisia
CNRS, Laboratoire de Synth ꢀe se et Physico-Chimie de Mol ꢁe cules d'Int ꢁe r ^e t Biologique, SPCMIB, UMR 5068, 118, Route de Narbonne, F-31062 Toulouse
b
Cedex 9, France
c
Universit ꢁe de Toulouse, UPS, Laboratoire de Synth ꢀe se et Physico-Chimie de Mol ꢁe cules d'Int ꢁe r ^e t Biologique, SPCMIB, UMR 5068, 118, Route de Narbonne,
F-31062 Toulouse Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 September 2014
Received in revised form
The goal of this study is to develop a novel brain receptor imaging agent. This study reports the synthesis,
characterization and the biological evaluation of 1-((2-methoxyphenyl) piperazine)ferrocenecarbox-
99m
99m
amide labeled with technetium-99 m (
Tc-MP). The
Tc-complex, characterized by HPLC (20e50% ACN of 0 at 5 min then
0% ACN of 5 at 17 min to finally with 50 at 20% ACN of 17 at 20 min), is stable, neutral and lipophilic
enough to cross the bloodebrain barrier which was confirmed by octanol/water partition coefficient
Tc-MP was obtained quickly (radiolabelling
6
May 2015
99m
time < 5 min), in 90% yield. The
Accepted 8 May 2015
Available online 9 May 2015
5
(
LogP ¼ 1.82). In vivo biodistribution indicated that this complex had exceptional brain uptake (2.47% ID/
Keywords:
g at 5 min and 0.75% ID/g at 60 min). The distribution of the activity at 15 min post-injection in various
rat brain regions showed a higher accumulation in the hippocampus area. After blocking with 8-
hydroxy-2-(dipropylamino) tetralin, the uptake of hippocampus was decreased significantly from
Radiopharmaceuticals
9
9m
Tc-tricarbonyl complex
-(2-methoxyphenyl) piperazine
1
0
.87% ID/g to 0.21% ID/g at 15 min p.i., while the cerebellum had no significant decrease.
The new 9 Tc-cyclopentadienyltricarbonyl technetium complex reported here showed promising
9m
biological results, making it an interesting starting point for the development of a new 9 Tc-complex as
9m
brain receptor imaging agent.
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
ideal radionuclide in the diagnostic nuclear medicine. To date most
Tc compounds assigned as CNS receptor-targeted agents are
3
þ
Currently, research is concentrated on the development of ra-
square-pyramidal complexes of the oxo core [Tc]O]
[1,2].
diotracers marked by technetium-99 m because it is available as a
generator product, has a short half-life of 6 h and emits suitable
However, the in vivo behavior of such complexes may be influenced
by the Tc]O unit offering a free position trans to the oxo ligand for
further reaction in vivo as observed especially for the ‘3 þ 1’ com-
plexes [3,4]. To reduce this in vivo reactivity, we consider oxo-free
Tc complexes containing the metal in lower oxidation states to be
interesting alternatives. In fact, bioorganometallic Tc(I) and Tc(III)
complexes described so far show excellent affinities to the target
receptor but suffer from insufficient brain uptake [5e7]. It is well
known that besides the thermodynamic stability of a complex its
kinetic stability or inertness is of equal and sometimes even of
greater importance for an application in vivo. The most promising
organometallic core for the labeling of biomolecules is the
technetium-tricarbonyl core [8,9]. The metal center is at the low
9
9m
gamma radiation. The almost ideal properties of
imaging and its availability still justify the interest in
Tc for SPECT
9
9m
Tc com-
plexes specific for brain receptor imaging. The widespread avail-
ability of single-photon-emission tomography (SPECT) facilities in
9
9m
comparison with that of PET facilities, in nowadays,
Tc is still the
*
Corresponding author.
