Novel 99mTc '4 + 1' peptide conjugates: Tuning the biodistribution by variation of coligands

Article abstract of DOI:10.1016/j.ejmech.2010.05.010

A sophisticated coligand strategy is presented for peptide-derived radioconjugates based on ^99mTc '4 + 1' mixed-ligand complexes. The new pharmacologically active coligands are assessed for ^99mTc- labeling of the RGD-peptide cyclo(Arg-Gly-Asp-d-Tyr-Lys) which is an established vehicle to target α_vβ₃ integrins playing a crucial part in tumor pathogenesis. Complexes of the general formula [ ^99mTc(NS₃R)X] were synthesized and evaluated, in which Tc(III) is coordinated by NS₃R, a derivative of the tetradentate chelator 2,2′,2″-nitrilotriethanethiol (NS₃), and by X, a monodentate binding isocyanide bearing the biomolecule. The novel tetradentate chelators (NS₃R = NS₃crown, NS₃en, NS ₃(COOH)₃) constitute NS₃ with a crown ether, an amine or a tricarboxylic acid as pharmacological modifiers. The isocyanides (X = L2-RGD, L2-Lys) contained the linker isocyanobutanoic acid (L2) coupled to N⁶-Lys of the RGD-peptide and additionally to a single Lys. The lipophilicity (distribution coefficient log D_O/W, pH = 7.4) of the RGD-containing radiotracers decreased in the order of the coligands NS₃crown (-1.7 ± 0.1), NS₃en (-2.7 ± 0.1) and NS₃(COOH)₃ (-3.3 ± 0.1). In the same order of the coligands, the biodistribution of the series [^99mTc(NS ₃R)(L2-RGD)] in normal rats showed a decrease of hepatobiliary and an increase of urinary excretion. The ratio of specifically to unspecifically uptaken activity (sum of surface bound and internalized activity) in α_vβ₃ integrin-expressing M21 cells was in the range of approximately 4-5 and comparable for all [^99mTc(NS ₃R)(L2-RGD)] tracers. The biodistribution of [ ^99mTc(NS₃en)(L2-RGD)] in ν/ν mice bearing M21 and M21L (control) tumor xenografts exhibited a specific tumor uptake with a low target-background ratio. The metabolic stability of the [ ^99mTc(NS₃R)(L2-RGD)] tracers in normal rats was high, since 75-87% of the radioactivity in the plasma extract was assigned to the injected radiotracers 60 min after intravenous application in a rat. The hypothetical metabolites [^99mTc(NS₃R)(L2-Lys)] were not found. These results demonstrate a considerable improvement of in vivo properties of ^99mTc '4 + 1' peptide conjugates and open up the possibility of applying the labeling approach for further radiodiagnostic peptides.

Full text of DOI:10.1016/j.ejmech.2010.05.010

European Journal of Medicinal Chemistry 45 (2010) 3645e3655
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 99mTc ‘4 þ 1’ peptide conjugates: Tuning the biodistribution by variation
of coligands
Jens-Uwe Kunstler a, Gesine Seidel b, Ralf Bergmann b, Ewa Gniazdowska c, Martin Walther b,
Eik Schiller b, Clemens Decristoforo d, Holger Stephan b, Roland Haubner d, Jorg Steinbach b,
,
Hans-Jurgen Pietzsch b
*
a Biotectid GmbH, 04103 Leipzig, Germany
b Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, PF 510 119, 01314 Dresden, Germany
c Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Poland
d Clinical Department of Nuclear Medicine, Innsbruck Medical University, 6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
A sophisticated coligand strategy is presented for peptide-derived radioconjugates based on 99mTc ‘4 þ 1’
Article history:
mixed-ligand complexes. The new pharmacologically active coligands are assessed for 99mTc-labeling of
Received 3 March 2010
Received in revised form
4 May 2010
Accepted 5 May 2010
Available online 12 May 2010
the RGD-peptide cyclo(Arg-Gly-Asp-D-Tyr-Lys) which is an established vehicle to target avb3 integrins
playing a crucial part in tumor pathogenesis.
Complexes of the general formula [99mTc(NS3R)X] were synthesized and evaluated, in which Tc(III) is
coordinated by NS3R, a derivative of the tetradentate chelator 2,20,200-nitrilotriethanethiol (NS3), and by
X, a monodentate binding isocyanide bearing the biomolecule. The novel tetradentate chelators
(NS3R ¼ NS3crown, NS3en, NS3(COOH)3) constitute NS3 with a crown ether, an amine or a tricarboxylic
acid as pharmacological modifiers. The isocyanides (X ¼ L2-RGD, L2-Lys) contained the linker iso-
cyanobutanoic acid (L2) coupled to N6-Lys of the RGD-peptide and additionally to a single Lys.
The lipophilicity (distribution coefficient log DO/W, pH ¼ 7.4) of the RGD-containing radiotracers
decreased in the order of the coligands NS3crown (ꢀ1.7 ꢁ 0.1), NS3en (ꢀ2.7 ꢁ 0.1) and NS3(COOH)3
(ꢀ3.3 ꢁ 0.1). In the same order of the coligands, the biodistribution of the series [99mTc(NS3R)(L2-RGD)]
in normal rats showed a decrease of hepatobiliary and an increase of urinary excretion.
The ratio of specifically to unspecifically uptaken activity (sum of surface bound and internalized
activity) in avb3 integrin-expressing M21 cells was in the range of approximately 4e5 and comparable for
Keywords:
Technetium
‘4 þ 1’ Mixed-ligand complex
Peptide
RGD
all [99mTc(NS3R)(L2-RGD)] tracers. The biodistribution of [99mTc(NS3en)(L2-RGD)] in
n/n mice bearing
M21 and M21L (control) tumor xenografts exhibited a specific tumor uptake with a low target-back-
ground ratio.
The metabolic stability of the [99mTc(NS3R)(L2-RGD)] tracers in normal rats was high, since 75e87% of
the radioactivity in the plasma extract was assigned to the injected radiotracers 60 min after intravenous
application in a rat. The hypothetical metabolites [99mTc(NS3R)(L2-Lys)] were not found.
These results demonstrate a considerable improvement of in vivo properties of 99mTc ‘4 þ 1’ peptide
conjugates and open up the possibility of applying the labeling approach for further radiodiagnostic
peptides.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
complexes. The ‘4 þ 1’ framework [M(NS3)X] consists of Tc/Re(III)
coordinated by a tetradentate tripodal chelator NS3 (2,20,200-nitri-
In the search for new radioactive technetium and rhenium
labeled biomolecules for radiopharmaceutical purposes, our group
has focussed on the development of ‘4 þ 1’ mixed-ligand
lotriethanethiol) and a monodentate isocyanide or phosphine (X)
[1e3]. Radioconjugates containing ‘4 þ 1’ complexes attached to
biomolecules including serotonin receptor ligands [4], fatty acids
[5e9], quinazolines [10] and peptides [11e13] have been investi-
gated. These studies demonstrated a primarily lipophilic nature of
the ‘4 þ 1’ framework. This is an inherent characteristic, since the
metal is well shielded by the ‘4 þ 1’ donor atom arrangement. The
* Corresponding author. Tel.: þ49 351 260 2706; fax: þ49 351 260 3232.
