Molecular assembly of multifunctional 99mTc radiopharmaceuticals using "clickable" amino acid derivatives

Article abstract of DOI:10.1002/cmdc.201000342

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side-chain-functionalized N(a)-propargyl lysine derivatives, conjugates containing a ^99mTc-based imaging probe for SPECT and two different moieties (e.g., tumor-targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the CuI-catalyzed alkyneazide cycloaddition in efficient one-pot protocols. This strategy was successfully applied to the preparation of a ^99mTc-labeled conjugate comprising a tumor-targeting peptide sequence (bombesin(7-14)) and a low-molecular-weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin-releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional ^99mTc-based radiopharmaceuticals with significant potential for a multitude of applications.

Full text of DOI:10.1002/cmdc.201000342

MED
DOI: 10.1002/cmdc.201000342
99m
Molecular Assembly of Multifunctional Tc
Radiopharmaceuticals Using “Clickable” Amino Acid
Derivatives
Thomas L. Mindt,*[a, b] Harriet Struthers,[b] Bernhard Spingler,[c] Luc Brans,[d] Dirk Tourwꢀ,[d]
Elisa Garcꢁa-Garayoa,[e] and Roger Schibli*[b, e]
Synthetic strategies that enable the efficient and selective
combination of different biologically active entities hold great
promise for the development of multifunctional hybrid conju-
gates useful for biochemical and medical applications. Starting
from side-chain-functionalized N(a)-propargyl lysine deriva-
tives, conjugates containing a 99mTc-based imaging probe for
SPECT and two different moieties (e.g., tumor-targeting vec-
tors, pharmacological modifiers, affinity tags, or second imag-
ing probes) can be assembled using the CuI-catalyzed alkyne–
azide cycloaddition in efficient one-pot protocols. This strategy
was successfully applied to the preparation of a 99mTc-labeled
conjugate comprising a tumor-targeting peptide sequence
(bombesin(7–14)) and a low-molecular-weight albumin binder,
a pharmacological modifier that prolongs the blood circulation
time of the conjugate. Evaluation of the conjugate in vitro and
in vivo provided promising results for its use as an imaging
agent for the visualization of tumors positive for the gastrin-re-
leasing peptide receptor. The methodology presented herein
provides an attractive synthetic tool for the preparation of
multifunctional 99mTc-based radiopharmaceuticals with signifi-
cant potential for a multitude of applications.
Introduction
The radiopharmaceutical sciences are engaged in the develop-
ment of radioactively labeled compounds for applications in
nuclear medicine as imaging probes (employing diagnostic g-
and b+-emitting isotopes) or radiotherapeutics (using emitters
of a or bꢀ particles or Auger electrons).[1,2] The majority of radi-
oisotopes with decay properties suitable for diagnosis and par-
ticularly therapy are transition metals or elements that possess
metallic character. As a consequence, bifunctional chelating
agents (BFCA) are required for the stable complexation of the
radionuclide and its attachment to targeted (bio)molecules
(vectors) of interest. Besides efficient methodologies for the se-
lective conjugation of radionuclide chelates to vectors, it
would be desirable to have synthetic procedures at hand that
facilitate the simultaneous introduction of pharmacological
modifiers, which optimize the biological characteristics of a ra-
dioconjugate. Examples of other moieties of interest include
affinity tags (e.g., biotin for pre-targeting approaches) and
therapeutic agents (for the development of theranostics). Alter-
natively, the introduction of different imaging probes would
yield dual-mode imaging agents, the development of which is
of significant contemporary interest.[3–5] While the potential of
multifunctional radioconjugates is intriguing, their preparation
by conventional synthetic approaches is a challenging task.
Therefore, new and efficient synthetic strategies, such as click
chemistry, for the controlled assembly of multifunctional radio-
nuclide-containing conjugates are of paramount importance.