* Corresponding author. CNRS, Laboratoire de Synth eꢀ se et Physico-Chimie de
Mol eꢁ cules d'Int eꢁ r e^ t Biologique, SPCMIB, UMR 5068, 118, route de Narbonne, F-
1062 Toulouse Cedex 9, France.
*
3
E-mail address: nadia.malek@gmail.com (N.M. Saied).
http://dx.doi.org/10.1016/j.ejmech.2015.05.014
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
281
oxidation state I and the metal tricarbonyl monocationic core
2.3. Synthesis of 1-((2-methoxyphenyl) piperazine)
ferrocenecarboxamide 2
[
M(CO)3 ] has a low-spin d6 electronic configuration which gives
þ
9
9m
to this compound (M (CO)
3
-core (M ¼
Tc)) the property to be
chemically inert. It is very compact, owning an almost spherical
shape. If the octahedral coordination sphere is “closed” with an
appropriate ligand system, the metal center is efficiently protected
against further ligand attack or re-oxidation. Several research
teams including ours demonstrated that Tc(I) could be efficiently
stabilized by cyclopentadienyl (Cp) derivatives [10,11]. Cp units are
particularly attractive ligands since they lead to organometallic
2.3.1. Ferrocenoyl chloride 1
Under a nitrogen atmosphere, to a stirred solution of ferrocene
carboxylic acid (1.20 g, 5.2 mmol) in freshly distillated dichloro-
methane (10 ml), was added dropwise oxalyl chloride (4 ml,
ꢁ
46.8 mmol), at 0 C. The resulting mixture was stirred at ambient
temp. 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
9
9m
half-sandwich complexes of general formula [{R-Cp}
Tc(CO)
R ¼ organic part or biomolecule), so-called cytectrenes I and II
12,13] Furthermore, the stable piano stool CpM(CO) core
Tc) is one of the most promising
3
]
(
[
(
ꢁ
3
crystalline solid (1.25 g, 97%): mp 134 C.
9
9m
1
Cp ¼ cyclopentadienyl, M ¼
H-NMR (300 MHz, CDCl
3 5 5
): d 4.36 (s, 5H, C H ), 4.66 (s, 2H,
moieties for this purpose. Indeed, the formal oxidation state of the
metal is þI, and the CO ligands can stabilize low valent metal
centers by backbonding. Therefore, the metal is less susceptible to
oxidation, as the cyclopentadiene ligand, is small, with low mo-
lecular weight, and presents the additional possibility of being
coupled to targeting vectors [14].
C
5
H
4
), 4.94 (s, 2H, C ).
5 4
H
2.3.2. 1-((2-methoxyphenyl) piperazine)ferrocenecarboxamide 2
1-(2-methoxyphenyl) piperazine was obtained as active prin-
ꢁ
ciple from Sigma and stored at 4 C. The Chlorocarbonyl ferrocene
(FeCOCl) was synthesized in our laboratory [16,17], briefly activa-
The application of this kind of ‘CpTc(CO)
3
’ moiety-based small
tion of ferrocene carboxylic acid by oxalyl chloride in CH CL is
2
2,
complexes for brain imaging promising. We anticipated that the
substitution of both the ester function and the piperidine de-
rivatives of cytectrene I and II by an amido group and 1-(2-
methoxyphenyl) piperazine respectively, should (i) lead, in a few
steps, to a new cytectrene with suitable in vivo stability and lip-
ophilicity to cross the blood brain barrier (BBB), (ii) allow a better
brain targeting, (iii) offer the possibility to develop a multitude of
other cytectrenes based on this new model.
stirred in the dark for 3 h under dry N2. Its structure was confirmed
by proton nuclear magnetic resonance and mass spectroscopy. All
the other chemicals were used as supplied. A mixture of 1-(2-
methoxyphenyl) piperazine (0.422 ml; 2412 mmol) and FeCOCl
(500 mg; 2012 mmol) in dry THF (20 ml) and pyridine (0.162 ml;
2012 mmol) is stirred in the dark for 2 h under dry N . Removal of
2
volatiles was realized by evaporation under reduced pressure. The
resulting crude product was purified by chromatography (silica,
then a gradient of Hexane 60%)/AcOEt (40%)) to give the 1-((2-
methoxyphenyl) piperazine)ferrocenecarboxamide (82%) as or-
Here, we report the synthesis, radiolabeling and biological
evaluation of a novel 9 Tc-tricarbonyl complex as a potential brain
receptor imaging agent.