E-mail address: h.j.pietzsch@fzd.de (H.-J. Pietzsch).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.010

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J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
application to the 99mTc-labeling of comparatively hydrophilic
biomolecules like peptides is therefore limited. The concept of
‘4 þ 1’ radioconjugates opens the possibility of varying the bio-
behavior by using tetradentate coligands grafted with pharmaco-
kinetic modifiers. In the present paper we address these variation
possibilities and describe the synthesis and characterization of
three novel NS3-chelators modified with a crown ether, an amine or
a tricarboxylic acid (NS3crown, NS3en, NS3(COOH)3). These units
are intended to decrease the lipophilicity under physiological
conditions by a neutral hydrophilic, a positively charged and
a negatively charged group and may considerably improve the in
vivo characteristics of a labeled peptide bearing a 99mTc ‘4 þ 1’
mixed-ligand complex. The ‘4 þ 1’ complexes containing the new
coligands were assessed conjugated to the RGD-peptide cyclo(Arg-
compounds were obtained as aminium chlorides, since the crude
products were purified by extraction into the aqueous phase using
an acidified product solution. Therefore the obtained coligand may
contain a certain amount of sodium chloride which was judged as
marginal for the intended use of the coligands. The identity of the
obtained ligands was confirmed by 1H NMR and 13C NMR.
2.1.2. Isocyanide modification
The RGD-peptide c(RGDyK) and Lys were selected as molecules
for monodentate attachment to the ‘4 þ 1’ framework. The RGD-
peptide was obtained by customized standard peptide synthesis
and its identity and purity were confirmed by ESIeMS and RP-
HPLC. The monodentate ligands were formed by functionalization
via the N6 of Lys with 2,3,5,6-tetrafluorophenyl 4-iso-
cyanobutanoate (L2-BFCA) which was designed as a bifunctional
coupling agent (BFCA) [13]. For the modification of Lys, N2 was
protected by Fmoc, which was removed by piperidine. The obtained
conjugates L2-RGD and L2-Lys are shown in Schemes 2 and 3.
The prepared conjugates were purified by RP-chromatography
using a basic mobile phase, since isocyanides are unstable under
acidic conditions and therefore RP-chromatography using an acidic
TFA containing mobile phase is not possible. The conjugates were
then lyophilized. The obtained ESIeMS data of the products were
consistent with the molecular weights calculated for each
compound.
Gly-Asp-D-Tyr-Lys).
Radioconjugates containing the RGD-motive have exhibited
a great potential for radiopharmaceutical applications as reviewed
by Haubner [14], Meyer et al. [15], Dijkgraaf and Boerman [16],
Schottelius et al. [17] and Liu [18] for example. In particular, the RGD
interacts with the target avb3 integrin that is involved in patho-
physiological processes such as tumor metastasis, neoangiogenesis,
osteoporosis, restenosis and inflammation. RGD-based radiotracers
have already been studied with a large variety in the RGD-con-
taining unit, in a modifying unit such as carbohydrate or poly-
ethylene glycol, and in the labeling system including the nuclides
64Cu, 18F, 68Ga, 123I, 125I, 111In, 177Lu, 99mTc and 90Y [14e18].
In this paper, the cyclo(Arg-Gly-Asp-
D
-Tyr-Lys) was selected as
2.1.3. 99mTc-labeling
biomolecule since its radioconjugates, consisting of a radiometal
complex bound via Lys to the peptide, have been shown to target
integrins with high affinity over-expressed in carcinomas
The 99mTc-labeling by forming the ‘4 þ 1’ complexes [99mTc
(NS3en)(L2-RGD)],
(L2-RGD)],
[ [
99mTc(crown)(L2-RGD)], 99mTc(NS3(COOH)3)
[
99mTc(NS3en)(L2-Lys)],
[
99mTc(NS3crown)(L2-Lys)],
[12,19,20]. Further, the cyclo(Arg-Gly-Asp-
D-Tyr-Lys) exhibited
and [99mTc(NS3(COOH)3)(L2-Lys)], displayed in Fig. 1, was per-
formed in a single step procedure (Scheme 4) that was recently
described [24].
a high in vivo stability as deduced from previously described
metabolism studies [21,22].
The RGD-peptide was modified with 4-isocyanobutanoic acid
Accordingly, a labeling mixture was prepared containing eluate
of a 99Mo/99mTc generator, the NS3-chelator, the isocyanide modi-
fied biomolecule, SnCl2, Na2EDTA, mannitol, ascorbic acid and
ethanol, and allowed to react for about 60 min at room tempera-
ture. As previously described, this procedure enables a 99mTc-
labeling with a radiochemical yield higher than 95% without the
need for a purification step, if at least 50 nmol isocyanide and
500 nmol NS3-chelator are used [24]. Lower amounts of the ligand
were used in the present work, primarily because the synthesized
NS3-chelators contained a certain amount of NaCl that could not
completely separated by the described preparation method.
Therefore, the radioconjugates were purified by HPLC to give
a radiochemical purity of higher than 95%. The 99mTc-labeled
compounds used in animal studies were purified by RP-HPLC using
a mobile phase containing water, ethanol and TFA. Ethanol was
used in place of acetonitrile to prevent the contamination of the
injection solution with the toxic acetonitrile.
(L2) as linker conjugated to N6-Lys and then the conjugates [99mTc
(NS3crown)(L2-RGD)],
[
99mTc(NS3en)(L2-RGD)],
[
99mTc
(NS3(COOH)3)(L2-RGD)] were prepared. The compounds were
characterized by means of chromatography, determination of
distribution coefficients, biodistribution and metabolism studies.
Previous in vivo metabolism studies of radiolabeled RGD-deriva-
tives containing cyclo(Arg-Gly-Asp-D-Phe-Lys) in mice exhibited
the formation of one main metabolite of the respective radiotracer
but the metabolites were not identified [21,22]. For the metabolism
studies of the [99mTc(NS3R)(L2-RGD)] series the formation of [99mTc
(NS3R)(L2-Lys)] as metabolites was assumed. Therefore the conju-
gates
[
99mTc(NS3crown)(L2-Lys)],
[
99mTc(NS3en)(L2-Lys)] and
[
99mTc(NS3(COOH)3)(L2-Lys)] containing Lys in place of the RGD-
peptide were prepared and served as reference compounds.
2. Results and discussion
The radioconjugates were stable in the HPLC eluate and in the
injection solution within the test period of 24 h.
2.1. Chemistry
A comparison of the HPLC profiles of the 99mTc-containing
radiotracer with those of analogous Re compounds which eluted at
the same retention times gave a strong indication of the identity of
the formed radioconjugates.
2.1.1. Synthesis of coligands
The new tripodal tetradentate ligands to form the ‘4 þ 1’
complexes of the isocyanide functionalized biomolecules were
designed as N(CH2eCH2eSH)3-chelator (‘NS3-chelator’) bearing
three different pharmacokinetic modifiers e a crown ether, an
2.1.4. Rhenium reference compounds
amine and
a
tricarboxylic acid. These NS3-chelators were
The ‘4 þ 1’ Re compounds [Re(NS3R)(BM)], NS3R ¼ NS3crown,
NS3en, BM ¼ L2-RGD, L2-Lys, (Fig.1) weresynthesizedbyexchanging
the thiourea ligands of [Re(tu)6]Cl3 against NS3R and PMe2Ph
resulting in the formation of [Re(NS3R)(PMe2Ph)]. Then, the phos-
phinewasreplacedbytheappropriateisocyanideandpurifiedbyRP-
HPLC. In order to synthesize [Re(NS3(COOH)3)(L2-RGD)] and [Re
(NS3(COOH)3)(L2-Lys)], the same procedurewas applied, resulting in
synthesized by coupling (DCC-activation) the modifier molecules
to N(Sbz)3COOH followed by deprotection (Scheme 1).
