Synthesis and characterization of new 99mTc-radiopharmaceuticals with dithiobenzoate derivatives for the study of septic inflammatory processes

Article abstract of DOI:10.1002/jlcr.673

Improved methods for the preparation of ^99mTc-radiopharmaceuticals containing dithiobenzoate ligands, in sterile and pyrogen free conditions, are described. These procedures are based on the reaction of these ligands either with [^99mTc] pertechnetate in the presence of a strong reducing agent (HCl/ tertiary phosphine, SnCl₂·2H₂O), or with pre-reduced complexes obtained from different kit formulations. All the preparations led to the high-yield formation of the neutral and lipophilic ^99mTc-complex [^99mTc][Tc(S₃CPh)₂ (S₂CPh)], which is analogous to the corresponding compounds obtained with rhenium and the long-lived β-emitting isotope technetium- 99 g recently described. HPLC analysis and thin layer chromatography were used to confirm the characterisation of the resulting ^99mTc-radiopharmaceutical which was found to be potentially suitable for blood-cell labelling as applied to the diagnosis of inflammatory processes. Copyright

Full text of DOI:10.1002/jlcr.673

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 319–331.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.673
Research Article
Synthesis and characterization of new 99mTc-
radiopharmaceuticals with dithiobenzoate
derivatives for the study of septic
inflammatory processes
F. Mevellec1,y, A. Roucoux1,*, N. Noiret1, A. Moisan2,
H. Patin1 and A. Duatti3
1
!
Ecole Nationale Superieure de Chimie de Rennes, CNRS UMR 6052
" !
‘‘Syntheses et Activation de Biomolecules’’, Institut de Chimie de Rennes,
35700 Rennes, Beaulieu, France
2
! "
Centre Regional de Lutte Contre le Cancer, Eugene Marquis, 35062 Rennes,
France
3 Laboratory of Nuclear Medicine, Department of Clinical and Experimental
Medicine, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy
Summary
Improved methods for the preparation of 99mTc-radiopharmaceuticals contain-
ing dithiobenzoate ligands, in sterile and pyrogen free conditions, are
described. These procedures are based on the reaction of these ligands either
with [99mTc] pertechnetate in the presence of a strong reducing agent (HCl/
tertiary phosphine, SnCl2 ꢀ 2H2O), or with pre-reduced complexes obtained
from different kit formulations. All the preparations led to the high-yield
formation of the neutral and lipophilic 99mTc-complex [99mTc][Tc(S3CPh)2
(S2CPh)], which is analogous to the corresponding compounds obtained with
rhenium and the long-lived b-emitting isotope technetium-99 g recently
described. HPLC analysis and thin layer chromatography were used to
confirm the characterisation of the resulting 99mTc-radiopharmaceutical which
was found to be potentially suitable for blood-cell labelling as applied to the
!
*Correspondence to : A. Roucoux, Ecole Nationale Superieure de Chimie de Rennes, UMR CNRS
6052 ‘‘Syntheses et Activation de Biomolecules’’, Institut de Chimie de Rennes, 35700 Rennes,
"
!
Beaulieu, France. E-mail: alain.roucoux@ ensc-rennes.fr
ybio international/Schering S.A. E-mail fmevellec@cisbiointernational.fr
Copyright # 2003 John Wiley & Sons, Ltd.
Received 13 April 2002
Revised 13 September 2002
Accepted 3 October 2002

320
F. MEVELLEC ET AL.
diagnosis of inflammatory processes. Copyright # 2003 John Wiley & Sons,
Ltd.
