Synthesis and biological evaluation of new [Tc(N)(PS)]-based mixed-ligand compounds useful in the design of target-specific radiopharmaceuticals: The 2-methoxyphenylpiperazine dithiocarbamate derivatives as an example

Article abstract of DOI:10.1007/s00775-010-0712-4

This study presents the first application of a general procedure based on the use of the [Tc(N)Cl(PS)(PPh₃)] species (PS is an alkyl phosphinothiolate ligand) for the preparation of Tc(N) target-specific compounds. [Tc(N)Cl(PS)(PPh₃)

Full text of DOI:10.1007/s00775-010-0712-4

J Biol Inorg Chem (2011) 16:137–155
DOI 10.1007/s00775-010-0712-4
ORIGINAL PAPER
Synthesis and biological evaluation of new [Tc(N)(PS)]-based
mixed-ligand compounds useful in the design of target-specific
radiopharmaceuticals: the 2-methoxyphenylpiperazine
dithiocarbamate derivatives as an example
•
Cristina Bolzati Nicola Salvarese Davide Carta Fiorenzo Refosco
•
•
•
•
•
•
Alessandro Dolmella Hans Ju¨rgen Pietzsch Ralf Bergmann Giuliano Bandoli
Received: 23 July 2010 / Accepted: 19 September 2010 / Published online: 7 October 2010
Ó SBIC 2010
Abstract This study presents the first application of a
general procedure based on the use of the [Tc(N)Cl(PS)
(PPh3)] species (PS is an alkyl phosphinothiolate ligand)
for the preparation of Tc(N) target-specific compounds.
[Tc(N)Cl(PS)(PPh3)] selectively reacts with an appropriate
dithiocarbamate ligand (S^Y) to give [Tc(N)(PS)(S^Y)]
compounds. 1-(2-Methoxyphenyl)piperazine, which dis-
plays a potent and specific affinity for 5HT1A receptors,
was selected as a functional group and conjugated to the
dithiocarbamate unit through different spacers (Ln).
5HT1A receptor was obtained for [99mTc(N)(PSiso)L3]
(IC50 = 1.5 nM); a reduction of the affinity was observed
for the other complexes as a function of the shortening of
the alkyl chain interposed between the dithiocarbamate and
the pharmacophore. Negligible brain uptake was found
from in vivo distribution data of [99mTc(N)(PSiso)L3]. The
key finding of this study is that the complexes maintained
good affinity and selectivity for 5HT1A receptors, and the
IC50 value for [99gTc(N)(PSiso)L3] being comparable to the
IC50 value found for WAY 100635. This result confirmed
the possibility of preparing [99mTc(N)(PS)]-based target-
specific compounds without affecting the affinity and
selectivity of the bioactive molecules for the corresponding
receptors.
[
99mTc(N)(PS)(Ln)] complexes were prepared in high yield
(more than 90%). The chemical identity of 99mTc com-
plexes was determined by high performance liquid chro-
matography comparison with the corresponding 99gTc
complexes. All complexes were found to be inert toward
transchelation with an excess of glutathione and cysteine.
No notable biotransformation of the native compound into
different species by the in vitro action of the serum and
liver enzymes was shown. Nanomolar affinity for the
Keywords Technetium ꢀ Phosphinothiolate ꢀ
Dithiocarbamate ꢀ 5HT1A receptor ꢀ Brain
Introduction
In recent years, the chemistry of potential 99mTc radio-
pharmaceuticals has been expanded owing to the intro-
duction of an efficient method for the production, at tracer
level, of 99mTc species containing the terminal Tc:N
multiple bound, in sterile and pyrogen-free conditions. It
was found that the resulting [Tc(N)]2? core could be
viewed as a true inorganic functional group exhibiting very
high stability under a wide range of experimental condi-
tions [1].
C. Bolzati (&) ꢀ F. Refosco
ICIS-CNR,
Corso Stati Uniti, 4,
Padua, Italy
e-mail: bolzati@icis.cnr.it
C. Bolzati ꢀ N. Salvarese ꢀ D. Carta ꢀ A. Dolmella ꢀ G. Bandoli
Department of Pharmaceutical Sciences,
University of Padua,
Via Marzolo, 5,
Padua, Italy
To explore the utility of the [Tc(N)]2? core in the design
of either essential or target-specific radiopharmaceuticals,
recently we devised a general procedure for the preparation
of a new class of disymmetrical 99mTc(N) complexes [2–4].
This procedure is based on the chemical property of the
H. J. Pietzsch ꢀ R. Bergmann
Forschungszentrum Dresden-Rossendorf,
Institute of Radiopharmacy,
PF 510 119, 01314 Dresden,
Germany
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monosubstituted [Tc(N)Cl(PS)(PPh3)] species, where PS is
an alkyl phosphinothiolate ligand, which rapidly reacts with
an appropriate bidentate mononegative coligand (S^Y), such
as a dithiocarbamate, to give neutral pentacoordinate nitrido
compounds of the type [Tc(N)(PS)(S^Y)].
final complex within the limit required to cross the BBB
(700 Da).
This work was performed to evaluate the applicability of
this new labeling procedure to the preparation of
[Tc(N)(PS)]-based target-specific compounds. In addition,
considering the ability of some [99mTc(N)(PS)(S^Y)]
compounds to cross the BBB, we also considered the
possibility to prepare a Tc agent useful in assessment of
central nervous system receptors.
These compounds, analogously to other mixed-ligand
complexes, are characterized by high chemical flexibility
due to the possibility of changing the substituents at the P
atoms and/or at the bidentate coligand. This property may
be employed to design different Tc:N mixed compounds
and to modulate their biological behaviors. In particular,
the bidentate coligand S^Y can be easily exploited to carry
selected functional groups or vectors for designing target-
specific radiopharmaceuticals. Furthermore, the restricted
molecular mass (about 300 Da) and the low steric hin-
drance of the [Tc(N)(PS)]? building block allow (1) the
control of the size of the final compound and (2) the
reduction of possible interactions between the
[Tc(N)(PS)]? molecular fragment and the bioactive moi-
ety, thus leaving the receptor binding domain to fit closely
the receptor site.
The fragment of WAY 100635, 1-(2-methoxy-
phenyl)piperazine (2-MPP), which displays a potent and
specific affinity for 5HT1A serotonergic receptors involved
in several neurological disorders, such as schizophrenia,
anxiety, and depression [5–10], was selected as the target
molecule. This pharmacophore can be easily derivatized, as
evidenced by previous studies [11–22]. In this work the 2-
MPP fragment was conjugated to the technetium chelate
dithiocarbamate unit through different group spacers. The
ligands used in this study are reported in Fig. 1.
The synthesis of [99m/99gTc(N)(PS)(Ln)] complexes and
their in vitro stability as well as their biological in vitro and
in vivo assays were investigated. The log k00 values of the
compounds as a measure of the relative lipophilicity of the
complexes were calculated by reverse-phase (RP) high-
performance liquid chromatography (HPLC) using various
mixtures of MeOH/phosphate buffer (pH 7.4) as the eluent,
and were compared with the corresponding log P values.
The log k00 value of the commercially available perfusion
agent [99mTc]-ethyl cysteinate dimer (ECD) was measured
under the same experimental conditions [3] and was used
as a reference. Stability studies were performed by con-
sidering (1) stability toward transchelation with glutathione
and cysteine, (2) binding to the serum proteins, and (3)
stability in human and rat sera as well as rat brain and liver
homogenates.
In a recent paper we reported the in vivo biodistribution
data of some representative [99mTc(N)(PS)(S^Y)] com-
pounds. These compounds displayed promising biological
properties characterized by a transient brain uptake and
encouraging brain/blood ratio (approximately 4), to indi-
cate the ability of this class of mixed complexes to move
freely in and out of the intact blood–brain barrier (BBB)
[3]. This behavior was attributed to the absence of an
efficient trapping mechanism that prevents the complex
from crossing the BBB in the opposite direction. To
increase brain retention for longer time intervals, the
bidentate coligand S^Y can be modified to carry appro-
priate functional groups which are involved in the trapping
mechanism such as receptor-specific interactions. More-
over, taking advantage of the nature of the [Tc(N)(PS)]?
building block, one can maintain the molecular mass of the
The in vitro affinity for the 5HT1A receptors of the
99gTc(N) complexes was assessed by measuring the ability
Fig. 1 Phosphine thiolate and 1-(2-methoxyphenyl)piperazine dithiocarbamate used in the experiments
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139
of the compounds to compete with [3H]-8-hydroxy-N,N-
dipropyl-2-aminotetralin (8-OH-DPAT) binding in isolated
membranes from rat cerebral cortex.
Electrospray ionization (ESI) mass spectrometry (MS)
measurements were performed with an ESI time-of-flight
Mariner biospectrometry workstation (PerSeptive Biosys-
tems, Stafford, TX, USA).
The biodistribution profile of the best radiolabeled
compound and its in vivo stability were evaluated in female
Sprague–Dawley rats. The effects of the cyclosporin A
(CsA), a wide-spectrum P-glycoprotein (P-gp) modulator,
on the pharmacokinetic properties of [99mTc(N)(PSiso)
(L3)] complex were evaluated too.
Chromatographic separations were accomplished on a
SiO2 column (30 cm 9 1 cm, 70–230 mesh, Aldrich).