The synthesis of N(Sbz)3COOH has been previously described
[11]. The linker H2NeC(eCH2eOeCH2eCH2eCOOeMe)3 was
prepared as described by Newkome et al. [23]. Finally, NS3crown,
NS3en and NS3(COOH)3 were obtained (Scheme 1). These

J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
3647
Scheme 1. Reaction routes to modified NS3-ligands.
esterification of the three carboxylic acid groups that afforded
a sapofinication of the isocyanide containing complex (Scheme 5).
The obtained ESIeMS data of the products were consistent with the
molecular weights calculated for each compound.
in the order of the modifiers crown ether, ethylenediamine and
tricarboxylic acid. In addition, the analogous complex containing
NS3COOH as coligand exhibited a log DO/W of ꢀ2.3 at pH 7.4, which
shows that
[
99mTc(NS3(COOH)3)(L2-RGD)] is considerably less
lipophilic [12]. The determined distribution coefficients for [99mTc
(NS3en)(L2-RGD)] and [99mTc(NS3(COOH)3)(L2-RGD)] are in the
range of RGD-peptides labeled with 99mTc using the HYNIC
approach [12,25] and are lower compared to the cyclo(Arg-Gly-
2.1.5. Lipophilicity
The lipophilicity of the labeled RDG-peptides was assessed by
measuring their distribution in an octanol/water system buffered at
pH 7.4. The results are shown in Table 1. The lipophilicity decreased
Asp-
a
D-Tyr-Lys) derivatives labeled via a
99mTc-tricarbonyl or
99mTc-nitrido complex as previously reported [12].
2.2. Pharmacology
2.2.1. Overall biodistribution
Selected biodistribution and elimination data after i.v. injection
into
rats
of
the
radioconjugates
[
99mTc(NS3R)BM],
Scheme 2. Introduction of isocyanide onto RGD-peptide.
Scheme 3. Introduction of isocyanide onto Lys.

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J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
Fig. 1. 99mTc and Re ‘4 þ 1’ complex-bearing RGD-peptide or Lys (M ¼ 99mTc or Re).
Scheme 5. Exemplary reaction route to rhenium-reference compounds.
NS3R ¼ NS3crown, NS3en, NS3(COOH)3, BM ¼ L2-RGD, L2-Lys, are
shown in Figs. 2e5. The values of %ID, %ID/g, and SUV (ID ¼ injected
dose; SUV ¼ standardized uptake value) are reported in detail in
the Supporting Information. The SUV as a measure for the activity
concentration was used to normalize the biodistribution data and
calculated as described in the experimental section.
The biodistribution data show distinctive differences between
the activity uptake into the kidney and liver, respectively the
hepatobiliary and urinary excretion. For the RGD conjugates, in the
order of the modifiers crown ether, ethylenediamine and tricar-
boxylic acid, a decrease of the hepatobiliary and an increase of the
urinary excretion were observed. This is consistent with the order
of the determined distribution coefficients. The conjugates had
a rapid clearance demonstrated by a decrease of the activity
contents in the organs between 5 and 60 min p.i. and an increase in
the intestinal uptake.
(NS3crown)(L2-Lys)] was different, showing a quick excretion by
the kidney and liver at about the same rate.
The activity concentration (SUV) of the investigated radio-
conjugates in the kidneys was the highest of all investigated organs
and higher for the Lys than for the RGD conjugates. The SUV were
lowest for the brain and testis and were roughly at blood level for
the other organs (data shown in the Supporting Information).
The investigated conjugates exhibited a minimal 99mTc uptake
into the stomach and the thyroid (data shown in the Supporting
Information) indicating that there is no or marginal dissociation
of 99mTc from the complex to produce 99mTcOꢀ4 .
Previous investigations using 99mTc ‘4 þ 1’ complexes con-
taining NS3 or NS3COOH as coligands attached to cyclo(Arg-Gly-
Asp-D-Tyr-Lys) [12] or bombesin derivatives [13] showed an
extensive hepatobiliary excretion. Thereby, no pharmacological
effect was observed to be caused by the one carboxylic group
proposed as pharmacokinetic modifier in the radioconjugates
containing NS3COOH compared to the conjugates containing
NS3 [13].
In the present study, the more comprehensive pharmacokinetic
modifiers display a considerable ability to shift the excretion
pathway to a roughly similar rate of renal and hepatobiliary
excretion for the tracers containing the coligands NS3crown and
NS3en and further to a predominant renal excretion for compounds
containing NS3(COOH)3.
The Lys-containing radioconjugates exhibited a faster clearance
compared to the labeled RGD derivatives. The grading between the
conjugates containing NS3en and NS3(COOH)3 were comparable for
Lys and RGD. The biodistribution characteristic for
[
99mTc
Table 1
Distribution coefficients (log DO/W) of the RGD-containing radioconjugates at pH 7.4
(mean ꢁ SD, n ¼ 4).
Radioconjugate
log DO/W
[
[
[
99mTc(NS3crown)(L2-RGD)]
ꢀ1.7 ꢁ 0.1
ꢀ2.7 ꢁ 0.1
ꢀ3.3 ꢁ 0.1
99mTc(NS3en)(L2-RGD)]
99mTc(NS3(COOH)3)(L2-RGD)]
Scheme 4. 99mTc-labeling reaction (BM ¼ biomolecule).

J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
3649
Fig. 2. Biodistribution of [99mTc(NS3crown)(L2-RGD)], diagonal stripes; [99mTc(NS3en)
(L2-RGD)], no stripes; [99mTc(NS3(COOH)3)(L2-RGD)], horizontal stripes, in rats after
single intravenous application in a tail vain. Data are % ID (mean ꢁ SD of 4 animals per
group).
Fig. 4. Biodistribution of [99mTc(NS3crown)(L2-Lys)], diagonal stripes; [99mTc(NS3en)
(L2-Lys)], no stripes;
99mTc(NS3(COOH)3)(L2-Lys)], horizontal stripes, in rats after
[
single intravenous application in a tail vain. Data are % ID (mean ꢁ SD of 4 animals per
group).
2.2.2. Cell uptake study
4e5 and also comparable for the investigated tracer series. These
findings indicate similar receptor binding and internalization
behavior of the investigated compounds.
Results of the cell binding study are summarized in Fig. 6. The
uptake into M21 cells expressing the avb3 integrin was compared to
the unspecific uptake in M21 cells blocked with an excess of the
non-labeled RGD-peptide, M21L tumor cells without avb3 integrin
expression and blocked M21L cells. The highest specific uptake
including surface bound and internalized radioactivity was found
2.2.3. Studies in tumor mice
The biodistribution of [99mTc(NS3en)(L2-RGD)] was investigated
[ [
99mTc(NS3en)(L2-RGD)] compared to 99mTc(NS3(COOH)3)
in n/n mice bearing M21 and M21L (control) tumor xenografts. The
for
(L2-RGD)] and [99mTc(NS3crown)(L2-RGD)]. However, the non-
specifically internalized activity was also highest for [99mTc(NS3en)
(L2-RGD)]. The ratio of specifically to unspecifically internalized
activity was in the range of about 6e8 and comparable for all
tracers. The ratio of specific to unspecific uptake (sum of surface
bound and internalized) activity was in the range of approximately
radioactivity content in the M21 xenograft was 3-fold the activity
in the control xenograft, proving a specific uptake of the tracer
(Table 2). However, the target-background ratios were low and the
radioactivity excretion into the intestine was very high. A consid-
erable washout of the tumor-enriched activity was also observed
4 h after application.