9m
ꢁ
ange powder: mp 134 C.
1
H NMR (DMSO-d
6
):
d
H
(ppm) ¼ 3.00 (m, 4H, CH
2
); 3.82 (s, 7H,
); 4.41 (m, 2H, CH3-4 Ar Cp); 4.60
2
. Materials and methods
2 ꢀ CH þ OCH ); 4.28 (s, 5H, CH
2
3
Ar Cp
13
(
m, 2H, CH2-5 Ar Cp); 6.95 (m, 4H, CHAr Ph); C NMR (DMSO-d
6
):
d
C
2
.1. Materials and equipment
(ppm) ¼ 50.5 (4C, CH ); 55.3 (1C, OCH ); 69.2, 69.4, 70.2 (9C, CH
2
3
Ar
Cp); 78.0 (1C, CAr Cp); 111.9, 118.3, 120.8, 122.8 (4C, CHAr Ph); 140.8,
þ
All purchased chemicals were of the highest purity commer-
152.0 (2C, CAr Ph); 168.2 (1C, C]O); MS (CI/NH ): [MþH] ¼ 405; IR
3
ꢂ1
cially available and used without further purification. Analytical
grade solvents were used and not further purified unless specified.
Reactions were monitored by TLC on Kieselgel 60 F254 (Merck) on
aluminium support under UV light (254 nm). Technetium-99 m as
sodium pertechnetate [Na
saline. Chromatographic purification was conducted using “gravity”
silica gel obtained from Merck. Re (CO) Cl was purchased from
Aldrich Chem. Co. [Re(CO) Cl ][NEt [15] was prepared as previ-
(KBr): nC
]
O
¼ 1606 cm ; C
23 19
H FeNO calcd. C, 65.36; H, 5.98; N,
6.93%; found: C, 64.97; H, 5.97; N, 6.69%.
The purity of the 1-((2-methoxyphenyl) pip eꢁ razine)ferrocene-
carboxamide was achieved by reverse-phase high performance
liquid chromatography (HPLC). The fractions were recorded by an
on-line UV-detector measuring the absorbance at 254 nm.
9
9m
4
TcO ] was obtained in physiological
5
3
3
4
]
2
2.4. Synthesis of rhenium complex: Re-MP
ously reported. Liquid Chromatography (HPLC) analysis was per-
formed on a SCL-10Avp SHIMADZU HPLC system coupled to a UV-
Absorbance detector from ICS and a Gabi gamma detector from
Raytest. Separations were achieved on a reverse phase C-18 column
A solution of [Re(CO) Cl ][NEt ] (30 mg, 0.047 mmol) and 1-
3
3
4 2
((2-methoxyphenyl) piperazine)ferrocenecarboxamide (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
2
50 ꢀ 4.6-mm (Shim-pack VP-ODS, SHIMADZU) eluted with a bi-
nary gradient system at a flow rate of 1 ml/min. Mobile phase A was
water containing 0.1% trifluoroacetic acid, while mobile phase B
was acetonitrile (ACN) containing 0.1% trifluoroacetic acid. Gamma
camera NaI (GAEBE) brand square head 20 cm/20 cm with photo
multiplier low-energy high-resolution, collimator collateral chan-
nels, acquisition matrix 256/256.
ꢁ
stirred vigorously during 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 Na SO and evaporated. The crude product
2
4
was purified by column chromatography on silica gel (eluent: pe-
troleum ether/ethyl acetate: 80/20 then 70/30) to obtain the
desired complex Re-MP as an orange powder (7 mg, 29% yield).
2.2. Animals
¼ 1938 and 2027 cmꢂ1
.
Albino Wistar male rats (Pasteur Institute, Tunisia), with a weight
IR (KBr): nC
]
O
of 250e300 g, were used in all experiments. Animals were housed
for one day before the onset of the experiments in our laboratory
housing facility. They were carried for in accordance with the
principles of the guide to the care and use of experimental animals.
