S-trityl protection of bis-amino bis-thiol (BAT) chelator enables flexible derivatisation and facile labelling with technetium-99m

Article abstract of DOI:10.1016/S0040-4039(03)00224-7

We have coupled S,S′-bis-trityl N-BOC protected 1,2-ethylenedicysteamine, a bis-amino bis-thiol (BAT) tetraligand, via a propylene or ethylene spacer to several biologically active molecules including 2-nitroimidazole, desethylflumazenil, a beta-CIT analo

Full text of DOI:10.1016/S0040-4039(03)00224-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2597–2600
S-Trityl protection of bis-amino bis-thiol (BAT) chelator enables
flexible derivatisation and facile labelling with technetium-99m
Bernard J. Cleynhens, Guy M. Bormans, Hubert P. Vanbilloen, Dominique V. Vanderghinste,
Davy M. Kieffer, Tjibbe J. de Groot and Alfons M. Verbruggen*
Laboratory for Radiopharmaceutical Chemistry and Nuclear Medicine, University of Leuven, Herestraat 49, B-3000 Leuven,
Belgium
Received 15 January 2003; accepted 21 January 2003
Abstract—We have coupled S,S%-bis-trityl N-BOC protected 1,2-ethylenedicysteamine, a bis-amino bis-thiol (BAT) tetraligand, via
a propylene or ethylene spacer to several biologically active molecules including 2-nitroimidazole, desethylflumazenil, a beta-CIT
analogue, glucose and 2-(2%-hydroxy-4%-aminophenyl)-1,3-benzothiazole. The conjugates were efficiently labelled with technetium-
99m by consecutive heating of the S,S%-bis-trityl protected ligand in hydrochloric acid followed by neutralisation and heating in
the presence of 99mTc-tartrate. The S,S%-bis-trityl BAT chelator is an interesting synthon that allows both flexible derivatisation
with various biologically active molecules and facile labelling with technetium-99m. © 2003 Elsevier Science Ltd. All rights reserved.
Bis-amino bis-thiol (BAT, N2S2) tetraligands are fre-
quently used bifunctional chelating agents which form
stable neutral complexes with technetium-99m and can
be conjugated to biologically active molecules.1–5 The
latter may exhibit affinity for specific targets such as
receptors, transporters or enzymes and can vectorise the
technetium-99m radiolabel to tissues expressing these
targets after intravenous administration of the radiola-
belled conjugate. The conjugation chemistry of this
kind of technetium binding bifunctional ligands is often
hampered by the instability of the thiol groups. The
thiols must be protected during conjugation and stor-
age, since in unprotected form they are easily oxidised
to the corresponding disulfide. A frequently used
method of thiol protection in BAT-type compounds is
their conversion to a disulfide, which can be reduced to
the corresponding bis-thiol using LiAlH4 or NaBH4
prior to labelling with technetium-99m.6–8 Alternatively,
the thiol groups have been protected with a p-methoxy-
benzyl group, which is removed using a mixture of
CF3COOH, methanesulfonic acid and anisole,9 or a
trityl group in which case the thiols are deprotected
mostly with CF3COOH under cation trapping condi-
tions (triethylsilane and anisole)10 (Fig. 1). Such depro-
tection methods are laborious and it is difficult to
isolate and store the deprotected ligands. The time
between deprotection and labelling with technetium-
99m should be kept as short as possible to avoid
disulfide formation.
Therefore, we have developed a method in which
deprotection of the ligand and labelling with 99mTc
Figure 1. Synthesis of Technepine 1 by deprotection of the
S,S%-bis-trityl protected precursor followed by labelling with
99mTc.10
* Corresponding author. Tel.: +32-16-34 37 30; fax: +32-16-34 38 91;
e-mail: alfons.verbruggen@uz.kuleuven.ac.be
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00224-7