Fig. 3. Biodistribution of [99mTc(NS3crown)(L2-RGD)], diagonal stripes; [99mTc(NS3en)
(L2-RGD)], no stripes; [99mTc(NS3(COOH)3)(L2-RGD)], horizontal stripes, in rats after
single intravenous application in a tail vain. Data are SUV (mean ꢁ SD of 4 animals per
group).
Fig. 5. Biodistribution of [99mTc(NS3crown)(L2-Lys)], diagonal stripes; [99mTc(NS3en)
(L2-Lys)], no stripes; [
99mTc(NS3(COOH)3)(L2-Lys)], horizontal stripes, in rats after
single intravenous application in a tail vain. Data are SUV (mean ꢁ SD of 4 animals per
group).

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J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
Fig. 6. Internalization and binding studies of
[ [
99mTc(NS3crown)(L2-RGD)], 99mTc
(NS3en)(L2-RGD)] and [99mTc(NS3(COOH)3)(L2-RGD)] in M21 and M21L cells (blocked:
co-incubation with 10 mM c(RGDfV); sb: surface bound, internal.: internalized). Data
are % of total activity added/mg protein (mean ꢁ SD of 3).
2.2.4. In vivo stability
The
metabolic
stability
of
[
99mTc(NS3R)(L2-RGD)],
NS3R ¼ NS3crown, NS3en, NS3(COOH)3 was assessed 60 min after
intravenous application of the radioconjugates in rats. The blood
samples were centrifuged to obtain the plasma and this was treated
with acetonitrile and TFA to precipitate the proteins. The obtained
supernatant was referred to as the plasma extract. The mean
activity in the plasma was in the range of 83e92% of arterial blood
activity; the activity of the plasma extract contained 20e50% of the
plasma activity. The extracts and urine were analyzed by RP-HPLC
for radiometabolites (Figs. 7e9). The relative activity in the plasma
extracts assigned to the radiotracer 60 min after intravenous
application was 87% for [99mTc(NS3(COOH)3)(L2-RGD)], 75% for
Fig. 7. Radiochromatograms (HPLC system 5) of [99mTc(NS3crown)(L2-RGD)], [99mTc
(NS3crown)(L2-Lys)] and rat plasma extract, urine, excrement extract 60 min after i.v.
injection of [99mTc(NS3crown)(L2-RGD)].
[
99mTc(NS3en)(L2-RGD)] and 78% for [99mTc(NS3crown)(L2-RGD)].
In urine, 78% for [99mTc(NS3(COOH)3)(L2-RGD)], 92% for [99mTc
(NS3en)(L2-RGD)] and 91% for
99mTc(NS3crown)(L2-RGD)] of
[
excreted radioactivity were intact radiotracer. The contents of the
original tracer compared to the whole radioactivity in the excre-
ment extracts were 81% for [99mTc(NS3(COOH)3)(L2-RGD)], 85% for
[
99mTc(NS3en)(L2-RGD)] and 81% for [99mTc(NS3crown)(L2-RGD)].
Formation of [99mTc(NS3R)(L2-Lys)], NS3R ¼ NS3crown, NS3en,
NS3(COOH)3 as metabolites in the plasma extract, urine or excre-
ment extract could be excluded by comparing with their
chromatograms.
Table 2
Biodistribution of [99mTc(NS3en)(L2-RGD)] in
n/n mice bearing M21 or M21L tumor
xenografts after single intravenous application in
a
tail vain. Data are %ID/g
(mean ꢁ SD of 3 or 4 animals per group).
Xenograft
Time p.i. (min)
%ID/g
M21
60
M21/M21L
M21
240
60
Mean
SD
Mean
SD
Mean
SD
Blood
Pancreas
Spleen
Kidneys
Muscle
Heart
Lung
Liver
Femur
Intestine
Stomach
M21 tumor
M21L tumor
0.96
0.35
0.74
2.68
0.46
0.64
1.77
2.22
0.52
19.3
1.57
1.50
e
0.15
0.07
0.14
0.64
0.08
0.12
0.38
0.62
0.31
3.6
1.17
0.42
0.95
3.36
0.51
0.75
2.02
0.75
0.42
19.5
1.71
1.70
0.52
0.14
0.02
0.09
0.21
0.12
0.09
0.02
0.09
0.10
3.1
0.17
0.13
0.25
0.98
0.12
0.17
0.59
0.61
0.17
13.4
0.69
0.40
e
0.01
0.01
0.01
0.10
0.02
0.04
0.18
0.50
0.06
5.6
0.29
0.37
e
0.04
0.13
0.23
0.20
0.09
e
Fig. 8. Radiochromatograms (HPLC system 5) of
[ [
99mTc(NS3en)(L2-RGD)], 99mTc
(NS3en)(Lys-L2)] and rat plasma extract, urine, excrement extract 60 min after i.v.
injection of [99mTc(NS3en)(L2-RGD)].

J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
3651
physiological conditions by a neutral hydrophilic, a positively
charged and a negatively charged group. Complexes containing the
new coligands could be synthesized and were assessed as conju-
gated to an RGD-peptide. The lipophilicity (distribution coefficient
log DO/W, pH ¼ 7.4) of the RDG-containing radiotracers decreased in
the order of the coligands NS3crown, NS3en and NS3(COOH)3. The
new coligands shift the excretion pathway to a roughly similar rate
of renal and hepatobiliary excretion for the tracers containing
NS3crown and NS3en, and further to a predominant renal excretion
for compounds containing NS3(COOH)3. This marks a considerable
improvement of in vivo properties of 99mTc ‘4 þ 1’ peptide conju-
gates, since those tracers prepared up to now contained unmodi-
fied NS3 or only a single free carboxylic group as modifier and
showed an extensive hepatobiliary excretion [12,13]. The effect of
the coligands was confirmed by a comparable grading between the
biodistribution data of smaller tracers bearing only Lys in place of
the RGD-peptide with the exception of [99mTc(NS3crown)(L2-Lys)]
which was quickly excreted by the kidney and the liver at about the
same rate. The biodistribution of [99mTc(NS3en)(L2-RGD)] in mice
bearing M21 and M21L (control) tumor xenografts exhibited
a specific tumor uptake. However, the target-background ratios
were low which demands more comprehensive structural changes
when aiming to develop a radiopharmaceutical based on a ‘4 þ 1’
complex containing RGD-peptide. The stability studies in normal
rats indicated a suitable metabolic stability of cyclo(Arg-Gly-Asp-D-
Tyr-Lys) and the novel modified 99mTc ‘4 þ 1’ complex units. The
presented coligand strategy offers new properties of 99mTc ‘4 þ 1’
mixed-ligand conjugates, making them more attractive for appli-
cation to radiopharmaceutically relevant biomolecules.
Fig. 9. Radiochromatograms (HPLC system 5) of [99mTc(NS3(COOH)3)(L2-RGD)], [99mTc
(NS3(COOH)3)(L2-Lys)] and rat plasma extract, urine, excrement extract 60 min after i.
v. injection of [99mTc(NS3(COOH)3)(L2-RGD)].
4. Experimental
4.1. Materials
In vivo metabolism studies of radiolabeled RGD-derivatives
containing cyclo(Arg-Gly-Asp-D-Phe-Lys) in mice have been
The RGD-peptide cyclo(Arg-Gly-Asp-
D
-Tyr-Lys) ꢂ
2 TFA, c
reported [21,22]. Accordingly, 24% intact radiotracer was found in
the plasma extract 30 min p.i. of 99mTc-DKCK-RGD and one radio-
metabolite was detected [21]. For [18F]Galacto-RGD, 86% intact
radiotracer was found in the plasma extract 120 min p.i. and also
only one radiometabolite was detected [22]. The differences in the
stability data suggested that the instability of 99mTc-DKCK-RGD is
mainly caused at the site of the radiolabel and not by metabolism of
(RGDyK), was obtained by customized synthesis from Biosyntan
GmbH (Germany). Fmoc-Lys was obtained from Bachem. Solvents
for HPLC were of HPLC grade and purchased from Merck or Fisher
Scientific. All other reagents and solvents were of analytical grade
unless stated otherwise and purchases from Merck, SigmaeAldrich
or Fisher Scientific and used without further purification.