Thin-layer-chromatography (TLC) and HPLC analyses
were used to evaluate the radiochemical yield (RCY) and
the stability of the compounds was evaluated as the radio-
chemical purity (RCP). TLC analysis of [99g/99mTc(N)
(PScy)(L1–2)] complexes was performed using RP-C18
F254S plates (Merck, Milan, Italy). TLC analysis of
Materials and methods
[
99/99mTc(N)(PSiso)(L1–3)] was performed using SiO2 F254S
Caution!
plates (Merck, Milan, Italy). The relevant mobile phases are
reported in Table 1. The activity on the plates was detected
and measured using a CycloneÒ instrument equipped with a
phosphorus imaging screen and the OptiQuant image
analysis program (Packard, Meridian, CT, USA).
HPLC studies were performed using a Beckman
System Gold instrument equipped with a model 126
programmable solvent module, a 210A sample injection
valve, a 166 scanning detector module, and a model
B-FC-3200 Bioscan radioisotope detector. The flow rate
was 1 mL/min.
99gTc is a weak b-emitter (Eb = 0.292 MeV, t1/2 = 2.1 9
105 years). All manipulations were carried out in labora-
tories approved for low-level-radioactivity use. Handling
milligram amounts of 99gTc does not present a serious
health hazard since common laboratory glassware provides
adequate shielding and all work is performed in approved
and monitored hoods and glove boxes. Bremsstrahlung is
not a significant problem owing to the low energy of the
b-particles; however, proper radiation safety procedures
must be followed at all times, and particular care should be
taken when handling solid samples.
The protein binding affinity and the stability in the
different tissues were evaluated through chromatographic
methods using HPLC and a size-exclusion Microspin G50
column (Amersham Biosciences). Analytical RP HPLC
was performed with a Symmetry 300 C4 precolumn (5 lm,
3.9 mm 9 20 mm) and a Symmetry 300 C4 column (5 lm,
4.6 mm 9 150 mm). Solvent A was H2O (0.1% trifluoro-
acetic acid, pH 3.0), and solvent B was CH3CN (0.1%
trifluoroacetic acid). The gradient was as follows: 0–2 min,
15% solvent B; 2–20 min, 65% solvent B ; 20–22 min,
15% solvent B; 22–25 min, 15% solvent B. The flow rate
was 1 mL/min.
All chemicals and reagents were purchased from Aldrich
Chemicals. The solvents were reagent grade and were used
without further purification. The phosphine thiol ligands
were purchased from Argus Chemicals (Prato, Italy).
Dithiocarbamate lihands L1–L3 were synthesized as reported
later. Owing to the tendency of the phosphine thiol ligands to
oxidize, all the solvents used in reactions with PSH were
previously degassed to remove dissolved trace oxygen.
Commercially available NH4[99gTcO4] (Oak Ridge
National Laboratories) was purified from the black con-
taminant (99gTcO2 nH2O) by addition of H2O2 and NH4OH
solutions followed by recrystallization as [NH949gTcO4]
from hot water. 99mTc as Na[99mTcO4] was eluted from a
99Mo/99mTc generator (Elumatic III, IBA CIS bio, France)
using 0.9% saline.
The log k0 values were determined according to the lit-
erature method [3].
Measured physical properties of [99mTc(N)(PS)(L1–3)]
agents including HPLC capacity factors are reported in
Table 1.
[
99g/99mTc(N)(PScy)Cl(PPh3)] and [99g/99mTc(N)(PSi-
3
so)Cl(PPh )] were prepared according to the literature
Ligand synthesis
methods [3, 4, 23].
Elemental analyses (C, H, N, S) were performed using a
Carlo Erba 1106 elemental analyzer. Fourier transform
(FT) IR spectra were recorded with a Nicolet 510P FT
spectrometer, in the range 4,000–200 cm-1 and in a KBr
mixture using a Spectra-Tec diffuse-reflectance collector
Sodium 4-(2-methoxyphenyl)piperazine-1-dithiocarbamate
In a ice-bath cooled vessel, 1.6 mmol of 1-(2-methoxy-
phenyl)piperazine hydrochloride was suspended in water
(10 mL) and an aqueous solution of NaOH (1.6 mmol/
1.5 mL) was slowly added followed by dropwise addition
of carbon disulfide (1.6 mmol/100 lL, 4 9 25 lL). The
reaction mixture was stirred at room temperature overnight
to obtain a yellow–orange solution. The solvent was
1
accessory. H, 13C, and 31P NMR spectra were acquired at
room temperature in CDCl3 with a Brucker 300 instrument,
using SiMe4 as the internal reference (1H and 13C) and 85%
aqueous H3PO4 as the external reference (31P).
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1,2
Table 1 High-performance liquid chromatography (HPLC),
Beckman column (250 mm 9 4.6 mm)
reverse-phase (RP) C18 Beckman precolumn (45 mm 9 4.1 mm) and RP-C18
Complexes
HPLC, Rt
TLC
RCY (%)
Method 1
log P
log k0
Mr
99gTc
99mTc
Rf
Method 2
[Tc(N)(PScy)(L1)]
[Tc(N)(PScy)(L2)] a/b
[Tc(N)(PSiso)(L1)]
[TcN(PSiso)(L2)] a/b
[TcN(PSiso)(L3)] a/b
[Tc(ECD)]
117.0
19.4/12.3
117.3
19.7/12.6
214.9
29.1/11.6; 14.6
211.6/15.1; 17.4
10.31
10.56
20.5
20.21
30.41
52.02 5.02
93.01 2.31
94.56 1.23
90.30 2.36
94.50 0.96
6.78
637.82
679.86
556.68
599.73
627.78
7.86/8.38
4.10
90.51 1.05
89.30 1.94
91.01 1.96
0.90 0.08
0.97 0.05
1.11 0.03
1.64
28.9/11.3;
211.3/14.8
4.48/4.86
4.96/5.38
2.44
3
3
24.5
[Tc(N)(PSiso)2]
[Tc(N)(L1)2]
[Tc(N)(L2)2]
319.1
38.9
310.3
317.4
20.43
20.54
10.63
20.28
1
90.00 2.31
89.36 3.20
91.00 1.56
88.00 2.87
[Tc(N)(L3)2]
2
3
Mobile phase: A, 0.02 M phosphate buffer pH 7.4; B, CH3OH; 0–30 min, 85% B (isocratic). 0–30 min, 75% B (isocratic). RP-C18 Waters
Symmetry ShieldTM precolumn (3.9 mm 9 20 mm, 5 lm), RP-C18 Waters Symmetry ShieldTM column (4.6 mm 9 250 mm, 5 lm). Mobile
phase: A, 0.01 M NH4Ac pH 7, B, CH3CN; 0–20 min, 60% B (isocratic), 20–30 min, 100% B (gradient), 30–40 min, 100% B (isocratic),
40–45 min, 60% B (gradient). Thin-layer chromatography (TLC): 1RP-C18 mobile phase CH3CN/H2O 90:10; 2SiO2 mobile phase EtOH/CHCl3/
3
C6H6 (0.1:2:1.5); SiO2 mobile phase EtOH/CHCl3/C6H6 (0.3:2:1.5)
a/b isomeric forms, RCY radiochemical yield, ECD ethyl cysteinate dimer, PS alkyl phosphinothiolate
removed under reduced pressure and the resulting crude
residue was recrystallized from EtOH/Et2O to give white
crystals of 4-(2-methoxyphenyl)piperazine-1-dithiocarba-
mate as the sodium salt (L1). Elemental analysis for
C12H15N2NaOS2 (Mr = 290.38), calcd (%): C, 49.63; H,
5.21; N, 9.65; S, 22.08. Found (%): C, 48.53; H, 4.02; N,
9.00; S, 22.58.
The solvent was evaporated and the yellow residue was
suspended in water (50 mL) and extracted with CH2Cl2
(3 9 50 mL). The combined organic layers were dried with
anhydrous Na2SO4, and evaporated to obtain 2-bromo-
N-(4-nitrobenzylidene)ethylamine (1) as a yellow oil. Yield
4.97 g, 95%.
Elemental analysis for C9H9BrN2O2 (Mr = 255.98),
calcd (%): C, 42.05; H, 3.53; N, 10.90; O, 12.45. Found
ESI(-)–MS m/z: 267.02 [M–Na?]- (100%).
FT IR (cm-1): [3,000–2,900, w, m(CH)], [1,501 and
1,465, m broad, m(C=C and C=S and CN)], [1,437, m,
m(CH)], [1,215, s, m(C–O)], [738, s, m(arom)].
(%): C, 42.53; H, 4.65; N, 8.98; O, 12.58.
1H NMR (CDCl3, d): 3.74 (t, 2H, CH2Br, JHH
3
=
=
3
6.0 Hz); 4.10 (td, 2H, CH2CH2Br, JHH = 6 Hz, JHH
4
1H NMR (D2O, d): 3.00 (m, 4H, CH2N(Ph)CH2pipera-
zine); 3.80 (s, 3H, OCH3); 4.42 (m, 4H, S2CNCH2CH2N);
7.00 (m, 4H, Harom).
1.2 Hz); 7.94 (m, 2H, Harom,ortho); 8.29 (m, 2H, Harom,meta
3JHH = 8.8 Hz); 8.39 (m, 1H, CH=N, JHH = 1.2 Hz).