The purity of the Re-MP was achieved by reverse-phase high per-
formance liquid chromatography (HPLC). The fractions were recorded
by an on-line UV-detector measuring the absorbance at 254 nm.

282
N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
2
.5. Synthesis and characterization of the 99mTc-complex
coefficient was calculated using the formula: log P ¼ counts in n-
octanol/counts in buffer. The reported value represents the average
of triplicate measurements.
A 2.5 mg aliquot of 1-((2-methoxyphenyl) piperazine)ferroce-
necarboxamide and 5 mg of Mn(CO)
5
Br dissolved in DMSO (300 mL)
were added to 300 L of sodium pertechnetate eluate (~300 MBq).
m
2.10. Biodistribution of 99mTc-MP in healthy Wistar rats
The reaction mixture was purged with nitrogen, then heated by
microwave irradiation (900 W) for 5 periods of 40 s (a 30 s period
was observed between two irradiations). Without optimizing the
conditions, the resulting Tc-complex was obtained in 90% yield.
The radiotracer 99mTc-MP (300
mL diluted in saline-EtOH (80/
20), 20 MBq) was injected via a lateral tail vein. At different in-
tervals after injection, the animals (n ¼ 5) were sacrificed by cer-
vical dislocation. Organs of interest, samples of blood were
collected, weighed and counted. Bladder and excreted urine were
not weighed. The calculation for blood was based upon measured
activity, sample weight and body composition data (considering
that blood comprise 7% of body weight). Results were expressed as
9
9m
After HPLC purification, complex
radiochemical purity (>98%).
Tc-MP was obtained with high
2.6. ITLC analysis
Labeling yield and stability of the 99mTc-MP during time, was
percent dose per gram of tissue (%ID g ).
ꢂ1
determined with instant thin layer chromatography (ITLC). In ITLC-
SG strips (ITLC-SG; Gelman), samples of the preparation containing
labeled compound were applied at approximately 1 cm from the
bottom (baseline) of the ITLC strips and were directly placed on an
air-tight containers with Hexane/AcOEt (6:4 v/v) as solvent. The
development time was 2e3 min, then the strips were mounted on a
radio-chromatograph (Minigita) and counting of radioactivity was
performed. The strips were then cut in several fractions corre-
sponding to 9 Tc-activity detected picks and the radioactivity was
determined in a well gamma counter. Radiolabeled compound
migrates with the front line, whereas free pertechnetate remains at
the origin. The chromatography was repeated at 30 min,1 h, 2 h, 4 h
and 24 h to check the label stability. Results are expressed as per-
centage of total 9 Tc-activity.
2.11. Regional brain distribution and blocking experiment in rats
Rat (200 g, 5 animals per group) was injected through the tail
9
9m
vein with 0.3 ml (~20 MBq) of
Tc-MP complex. The animals were
sacrificed at 15 min post-injection time. The brain was rapidly
removed, chilled and dissected. Samples from different brain regions
(cortex, hippocampus and cerebellum) were collected, weighed and
counted. The percentage dose/g of each sample was calculated by
comparing sample counts with the counts of injected dose. In order
9m
9
9m
to further confirm that the radiolabeled
Tc-MP had specific re-
ceptor binding, blocking study was performed by conducting the
biodistribution experiment in the presence of 8-Hydroxy-2-(dipro-
pylamino) tetralin (8-OH-DPAT, 5-HT1A agonist, 10 mg/kg body
9m
weight) as blocking agent. 10 min after the first injection of blocking
.7. Stability of 99mTc-MP in serum
agent,
99m
Tc-MP complex was injected (~20 MBq, 0.3 ml). Rats were
2
sacrificed at 15 min post-injection time (n ¼ 5). Results were
expressed as the percentage of the injected dose per gram tissue (%
ID/g). Averages and standard deviations were calculated. The average
ratios and standard deviations of % ID/g tissue of the different brain
regions to cerebellum were calculated [19].