Key Words: Technetium-99m; radiopharmaceuticals; dithiobenzoate ligands
Introduction
Nuclear medicine is an area of research having a specific interest in the
design, synthesis and clinical application of new radiopharmaceuticals
for diagnosis and therapy.1 In particular, there is a growing interest for
the development of new tracers for blood-cell labelling which may be
useful in the study of the mechanism of immune response or to visualize
inflammatory lesions.2 This approach has been successfully applied
during the last 20 years using 111In-oxine and 99mTc-HMPAO
(HMPAO=hexamethylpropyleneamine oximate).3 However, these
well-known blood-cell labelling agents suffer from various disadvan-
tages, the most significant being their lack of selectivity, which
necessarily requires a first separation step of the target blood cells
before labelling. Thus, current efforts are focused on the design and the
preparation of more specific blood-cell labelling agents. In our
laboratory, we found that the class of nitrido technetium-99m
complexes with dithiocarbamate ligands showed promising results for
this purpose. Specifically, the complex bis-(N-ethoxy, N-ethyldithiocar-
bamate)nitridotechnetium(V) [99mTcN(NOET)2] allows in vitro specific
polynuclear cells labelling in the whole blood.4 whereas similar
disubstituted complexes with dithiocarboxylates having aliphatic linear
side-chains induce a high lymphocyte selectivity in the same conditions.5
[
99mTcN(NOET)2]6 is an example of a large class of Tc-99m
complexes, including also xanthates7 and dithiocarboxylates,8 charac-
terised by the presence of a terminal TcꢁN multiple bond. It was found
that [99mTcN(NOET)2] exhibits high myocardial localization both in
animals and in humans, and it is currently under phase III clinical trials
for receiving final approval as heart imaging agent.9 The efficient
preparation of the TcꢁN group, at no carrier-added level and under
sterile and pyrogenic free conditions, has been recently made possible
after the introduction by Pasqualini et al.10 of a suitable kit formulation
based on the initial reaction of pertechnetate ([99mTc]TcO4ꢂ) with N-
methyl-S-methyl dithiocarbazate [MDTCZ=H2N–N(Me)C(=S)SMe]
or, alternatively, with succinic dihydrazide [SDH=H2N–NH–C(=O)–
(CH2)2–(O=)C–NH–NH2] as sources of nitride nitrogen atoms, in the
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND CHARACTERIZATION OF NEW 99MTC-
321
presence of a reducing agent such as SnCl2ꢀ2H2O or HCl and tertiary
phosphines. The initial step is then followed by the addition of the
appropriate ligand to afford the final radiopharmaceutical having the
nitrido core.
In this paper, we report the synthesis of the complex
[
99mTc(S3CPh)2(S2CPh)] using different kit methods and by varying
the nature of the counter ion in the starting ligand PhCSꢂ2 X+ L1–L4
[X=MgBr, Na, NH2(CH2)5, NMe4]. The preparation and characteriza-
tion of the complexes [M(S3CPh)2(S2CPh)] (M=99gTc, Re) in macro-
scopic amounts, using the long-lived isotope Tc-99 g and the stable
rhenium congener, has been recently described by our group.11 These
latter species have been utilized here as reference compounds to obtain
the full elucidation of the chemical nature of the new 99mTc-radio-
pharmaceutical by HPLC analysis and thin layer chromatography.
Experimental
Materials
Tetrahydrofuran (THF) was distilled twice under nitrogen from
sodium/benzophenone before use, while diethyl ether, dichloromethane
and petroleum ether were distilled using the same procedure from
calcium chloride. Bromobenzene, magnesium turnings, carbon disulfide,
tetramethylammonium hydroxyde, piperidine, triphenylphosphine and
stannous chloride dihydrate were commercially available (Aldrich,
St Quentin Fallavier, France) and used as purchased. Sodium tris
(m-sulfonatophenyl)phosphine [P(m-C6H4SO3)3]Na3 (TPPTS) was
synthesized as reported previously.12 Sodium [99mTc]pertechnetate, in
physiological solution, was obtained from a 99Mo/99mTc generator ELU
III (CIS bio international/Schering SA, Gif sur Yvette, France). All
other chemicals were of laboratory grade and used without further
purification. 1,2-diaminopropane-N-N-N0-N0-tetraacetic acid (PDTA)
and kits for the preparation of the TcꢁN group (99mTc-MDTCZ and
99mTc-SDH) and of 99mTc-Gluconate were generously offered by CIS
bio international/Schering SA (Gif-sur-Yvette, France).
Ligands
The magnesium bromide salt, PhCSꢂ2 MgBr+ L1, was prepared in
anhydrous THF as reported previously,13 and isolated by filtration at
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322
F. MEVELLEC ET AL.
CS2 NMe4
L4
NMe4OH
Mg
CS2
-15°C
HCl
NaOH
Br
MgBr
CS2MgBr
CS2H
CS2 Na
L2
THF/N2
L1
Piperidine
Et2O
CS2 H2 N
L3
Scheme 1. Synthesis of dithiobenzoic ligands
Table 1. Ligand synthesis
Ligands
Conter ion
Rfa
M.p. (8C)
yield(%)
L1
L2
L3
L4
MgBr
Na
H2N(CH2)5
NMe4
0.85
0.81
0.83
0.74
Oxidation in air
92-94
96-98
82
94
76
52
98-100
a eluent : acetone
–158C, washed with cold diethyl ether and dried under reduced pressure.