4
13C NMR (CDCl3, d): 32.0 (CH2Br); 62.1 (CH2CH2Br);
123.5, 128.9 (Harom,ortho, meta); 140.8, 148.9 (O2N–C and
C–CH=N–); 160.6 (CH=N).
13C NMR (D2O, d): 49.94, 50.47 (Cpiperazine); 54.79 (OCH3);
111.43–118.72–120.77–124.41 (Carom 2-methoxyphenyl); 141.22,
152.57 (N–Cquat.arom and CH3O–C); 208.54 (s, CS2).
2-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-
(4-nitrobenzylidene)ethylamine
Synthesis of sodium 2-(4-(2-methoxyphenyl)piperazin-1-yl)
ethyldithiocarbamate (L2)
2-MPP (6.62 g, 28.9 mmol) and triethylamine (8 mL,
0.057 mmol) were dissolved in CH2Cl2 (150 mL). To this
solution, 1 (4.97, 19.3 mmol) was added and the reaction
mixture was stirred at room temperature overnight. The
solvent was evaporated and the yellow residue was sus-
pended in water (50 mL) and extracted with CH2Cl2
(3 9 50 mL). The combined organic layers were dried with
Na2SO4, evaporated, and the resulting yellow oil was treated
with Et2O to obtain 2-(4-(2-methoxyphenyl)piperazin-1-yl)-
N-(4-nitrobenzylidene)ethylamine (2) as a pale-yellow
powder. Yield 5 g (14 mmol) 72.8%.
For L2 the general synthesis pathway is outlined in
Scheme 1.
2-Bromo-N-(4-nitrobenzylidene)ethylamine
2-Bromoethylamine hydrobromide (5.78 g; 28.2 mmol)
and triethylamine (3.92 mL, 28.2 mmol) were suspended
in CH2Cl2 (100 mL). To this solution p-nitrobenzaldehyde
(3 g; 19.8 mmol) was added, and the reaction mixture was
stirred at room temperature overnight.
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141
Scheme 1 Pathway of
synthesis of sodium 2-(4-(2-
methoxyphenyl)piperazin-1-
yl)ethyldithiocarbamate (L2),
RT room temperature
Elemental analysis for C20H24N4O3 (Mr = 368.18),
calcd (%): C, 65.20; H, 6.57; N, 15.21; O, 13.03. Found
(%): C, 65.73; H, 6.99; N, 15.11; O, 13.28.
with Na2SO4, and evaporated to obtain 2-(4-(2-methoxy-
phenyl)piperazin-1-yl)ethylamine (3) as a yellow oil. Yield
3.00 g (12.7 mmol) 96.2%.
1H NMR (CDCl3, d): 2.81 (m, 6H, NCH2CH2Nlinear chain
and NCH2CH2Npiperazine); 3.12 (m, 4H, NCH2CH2Npiperazine);
3.88 (s, 3H, OCH3); 3.89 (m, 2H, NCH2CH2Nlinear chain);
Elemental analysis for C13H21N3O (Mr = 235.17), calcd
(%): C, 66.35; H, 8.99; N, 17.86; O, 6.80. Found (%): C,
65.98; H, 9.20; N, 17.01; O, 7.00.
6.94 (m, 4H, Harom); 7.91 (m, 2H, Harom
,
2J =
1H NMR (CDCl3, d): 2.51 (t, 2H, H2NCH2CH2
m-NO2
8.8 Hz); 8.28 (m, 2H, Harom o-NO2) 8.41 (m, 1H, CH=N).
13C NMR (CDCl3, d): 50.2, 53.4 (Cpiperazine); 55.0
(OCH3); 58.3, 59.0 (NCH2CH2Nlinear chain); 110.8–
117.8–120.6–122.6 (Carom 2-methoxyphenyl); 123.5, 128.4
(Carom,ortho,meta p-nitrophenyl); 140.9, 148.7 (O2N–C and
C–CH=N); 141.3, 151.9 (N–Cquat.arom and CH3O–C); 159.6
(CH=N).
Npiperazine,
3JHH = 6.1 Hz); 2.67 (m, 4H, CH2N(CH2)
CH2piperazine); 2.85 (t, 2H, H2NCH2CH2N, 3JHH = 6.1 Hz);
3.10 (m, 4H, CH2N(o-MeO-Ph)CH2); 3.86 (s, 3H, OCH3);
6.93 (m, 4H, Harom).
13C NMR (CDCl3, d): 38.3 (NH2CH2CH2); 50.3 and
53.1 (Cpiperazine); 55.0 (OCH3); 60.6 (NH2CH2CH2N);
110.8, 117.8, 120.6, 122.5 (Carom 2-methoxyphenyl); 141.0,
152.0 (N–Cquat.arom and CH3O–C).
2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethylamine
Sodium 2-(4-(2-methoxyphenyl)piperazin-1-
yl)ethyldithiocarbamate
2 (4.7 g; 13.2 mmol) was dissolved in CH2Cl2 (100 mL)
and 1 M HCl (50 mL) was added.
The reaction mixture was stirred at room temperature for
2 h. Then, the two layers were separated and the organic
phase was treated with 1 M HCl (3 9 50 mL).
3 (3 g; 12.7 mmol) was suspended in water (10 mL) and a
NaOH solution (0.507 g; 12.7 mmol in 50 mL of water)
was slowly added followed by acetone (60 mL). The
resulting mixture was stirred at room temperature until it
became clear, then it was cooled in an ice bath, and carbon
disulfide (0.80 mL, 12.7 mmol) was added dropwise. The
reaction mixture was stirred overnight, then the solvents
were evaporated, and the resulting yellow–orange crude
The pH of the combined hydrophilic layers was adjusted
to 9 through the addition of an aqueous solution of 2 M
NaOH, and a pale-yellow suspension was observed.
Finally, the resulting free base was extracted with CH2Cl2
(3 9 50 mL). The combined CH2Cl2 layers were dried
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residue was dissolved in the minimum amount of CH2Cl2
and treated with Et2O. A pale-yellow powder of sodium
2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyldithiocarbamate
(4, L2) was collected. Yield 3.85 g (11.5 mmol) 90.86%.
Elemental analysis for C14H20N3OS2Na (Mr = 333.6),
calcd (%): C, 50.40; H, 6.04; N, 12.65; S, 19.22. Found
(%): C, 50.33; H, 6.05; N, 12.40; S, 19.29.
by a dinitrogen stream and the crude oily residue was
treated with n-hexane and with Et2O to remove PPh3 and
the starting material, respectively.
Column chromatography was necessary to separate the
pure species.
[
99gTcN(PScy)(L1)]
ESI(-)–MS m/z: 310 [M–Na]- (100%).
FT IR (cm-1): [3392, s, m(NH)], [3,000–2,900, w,
m(CH)], [1,498 and 1,458, s, m(C=C and C=S and CN)],
[1,377, m, m(CH)], [1234, s, m(C–O)], [749 m(arom)].
1H NMR (D2O, d): 2.63 (m, 4H, CH2N(CH2)CH2piperazine
and S2CNHCH2CH2N); 2.98 (m, 4H, CH2N(Ph)
The crude oily residue was dissolved in CH2Cl2 (1 mL)
and purified by chromatography on a SiO2 column previ-
ously conditioned with CH2Cl2. The column contents were
eluted with CH2Cl2. A yellow band was collected. The
eluent was completely evaporated with a gentle dinitrogen
stream. The final product was isolated as a pure yellow
powder upon treatment of the oil residue with Et2O and
n-hexane. Yield 80%. The complex is soluble in chlori-
nated solvents and DMSO and insoluble in Et2O, MeCN,
alcohols, and water. Elemental analysis for C26H41
N3OPS3Tc (Mr = 637.82), calcd (%): C, 48.97; H, 6.48; N,
6.59; S, 15.08. Found (%): C, 49.00; H, 6.50; N, 6.61; S,
15.05. ESI(?)–MS m/z: 638.74 [M?H]? (100%). FT IR
(cm-1): [3,000–2,900, w, m(CH)], [1,501, s, and 1438, s
broad, m(C=C and C=S and CN)], [1,238, s, m(C–O)],
[1,062, m, m(Tc:N)], [748, s, m(arom)]. 31P NMR (CDCl3,
d): 92.11 (s, br).1H NMR ((CDCl3, d): 1.09–2.16 (m, 22H,
3
CH2piperazine); 3.67 (t, 2H, H2NCH2CH2N, JHH = 6.8 Hz);
3.76 (s, 3H, OCH3); 7.01 (m, 4H, Harom).
13C NMR (D2O, d): 38.2 (CS2NH2CH2CH2N); 50.2 and
52.8 (Cpiperazine); 55.0 (OCH3); 56.2 (CS2NH2CH2CH2N);
38.2 (NH2CH2CH2); 110.8, 117.8, 120.6, 122.8 (Carom
2-methoxyphenyl); 141.1, 152.3 (N–Cquat.arom and CH3O–C);
209.6 (s, CS2).
Sodium 4-(4-(2-methoxyphenyl)piperazin-1-
yl)butyldithiocarbamate
Sodium 4-(4-(2-methoxyphenyl)piperazin-1-yl)butyldithio-
carbamate (L3) was prepared by reaction of the appropriate
amine prepared according to the literature [17] with an
equivalent amount of carbon disulfide in NaOH solution.