Na99mTcO4 was eluted from a commercial 99Mo/99mTc generator
(Covidien). The coupling agent 2,3,5,6-tetrafluorophenyl 4-iso-
cyanobutanoate (L2-BFCA) was synthesized according to a previ-
ously published method [13]. 3-(Benzylthio)-2-(bis(2-(benzylthio)
ethyl)amino)propanoic acid (N(Sbz)3COOH) was synthesized
according to the previously published procedure [11]. Tris(2-
cyclo(Arg-Gly-Asp-
18F]Galacto-RGD, there was no main metabolite detected for [99mTc
(NS3crown)(L2-RGD)] and [99mTc(NS3en)(L2-RGD)] (Figs. 7 and 8).
99mTc(NS3(COOH)3)(L2-RGD)] caused
main metabolite at
a retention time of about 24.3 min (Fig. 9).
D
-Phe-Lys). In contrast to 99mTc-DKCK-RGD and
[
[
a
The observed metabolic stability of tracers in the series [99mTc
(NS3R)(L2-RGD)] is roughly in the same range as reported for [18F]
Galacto-RGD. These investigations confirm a suitable metabolic
stability of the used cyclic RGD-peptide, the 99mTc ‘4 þ 1’ complex
unit as also shown previously [2,11], and further of its novel
versions grafted with the described pharmacokinetic modifiers.
methoxycarbonylethoxymethyl)aminomethane
(H2NeC
(eCH2eOeCH2eCH2eCOOeMe)3) was synthezised according to
Newkome et al. [23]. The synthesis of the hexathiourea Re(III)
precursor complex [Re(tu)6]Cl3 ꢂ 4H2O (tu ¼ SC(NH2)2) was carried
out according to Gambino et al. [26]. The Re(III)-dimethyl phenyl
phosphino complex [Re(NS3)(PMe2Ph)] was prepared as published
by Spies et al. [27].
3. Conclusion
4.2. Instrumentation
The concept of applying ‘4 þ 1’ mixed-ligand complexes for
99mTc-labeling of radiopharmaceutically interesting peptides is
challenged by the lipophilic nature of the ‘4 þ 1’ complex unit
resulting in an unwanted extensive hepatobiliary excretion. The
mixed-ligand framework carries the possibility that hydrophilic
coligands may have a compensatory effect. The presented novel
NS3-chelators grafted with pharmacokinetic modifiers e a crown
ether, an amine and a tricarboxylic acid (NS3crown, NS3en,
NS3(COOH)3) were intended to decrease the lipophilicity under
Electrospray ionization mass spectrometry (ESIeMS) was per-
formed using a Micromass Tandem Quadrupole Mass Spectrometer
(Quattro LC). Nuclear magnetic resonance spectra were recorded on
a 400 MHz Varian Inova 400 spectrometer. Chemical shifts are
reported as
d in ppm relative to the residual solvent signal or TMS
(1H: 400 MHz, 13C: 101 MHz). TLC strips were scanned using a Rita
linear radioanalyser (Raytest). Shown chromatograms were
normalized to the peak height. Lyophilization was performed with

3652
J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
a freeze dryer Alpha 1e4 (Christ). Submilligram peptide amounts
were weighted using a micro balance M2P (Sartorius). Distribution
coefficients were radiometrically determined using the NaI(Tl)
scintillation counter Isomed (Nuklearmedizintechnik Dresden).
RP-HPLC analyses and purification were performed on a Smart-
line system (Knauer) equipped with a pump 1000, a manager 5000,
purified by column chromatography (silica gel 60, eluent: tri-
chloromethane/ethylacetate, 5/1, v/v, Rf ¼ 0.6). A colorless viscous
oil was obtained. Yield: 35%; 1H NMR (CDCl3): 1.41 (s, 9H, CeCH3),
2.29e2.38 (m, 4H, CH2), 2.49e2.55 (m, 3H, NCH2) 2.62e2.68 (m, 2H,
SCH2CH), 3.08e3.23 (m, 1H, NCHCO), 3.20e3.24 (m, 4H, SCH2CH),
3.62 (s, 4H, SCH2Ph), 3.66 (s, 2H, SCH2Ph), 3.68 (m, 1H), 5.10 (s, 1H,
NHeCOO), 7.17e7.28 (m, 15H, phenyl), 7.83 (s, 1H, NHeCH2); 13C
NMR (CDCl3): 28.7, 29.1, 30.6 (SCH2C), 36.5 (SCH2C), 38.0 (SCH2Ph),
39.7, 40.9, 50.2, 64.7 (NCHCO), 79.4, 127.3, 128.7, 129.0, 129.1, 138.3,
138.5, 156.2, 172.6; ESIeMS (m/z) found (calcd): 653.9 (654.3) for
[M þ H]þ (¼[C35H48N3O3S3]þ).
a PDA detector 2800 and a homemade g-ray detector (Bohrloch, NaI
(Tl) crystal). Shown chromatograms were normalized to the peak
height. The gradient and HPLC conditions were as follows: eluent A:
NH3 (0.01 M) in acetonitrile, eluent B: NH3 (0.01 M) in water, eluent
C: 0.1% (v/v) trifluoroacetic acid (TFA) in acetonitrile; eluent D: 0.1%
(v/v) TFA in water, eluent E: 0.1% (v/v) TFA in ethanol, eluent F:
0.04% (v/v) TFA in acetonitrile, eluent G: 0.05% (v/v) TFA in water.
Synthesis
of N-(2-aminoethyl)-3-(benzylthio)-2-(bis(2-(ben-
zylthio)ethyl)amino)pro-panamide (N(Sbz)3en): Trifluoroacetic acid
(0.8 mmol) was added to N(Sbz)3(boc)en (0.05 mmol) dissolved in
trichloromethane (2 mL) and stirred at r. t. for 12 h. Afterwards, the
reaction mixture was neutralized using a saturated aqueous
NaHCO3-solution followed by 3-fold extraction with dichloro-
methane. The combined organic phases were dried with MgSO4
and the solvent was removed in vacuum. A colorless oil was
obtained. Yield: 90%; 1H NMR (CDCl3): 2.30e2.38 (m, 4H, CH2),
2.49e2.56 (m, 3H, NCH2) 2.62e2.73 (m, 4H, SCH2CH), 3.09e3.14 (m,
2H), 3.25e3.27 (m, 2H, SCH2CH), 3.61 (s, 4H, SCH2Ph), 3.67 (s, 2H,
SCH2Ph), 3.68 (m, 1H), 7.16e7.29 (m, 15H, phenyl), 7.76 (s, 1H,
NHeCH2); 13C NMR (CDCl3): 29.1, 30.7 (SCH2C), 36.5 (SCH2C), 38.0
(SCH2Ph), 41.9, 42.8, 50.3, 64.8 (NCHCO), 127.3, 128.7, 128.8, 129.0,
129.1, 138.3, 138.5, 172.3; ESIeMS (m/z) found (calcd): 553.8 (554.2)
for [M þ H]þ (¼[C30H40N3OS3]þ).