The in vitro stability of the purified complex 99mTc-MP was
evaluated at different time points using the following procedure: in
9
9m
a borosilicated vial,
Tc-MP (100 mL) was added to 0.9 ml of fresh
ꢁ
rat serum at 37 C. Aliquots were withdrawn in duplicate during the
incubation at different time intervals till 24 h and subjected to
chromatography using (ITLC-SG; Gelman) paper and Hexane/AcOEt
2.12. Rats gamma camera imaging experiments
(
6:4 v/v) as solvent. Any increase in the free pertechnetate was
considered as the degree of degradation.
Two Albino Wistar male rats (Pasteur Institute, Tunisia), with a
weight of 250e300 g were used for the experiments. For each
gamma experiment, the rat was initially anesthetized with keta-
mine. An intravenous perfusion line, filled with saline (0.9% w/v),
was used for bolus radioligand (1.60e2.40 mCi) injection. Gamma
serial dynamic images were obtained on an Advance gamma
camera NaI (Gaebe). Scans were acquired for up to 30 min in 20
frames and were corrected for attenuation and scatter. Gamma
images were co-registered to scans and decay-corrected.
2.8. Protein binding assay
The protein binding assay of the purified complex 99mTc-MP was
evaluated at different time points using the following procedure: in
9
9m
a borosilicated vial,
Tc-MP (100 mL) was added to 5 ml of fresh
ꢁ
rat plasma at 37 C. Aliquots were withdrawn during the incubation
at different time intervals till 24 h and plasma was separated from
blood samples by centrifugation (3000 rpm for 4 min) and indi-
vidual fractions were counted in a well counter.
2.13. Statistical analysis
The plasma aliquots were treated with acetonitrile to precipitate
proteins. After centrifugation (2000 rpm for 5 min), the activities of
both phases (supernatant and precipitate) were measured sepa-
rately. The reported value represents the average of triplicate
measurements [18].
Experiments were performed in triplicate unless stated other-
wise. Results were reported as mean ± standard deviation. Differ-
ences between the data were evaluated with the student t test. The
level of significance was set at P < 0.05.
2.9. Partition coefficient determination
3. Results and discussions
Lipophilicity was studied through the partition coefficient be-
tween n-octanol and phosphate buffer (0.125 M, pH 7.4). In a
3.1. Chemical synthesis of 1-((2-methoxyphenyl) piperazine)
ferrocenecarboxamide
centrifuge tube containing 500
mL of each phase, 100 mL of the
purified 9 Tc complex solution was added, and the mixture was
9m
The synthesis of 1-((2-methoxyphenyl) piperazine)ferrocene-
carboxamide was accomplished using a nucleophilic addition re-
action of 1-(2-methoxyphenyl) piperazine on the chlorocarbonyl
ferrocene in the presence of THF and Pyridine (Fig. 1).
vortexed one minute at ambient temperature, followed by centri-
fugation at 5000 rpm for 5 min. 100
mL-aliquots of both buffer and
organic layers were count using a well counter. The partition

N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
283
Fig. 1. Structures and reaction scheme for the preparation of 1-((2-methoxyphenyl) piperazine)ferrocenecarboxamide.
The purity of the 1-((2-methoxyphenyl) piperazine)ferrocene-
carboxamide was confirmed using C18 reverse-phase HPLC. Only
one peak was seen with a retention time of 9.7 min (Fig. 2).
the radiochromatogram (Fig. 4).
Rhenium complex, “cold” metallic analogue to desired 99mTc-
MP complex, was prepared via a single-ligand transfer reaction
using [Re(CO)
source. The reaction was carried out by heating a DMF solution of 1-
(2-methoxyphenyl) piperazine)ferrocenecarboxamide derivative
3 3 4 2
Cl ][NEt ] , [15] as tricarbonylrhenium(I) entity
3
.2. Radiochemical analysis of 99mTc-MP
(
The labeled complex, as shown in the following scheme (Fig. 3).