Elemental analysis for C16H24N3NaOS2 (Mr = 361.50),
calcd (%): C, 53.11; H, 6.64; N, 11.62; S, 17.740 found
(%): C, 54.23; H, 7.01; N, 11.98; S, 18.74.
Hcyclohexyl); 1.93, 2.19 (2 m, 2H, SCH2CH2P); 2.89 (m, 2H,
CH2S); 3.18 (m, 4H, PhNCH2CH2NCS2); 3.89 (s, 3H,
OCH3); 4.10, 4.35 (2 m, 4H, CH2NCS2); 6.88–7.26 (m, 4H,
1
H
arom).13C NMR (CDCl3, d): 25.88 (d, CH2P, JCP
=
21.9 Hz); 26.14–27.26, 29.08 (m, Hcyclohexyl); 30.78
(d, SCH2, 2JCP = 8.3 Hz); 33.24, 33.86 (2 d, PCH); 47.49,
48.06 (2 s, CH2NCS2); 50.05 (bs, CH2CH2NCS2); 55.48
(2 s, OCH3); 111.40, 118.73, 121.14, 124.10, 139.78,
152.26 (6 s, Carom); 215.47 (s, CS2).
ESI(-)–MS m/z: 338 [M–Na]- (100%).
1H NMR (dimethyl sulfoxide, DMSO, d) 1.4–1.7 (m,
–CH2–CH2CH2–CH2–, 4H), 2.4 (t, –CH2–CH2–Npip–, 2H),
2.6 and 3.0 (m, Hpiperazine, 8H), 3.4 (t, CN–CH2–, 2H), 3.9
(s, –OCH3, 3H), 6.9 (m, –C6H5, 4H).
[
99gTc(N)(PScy)(L2)]
13C NMR (DMSO, d): 24.8 and 26.5 (2 s,
CS2NH2CH2CH2CH2CH2N); 40.2 (CS2NH2CH2CH2CH2
CH2N); 50.7 and 52.9 (Cpiperazine); 55.4 (OCH3); 59.2
(CS2NH2CH2 CH2CH2CH2N); 111.4, 117.9, 120.8, 123.1
(Carom 2-methoxyphenyl); 141.5, 152.7 (N–Cquat.arom and
CH3O–C); 209.2 (s, CS2).
The yellow solution was evaporated and the resulting oily
residue was dissolved in CHCl3 (1 mL) and purified by
chromatography on a SiO2 column previously conditioned
with CHCl3. The column contents were eluted with CHCl3
followed by a mixture of MeOH containing 2% of NH4OH
(20% v/v)/CHCl3 (2:98). A yellow band was collected. The
eluent was completely evaporated with a gentle dinitrogen
stream. The final product was isolated as a pure yellow
powder upon treatment of the oil residue with n-hexane.
Yield 70%. The complex is soluble in alcohols, Et2O,
MeCN, and chlorinated solvents and insoluble in n-hexane
and water. Elemental analysis for C28H46N4OPS3Tc
(Mr = 679.86), calcd (%): C, 49.47; H, 6.82; N, 8.24; S,
14.15. Found: C, 50.40; H, 6.91; N, 8.32; S, 14.39.
ESI(?)–MS m/z: 680.77 [M?H]? (100%). FT IR (cm-1):
[3,390, s, m(NH)], [3,000–2,900, w, m(CH)], [1,501, s, and
Synthesis of [99gTc(N)(PS)(Ln)] complexes
[
99gTc(N)(PScy)(L1,2)]
To 0.065 mmol of [99gTc(N)(PScy)Cl(PPh3)] dissolved in
5 mL of CH2Cl2, 0.079 mmol of the appropriate dithio-
carbamate ligand (L1,2) dissolved in 5 mL of MeOH was
added. The reaction mixture was stirred at room tempera-
ture for 30 min. The initial yellow solution rapidly changed
to pale yellow. The solvents were completely evaporated
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143
1,449, s broad, m(C=C and C=S and CN)], [1,239, s, m(C–O)],
[1,063, m, m(Tc:N)], [748, s, m(arom)]. NMR and HPLC
analyses revealed that the isolated yellow product was a
mixture of two different compounds a and b (vide infra).
NMR (CDCl3, d): 0.8–1.30 (2 m, 24H, CH3CHP); 1.8–2.20
(2 m, 4H, SCH2CH2P); 2.25–2.50 (2 m, 4H, PCHCH3);
2.69 (m, 12H, S2CNCH2CH2N and CH2Npiperazine);
2.75–3.05 (m, 4H, PCH2CH2S); 3.10 (m, 8H, CH2Npiperazine);
3.75 (m, 4H, S2CNCH2); 3.87 (bs, 6H, CH3O); 6.94 (m,
8H, Harom). 13C NMR (CDCl3, d): 17.23, 17.42, 18.83,
19.34 (4 s, CH3CHP); 23.61, 23.85, 24.68, 24.99 (4 s,
1
31P NMR (CDCl3, d): 92.55 (s, br). H NMR (CDCl3, d):
1.10–2.18 (m, 44H, Hcyclohexyl); 1.94 and 2.17 (2 m, 4H,
SCH2CH2P); 2.71 (m, 12H, S2CNHCH2CH2N and CH2
N(CH2)CH2piperazine); 2.89 (m, 4H, CH2S); 3.10 (m, 8H,
CH2N(Ph)CH2piperazine); 3.73 (m, 4H, S2CNHCH2CH2N);
3.87 (s, 6H, OCH3); 6.84–7.06 (m, 8H, Harom). 13C NMR
(CDCl3, d): 25.90 (d, CH2P, 1JCP = 22.09 Hz); 26.02–27.28,
1
CH3CHP); 25.93, 26.33 (2 d, CH2P, JCP = 30.94 Hz);
31.10 (bs, SCH2); 40.48, 40.90 (2 s, CH2NHCS2); 50.50,
52.96 (2 s, NCH2CH2Npiperazine); 54.76, 54.97 (2 s,
NCH2CH2NHCS2); 55.36 (bs, OCH3); 111.15, 111.23,
118.20, 118.33, 120.99, 121.06, 123.14, 128.41, 140.97,
152.23 (10 s, Carom); 217.44, 217.67 (2 s, CS2).
2
29.08 (m, Hcyclohexyl); 30.65 (d, SCH2, JCP = 8.7 Hz);
33.54, 33.95 (2 d, PCH); 40.49, 40.56 (2 s, CH2NCS2);
50.05, 52.88 (2 s, NCH2CH2Npiperazine); 54.66, 54.97 (2 s,
NCH2CH2NHCS2); 55.48 (2 s, OCH3); 111.16, 111.20,
118.23, 118.40, 121.01, 121.10, 123.30, 128.60, 140.78,
152.26 (10 s, Carom); 215.20 (s, CS2).
[
99gTc(N)(PSiso)(L3)]
Yield 68%. The complex is soluble in alcohols, MeCN,
chlorinated solvents, and DMSO and insoluble in Et2O,
n-hexane, and water. Elemental analysis for C22H38
N4OPS3Tc (Mr = 627.78), calcd (%): C, 45.92; H, 6.74; N,
8.92; S, 15.32. Found (%): C, 45.99; H, 6.38; N, 8.63; S,
15.52. ESI(?)–MS m/z: 628.32 [M?H]? (100%). FT IR
(cm-1): [3,393, s, m(NH)], [3,000–2,900, w, m(CH)], [1,502,
s, and 1,450, s broad, m(C=C and C=S and CN)], [1243, s,
m(C–O)], [1,062, m, m(Tc:N)], [748, s, m(arom)]. NMR and
HPLC analyses revealed that the isolated yellow product was
a mixture of two different compounds a and b (vide infra).
[
99gTc(N)(PSiso)(L2–3)]
To 0.056 mmol of [99gTc(N)(PSiso)Cl(PPh3)] dissolved in
5 mL of CH2Cl2, 0.112 mmol of the appropriate dithio-
carbamate ligand (L2,3) dissolved in 5 mL of MeOH was
added. The reaction mixture was stirred at room tempera-
ture for 30 min. No color change was observed.
The solvents were completely evaporated by a gentle
dinitrogen stream and the yellow residue was dissolved
with CH2Cl2 and washed, three times, with an equal vol-
ume of water.
1
31P NMR (CDCl3, d): 102.00 (s, br). H NMR (CDCl3,
d): 0.6–1.36 (3 m, 24H, CH3CHP); 1.77 (bs, 8H, CH2
CH2CH2CH2); 1.83–2.15 (2 m, 5H, CH2P and PCHCH3);
2.31 (m, 3H, PCHCH3); 2.5 (bs, 4H, S2CNCH2CH2
CH2CH2Npiperazine); 2.77 (m, 8H, CH2Npiperazine); 2.92
(m, 4H, SCH2); 3.21 (m, 2H, CH2Npiperazine); 3.31 (bs, 4H,
CH2Npiperazine); 3.43 (m, 3H, CH2Npiperazine and CHHNCS2);
3.60 (bs, 2H CH2NCS2); 3.80 (m, 1H, CHHNCS2); 3.86
(bs, 6H, OCH3); 6.93 (m, 8H, Harom). 13C NMR (CDCl3,
d): 17.17, 17.45, 18.81, 19.34 (4 s, CH3CHP); 23.42, 23.73,
24.57, 24.64, 24.89 (5 s, CH3CHP and CH2alkyl); 25.70,
The organic layer was collected, dried with anhydrous
Na2SO4, and evaporated. A yellow residue was obtained.