System 1: Gemini 5
gradient elution using eluents A and B: 5e65% A in 40 min, 2 mL/
m
C18 110 Å (Phenomenex), 250 ꢂ 10 mm,
min; 220 nm.
System 2: Jupiter 4
gradient elution using eluents C and D: 20e80% C in 20 min, 2 mL/
m
Proteo 90 Å (Phenomenex), 250 ꢂ 10 mm,
min; 220 nm.
System 3: Jupiter 4
gradient elution using eluents C and D: 20e80% C in 20 min, 1 mL/
min; 220 nm and/or -detection.
System 4: Jupiter 4
Proteo 90 Å (Phenomenex), 250 ꢂ 10 mm,
gradient elution using eluents D and E: 20e80% E in 20 min, 2 mL/
min; -detection.
System 5: Zorbax 300SB-C18 5
gradient elution using eluents F and G: 10e70% F in 40 min, 2 mL/
min; -detection.
m
Proteo 90 Å (Phenomenex), 250 ꢂ 4.6 mm,
g
m
g
mm (Agilent), 250 ꢂ 9.4 mm,
g
Synthesis of 5-(2-(benzylthio)ethyl)-6-(benzylthiomethyl)-9,9-bis
((2-carboxyethoxy)me-thyl)-7-oxo-1-phenyl-11-oxa-2-thia-5,8-dia-
zatridecane-13-carboxylic acid (N(Sbz)3(COOH)3): N(Sbz)3COOH
(2.17 mmol) were dissolved in trichloromethane (20 mL) and
cooled at approximately 0 ꢃC. The solution of N,N0-dicyclohex-
ylcarbodiimide (DCC, 2.17 mmol) dissolved in trichloromethane
(20 mL) were added dropwise and the reaction mixture was
4.3. Methods
4.3.1. Synthesis of NS3crown, NS3en and NS3(COOH)3
4.3.1.1. Coupling. Synthesis of 3-(benzylthio)-2-(bis(2-(benzylthio)
ethyl)amino)-1-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)
propan-1-one (N(Sbz)3crown): N(Sbz)3COOH (0.2 mmol) and
hydroxybenzotriazole (HOBt, 0.2 mmol) were dissolved in tetra-
hydrofurane (THF, 3 mL) and cooled at approximately 0 ꢃC. The
solution of N,N0-dicyclohexylcarbodiimide (DCC, 0.2 mmol) dis-
solved in THF (2 mL) was added dropwise and the reaction mixture
was stirred at 0 ꢃC for 15 min. Afterwards, 1-aza-18-crown-6
(0.2 mmol) dissolved in THF (2 mL) were added and the reaction
mixture was stirred over night at r.t. The residue was filtered off and
the solvent of the supernatant was removed under vacuum. The
residual yellow oil was purified by column chromatography (silica
gel 60, eluent: trichloromethane/ethanol, 19/1, v/v). A pale yellow
oil was obtained. Yied: 50%; TLC (silica gel 60 F254s, eluent: tri-
chloromethane/ethanol, 19/1, v/v): Rf e 0.3; 1H NMR (CDCl3):
2.26e2.30 (t, 4H, CH2, J ¼ 8.2 Hz), 2.33e2.50 (m, 3H, NCH2)
2.51e2.59 (m, 3H, SCH2CH), 2.65e2.72 (m, 2H, SCH2CH), 2.94e3.00
(m, 1H, NCHCO), 3.30e3.37 (m, 1H, SCH2CH), 3.53e3.70 (m, 26H,
CON(CH2)2, SCH2Ph), 3.81e3.88 (m,1H), 7.17e7.28 (m,15H, phenyl);
13C NMR (CDCl3): 27.6, 30.8 (SCH2C), 36.5 (SCH2C), 37.4 (SCH2Ph),
47.7, 48.7 (CON(CH2)2), 51.3, 62.5 (NCHCO), 69.7, 70.6, 70.7, 70.8,
70.9, 71.0, 127.2, 128.7, 129.0, 129.1, 138.6, 138.9, 170.9. ESIeMS (m/z)
stirred at
0
ꢃC for 15 min. Afterwards, H2NeC
(eCH2eOeCH2eCH2eCOOeMe)3 (2.17 mmol) dissolved in tri-
chloromethane (20 mL) were added and the reaction mixture was
stirred over night at 0 ꢃC. The residue was filtered off and the
solvent of the supernatant was removed under vacuum. For
saponification, the obtained conjugate (1 mmol) was dissolved in
dioxane (10 mL) and aqueous sodium hydroxide solution (1 M,
10 mL) were added. After stirring over night, the reaction mixture
was acidified using glacial acetic acid. Subsequently, the reaction
mixture was extracted with trichloromethane (3 ꢂ 20 mL). The
combined organic phases were dried using MgSO4 and then the
solvent removed in vacuum. A yellow viscous oil was obtained.
Yield: 60%; 1H NMR (CDCl3): 2.37 (m, 6H, CH2eCO), 2.48 (m, 4H,
CH2) 2.58 (m, 4H, SCH2CH), 2.99e3.04 (m, 1H), 3.17e3.19 (t, 1H,
J ¼ 8 Hz) 3.57e3.66 (m, 22H, SCH2Ph, OCH2), 7.18e7.27 (m, 15H,
phenyl), 7.36 (s, 1H, NH); 13C NMR (CDCl3): 29.1, 30.6 (SCH2C), 35.0
(SCH2C), 36.5 (SCH2Ph), 37.9, 59.7, 65.2 (NCHCO), 66.9 (CeOeC),
69.1 (CeOeC), 127.2, 127.3, 128.7, 129.0, 138.6, 171.8, 176.2 (COOH);
ESIeMS (m/z) found (calcd): 829.3 (829.3) for [MeH]ꢀ
(¼[C41H53N2O10S3]ꢀ).
found (calcd): 779.5 (779.3) for [N(Sbz)3crown
þ
Na]þ
(¼[C40H56N2NaO6S3]þ).
4.3.1.2. Deprotection of the tristhiolate ligands. A solution of
0.05 mmol of N(Sbz)3crown, N(Sbz)3en or N(Sbz)3(COOH)3 in dry
THF (1 mL) was added dropwise to liquid ammonia (about 5 mL,
eꢀ50 to ꢀ30 ꢃC). Sodium was added in small pieces under gentle
stirring until the solution remained deep blue for 15 min. After-
wards the solvents were removed using an argon stream. Water
(5 mL) was added to the residue and the solution was acidified with
hydrochloric acid (1 M) to pH 3e4. The solution was extracted with
ethyl acetate (3 ꢂ 5 mL) to remove organic side products. The
solvent of the aqueous phase (water) was removed in vacuum. The
Synthesis of tert-butyl 2-(3-(benzylthio)-2-(bis(2-(benzylthio)
ethyl)amino) propanamido) ethylcarbamate (N(Sbz)3(boc)en): At
0 ꢃC, a solution of DCC (0.5 mmol) in trichloromethane (2 mL) was
added dropwise to a solution of N(Sbz)3COOH (0.5 mmol) dissolved
in trichloromethane (6 mL) and stirred for 15 min. Afterwards,
N-boc-ethylenediamine (0.55 mmol) dissolved in trichloromethane
(2 mL) were added dropwise and the reaction mixture was stirred
at r.t. for 12 h. The precipitated byproduct was filtered off and the
solvent was removed in vacuum. The residual yellow oil was

J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
3653
residue was resuspended in methanol and then filtered off to
4.3.5. Synthesis of [Re(NS3R)X]; NS3R ¼ NS3crown, NS3en,
separate the product from sodium chloride. The solvent (methanol)
of the filtrate was removed under reduced pressure. White
powders or colorless oils were obtained. Yield: e100% (partly
contaminated with sodium chloride).