and the Re(I) precursor, in the presence of a 0.1 N HCl solution, at
160 C for 2 h. After chromatography purification, rhenium com-
ꢁ
This reaction was accomplished in one pot, reduction, carbonyla-
tion and cyclopentadienylation of 99mTcO4ꢂ in a rapid and rela-
tively mild manner. This transformation is referred to as a double-
ligand transfer (DLT) reaction [20] because two different ligands, Cp
and CO, from two different metal atoms, Fe and Mn, were trans-
ferred to a third metal atom 99mTc. Exchange mechanism between
iron and technetium has been discussed by many authors. High
temperature destabilizes the cyclopentadienyl nucleus and pro-
motes the exchange between the central atoms. The destabilization
of the complex ferrocene and the release of iron in the reaction
medium presents a major advantage in the reduction of pertech-
netate (99mTcO4ꢂ) passing an oxidation state þ VII (very stable
complex) to an oxidation state þ I. This state oxidation (þI) is sta-
bilized by the bonds of the cyclopentadienyl aromatic structure.
The product was analyzed by HPLC [Shim-pack VP-ODS C18
column; mobile phase: 0e5 min, acetonitrile (20e50%)þ0.1% TFA
plexes, Re-MP, was obtained in modest yield, to 29%. The chemical
identification of the radiotracer was accomplished by HPLC-
comparison of its chromatogram with that of the rhenium
analogue, as underlined in Fig. 5. Similar retention times were
9
9m
observed (13 min for
Tc-MP.13.7 min for Re-MP), confirming the
iso-structurality of both complexes.
3.3. ITLC analysis
ITLC analysis of 99mTc-MP showed a high labeling efficiency
(>95%) and this value was still unchanged up to 24 h. No significant
differences were observed between radiochromatographic analysis
9
9m
and counting of the cut ITLC strips.
Tc-MP migrated of the
mobile phase (Rf ¼ 0.9) and pertechnetate remained at the origin.
(
A) and ultrapure water (80e50%)þ0.1% TFA (B); 5e15 min, 50% (A)
3.4. Stability of 99mTc-MP in serum
ꢂ1
and 50% (B); 15e17 min, 50e20% (A), 50e80% (B); flow: 1 ml min
;
detector NaI crystal]. Radiochemical purity was confirmed by a
single peak at 13 min corresponding to the 99mTc-MP, illustrated in
The complex showed excellent in vitro stability under physio-
9
9m
logical conditions in rat plasma with >99% of the
Tc-MP
Fig. 2. Chromatogram of the 1-((2-methoxyphenyl) piperazine)ferrocenecarboxamide on C18 column HPLC reverse-phase. Column: C18 Shim pack VP-ODS. Mobile phase: 0e5 min
0e50% Acetonitrile þ0.1%TFA (A). Water þ0.1% TFA (B) 80e50%. 5e15 min 50% A and 50% B, 15e17 min 50e20% A 50e80 % B. Flow rate: 1 ml/min.
2

284
N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
Fig. 3. Synthesis of radiolabeled complex 99mTc-MP.
compound remaining intact after 24 h. Consequently, no reox-
idation to pertechnetate was observed, even after 24 h of incuba-
tion, as indicated by ITLC analysis. The data suggested high in vitro
stability of the complex (Fig. 6).
Consequently, the radiocomplexes had 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 log P (oct/buffer). A value of 1.82 was found, indi-
9
9m
3.5. Protein binding study
cating that
Tc-MP is lipophilic. Interestingly, this value is
within the range for related radiocomplexes able to cross the
The assays with rat plasma were performed to determine the
blood brain barrier (logPo/w ¼ 0.5e2.5) [22,23].
percentage of protein binding. After incubation of the radiotracer in
blood samples, plasma was separated from blood samples by
centrifugation and treated with acetonitrile to precipitate the
proteins. Two collected fractions (supernatant and protein) were
measured by well gamma counter. The 99mTc-complex activity has
been found to be higher in plasma fraction compared to the blood
cell one. The protein binding study depicted low binding of the
complex with blood protein. Only 10% of the complex was bound to
serum proteins after 24 h of incubation (Fig. 7). The major part of
the circulating radioactivity corresponds to free complex, which is a
favorable feature, in terms of its application in the imaging of 5-
HT1A receptor.