This was treated with Et2O and n-hexane, dissolved with
CHCl3, and purified by chromatography on a SiO2 column
previously conditioned with CHCl3. The column contents
were eluted with CHCl3. The eluent was completely
evaporated with a gentle dinitrogen stream. The final
product was isolated as a pure yellow powder upon treat-
ment of the oil residue with Et2O.
1
26.18 (2 d, CH2P, JCP = 28.68 Hz); 26.98, 27.19 (2 s,
[
99gTc(N)(PSiso)(L2)]
CH2alkyl); 31.12, 31.22 (2 s, SCH2); 45.01 (bs, CH2NCS2);
49.60, 50.02 (2 s, CH2piperazine); 53.17, 53.33 (2 s,
CH2piperazine); 55.34, 55.39 (2 s, OCH3); 57.43, 58.35 (2 s,
S2CNCH2CH2CH2CH2Npiperazine); 110.97, 111.10, 118.49,
118.88, 120.81, 121.21, 123.18, 140.77, 141.01, 152.12,
152.27 (11 s, Carom); 215.04, 215.86 (2 s, CS2).
Yield 60%. The complex is soluble in alcohols, MeCN,
chlorinated solvents, and DMSO and insoluble in Et2O,
n-hexane, and water. Elemental analysis for C22H38N4
OPS3Tc (Mr = 599.73), calcd (%): C, 44.06; H, 6.39; N,
9.34; S, 16.04. Found (%): C, 43.99; H, 6.38; N, 9.23; S,
16.22. ESI(?)–MS m/z: 600.64 [M?H]? (100%). FT IR
(cm-1): [3,393, s, m(NH)], [3,000–2,900, w, m(CH)], [1,501,
s, and 1,448, s broad, m(C=C and C=S and CN)], [1,240, s,
m(C–O)], [1,062, m, m(Tc:N)], [745, s, m(arom)]. NMR
and HPLC analyses revealed that the isolated yellow
product was a mixture of two different compounds a and
Synthesis of [99mTc(N)(PS)(Ln)] complexes
Method 1
The preparation requires the preliminary formation of the
precursor complex [99mTc(N)(PS)X(PPh3)] (PS is PScy,
PSiso) as previously reported [3], followed by its
1
b (vide infra). 31P NMR (CDCl3, d): 102.54 (s, br). H
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J Biol Inorg Chem (2011) 16:137–155
conversion, at room temperature, into the final disymmet-
rical compound by reaction with 0.200 mL of saline con-
taining 0.250–0.100 mg of the selected bidentate ligand
(Ln). The pH of the reaction mixture, measured at the end
of the reaction, was in the range 2–3.
both the organic and the aqueous phases were collected and
counted with a c-counter. Partition coefficients, expressed
as (activity concentration in n-octanol)/(activity concen-
tration in water), are reported in Table 1.
A high yield of [99mTc(N)(PSiso)L1] was obtained by
adding to the precursor complex 0.250–0.100 mg of L1
dissolved in 0.200 mL of Na2HPO4 (0.5 M, pH 8). The
reaction pH measured at the end of the reaction was 7.
The RCY of the complexes, evaluated by TLC and
HPLC, was found to be always more than 90%.
In vitro studies
Stability studies
The in vitro stability of the complexes was evaluated by
monitoring the RCP at different time points (15 min, 1, 3,
and 24 h) using the following procedures. In a propylene test
tube an aliquot (50 lL) of HPLC-purified 99mTc complex
was added to 950 lL of (1) saline, (2) phosphate buffer
(0.10 M, pH 7.4), and (3) b-HPC saline solution (0.5 mg/
mL). The resulting mixture was incubated at 37 °C for 24 h.
The RCP was determined by TLC and HPLC.
Method 2
Na99mTcO4 (0.250 mL,50 MBqto3 GBq)wasaddedtoavial
containing succinic dihydrazide (SDH; 5 mg), PPh3 (3 mg),
0.1 mL of HCl (1 M), 1.5 mL of EtOH and PSisoH 0.025 mg
dissolved in 0.200 mL of EtOH. The vial was heated at 50 °C
for 15 min. After cooling, the selected Ln ligand (0.250–
0.100 mg) was added. The reaction mixture was left at room
temperature for 15 min. RCYs evaluated by TLC and HPLC
were comparable to those obtained by applying method 1.
Glutathione and cysteine challenge
An aliquot (50 lL) of a freshly prepared aqueous solution
of glutathione (10 mM) or, alternatively, of L-cysteine
(10 mM, 1 mM) was added to a propylene test tube, con-
taining phosphate buffer (250 lL, 0.2 M, pH 7.4), water
(100 lL), and 99mTc complex (100 lL). The mixture was
vortexed and incubated at 37 °C for 24 h. A control reac-
tion, with an equal volume of water added instead of glu-
tathione or cysteine solution, was conducted in parallel. At
0.25, 1, 3, and 24 h, aliquots of the reaction mixture were
analyzed by TLC and HPLC.
Solution stability of the 99mTc(N) complexes
Aliquots of each reaction mixture containing the relevant
99mTc(N) complex were analyzed by HPLC at 1, 8, and
18 h after labeling.
Purifications
To eliminate the excess of the cold ligands, the final 99mTc(N)
compounds were purified by HPLC. The desired fraction was
diluted with water (8 mL) and loaded onto a Sep-Pak RP-C18
cartridge previously activated with 95% EtOH (5 mL) and
water (5 mL). The cartridge was rinsed with water and the
99mTc species was eluted using 1 mL of EtOH (1 9 0.40 mL,
1 9 0.60 mL). The second fraction, containing all the
activity, was diluted with saline to obtain an aqueous solution
with 10% of EtOH and utilized for in vitro and in vivo studies
or, alternatively, with a physiological solution containing
0.5 mg/mL of hydroxypropyl beta-cyclodextrin (b-HPC).
Serum stability and protein binding
The serum stability and the affinity of the complexes for
the serum protein were evaluated after 0.25, 1, and 3 h of
incubation at 37 °C in human and in mouse serum as well
as in rat serum by chromatographic methods using the
procedures reported below.
In a propylene test tube an aliquot (100 lL) of HPLC-
purified 99mTc complex was added to 900 lL of human or
rat serum. One hundred and fifty microliters of each sample
was withdrawn and diluted in 1,350 lL of phosphate buffer
(0.02 M, pH 7.4). Aliquots of this solution were treated and
analyzed as follows:
As
a
reference,
symmetrical
bisubstituted
[
99mTc(N)(PS)2] and [99mTc(N)(L)2] compounds were
prepared according to the literature methods [3].
Method a: One hundred microliters was analyzed by
HPLC, without further purification, using a RP Symmetry
C4 column
Determination of log P values
Method b: Twenty-five microliters were loaded on a
prespun (2,000g for 1 min) Sephadex G-50 minicolumn.
The column was centrifuged at 2,000g for 1 min. The
collected eluate and the column were counted in a c-
counter. The protein-bound complex was calculated as the
percentage of the total activity. As a blank, 25 lL of
n-Octanol/water partition coefficients were determined by
vortex mixing (20 min) 3 mL of n-octanol, 3 mL of
phosphate buffer (0.02 M, pH 7.4), and 100 lL of the
radiolabeled compound purified as described already. After
centrifugation (3,000g for 10 min), aliquots (100 lL) of
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145
99mTc(N) complex was measured after incubation in
Biodistribution was carried out in rats. Before injection,
99mTc complexes were purified using the procedure outlined
earlier. After purification, the activity recovered was further
diluted with saline to obtain a final solution that was 10% in
EtOH content. Female Sprague–Dawley rats weighing
180–200 g were anesthetized with an intraperitoneal
injection of a mixture of Zoletil (40 mg/kg) and xylazine
(2 mg/kg). A jugular vein was surgically exposed and
100 lL (300–370 kBq) of the solution containing the
radioactive complex was injected. The rats (n = 3) were
killed by cervical dislocation at different times after injec-
tion. The blood was withdrawn from the heart with a syringe
immediately after death and quantified. Organs were
excised, rinsed in saline, weighed, and the activity was
counted in a NaI well counter. The results were expressed as
the percentage of injected dose per gram (%ID/g) and are
summarized in Table 2.
phosphate-buffered saline at 37 °C for 60 min.
Stability in brain and liver homogenates
In a propylene test tube an aliquot (50 lL) of HPLC-purified
99mTc complex was added to 450 lL of homogenate of rat
brain and to 450 lL of homogenate of rat liver. The resulting
mixtures were incubated at 37 °C for 3 h. At 0.25, 1, and 3 h,
aliquots (200 lL) of each solution were withdrawn, diluted
with phosphate buffer (800 lL, 0.2 M, pH 7.4), and treated
using an OASIS HLB Hydrophilic Lipophilic Balanced
extraction cartridge (see earlier) before HPLC injection.
Receptor binding assay
The binding assay was performed using the experimental
procedure described in the literature [16].