We have shown that the CuI-catalyzed alkyne–azide cycload-
dition (CuAAC),[6,7] a transformation known as a click reaction,[8]
can be used for the efficient labeling of (bio)molecules with
99mTc. By reacting appropriate alkyne pro-chelators with azide-
functionalized derivatives of (bio)molecules, the synthesis of
the ligand system and its selective conjugation can be accom-
plished simultaneously in a single step without protecting
groups.[9] The conjugates obtained are functionalized with a tri-
dentate ligand system containing a 1,4-disubstituted 1,2,3-tria-
zole that can be radiolabeled directly in situ with the organo-
metallic core [99mTc(CO)3(H2O)3]+.[10] Given the efficiency of this
one-pot, two-step protocol, we termed this strategy “click-to-
chelate”. In the meantime we have demonstrated that this
[a] Prof. T. L. Mindt
Department of Radiology and Nuclear Medicine
Division of Radiological Chemistry
University Hospital Basel, 4031 Basel (Switzerland)
Fax: (+41)61-265-5559
E-mail: TMindt@uhbs.ch
[b] Prof. T. L. Mindt, Dr. H. Struthers, Prof. R. Schibli
Department of Chemistry and Applied Biosciences
Institute of Pharmaceutical Sciences
ETH Zurich, 8093 Zurich (Switzerland)
Fax: (+41)44-633-1367
E-mail: roger.schibli@pharma.ethz.ch
[c] Priv.-Doz. Dr. B. Spingler
Institute of Inorganic Chemistry
University of Zurich, 8057 Zurich (Switzerland)
[d] Dr. L. Brans, Prof. D. Tourwꢀ
Department of Organic Chemistry, Faculty of Sciences
Vrije Universiteit Brussels, 1050 Brussels (Belgium)
[e] Dr. E. Garcꢁa-Garayoa, Prof. R. Schibli
Center for Radiopharmaceutical Science ETH-PSI-US
Paul Scherrer Institute, 5232 Villigen-PSI (Switzerland)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000342.
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Multifunctional 99mTc Radiopharmaceuticals
strategy can be applied to the preparation of a variety of 99mTc-
based single photon emission computed tomography (SPECT)
probes suitable for applications in vitro and in vivo.[11–13] We
now report an extension of our click chemistry strategy. The
use of readily available N(a)-propargyl amino acid derivatives
as click substrates not only offers a convenient and efficient
method for labeling targeted (bio)molecules with [99mTc(CO)3]+
(e.g., R=H, Figure 1), it also provides a means for the concur-
rent introduction of a second entity of interest (R=various en-
tities attached by functionalization of the side chain, Figure 1).
The possibility of combining two different moieties with bio-
logical function and a chelating system in a single step is an
attractive feature for the development of metallic radiotracers
and opens up numerous potential applications.
Scheme 1. Synthesis of N(a)-propargyl amino acid derived tridentate chelat-
ing systems and their complexes with [M(CO)3]+ (M=99mTc, Re). a) For
GlyOMe, ValOMe and ProOMe: MeOH, K2CO3, RT; for Gln(Et)OBn and
Lys(Ac)OMe: THF, DIPEA, 508C, 1–2 days; b) for 2, 3, 6: tBuOH/H2O (1:1),
20 mol% Cu(OAc)2, 40 mol% sodium ascorbate, RT, 18 h; for 4, 5: tBuOH/
H2O (1:1), excess Cu(0), RT, 20 h; c) for M=Re: [Re(CO)3Br3][Et4N]2, alcohol/
H2O, 50–758C, 0.5–4 h; for M=99mTc : [99mTc(CO)3(H2O)3]+, PBS pH ~7, 1008C,
30 min.
characterized (NMR, IR, MS). In the case of L=L4, L5, and L6,
crystals of the corresponding rhenium complexes suitable for
X-ray analysis could be obtained. Figure 2 shows the ORTEP
plots of the three complexes, confirming the coordination by
the N(3) atom of the 1,2,3-triazole heterocycle, and the N(a)
and carboxylate of the amino acid as depicted in Scheme 1. All
amino acid derived chelators could also be readily radiolabeled
with [99mTc(CO)3(H2O)3]+ in aqueous phosphate buffer at 1008C
and pH ~7. Quantitative formation of radionuclide complexes
was achieved in all cases at ligand concentrations in the micro-
molar range (10ꢀ5–10ꢀ6 m), which again demonstrates the ex-
cellent efficiency of 1,2,3-triazole-containing tridentate chelat-
ing systems for complexation of the 99mTc–tricarbonyl core.
Identity of the [99mTc(CO)3(L)] (L=L2–L6) complexes was con-
firmed in each case by comparison of the g-HPLC with the UV
trace of the corresponding rhenium analogues, a procedure
which is common practice with 99mTc complexes on a no carrier
added (n.c.a.) level.
Figure 1. N(a)-propargyl derivatives of amino acids as CuAAC substrates for
the one-pot assembly of multifunctional 99mTc radioconjugates. Squiggled
lines represent different spacers and R (residue) stands for amino acid specif-
ic side chains to which different entities can be conjugated.