The tetramethylammonium salt L4, PhCSꢂ2 NMe4+, was precipitated by
addition of an equivalent amount of tetramethylammonium hydroxide
to a pink solution of dithiobenzoic acid, under vigorous stirring, and
then isolated by filtration, washed with diethyl ether and dried under
reduced pressure. PhCS2H was previously obtained by addition of 20 ml
of HCl (1 mol ꢂ1) to the reaction solution containing the magnesium
bromide salt at 08C and then isolated after extraction with diethyl ether.
The sodium salt L2, PhCS2ꢂNa+, was prepared using a similar
procedure by addition of an equivalent amount of aqueous sodium
hydroxide (1.25 mol dmꢂ3), and precipitated after concentration of the
resulting
solution.
Similarly,
the
piperidinium
salt
L3,
PhCSꢂ2 (C5H10)NH+2 , was precipitated from PhCS2H, by addition of
an equivalent amount of piperidine, and then isolated by filtration,
washed with diethyl ether and dried under vacuum. The preparations of
the various salts of the ligand PhCSꢂ2 are summarized in Scheme 1.
Analytical data are reported in Tables 1–3. All the products were stored
at –128C under a nitrogen atmosphere.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND CHARACTERIZATION OF NEW 99MTC-
323
Table 2. 1H NMR and 13C NMR of ligands
Ligands (sol- 1H NMR [ppm, peak multiplicity, (number of hydrogens)]a
vent)
L1 : dmso-d6
Aromatic hydrogens: 7.19, t, J=7.6 Hz(2 H) 7.29, t, J=7.6 Hz(1 H)
8.15, d, J=7.6 Hz(2 H)
L2 : D2O
Aromatic hydrogens: 7.28, t, J=7.1 Hz (2 H) 7.39, t, J=7.1 Hz
(1 H) 7.89, d, J=7.1 Hz (2 H)
L3 : CDCl3
CH2: p, J=5.0 Hz(2 H) CH2: p, J= 6.0 Hz(4 H) NCH2: 3.22, s
(12 H)
Aromatic hydrogens: 7.35, t, J=7.5 Hz(2 H) 7.46, t, J=7.0 Hz(1 H)
8.46, d, J=7.5 Hz(2 H) NH: 8.12,s(1 H)
NCH3: 3.10, s, 12 H Aromatic hydrogens: 7.14, t, J=7.1 Hz(2 H)
7.29, t, J=7.1 Hz(1 H) 8.15, d, J=7.1 Hz(2 H)
L4 : dmso-d6
13C NMR (ppm)
Aromatic carbons : 125.8, 125.9, 128.5, 152.7 CS2: 231.0
Aromatic carbons : 126.3, 127.9, 130.6, 153.9 CS2: 228.4
CH2 : 22.2 ; 22.8 NCH2 : 45.4
L1 : dmso-d6
L2 : D2O
L3: CDCl3
Aromatic carbons: 126.9, 127.3, 130.8, 150.8 CS2: 254.8
NCH3 : 54.3 Aromatic carbons: 126.2, 126.3, 128.0, 152.9 CS2: 250.2
L4 : dmso-d6
a m=multiplet, t=triplet, d=doublet and p=pentuplet
Methods
1H and 13C NMR spectra were recorded in CDCl3, D2O or dmso-d6
solutions on a Bruker ARX 400 instrument. Values for the chemical
shift were determined using CHCl3 as a reference. Elemental analysis of
C, H and S were performed by I.C.S.N. (Gif-sur-Yvette, France) on a
Carlo Erba 1106 analyser. FT IR spectra were recorded on a Nicolet
205 instrument in KBr pellets within the range 4000–500 cmꢂ1
.
Radiochemical purity (RCP) of radioactive compounds was mea-
sured by thin-layer chromatography (TLC) on aluminium-backed silica-
gel plates (F254, Merck) using a mixture of petroleum ether and
dichloromethane (60:40) as mobile phase. Quantitative evaluation of
radioactivity profiles was obtained by applying the following method.