5HT1A receptor binding assay Competitive binding
assays were performed in triplicate, at 20 °C for 120 min, on
rat hippocampus homogenate (about 0.5 mg/mL protein)
using [3H]8-OH-DPAT (4.6 TBq/mmol from NEN) as the
radioligand. Nonspecific binding was defined as the amount
of [3H]8-OH-DPAT bound in the presence of 10 lM sero-
tonin (Sigma). Displacement was performed using different
concentrations (10 lM to 1 nM) of the 99gTc(N) complex
(1 mM in DMSO, followed by further dilutions with buffer).
The incubation was terminated by rapid filtration
through GF/B glass-fiber filters (Whatman), using a 30-port
Brandel cell harvester. The filters were rapidly washed with
4-mL portions of ice-cold buffer, transferred into 4 mL
scintillation fluid (Ultima Gold, Packard), and analyzed
for radioactivity by scintillation counting (Beckman LS
600-LL).
Effects of CsA on the pharmacokinetic properties
of [99mTc(N)(PSiso)(L2–3)] complexes
Rats (n = 6) were treated with a solution of CsA (20 mg/
kg in 250 lL of DMSO intraperitoneally) 60 min before
the radiotracer administration. At 2, 20, and 60 min after
injection, the rats (n = 3) were killed. As a blank, a group
of rats (n = 6) received the carrier vehicle solution
(DMSO) by intraperitoneal injection 60 min before radio-
tracer administration. The results are summarized in
Table 2.
Metabolites in the urine
The urine was collected from the bladder and loaded
directly onto an activated OASIS HLB extraction cartridge.
The complex was eluted with a mixture of EtOH and saline
(90:10; 0.5 mL) and analyzed by TLC and HPLC.
5HT2A receptor binding assay The 5HT2A binding
assays were performed in triplicate, at 20 °C for 120 min,
on rat cortex homogenate (about 0.5 mg/mL protein) using
[3H]ketanserin (2.3 TBq/mmol from NEN) as the specific
radioligand. Nonspecific binding was defined as the
amount of [3H]ketanserin bound in the presence of 1 lM
mianserin (Sigma). Displacement was performed using
different concentrations (10 lM to 1 nM) of the 99gTc(N)
complex (1 mM in DMSO, further dilutions with buffer).
Filtration and counting of the samples were the same as
described earlier.
Results
Synthesis of [99gTc(N)(PS)(Ln)] compounds
According to the synthetic pathway outlined in Scheme 2,
neutral disymmetrical nitrido complexes
[
99gTc(N)
(PS)(Ln)] were easily prepared via ligand-exchange reac-
tion treating the selected monosubstituted [99gTc(N)(PS)
Cl(PPh3)] compounds with a suitable dithiocarbamate
ligand (Ln) in CH2Cl2/MeOH solutions at room tempera-
ture. In these reactions [99gTc(N)(PS)Cl(PPh3)] selectively
and instantaneously reacted with Ln to afford the final
neutral five-coordinate mixed-substituted compounds in
good yields.
In vivo studies
Biodistribution
Animal experiments were carried out in compliance with
the relevant national laws relating to the conduct of animal
experimentation.
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The synthesis and characterization of monosubstituted
99gTc(N)(PS)Cl(PPh3)] complexes were previously repor-
[
ted [4].
Physicochemical characterization of the isolated
99gTc(N)(PS)(Ln)] disymmetrical compounds was in
[
agreement with the proposed formulations. FT IR spectra
exhibited the characteristic m(Tc:N) stretching vibration
at 1063/1,062 cm-1, along with typical absorption of
coordinated alkyl phosphine in the 3,000–2,900-cm-1
region and of the dithiocarbamate ligand in the region
around 1,500 and 750–745 cm-1 characteristic for –CS2
stretching and aromatic bending, respectively.
ESI(?) mass spectra of disymmetrical compounds
showed the protonated ([M?H]?) molecular ion peak
without detectable fragmentation.
As expected, 31P NMR spectra showed a broadened
singlet in the 102.5–92.1-ppm region diagnostic for the
formation of the mixed-substituted complexes [4]. Both 1H
and 13C NMR spectra showed complicated patterns con-
sistent with the molecular structure depicted in Scheme 2.
For [99gTc(N)(PS)(L2,3)], two distinct species were
observed both in NMR spectra and in HPLC profiles. Upon
the coordination, the presence in L2 and L3 of the chiral
center on the N-dithiocarbamate made possible the for-
mation of two distinct isomeric forms (syn and anti) orig-
inating from the different orientation of the N-substituted
pendant group with respect to the central Tc:N terminal
core. HPLC profiles showed in both cases an isomeric ratio
of approximately 60:40.
In this case, the rapid isomeric conversion made impos-
sible the separation and full characterization of each species.
Synthesis of [99mTc(N)(PS)(Ln)] compounds
The preparation of disymmetrical [99mTc(N)(PS)(Ln)]
complexes was carried out as outlined in Scheme 3.
According to ‘‘Method 1,’’ the synthesis required a three-step
approach which entailed the preliminary formation of the
well-characterized monosubstituted [99mTc(N)(PS)X(PPh3)]
compound [3] followed by its conversion into the final com-
plex by the addition of a suitable dithiocarbamate ligand.
Using this procedure, we optimized the RCY of the
radioactive complexes considering different reaction
parameters such as the amount of ligand and the influence
of the pH as well as of the incubation time. Considering the
data for other disymmetrical [99mTc(N)(PS)(L)] com-
pounds, the temperature was kept at roon temperature [3].
Among these reaction parameters, the pH was found to
be a crucial factor in determining the formation of the
complexes in high yield. Figure 2a displays the variation of
the percentage RCY (%RCY) of the different complexes
evaluated, after 60 min at room temperature, at different
pH values (in the range 2–9). For [99mTc(N)(PS)(L1)]
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J Biol Inorg Chem (2011) 16:137–155
147
Scheme 2 Synthesis of
99gTc(N)(PS)(Ln)] complexes
[
Scheme 3 Synthesis of
99mTc(N)(PS)(Ln)] complexes.
[
PSH phosphino thiol ligand,
SDH succinic dihydrazide, RCY
radiochemical yield
a high %RCY was achieved at a pH value of 6–7. For
99mTc(N)(PS)(L2,3)], a high %RCY was obtained at a low
considerable amount of the symmetrical bisubstituted
99mTc(N)(PS)2] species was observed.
[
[
pH value, in the range 2–3. For these latter compounds, a
significant reduction of the %RCY was observed by
increasing the pH to 9. At this pH, the formation of a
The variation of the %RCY of [99mTc(N)(PS)(Ln)],
evaluated after 60 min at room temperature, using a dif-
ferent amount of the selected dithiocarbamate ligand is
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Fig. 2 a Variation of the
percentage radiochemical yield
(%RCY) of the different
(a) 100
90
[
99mTc(N)(PSiso)(Ln)]
80
70
60
50
40
30
20
complexes as function of pH.
One milligram of the selected
Ln was dissolved in (1)
0.250 mL of saline, final pH
2/3; (2) 0.250 mL of 0.5M
NaAc pH 7, final pH 5; (3)
0.250 mL of 0.5 M Na2HPO4
pH 8, final pH 6/7;
(4) 0.250 mL of 0.5 M
-
HCO3-/CO3 pH 9, final pH 9;
%RCY was evaluated after
60 min at room temperature.
b Variation of %RCY of the
different [99mTc(N)(PSiso)(Ln)]
complexes as a function of the
ligand concentration. %RCY
was evaluated after 60 min at
room temperature. c Variation
of %RCY of the different
2
3
5
7
9
pH
[99mTc(N)(PSiso)(L1)]
[99mTc(N)(PSiso)(L2)]
[99mTc(N)(PSiso)(L3)]
100
(b)
90
80
70
60
50
40
30
[
99mTc(N)(PSiso)(Ln)]
complexes over time
1
0,5
0,25
0,10
0,05 mg
[99mTc(N)(PSiso)(L1)]
[99mTc(N)(PSiso)(L2)]
[99mTc(N)(PSiso)(L3)]
100
(c)
90
80
70
60
50
40
30
5 min
15 min
[99mTc(N)(PSiso)(L1)]
30 min
60 min
time
[99mTc(N)(PSiso)(L2)]
[99mTc(N)(PSiso)(L3)]
reported in Fig. 2b. In all cases, high radiolabeling effi-
ciency was observed using a relatively low amount of
ligand, in the range 0.250–0.100 mg.
A quick and quantitative conversion into neutral di-
symmetrical nitrido species occurred within 15 min at
room temperature (Fig. 2c). No variation of the RCY of the
123

J Biol Inorg Chem (2011) 16:137–155
149
final complex was observed when the incubation time was
extended from 15 to 60 min.
[
99mTc]ECD measured under the same experimental con-
ditions. log k00 is obtained from log k0, where k0 is the
capacity factor measuring the partitioning of the compound
between a nonpolar stationary phase and a polar0 mobile
phase [3, 24]. It was shown previously that log k0 values
are well correlated with log P values (P is the water/oct-
anol partition coefficient) for lipophilic compounds [24].
The results are reported in Table 1 and are compared with
the corresponding log P values.