NS3(COOH)3; X ¼ L2-RGD, L2-Lys
The isocyanide L2-RGD or L2-Lys (2
mmol) was dissolved in
a mixture of methanol (0.8 mL) and water (0.2 mL). An equimolar
amount of either [Re(NS3crown)(PMe2Ph)], [Re(NS3en)(PMe2Ph)]
or [Re(NS3(COOMe)3)(PMe2Ph)] dissolved in MeOH (0.2 mL) was
added. After stirring for 1e2 h at r.t., the solvent was removed
under vacuum. In order to prepare [Re(NS3(COOH)3)X], aqueous
NaOH (1 M, 1 mL) was added to the residue and the suspension was
stirred for 1 h followed by removing the solvent under vacuum. The
crude products were purified by HPLC (system 2). Green powders
were obtained after lyophilization. Yield: 30e40%; HPLC (system
3): tR ¼ 10.8 min for [Re(NS3crown)(L2-RGD)], tR ¼ 8.2 min for [Re
(NS3en)(L2-RGD)], tR ¼ 9.5 min for [Re(NS3(COOH)3)(L2-RGD)],
tR ¼ 9.2 min for [Re(NS3crown)(L2-Lys)], tR ¼ 4.5 min for [Re(NS3en)
(L2-Lys)], tR ¼ 10.2 min for [Re(NS3(COOH)3)(L2-Lys)]; ESIeMS (m/
z) found (calcd): 1383.5, 1385.6 (1383.5, 1385.5) for [Re(NS3crown)
(L2-RGD)þH]þ (¼[C51H82N12O15ReS3]þ), 1180.6, 1182.6 (1180.3,
1182.3) for [Re(NS3en)(L2-RGD)þH]þ (¼[C41H65N13O10ReS3]þ),
1457.8, 1459.8 (1457.4, 1459.4) for [Re(NS3(COOH)3)(L2-RGD)þH]þ
(¼[C52H80N12O19ReS3]þ), 909.6, 911.7 (909.3, 911.3) for [Re
(NS3crown)(L2-Lys)þH]þ (¼[C30H55N5O9ReS3]þ), 706.5, 708.5
(706.2, 708.2) for [Re(NS3en)(L2-Lys)þH]þ (¼[C20H38N6O4ReS3]þ),
983.5, 985.5 (983.2, 985.2) for [Re(NS3(COOH)3)(L2-Lys)þH]þ
(¼[C31H53N5O13ReS3]þ), two signals corresponding to 185Re and
187Re.
3-Mercapto-N,N-bis(2-mercaptoethyl)-1-oxo-1-(1,4,7,10,13-pentaoxa-
16-azacyclooctadecan-16-yl)propan-2-aminium-chlorid (NS3crown): 1H
NMR (D2O): 2.71e2.81 (m, 4H), 2.91e2.97 (m, 1H) 3.13e3.26 (m,
3H), 3.37e3.44 (m, 2H), 3.51e3.64 (m, 22H), 3.68e3.76 (m, 2H),
4.74e4.77 (m, 1H, CHCON(CH2)2); 13C NMR (D2O): 18.0, 22.2, 47.0,
49.4, 53.7, 63.2, 63.3, 68.4, 68.8, 69.8, 167.0.
1-(2-Ammonioethylamino)-3-mercapto-N,N-bis(2-mercaptoethyl)-
1-oxopropan-2-aminium-dichlorid (NS3en): 1H NMR (D2O):
2.87e3.07 (m, 4H), 3.10e3.35 (m, 4H), 3.44e3.74 (m, 6H), 4.33e4.36
(m, 1H, CHCONH); 13C NMR (D2O): 18.8, 22.0, 37.7, 38.9, 54.5, 66.5,
167.2.
1-(1,3-Bis(2-carboxyethoxy)-2-((2-carboxyethoxy)methyl)propan-
2-ylamino)-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan-2-
aminium-chlorid (NS3(COOH)3): 1H NMR (CD3OD): 2.52 (m, 6H,
CH2COOH), 2.58e2.81 (m, 10H) 3.44 (t, 1H, J ¼ 6.84 Hz, CHCONH),
3.69 (m, 12H, CH2O), 7.56 (s, 1H, CONH); 13C NMR (CD3OD): 21.9,
22.7, 34.6, 54.1, 60.3, 66.9, 67.5, 68.7, 171.8, 174.2.
4.3.2. Synthesis of the isocyano-RGD derivative L2-RGD
The RGD-peptide cyclo(Arg-Gly-Asp-
(10
Triethylamin (35
D
-Tyr-Lys)
ꢂ
2
TFA
m
mol) was dissolved in 0.5 mL N,N-dimethylformamide (DMF).
m
mol, reagent grade) and L2-BFCA (9
m
mol) dis-
solved in DMF (0.1 mL) were added. After being stirred for 12 h at r.
t., the solvent was removed under vacuum and the residue was
purified by HPLC (system 1). A white powder was obtained after
lyophilization. Yield: 40e50%; HPLC (system 1): tR ¼ 16.5 min;
4.3.6. Preparation of 99mTc ‘4 þ 1’ conjugates [99mTc(NS3R)X];
NS3R ¼ NS3crown, NS3en, NS3(COOH)3; X ¼ L2-RGD, L2-Lys
EDTA solution (100
solution (100
(200 L, 200e400 nmol NS3en, NS3crown or NS3(COOH)3 dissolved
in a mixture of 100 L ethanol and 100 L water), and ascorbic acid
solution (1 mg in 50 L water) were transferred into a vial con-
taining L2-RGD or Lys-L2 (100e200 nmol), respectively. SnCl2
solution (0.1 mg in 20 L ethanol) was added followed by the
mL, 10 mg Na2EDTA in 1 mL water), mannitol
mL, 50 mg mannitol in 1 mL water), NS3-solution
ESIeMS (m/z) found (calcd): 715.4 (715.5) for [M
(¼[C32H46N10O9]þ).
þ
H]þ
m
m
m
m
4.3.3. Synthesis of the isocyano-lysine derivative L2-Lys
Fmoc-Lys (40
and water (0.8 mL). Triethylamin (0.2 mmol, reagent grade) and L2-
BFCA (40.5 mol, dissolved in 0.2 mL DMF) were added. After being
mmol) was dissolved in a mixture of DMF (2 mL)
m
addition of 99mTcOꢀ4 solution (1e2 mL, 0.5e1 GBq). Ethanol was
added to adjust the content of it in the reaction mixture to
approximately 25% of the mixture volume. After a reaction time of
30e60 min, the radiochemical yield was 60e80%. The radio-
conjugates were purified by HPLC (system 4) to get a radiochemical
purity of higher than 95%. To prepare the injection solution for
biodistribution studies, the HPLC-eluate was heated to about 50 ꢃC
and the volume was reduced using an argon gas stream. Afterwards
the solution was diluted with phosphate buffered saline and the pH
value was adjusted to 7.4, ethanol was added to adjust its content to
about 10% yielding a radioconjugate solution of about 50 MBq in
0.5 mL. HPLC (system 3): tR ¼ 10.7 min for [99mTc(NS3crown)(L2-
RGD)], tR ¼ 8.0 min for [99mTc(NS3en)(L2-RGD)], tR ¼ 9.6 min for
m
stirred for 12 h at r.t., the solvent was removed under vacuum. In
order to deprotect the compound, the residue was dissolved in DMF
(1 mL), piperidine (0.5 mL) was added and it was placed in an
ultrasound bath for 30 min. Afterwards the solvent was removed
under vacuum. The crude product was purified by HPLC (system 1).