After development of the chromatogram, the plates were dried under an
air stream and protected with an adhesive tape to avoid contamination.
The chromatograms were then placed in close contact with a Fuji
imaging plate (BAS-IIIS), in a dark box, for 5–10 min depending on the
initial activity. Location of the radioactivity as a dark spot was
accomplished with a Fujix Bas 1000 bio-imaging analyser (Fujix Bas
1000). The original chromatographic strip was then cut in correspon-
dence of each spot and radioactivity was measured with a Kontron
GAMMAMATIC g-counter. RCP values were calculated as the ratio of
migrated radioactivity to the total radioactivity on the strip.
Copyright # 2003 John Wiley & Sons, Ltd.
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324
F. MEVELLEC ET AL.
Table 3. Elemental analysis and Infrared of dithiobenzoate ligands
Ligands
Assayed values (%)
Calculated values (%)
C
H
S
C
H
S
L1 : C7H5BrMgS2
L2 : C7H5NaS2
L3 : C12H15NS2
L4 : C11H7NS2
32.54
47.73
60.17
58.34
2.12
2.92
7.16
7.54
24.81
36.38
26.71
27.13
32.70
47.70
60.20
58.10
2.00
2.90
7.16
7.50
24.90
36.40
26.70
28.20
Infrared (KBr pellets) : cmꢂ1 (intensity)
L1
L2
L3
1609(m), 1444(m), 1304(w), 1216(s), 1173(m), 1077(m), 1008(s,
n
CꢂS), 980(s), 967(m), 904(s), 757(s), 683(s), 653(m).
1629(s), 1438(m), 1316(w), 1223(s), 1168(m), 1077(m), 1014(s,
CꢂS), 910(m), 906(m), 765(s), 692(m), 655(m)
n
2955(w), 1584(f), 1443(m), 1384(w), 1305(w), 1263(w), 1208(m),
1177(w), 1082(m), 1004(s, nCꢂS), 988(s), 944(w), 926(w), 906(w),
767(m), 694(m), 655(m)
L4
1487(s), 1437(m), 1403(w), 1216(w), 1168(m), 1077(m), 1014(s,
nCꢂS), 956(s), 948(s), 906(m), 759(s), 694(m)
The complexes [M(S3CPh)2(S2CPh)] (M=99mTc, 99gTc and Re)
chromatograms were performed by HPLC (System Gold, Beckman),
equiped with a Spectra-Physics SP 8800 ternary pump. Injections were
performed automatically with a Spectra-Physics autosampler with 20-ml
Rheodine injector. Elutions were run in the isocratic mode on a ODS
Beckman Column (4.6 ꢃ 250 mm, d.p. 5 mm) equiped with a ODS pre-
column (4.6 ꢃ 45 mm, dp 5 mm) using He-degassed tetrahydrofuran
(THF)/H2O gradient (0–3 min 70% H2O; 3.1–17 min 100% THF ;
17.1–30 min 70% H2O). The flow was 1 ml/min and the injection volume
varied between 10 and 100 mL depending on the activity containing in
the vial. The detector consist of a ABI Kratos UV-Vis detector
(Spectroflow 783) coupled to a 10-ml loop flow-through gamma detector
(LB2040 Berthold Spectrometer). Output signals were analysed by a
Spectra-Physics SP4290 dual-channel integrator. The software
program controlling the apparatus is from Beckman (System Gold
Package). Retention time differences (16.1 and 15.6 min) between
gamma emitting compounds and UV-Vis complexes is due to the
answer delay resulting from the coupling through a loop-flow of two
different detectors.
Elution yields of the different radioactive entities were satisfactory
(>95%), thus indicating that these compounds were not retained in the
column or the HPLC system (Table 4).
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND CHARACTERIZATION OF NEW 99MTC-
325
Table 4. HPLC and TLC analysis of [M(S3CPh)2(S2CPh)] (M = 99mTc, 99gTc,
185/187
Re)
[
99mTcO4]
[
99mTc(S3CPh)2
(S2CPh)]
[
99gTc(S3CPh)2
(S2CPh)]
[Re(S3CPh)2(S2CPh)]
RCP (%)a
Rt (min)b
Rfc
>95
3.44
0
>95
16.1
0.62
>90
15.6
0.62
>90
15.6
0.62
a Chromatographic purity determined by HPLC analysis.
b retention time.
c TLC analysis on SiO2 normal plates eluated with CH2Cl2/petroleum ether 30/70.