Alternatively, complexes were obtained with compara-
ble RCY using a two-step procedure as indicated in
‘‘Method 2.’’ Using this method, we obtained the inter-
mediate monosubstituted [99mTc(N)(PS)X(PPh3)] in only
15 min at 50 °C by adding pertechnetate to a vial con-
taining an alcohol solution of SDH and PPh3 at pH 2 and
PSisoH.
The final compound was obtained, at room temperature,
by the reaction of the intermediate complex with the suit-
able dithiocarbamate ligand.
Measurements of log P showed that the lipophilicity of
99gTc(N)(PSiso)(Ln)] is in the appropriate range necessary
[
for crossing the BBB.
The chemical identity of 99mTc complexes was deter-
mined by HPLC comparison of their chromatographic
properties with those of the corresponding well-character-
ized 99gTc complexes (see Table 1; Fig. 3).
Staring from the less lip0ophilic compound, the corre-
sponding log P and log k0 values are as follows: for
[
99mTc(N)(PSiso)(L1)], log P = 0.90 0.08, log k0
=
0
4.10; for [99mTc(N)(PSiso)(L2)], log P = 0.97 0.05,
The HPLC profile of [99mTcN(PS)(L2,3)] displays the
presence of two separate peaks, a and b, which overlap
those obtained with the [99gTcN(PS)(L2,3)] analogue. The
isomeric ratios, evaluated by TLC and HPLC, are almost
the same as those estimated for the 99gTc complexes,
approximately 60:40. The separation of the pure isomeric
species was unsuccessful. Thus, in vitro and in vivo studies
were performed using the unresolved mixture.
log k0 = 4.48/4.86; for [99mTc(N)(PSiso)(L3)], log P =
0
0
1.11 0.03, log k0 = 4.96/5.38.
Stability studies
Neutral disymmetrical [99mTc(N)(PSiso)(L1–3)] complexes
remained stable in reaction solution for more than 18 h
after labeling and for 6 h, after HPLC purification, in EtOH
solution or in aqueous solution containing 10% EtOH.
Transformation of the mixed complexes into more hydro-
philic compounds was observed after dilution with saline
or phosphate buffer (0.02 M, pH 7.4), to obtain a final
solution containing 5% EtOH. The corresponding RCP
evaluated by TLC and HPLC was found reduced to 70% at
1 h. In these latter conditions, a significant increase of the
stability (RCP 90% after 6 h) in aqueous solution was
achieved by adding a small amount of b-HPC (1.5 mg/mL)
to the complex solution.
The log k00 values of the compounds as a measure of the
relative lipophilicity of the complexes were calculated by
RP HPLC using various mixtures of MeOH/phosphate
buffer (pH 7.4) as the eluent, and were compared with
log k00 of the commercially available perfusion agent
In vitro stability studies and challenge with glutathione
and cysteine
The transchelation with an excess of free glutathione and
cysteine was investigated for all [99mTc(N)(PSiso)(Ln)]
complexes.
No significant changes in RCPs of the complexes were
observed in challenge experiments after 24 h of incubation
at 37 °C with glutathione (10 mM), whereas a slight dif-
ference in stability was observed after treatment with
cysteine. In all cases, the formation of a more hydrophilic
compound was observed in the HPLC profile of the com-
plexes after incubation with an excess of 10 mM cysteine
as consequence of a transchelation reaction between ligand
and cysteine. The stability was found to decrease in the
order
99mTc(N)(PSiso)(L3)]. Nevertheless, studies carried out
[
99mTc(N)(PSiso)(L1)] [ [99mTc(N)(PSiso)(L2)] [
Fig. 3 Reverse-phase (RP) high performance liquid chromatography
(HPLC) comparison of [99mTc(N)(PSiso)(L2)] complexes with the
[
[
99gTc(N)(PSiso)(L2)] analogue
with 1 mM cysteine evidenced a reduction of the
123

150
J Biol Inorg Chem (2011) 16:137–155
transchelation rate of the complexes, which therefore return
to stability.
of the compounds, expressed as IC50 values, are reported in
Table 4. [99mTc(N)(PSiso)(L3)] has the highest affinity and
selectivity for the 5HT1A receptors (IC50 for 5HT1A
1.5 0.1 nM; competitor 5HT2A 570 30). A reduction
of the affinity for the other complexes as a function of the
reduction of the alkyl chain length interposed between the
dithiocarbamate unit and the bioactive molecule was
observed.
The binding to the serum proteins and biotransformation
of the agents in blood and tissue homogenate were assessed
in vitro by incubating the purified compound at 37 °C in
human and rat sera as well as in rat liver and rat brain
homogenates. The affinity of the complexes for the serum
protein was evaluated at 2, 60, and 360 min using size-
exclusion chromatography (microspin columns). The
results are summarized in Table 3.
Biodistribution
In general, after 15 min incubation an high and persis-
tent percentage of the radiocomplex was found associated
with the plasma proteins. For the various compounds the
different affinity for the plasma proteins was in agreement
with their lipophilic character. The in vitro metabolism
studies were assessed by HPLC evaluating the variation of
the %RCP of the complexes incubated with the different
media over time.
In vivo studies of [99mTc(N)(PSiso)(L2)] and [99mTc(N)
(PSiso)(L3)] were performed in female Sprague–Dawley
rats.
The radiolabeled compounds were purified by HPLC
before injection to remove excess unlabeled cold ligand.
Table 2 shows the distribution of the complexes expressed
as the percentage of injected dose per gram of tissue. The
RCPs of the purified complexes were verified by HPLC
prior to in vivo administration as well as during the utili-
zation period, to guarantee better than 90% purity of the
injected tracer. For [99mTc(N)(PSiso)(L3)] the effect of
CsA on the pharmacokinetic properties of the complex was
also investigated (Table 2).
The data clearly indicated that no biotransformation of
the native compounds in different species was observed
after incubation in both human and rat sera (Fig. 4).
A slight reduction of the %RCP of the complexes was
detected after exposure to liver and brain homogenates
(Fig. 5). In particular, in rat liver homogenate the HPLC
chromatogram of [99mTc(N)(PSiso)(L2,3)], collected after
3 h of incubation, displayed the presence of a more hydro-
philic species (20–30%), as a possible metabolized form.
The complexes exhibited a similar distribution pattern
characterized by a rapid blood clearance and organ
extraction. An appreciable heart uptake was observed at
2 min after injection of 1.90 0.39 and 2.58 0.44 %ID/
g for [99mTc(N)(PSiso)(L2)] and [99mTc(N)(PSiso)(L3)],
respectively. At 20 min after injection the activity was
found to be reduced to 0.43 0.05 and 0.82 0.09 %ID/
g for [99mTc(N)(PSiso)(L2)] and [99mTc(N)(PSiso)(L3)],
respectively.
Receptor binding
To evaluate the effect of the steric hindrance of the
[Tc(N)(PS)] moiety and the effect of the length and flexi-
bility of the spacer interposed between the pharmacophore
and the dithiocarbamate chelate unit, the in vitro binding
affinity of the different technetium complexes for the
5HT1A and 5HT2A receptors was determined.
For the 5HT1A receptors the [99mTc(N)(PS)(Ln)] affinity
was assessed by measuring the ability of the compounds to
compete with [3H]8-OH-DPAT binding in rat hippocampal
homogenates.
As expected, the complexes have a high liver uptake,
which quickly decreased over time and at 20 min after
injection a significant amount of the injected dose cleared
into the intestine. A low kidney uptake followed by a rapid
washout was observed. The activity extracted in the urine
was extremely low, suggesting that these compounds were
mainly eliminated through the hepatobiliary system.
For [99mTc(N)(PSiso)(L3)], the HPLC analysis of the
urine collected at 1 h after injection revealed that 70% of the
total injected activity was found to be coincident with the
Rat cortex homogenates were used for 5HT2A receptor
binding assays with [3H]ketanserin as a specific radioli-
gand. The in vitro affinity for 5HT1A and 5HT2A receptors
Table 3 Percentage of protein binding of the Tc(N) complexes evaluated over time in human serum (HS) and rat serum (RS) by size-exclusion
chromatography
Protein binding in HS (%)
Protein binding in RS (%)
15 min
60 min
180 min
15 min
60 min
180 min
[
[
[
99mTc(N)(PSiso)(L1)]
99mTc(N)(PSiso)(L2)]
99mTc(N)(PSiso)(L3)]
52.29 0.30
21.14 0.67
38.26 0.24
52.87 0.21
20.44 1.36
34.79 0.22
52.44 0.49
19.76 0.13
33.72 0.33
53.12 4.84
13.94 0.25
40.73 0.46
52.53 4.42
14.17 0.47
44.58 1.10
50.16 3.48
15.03 0.86
47.23 0.89
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J Biol Inorg Chem (2011) 16:137–155
151
Radio
Retention Time
Area Percent
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
5
10
15
20
25
30
Minutes
0.35
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Radio
Retention Time
Area Percent
Radio
Retention Time
Area Percent
Radio
Retention Time
1.8
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Area Percent
0.30
0.25
0.20
0.15
0.10
0.05
0.00
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0
5
10
15
20
25
30
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Minutes
Minutes
Minutes
Radio
Retention Time
Area Percent
Radio
Retention Time
Area Percent
Radio
Retention Time
Area Percent
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
5
10
15
20
25
30
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Minutes
Minutes
Minutes
Fig. 4 HPLC profiles of [99mTc(N)(PSiso)(L3)] after 0.25, 1, and 3 h of incubation in human (above) and rat (below) serum. The analysis was
performed using a RP C4 column as described in ‘‘Materials and methods’’
native compound and only a small amount of the radioac-
tivity, approximately 20%, was eluted as more hydrophilic
species, evidencing good in vivo stability of the complex.