A white powder was obtained after lyophilization. Yield: 30e40%;
HPLC (system 1): tR ¼ 8.6 min; ESIeMS (m/z) found (calcd): 242.2
(242.3) for [M þ H]þ (¼[C11H20N3O3]þ).
4.3.4. Synthesis of [Re(NS3R)(PMe2Ph)]; NS3R ¼ NS3crown, NS3en,
NS3(COOMe)3
The reaction mixture containing the precursor [Re(tu)6]
Cl3 ꢂ H2O (0.1 mmol), dissolved in methanol (5 mL), dimethyl
phenyl phosphine (0.2 mmol) and 0.1 mmol of the respective tet-
radentate ligand NS3crown, NS3en or NS3(COOH)3 was heated
under reflux for 3 h. The acidification during the reaction resulted
in esterification of the carboxyl groups of NS3(COOH)3 whereby [Re
(NS3(COOMe)3)(PMe2Ph)] was formed. Afterwards the solvent was
removed in vacuo and the crude product was purified by column
chromatography (silica gel 60, THF/n-hexane: 4/3, Rf ¼ 0.5e0.7).
After removal of the solvent the products were obtained as green
solid. Yield: 80e90%; ESIeMS (m/z) found (calcd): 807.4, 807.6
[
99mTc(NS3(COOH)3)(L2-RGD)], tR ¼ 9.1 min for [99mTc(NS3crown)
(L2-Lys)], tR ¼ 4.4 min for [99mTc(NS3en)(L2-Lys)], tR ¼ 10.2 min for
[
[
99mTc(NS3(COOH)3)(L2-Lys)]; HPLC (System 5): tR ¼ 23.9 min for
99mTc(NS3crown)(L2-RGD)], tR ¼ 17.4 min for [99mTc(NS3en)(L2-
RGD)], tR
¼
21.6 min for
[
99mTc(NS3(COOH)3)(L2-RGD)],
tR ¼ 22.5 min for [99mTc(NS3crown)(L2-Lys)], tR ¼ 14.1 min for
[
99mTc(NS3en)(L2-Lys)], tR ¼ 20.0 min for [99mTc(NS3(COOH)3)
(L2-Lys)].
4.3.7. Determination of distribution coefficients
Distribution coefficients log Do/w were determined by a distri-
bution experiment using an 1-octanol/buffer system. The buffer
solution containing the 99mTc-labeled compound was prepared by
using the HPLC-eluate containing the purified radioconjugate,
removing of the solvent by heating the vial at about 50 ꢃC when
applying an argon gas steam, redissolving in phosphate buffer
(807.2,
809.2)
for
[Re(NS3crown)(PMe2Ph)
þ
H]þ
(¼[C27H47N2O6PReS3]þ), 604.2, 606.2 (604.1, 606.1) for [Re(NS3en)
(PMe2Ph) þ H]þ (¼[C17H30N3OPReS3]þ), 923.5, 925.6 (923.2, 925.2)
for [Re(NS3(COOMe)3)(PMe2Ph) þ H]þ (¼[C31H51N2O10PReS3]þ),
two signals corresponding to 185Re and 187Re.

3654
J.-U. Kunstler et al. / European Journal of Medicinal Chemistry 45 (2010) 3645e3655
(0.1 M) and adjustment of the pH value to 7.4. The buffer solution
(0.5 mL) containing approximately 0.5 MBq 99mTc-tracer and
1-octanol (0.5 mL) were mixed and mechanical shaken in a micro-
centrifuge tube for 15 min and then centrifuged for phase separa-
tion. The activity concentrations of both phases were determined
radiometrically using a NaI(Tl) scintillation counter and the distri-
bution coefficients were calculated. The results are the average
values of four independent experiments.
the tumor uptake of [99mTc(NS3crown)(L2-RGD)], each mouse was
injected with 1 MBq (roughly 4 ꢂ 10ꢀ13 mol) of the radioconjugate
into the tail vein and sacrificed by cervical dislocation at 1 h and
4 h p.i.
4.3.10. In vivo stability
The in vivo stability of [99mTc(NS3crown)(L2-RGD)], [99mTc
(NS3en)(L2-RGD)] and [99mTc(NS3(COOH)3)(L2-RGD)] was studied
using rat arterial blood samples, urine and faeces at 1 h after i.v.
injection of the radiotracer solution (0.5 mL) containing about
20 MBq (roughly 7 ꢂ 10ꢀ12 mol). The samples were centrifuged to
precipitate cells and corpuscular parts. A solution consisting of
4.3.8. Internalization and binding studies in avb3-positive and
avb3-negative cells
avb3-positive M21 and -negative M21L human melanoma cells
were grown in culture until sufficient number of cells were avail-
able. For internalization experiments cells were collected to
a concentration of 2 ꢂ 106 cells/mL in RPMI1640 containing 1%
Glutamine and 1% PBS. Each 1 mL was pipetted into a separate tube.
After addition of 99mTc-labeled peptide (>100 000 cpm, 1 nM) cells
were incubated at 37 ꢃC for 90 min in triplicates with either PBS/
acetonitrile (50 mL), water (45 mL) and trichloroacetic acid (5 mL)
was added to the supernatant (200 mL) and then the mixture was
cooled at ꢀ20 ꢃC. After centrifugation, the supernatant was
analyzed by HPLC (system 5). To identify potential radio-
metabolites, the obtained chromatograms were compared with
chromatograms of the injection solution and the reference
0.5%BSA buffer alone (150
mL, total series), or with c(RGDyK)
compounds
[
99mTc(NS3crown)(L2-Lys)],
[
99mTc(NS3en)(L2-Lys)]
(10 M) in PBS/0.5%BSA buffer (150
m
mL, non-specific series). Incu-
and [99mTc(NS3(COOH)3)(L2-Lys)].
bation was interrupted by centrifugation, removal of medium and
rapid rinsing twice with ice-cold TRIS buffered saline two times.
Thereafter, the cells were incubated twice at room temperature in
acid wash buffer (50 mM acetate buffer pH ¼ 4.2) for 15 min at
37 ꢃC. The supernatant was collected (membrane bound radio-
ligand fraction) and the cells were washed with acid wash buffer.
Cells were lyzed by treatment in NaOH (1 M) and cell radioactivity
collected (internalized radioligand fraction). Protein concentration
in the NaOH fraction was determined using spectrophotometric
determination according to Bradford. The internalized and non-
internalized fractions were determined by measuring radioactivity
and the internalized fraction was expressed as % of total activity per
mg protein.
Acknowledgments
This work was supported by the Marie Curie Transfer of
Knowledge Scheme (POL-RAD-PHARM, contract no MTKD-CT-
2004-509224) part of the 6th EU Framework Programme for
Research and Development. The authors thank Mrs Regina Herrlich
and Mrs Andrea Suhr for their excellent technical assistance.
This work was initiated by an IAEA Coordinated Research Project
‘Development of Tc-99m based small biomolecules using novel
99mTc cores’. Special thanks go to Dr. M.R.A Pillai from the Depart-
ment of Nuclear Sciences and Applications of the IAEA for his
support and stimulation of cooperation activities.
4.3.9. Biodistribution
The animal studies were carried out according to the relevant
national regulations.
Appendix. Supporting Information
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.ejmech.2010.05.010.
The specified injected molar amounts of radioconjugates were
estimated considering the specific activity of 99mTc, the total
amount of Tc contained in the generator eluate [28], which was
eluatetd 24 h after its last elution and a time period of one 99mTc
half life between generator elution and application into the animal.
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