Preparation of [99mTc(S2CPh)(S3CPh)2]
The title compound was prepared by reacting the dithiobenzoate ligand
DTCX [PhCS2X L1–L4, X=MgBr, NMe4, Na (C5H10)NH2] with two
different kit formulations for producing the TcꢁN triple bond at
carrier-free level which have been described previously.6, 9, 10, 14 The two
formulations, 99mTc-MDTCZ and 99mTc-SDH, differ in the nature of
the donor of N3ꢂ groups. In 99mTc-MDTCZ this reagent is N-methyl S-
methyl dithiocarbazate [MDTCZ=H2N–N(Me)C(=S)SMe] while in
99mTc-SDH it is succinic dihydrazide [SDH=H2N.NH–C(=O)–
(CH2)2–(O=)C–NH–NH2].
99mTc-MDTCZ. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a
vial containing 1.0 mg MDTCZ, 0.1 mg of SnCl2 ꢀ 2H2O and 10.0 mg of
PDTA in a freeze-dried form. The mixture was heated at 1008C for
30 min and then cooled to room temperature. 20.0 mg of the appropriate
dithiobenzoate ligand DTCX, dissolved in 1.0 mL of physiological
serum, were finally added and the solution was heated at 1008C for
45 min.
99mTc ꢂ SDH. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a
vial containing 5.0 mg of SDH, 0.1 mg of SnCl2 ꢀ 2H2O and 5.0 mg of
PDTA in a freeze-dried form. The mixture was stirred at room
temperature for 30 min. 20.0 mg of the appropriate DTCX ligand,
dissolved in 1.0 mL of physiological serum were finally added, and the
solution was heated at 1008C for 45 min.
The following procedures have been also used to prepare the same
complex [99mTc(S2CPh)(S3CPh)2] using different reducing agents and
from the commercial kit 99mTc-Gluconate.15
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326
F. MEVELLEC ET AL.
PPh3/HCl. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a vial
containing 0.2 ml of an ethanolic solution of triphenylphosphine
(2 ꢃ 10ꢂ2 mol dmꢂ3), 0.2 mL of aqueous HCl (0.1 mol dmꢂ3) and
0.6 ml of physiological serum. The mixture was vortexed and then
heated at 1008C for 15 min. 10.0 mg of the appropriate ligand DTCX,
dissolved in 1.0 ml of physiological serum, were finally added to the hot
solution and the heating was continued for 30 min.
TPPTS/HCl. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a vial
containing 0.2 mL of an aqueous solution of TPPTS (2 ꢃ 10ꢂ2
mol dmꢂ3), 0.2 mL of aqueous HCl (0.1 mol dmꢂ3) and 0.6 mL of
physiological serum. The mixture was vortexed and then heated at
1008C for 15 min. 10.0 mg of the appropriate ligand DTCX, dissolved in
1.0 ml of physiological serum, were finally added to the hot solution and
the heating was further continued for 30 min.
SnCl2. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a vial 0.1 mg
of SnCl2ꢀ2H2O and 10 mg of 1,2-diaminopropane-N-N-N0-N0-tetraacetic
acid (PDTA). The mixture was vortexed and then heated at 1008C for
15 min. 20.0 mg of the appropriate DTCX ligand, dissolved in 1.0 mL of
physiological serum, were finally added to the hot solution and the
heating was further continued for 45 min.
99mTc-Gluconate. [99mTc]Pertechnetate (0.4–0.8 GBq) was added to a
vial containing 75.0 mg of calcium gluconate dissolved in 0.1 ml of
water, 0.75 mg of SnCl22H2O and 25.0 mg of sodium chloride. The
mixture was stirred for 10 min at room temperature. 20.0 mg of the
appropriate DTCX ligand, dissolved in 1.0 mL of physiological serum,
were finally added and the solution was heated at 1008C for 15 min.
Results and discussion
Ligands
Various salts of dithiobenzoic acid PhCS2X L1–L4 (DTCX, X=MgBr,
Na, NH2(C5H10), NMe4) were synthesized using well-established
procedures. Analytical, spectral and chromatographic data for these
compounds are given in Tables 1–3.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND CHARACTERIZATION OF NEW 99MTC-
327
[
99mTc(S2CPh)(S3CPh)2]
The preparation of the complex bis(trithioperoxybenzoate)(dithioben-
zoate)technetium-99m, [99mTc(S2CPh)(S3CPh)2], was accomplished
using various procedures and different salts of the ligand PhCSꢂ2 L.