3.7. Biodistribution of 99mTc eMP in healthy Wistar rats
The in vivo behavior of the 99mTc-MP was evaluated in healthy
Wistar rats at different time points, the results were shown in
Table 1. Interestingly, the complex exhibits a fast blood clearance
(0.53% and 0.27% at 5 and 60 min respectively) combined with a
good brain uptake at earlier post-injection time (2.47%ID/g 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 occurred mainly via the renal urinary pathway (2.39%
and 2.93% at 5 and 60 min respectively) and through the hep-
atobiliary 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 (2.47%ID/g). The
retention of the radioactivity in brain is fairly good at 60 min p.i.
3
.6. Lipophylicity study
The lipophilicity is an important parameter to predict and
interpret the biological activity of a molecule intended to be
9
9m
(0.75%ID/g). The ability of
Tc-MP of penetrating the blood brain
injected in vivo. It is generally accepted that only the lipid
barrier could be explained by its good properties in terms of charge,
small size, lipophilicity and in vitro, in vivo stability. The brain
9
9m
pathway provides access to the brain for
Tc-complexes [21].
Fig. 4. HPLC radiochromatogram of 99mTc-MP on C18 column HPLC reverse phase.

N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
285
Fig. 5. HPLC comparison of rhenium complex Re-MP and 99mTc complex 99mTc-MP.
uptake value of 99mTc-MP is higher comparable to those complexes
DADT (0.6%ID/g), DEEDA (1.63%ID/g), BMPBA (1.77%ID/g) [24e26].
The in vivo distribution of many compounds showed a moderate
brain uptake, it was clear that more extensive studies in animal
models will be needed to correlate the in vitro data to predict there
in vivo behavior in humans.
and the brain distribution of the radioactivity. Further studies are
ongoing to clarify this point.
3.8. Regional brain distribution and blocking experiment in rats
The distribution of the activity in various rats brain regions was
performed in normal rats (Table 2). The biodistribution of the ac-
tivity in selected regions of the brain was also investigated at 15 min.
The highest concentration of radioactivity was found in the hippo-
campus area where the 5-HT1A receptor density was important. For
cortex, region where the 5-HT1A receptor density was less impor-
tant than in the hippocampus, the radioactivity value is lower. For
The biodistribution of the activity in selected regions of the
brain was also investigated at 15 min (Fig. 8).
The highest concentration of radioactivity was found in the
hippocampus area where the 5-HT1A receptor density was impor-
tant. For cortex, region where the 5-HT1A receptor density was less
important than in the hippocampus, the radioactivity value is
lower. Nevertheless, the repartition of the radioactivity in the
different regions of the brain seems too homogeneous to establish a
clear correlation between the repartition of the 5-HT1A receptors
9
9m
Tc-MP, the radioactivity concentration of hippocampus (Hipp) at
15 min p.i. was 0.87% ID$gꢂ1. For cerebellum (CB), the uptake was
much lower than that of Hipp. The ratio of Hipp/CB was 4.83. For
Fig. 6. In vitro stability of 99mTc-MP in serum.

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N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
Fig. 7. Protein binding study of 99mTc- MP.
Table 1
Table 2
Biodistribution in healthy Wistar rats 5 min, 15 min, 30 min and 60 min after
Regional brain distribution and blocking studies of99mTc-MP in normal rats at 15 min
administration of9 Tc-MP.
9m
p.i. (%ID/g ± SD, n ¼ 5).