In both cases, only a negligible fraction of the injected
activity (at 2 min, 0.06 0.00 %ID/g for [99mTc(N)(PSiso)
(L2)] and 0.08 0.01 %ID/g for [99mTc(N)(PSiso)(L3)])
crossed the BBB, and no retention of the radioactivity was
detected.
No variation of the BBB penetration of [99mTc(N)(PSiso)
(L3)] was detected after CsA administration. The effects of
123

152
J Biol Inorg Chem (2011) 16:137–155
(a) 120
100
80
60
40
20
0
time
15 min
60 min
180 min
120
(b)
100
80
60
40
20
0
time
15 min
60 min
180 min
[99mTc(N)(PSiso)(L1)]
[99mTc(N)(PSiso)(L2)]
[99mTc(N)(PSiso)(L3)]
Fig. 5 Stability of [99mTc(N)(PSiso)(Ln)] complexes after incubation in brain (a) and liver (b) homogenates. The experiments were performed in
duplicate, the results are expressed as the %RCP
Table 4 IC50 (nM) values for
the compounds tested
Complex
IC50 (nM)
5HT1A [3H]-8-OH-DPAT
5HT2A [3H]ketanserin
5HT2A/5HT1A
[
[
[
[
99gTcN(PScy)(L1)]
99gTcN(PScy)(L2)]
99gTcN(PSiso)(L2)]
99gTcN(PSiso)(L3)]
2,800 10
100 1.0
145 1.0
1.5 0.1
6,200 100
11,370 400
290 10
2.21
113.7
2
570 30
380
CsA were evident on the pharmacokinetic profile of
99mTc(N)(PSiso)(L3)]; in fact, a notable increase of both
[Tc(N)Cl(PS)(PPh3)] is an active intermediate which
selectively reacts with an appropriate mononegative che-
late (S^Y) to form neutral [Tc(N)(PS)(S^Y)] compounds
characterized by a disymmetrical arrangement of two
bidentate ligands around the metal center.
[
liver and kidney uptake as well as a reduction in the cor-
responding washout were observed.
Neutral and lipophilic disymmetrical complexes of the
type [99gTc(N)(PS)(Ln)] were simply prepared in high yield
by ligand-exchange reactions of the corresponding
Discussion
In this study a new route for incorporating a bioactive
molecule into stable technetium-99m nitrido compounds
was reported. The method is based on the use of the
monosubstituted [Tc(N)Cl(PS)(PPh3)] species character-
ized by a bulky alkyl phosphinothiolate ligand (PS) coor-
dinated to the technetium nitrido group and two labile sites
usually filled by two monodentate ligands (halogen and
PPh3) which complete the square-pyramidal environment.
[
99gTc(N)Cl(PS)(PPh3)] (PS is PScy, PSiso) precursor
complex with the bifunctional ligand Ln. As indicated in
Scheme 2, the disymmetrical complexes have
99gTc(N)(PS)] moiety coordinated to the dithiocarbamate
a
[
Ln ligand.
For [99gTc(N)(PSiso)(L2,3)], upon coordination to the
99gTc(N)(PS)]? building block, Ln adopts two distinct
[
isomeric forms depending on the relative syn or anti
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J Biol Inorg Chem (2011) 16:137–155
153
orientation of the N-substituted pendant group with respect
Nanomolar affinities for the 5HT1A receptor were found
for [99gTc(N)(PSiso)(L3)] (IC50 = 1.5 nM). In this com-
pound the bioactive molecule is bound to the less sterically
hindered [Tc(N)(PSiso)] moiety and a four-member group
spacer is interposed between the dithiocarbamate chelating
system and the pharmacophore. Notably, the IC50 value of
this Tc-based compound is lower than the reported value
for the parent compound WAY 100635 (IC50 = 2.2 nM),
suggesting that the affinity of 2-MPP for the 5HT1A
receptor is not reduced by its functionalization with the
dithiocarbamate chelate and by the presence of the
to the Tc:N terminal group (see earlier).
At tracer level, disymmetrical
[
99mTc(N)(PS)(Ln)]
complexes were prepared using a multistep procedure
which required the addition at room temperature of the Ln
ligand to a mixture of the prereduced intermediate
[
99mTc(N)X(PS)(PPh3)]?/0 compounds. Table 1 summa-
rizes the chromatographic properties and the RCY values
obtained for the corresponding disymmetrical complexes.
RCY data evidenced the ability of the dithiocarbamate to
form the final complexes, exhibiting a high selectivity in
displacing the monodentate ligands (X- and PPh3) from
the [99mTc(N)X(PS)(PPh3)]?/0 intermediate compound by
using a relatively small amount (0.100 mg) of Ln.
[
99gTc(N)(PSiso)] building block.
For the other complexes, a reduction of the receptor
affinity as a function of the reduction of the alkyl chain
length interposed between the dithiocarbamate group and
the bioactive molecule was observed. A complete loss of
receptor affinity was exhibited by [99gTc(N)(PScy)(L1)].
This behavior is attributed to the absence of an appropriate
group spacer between the piperazine moiety and the
dithiocarbamate fragment and to the presence of the
bulkiest [99gTc(N)(PScy)] moiety.
The chemical identity of 99mTc complexes was deter-
mined by HPLC comparison with the well-characterized
99m/
99gTc analogues. After co-injection of the appropriate
99gTc couples, identical retention times were observed
(Fig. 2).
Stability studies displayed that these agents are stable
for hours in aqueous solution in presence of b-HPC and are
inert toward transchelation with an excess of free gluta-
thione. Nevertheless, [99mTc(N)(PSiso)(Ln)] complexes
were found to undergo challenge reactions with an excess
of cysteine (10 mM). Ligand-exchange reaction of the
dithiocarbamate ligand occurs with different rates
([99mTc(N)(PSiso)(L3)] [ [99mTc(N)(PSiso)(L2)] [ [99mTc
(N)(PSiso)(L1)]), depending on the nature of the dithio-
carbamate ligand (length of the spacer, flexibility of the
molecule, and presence of a chiral center at N-dithiocar-
bamic). For [99mTc(N)(PSiso)(L3)] and [99mTc(N)(PSi-
so)(L2)] the challenge reactions may be correlated to their
rapid isomeric conversion which could involve the disso-
ciation of Ln and the insertion of the cysteine [3, 22].
Hence, change of the inner coordination sphere of
the complexes with formation of the corresponding
These data clearly confirm the importance of an
appropriate group spacer between the biomolecule and the
functional chelator.
All complexes had a low binding affinity for the 5HT2A
receptor. [99gTc(N)(PSiso)(L3)] has a high 5HT1A affinity,
characterized by a 5HT2A/5HT1A ratio of 380.
Although the charge, the molecular weight, and the
lipophilicity of [99gTc(N)(PSiso)(L2,3)] are in the appro-
priate range required for crossing the BBB (log P in the
range 1.0–2.5; Mr \ 700), the in vivo biodistribution data
showed for both complexes there was a negligible brain
uptake at 2 min after injection along with no appreciable
retention of the activity. This fact indicated that attempts to
interpret the BBB transport of the Tc complexes on the
basis of rough physicochemical parameters are not
exhaustive. Hence, the in vitro/in vivo stability and the
plasma protein binding affinity of [99gTc(N)(PSiso)(L2,3)]
must be considered to explain the absence of brain uptake.
Stability studies established that the compounds are stable
in serum and undergo partial metabolic degradation, in
vitro after exposure to brain and liver homogenate and in
vivo forming a more hydrophilic compound, despite the
intact complexes in all situations being the dominant
species.
[
99mTc(N)(PSiso)(cys)] was observed. Monoanionic
bidentate ligands having [S-, NH2] as a coordinating pair
such as cysteamine and cysteine ethyl ester were found to
efficiently and selectively react with [99mTc(N)(PS)]? to
form stable mixed compounds [3].
These findings suggest that, as found for other TcN-based
mixed-ligand compounds, the arrangement of p-acceptor
and p-donor coordinating atoms around the [Tc:N]2? core
is responsible for the high stability and kinetic inertness of
the resulting mixed compound [3, 25]. No noteworthy in
vitro serum protein binding and low biotransformation of the
native compound into different species by the in vitro action
of the serum and liver enzymes was evidenced.
In vitro, protein binding assays displayed a high affinity
of the complexes for the plasma proteins; in fact, only 50%
of [99gTc(N)(PSiso)(L3)] was found as free complex. Fre-
quently, in vivo, this behavior limits the bioavailability of
the agent, reducing its brain penetration.
The in vitro affinity for the 5HT1A and 5HT2A receptors
of the compounds expressed as values of IC50 is reported in
Table 2.
Although it is well known that the protein binding can
be a reversible process, it is important that for compounds
123

154
J Biol Inorg Chem (2011) 16:137–155
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building block, which, once formed, selectively reacts with
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