This study was undertaken with the aim to find the more suitable
procedure for obtaining this radiopharmaceutical in high yield and in
conditions as close as possible to physiological conditions. The main
interest in the compound [99mTc(S2CPh)(S3CPh)2] comes from the fact
that in vitro experiments showed that it is very efficiently and selectively
retained into leukocytes when incubated with whole blood. Further, it
was found that the activity was stably incorporated thus suggesting that
this tracer has a peculiar affinity for these blood constituents. The solid
structure of the analogous complexes, [M(S2CPh)(S3CPh)2], prepared in
macroscopic amounts respectively, with the long-lived Tc-99 isotope
and cold rhenium, has been determined by X-ray diffraction methods.11
The central metal lies in a distorted trigonal prism environment made
up by the six terminal sulfur atoms. These complexes were utilized to
establish the chemical identity of the corresponding Tc-99m compound
through the comparison of their chromatographic properties. HPLC
and TLC chromatographic data are illustrated in Table 4.
The need to investigate different reaction procedures for obtaining an
efficient
method
for
the
preparation
of
the
complex
[
99mTc(S2CPh)(S3CPh)2], at no carried-added level, was dictated by the
very complicated reactivity of dithiocarboxylate ligands which may give
rise to a wide variety of final products.12 A first attempt was undertaken
by reacting the ligands L1–L4 with the [99mTcꢁN]2+ core prepared
through a well-established procedure described a few years ago.10 Such
an attempt was justified by the fact that it was previously observed that
thiol and dithiol ligands were able to remove easily the TcꢁN multiple
bond to afford the corresponding S-substituted complexes.16 Thus, the
reaction was carried out by first producing the TcꢁN group through the
reaction of [99mTc]pertechnetate with S-methyl-N-methyldithiocarba-
zate (MDTCZ), or alternatively succinic dihydrazide (SDH), as source
of nitride nitrogen atoms (N3ꢂ), in the presence of a reducing agent such
as SnCl2 ꢀ 2H2O or a combination of HCl and a tertiary phosphine.
Another approach to the synthesis of the complex [99mTc(S2CPh)
(S3CPh)2] was undertaken when 99mTc-gluconate was used as pre-
reduced substrate in the reaction with DTCX. In this prepara-
tion, a commercial kit formulation for 99mTc-gluconate15 was
utilized, and successively mixed with the ligands DTCX. Finally,
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328
F. MEVELLEC ET AL.
a
b
c
0.9% NaCl solution
99m
[
-
+
TcO ]
Reductor
complexant
+
+
Ligand
Pre-reduced-99m-Tc-complex
4
T = r.t.-100°C
a) SnCl2,2H2O or PR3/HCl
t = 10-30 min
b) PDTA if presence of stannous chloride
c) MDTCZ or SDH or Gluconate
0.9% NaCl solution
[
99mTc(S3CPh)2(S2CPh)]
Pre-reduced-99m-Tc-complex + PhCS2X
X = MgBr, Na, NH2(CH2)5 and NMe4
T = 100°C
t = 15-45 min
Scheme 2. Synthesis of [99mTc(S3CPh)2(S2CPh)]
Table 5. Influence of the chemical nature of the ligand on RCP using the
MDTCZ/PDTA Kit
Ligand
NOETa
C10MgBra
L1
L2
L3
L4
Rf
RCPd (%)
0.62b
>90
0.75b
70
0.62c
70
0.62c
90
0.62c
70
0.62c
40
a Ligands used as reference (NOET: Et(OEt)NCS2Na and C10MgBr: CH3(CH2)8CS2MgBr).
b TLC ( SiO2), eluent CH2Cl2, Rf ( ꢄ 0.12).
c TLC ( SiO2), eluent petroleum ether/CH2Cl2 : 70/30, Rf ( ꢄ 0.12).
d Radiochemichal purity estimated.