Organs
5 min (%ID/g)
15 min (%ID/g)
30 min (%ID/g)
60 min (%ID/g)
Without blocking
With blocking
P value
Blood
Heart
Lungs
Liver
Spleen
Kidney
Brain
0.53±(0.03)
0.43±(0.01)
5.70±(0.04)
18.32±(0.04)
5.11±(0.02)
2.39±(0.03)
2.47±(0.04)
0.45±(0.04)
1.03±(0.02)
4.11±(0.02)
16.11±(0.03)
3.20±(0.03)
2.25±(0.04)
1.81±(0.01)
0.39±(0.02)
0.40±(0.05)
3.91±(0.04)
12.65±(0.03)
2.41±(0.02)
2.82±(0.05)
0.93±(0.04)
0.27±(0.01)
0.51±(0.01)
2.60±(0.05)
10.72±(0.02)
1.51±(0.05)
2.93±(0.02)
0.75±(0.03)
Hippocampus
Cortex
Cerebellum
Hippocampus/cerebellum
Cortex/cerebellum
0.87 ± 0.03
0.50 ± 0.04
0.18 ± 0.02
4.83
0.21 ± 0.03
0.35 ± 0.02
0.17 ± 0.02
1.23
0.003
0.045
0.401
2.77
2.05
In the blocking and without blocking experiments, the two-tailed paired Student's t
test was applied to compare the values between before and after blocking. A P value
of less than 0.05 was considered statistically significant.
ꢃ Expressed as % injected dose per gram (%ID/g ±SD, n ¼ 5).
cortex, the uptake was 0.5% ID$gꢂ1, in the middle of Hipp (0.87%
ID$gꢂ1) and CB (0.18% ID$gꢂ1). The distribution of radioactivity in
hippocampus, cerebellum and cortex was correlated very well with
the distribution of 5-HT1A receptors in brain. In order to further
change is probably due to the competition of 8-OH-DPAT binding to
the same 5-HT1A receptor in the brain. Based on the above in vivo
9
9m
results, we could conclude that complex
Tc-MP had like enough
specific binding to the 5-HT1A receptor and hopefully be developed
as potential technetium- 99 m receptor imaging agent.
9
9m
characterize the vivo brain uptake of this
Tc labeled complex in
rats, a blocking study was carried out to determine the changes of
regional brain uptake. After blocking by 8-hydroxy-2-(dipropyla-
mino) tetralin (8-OH-DPAT), the uptake of hippocampus was
decreased obviously from 0.87% ID$gꢂ1 to 0.21% ID$gꢂ1 at 15 min
p.i. (P ¼ 0.003), and the uptake of cortex was also decreased from
3.9. Rats gamma camera imaging
After injection of 99mTc-MP into a Wistar rats, there was a rapid
and moderate uptake of radioactivity into rat's brain and agree with
bio-distribution. The accumulation of radioactivity in the brain is
indicative of an efficient bloodebrain-barrier passage of labeled
compound (Fig. 9).
0
.5% ID$gꢂ1 to 0.35% ID$gꢂ1 at 15 min p.i. (P ¼ 0.045), but the
cerebellum had no significant decrease (P ¼ 0.40) (Table 2). The
ratios of Hipp/CB were changed from 4.83 to 1.23 at 15 min p.i. This
Fig. 8. Regional distribution in rat brain at 15 min p.i.

N.M. Saied et al. / European Journal of Medicinal Chemistry 97 (2015) 280e288
287
Fig. 9. Gamma camera of rat brain illustrates the distribution of 99mTc-MP accumulation (a) and dynamic studies in the various organs over time (b).
4
. Conclusion
and Addelmonen Kraiem (Centre gamma de medicine nucl eꢁ aire)
for their collaborations on using the Gamma camera NaI (GAEBE).
We designed and synthesized a novel brain receptor imaging
agent 9 Tc-MP with high radiolabeling yield (>90%). According to
9m
Appendix A. Supplementary data
9
9m
the results of in vivo biodistribution studies, we found that
Tc-MP
had favorable properties for further study. The preliminary biolog-
ical studies are interesting. Firstly, the Tc-complex is lipophilic
enough to cross the blood brain barrier, as expected (Log P ¼ 1.82).
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 (0.75% ID/g after 1 h).
Then, the distribution of the activity at 15 min post-injection in
various rat brain regions showed a higher accumulation in the
hippocampus area which is rich in 5HT1A receptors.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.014.
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