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occur in a one-pot two-step procedure that is amenable
to ‘kit’ formulation. The most simple BAT-ligand was
chosen as the model, i.e. 1,2-ethylenedicysteamine.
1,2-Ethylenedicysteamine (a, Scheme 1) was obtained in
high yields via a reductive dimerisation of thiazolidine
by the action of sodium in liquid ammonia, in a similar
way as described for the synthesis of 1,2-ethylenedicys-
teine (EC).11,12 Subsequent thiol protection of this BAT
compound was achieved by reaction with triphenyl-
methanol in trifluoroacetic acid. Trityl groups were
preferred over other S-protecting groups (such as ben-
zoyl, benzyl, benzamidomethyl, p-methoxybenzyl)13 as
they combine a good stability during conjugation with
the ease of removal prior to labelling with technetium-
99m.
The developed synthesis method of a is a good alterna-
tive to the conventional synthesis of BAT compounds.
An earlier method for the synthesis of a consisted of
reaction of 1,2-ethylenediamine with S-protected 2-
mercaptoacetyl chloride followed by reduction of the
amide groups with BH3.14 This reduction step is not
feasible when S-trityl protecting groups are used, as
they are not stable under these conditions.
One amino group of S,S%-bis-trityl-1,2-ethylenedicys-
teamine (b, Scheme 1) was protected with a tert-butyl-
oxycarbonyl (BOC) group by treatment with 1 equiv. of
(BOC)2O to afford a S,S%-bis-trityl, N-BOC protected
BAT synthon (c, Scheme 1). This synthon could
efficiently be coupled through a propylene or ethylene
spacer to several vectors including nitroimidazole (NI),
desethylflumazenil (FZ, acid analogue of flumazenil,
RO-15-3890),
3b-(4-chlorophenyl)tropane-2b-car-
boxylic acid (TROP), glucose (GLU) and 2-(2%-
hydroxy-4%-aminophenyl)-1,3-benzothiazole (BT) (Fig.
2).
Coupling to NI was done by reaction of c with 1-
bromo-3-chloropropane and subsequent N-alkylation
of 2-nitroimidazole yielding conjugate 1 (Scheme 1, Fig.
2). Coupling of c to TROP and FZ involved alkylation
of c with 3-bromo-1-propanol and subsequent ester
formation with the carboxylic acid of, respectively,
TROP and RO-15-3890 after activation with oxalyl
chloride to obtain 2 and 3.
Figure 2. Synthesised conjugates with the S,S%-bis-trityl pro-
tected BAT synthon.
Conjugate 4 was synthesised by reaction of c with
2-[2%-(3%%-tosyloxypropoxy)-4%-aminophenyl]-1,3-benzo-
thiazole. For preparation of a thiol protected BAT-glu-
EtOH and heating for 10 min in a boiling water-bath.
Neutralisation followed by addition of SnCl2, sodium
tartrate and 99mTc-generator eluate (containing 99mTcO−4)
and heating for 10 min in a boiling water-bath yielded
the corresponding Tc(V)O-BAT-vector complex.
Radio-LC-MS was used to confirm the identity of all
mentioned 99mTc-labelled conjugates.
cose conjugate, 2-iodoethyl-2,3,4,6-tetra-O-acetyl-b-D-
glucoside was reacted with c and the conjugate was
subsequently deacetylated using sodium methanolate to
obtain 5.
The resulting conjugates were deprotected and isolated
for characterisation (LC–MS) by reaction with tri-
fluoroacetic acid and anisole. For labelling with 99mTc,
however, we found that it was possible to deprotect a
sufficient fraction of the ligand by addition of 0.5 ml
HCl 0.5 M to a solution of 1 mg of the ligand in 0.3 ml
Mass spectrometric analysis of the reaction mixture
after deprotection of 1 (with HCl 0.5 M at 100°C)
showed the presence of (i) unmodified conjugate 1
(57%), (ii) the ligand without the N-BOC group (38%),
(iii) the ligand without the N-BOC and one of the trityl

B. J. Cleynhens et al. / Tetrahedron Letters 44 (2003) 2597–2600
2599
Scheme 1. Synthesis of S,S%-bis-trityl protected BAT conjugates.
groups (2.9%) and (iv) the ligand without one of the
trityl groups (1.4%) (Fig. 3). The conjugate with a fully
deprotected BAT was not detected.
step with HCl 0.5 M did not yield the desired complex.
This suggests that deprotection of the derivative still
containing one thiol protective group may proceed
further during the complexation step. In earlier studies
we have shown that S-benzyl or S-benzamidomethyl
protected mercaptoacetyltriglycine can be labelled
efficiently with 99mTc using a similar labelling proce-
Labelling with 99mTc requires preceding removal of at
least one trityl group as labelling starting from the
S,S%-bis-trityl protected ligand without the deprotection

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B. J. Cleynhens et al. / Tetrahedron Letters 44 (2003) 2597–2600
Acknowledgements
Financial support was provided by the Fund for Scien-
tific Research-Flanders, Belgium and by a grant of the
Onderzoeksraad K.U. Leuven (OT.00.39).
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In conclusion, we developed a new synthetic pathway
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protected form and labelled with 99mTc in a simple
one-pot two-step procedure. This labelling method is
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