[
99mTc(S2CPh)(S3CPh)2] was prepared through a simpler route invol-
ving the reaction of DTCX with [99mTc]pertechnetate in the presence of
SnCl2 or HCl/tertiary phosphine as reductants. All the procedures are
summarised in Scheme 2. It should be noted that, in all preparations
using stannous chloride, 1,2-diaminopropane-N-N-N0-N0-tetraacetic
acid (PDTA) was always added as complexing agent to avoid
precipitation of Sn2+ ions co-ordinated to the ligand DTCX.
The results are reported in Tables 5 and 6. The complex
99mTc(S2CPh)(S3CPh)2] was obtained as the main product in all
[
preparations, with a radiochemical purity (RCP) depending on both the
chemical nature of the ligand DTCX and the reaction procedure
performed. Starting with a precursor containing the TcꢁN multiple
bond, the highest yield (approximately 90%) was obtained with
MDTCZ as donor of N3ꢂ groups and using the sodium salt L2 of the
ligand DTCX (X=Na). Others salts gave lower RCP values. Similarly,
the preparation containing SDH as N3ꢂ donor produced unsatisfactory
results with a final yield lower than 60%.
The complex [99mTc(S2CPh)(S3CPh)2] could not be easily obtained
starting from [99mTc]pertechnetate and SnCl2 (RCP 5 20%). Never-
theless, the use of a tertiary phosphine and aqueous hydrochloric acid
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 319–331

SYNTHESIS AND CHARACTERIZATION OF NEW 99MTC-
329
Table 6. Different kit formulations for the synthesis of [99mTc(S3CPh)2(S2CPh)]
with PhCS2Na L2
KIT
medium / pH
T (8C)
t (min)
Rfg
PRC (%)
MDTCZ/PDTAa
pH4
100
100
100
100
100
100
45
30
30
15
45
45
0.62
0.62
0.62
0.62
0.62
0.62
85
71
94
>95
55
b
HCl/PPh3
Serum j
Serum j
Serum j
Serum j
Serum j
HCl/TPPTSc
SnCl2/Gluconated
SDHe
SnCl2/ PDTAf
5 20
a MDTCZ/PDTA kit (CIS bio international) and PhCS2NH2(CH2)5 L3.
b HCl 0.1 N (0.2 ml), PPh3 2.10ꢂ2 M / EtOH (0.2 ml).
c HCl 0.1 N (0.2 ml), TPPTS 2.10ꢂ2 M / H2O (0.2 ml).
d SnCl2/gluconate kit (CIS bio international) diluted 1/10 (1.0 ml).
e SDH kit (CIS bio international).
f SnCl2/ PDTA kit.
g TLC (SiO2), eluent petroleum ether/CH2Cl2 : 70/30, Rf ꢄ 0.12.
gave the 99mTc-radiopharmaceutical in higher yield (71% for PPh3 and
94% for TPPTS). The most quantitative reaction was obtained using
the 99mTc-gluconate kit. [99mTc(S3CPh)2(S2CPh)] was prepared with the
highest radiochemical yield (> 95%) through the reaction of 99mTc-
gluconate with PhCS2Na at 1008C for 15 min. In this reaction, the use of
the other salts of the same ligand produced lower yields.
Conclusion
In this work, the 99mTc-labelled compound [99mTc(S3CPh)2(S2CPh)]
analogous to the compounds [M(S3CPh)2(S2CPh)] (M=99Tc, Re),
previously isolated and characterized in macroscopic amounts, was
prepared using several procedures. The most efficient method was based
on the reaction of a lyophilized formulation of 99mTc-gluconate with the
sodium salt L2 of phenyldithiocarboxylic acid. The neutral and
lipophilic [99mTc(S3CPh)2(S2CPh)] was obtained in very high yield (>
95%) and characterized by TLC and HPLC chromatography. The
similarity between its chromatographic behaviour and that of the
analogous 99gTc and Re complexes clearly established the chemical
nature of this new radiopharmaceutical. The aim of these experiments
will be in carefully evaluating the possibility to introduce the new 99mTc-
labelled compound into the clinical practice as an attractive alternative
to the current employed leukocyte labelling agents.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 319–331

330
F. MEVELLEC ET AL.
Acknowledgements
We thank Mrs A. Trichet and Mrs S. Abgrall for technical assistance in
the radiopharmaceuticals preparation. CIS bio international/Schering
SA and Region Bretagne for financial support for this work. We are
also grateful to Dr. A. Boschi for HPLC analysis and the International
Society of Radiolabelled Blood Elements (ISORBE).
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