Results and Discussion
Chemical syntheses
Amino acids represent a structurally diverse pool of building
blocks for the development of ligand systems for complexation
of the tricarbonyl core [M(CO)3]+ (M=99mTc, Re). For example,
chelators derived from histidine, cysteine, lysine, or synthetic
analogues have all been reported in this context.[9,14,15] We rec-
ognized that N(a)-propargyl-functionalized amino acids are
readily available precursors for the “click-to-chelate” strategy
(Scheme 1).[12,13] To verify their general utility for the prepara-
tion of 99mTc-based radiotracers, we studied a series of N(a)-
propargyl amino acid derivatives. Thus, reaction of propargyl
bromide (1) with HGlyOMe, HValOMe, HProOMe, HGln(Et)OBn,
and HLys(Ac)OMe efficiently provided N(a)-alkylated propargyl
derivatives 2–6.[16,17] Subsequent CuAAC with model com-
pound benzyl azide (7) in aqueous media at room temperature
yielded 1,2,3-triazole-containing ligands L2–L6 in a single step
and high yields.[12] Reaction of the tridentate chelators with
While all investigated N(a)-1,2,3-triazolyl amino acid deriva-
tives were shown to be efficient chelators for [M(CO)3]+, only
those with a functionalizable side chain group (Lys, Gln/Glu)
could be considered for an extended “click-to-chelate” ap-
proach. In an initial series, we investigated the utility of pro-
tected glutamic acid derivatives. However, we observed that
intermediates obtained from protected HGlu(tBu)OMe accord-
ing to Scheme 1 spontaneously undergo intramolecular lac-
tamization (data not shown). We therefore focused on Lys de-
rivatives and investigated the chemistry required for the syn-
thesis of a common click precursor to which different entities
can be selectively conjugated via the N(e) amine (Scheme 2).
BocLysOMe 8 was converted into trifluoroacetate 9, and the
Boc group was cleaved using TFA. N(a) alkylation of intermedi-
[18]
[Re(CO)3Br3][Et4N]2 in alcohol or water according to previous-
ly described procedures resulted in hydrolysis of the ester
functionalities and gave complexes [Re(CO3)(L)] (L=L2–L6) as
single products (HPLC).[12] All rhenium complexes were fully
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T. L. Mindt, R. Schibli, et al.
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Scheme 2. Synthesis of a Lys-derived, common alkyne precursor for the ex-
tended “click-to-chelate” approach. a) trifluoroacetic acid anhydride, THF,
DIPEA, RT, 5 h; b) CH2Cl2/TFA (3:1), RT, 18 h; c) propargyl bromide (1), THF,
DIPEA, RT, 2 days; d) Boc2O, THF, DIPEA, 558C, 2 days; e) for 13: MeOH/
NH4HCO3(aq) 1m (1:1), 558C, 3 days; f) for 14: MeOH/K2CO3(aq) 0.5m (1:1),
758C, 3 h.
ate 10 with propargyl bromide (1) followed by reaction of the
resulting secondary amine 11 with Boc anhydride yielded com-
pound 12. Installation of the N(a)-Boc group became necessa-
ry, because attempted direct alkylation/acylation of the primary
N(e)-amine in the presence of the N(a) secondary amino group
of Lys derivatives yielded complex mixtures of isomeric prod-
ucts (data not shown). Selective removal of the N(e)-trifluoro-
acetate in the presence of the N(a)-Boc group was achieved
under carefully optimized reaction conditions.[20] Thus, treat-
ment of a solution of intermediate 12 in methanol with
NH4HCO3 provided methyl ester 13, whereas the use of K2CO3
gave intermediate 14. Compounds 13 and 14, each prepared
in five steps and good overall yield, were equally suitable as a
common alkyne precursor for our studies.
Figure 2. ORTEP-3[19] representations of the complexes A) [Re(CO)3(L4)] with
thermal ellipsoids shown at 20% probability, and B) [Re(CO)3(L5)] and
C) [Re(CO)3(L6)] with thermal ellipsoids shown at 50% probability. Only one
out of seven molecules in the asymmetric unit of [Re(CO)3(L4)] is shown. Hy-
drogen atoms and solvent molecules are omitted for clarity. Selected bond
lengths [ꢃ] and angles [8]: [Re(CO)3(L4)] Re(1)–C(3) 1.890(9), Re(1)–C(2)
1.920(8), Re(1)–C(1) 1.920(8), Re(1)–O(5) 2.110(4), Re(1)–N(2) 2.142(5), Re(1)–
N(1) 2.250(5), C(3)–Re(1)–C(2) 87.1(3), C(3)–Re(1)–C(1) 88.6(3), C(2)–Re(1)–C(1)
88.1(3), C(2)–Re(1)–O(5) 176.8(2), C(3)–Re(1)–N(1) 170.0(3), C(1)–Re(1)–N(2)
173.4(3). [Re(CO)3(L5)] Re(1)–C(1) 1.907(4), Re(1)–C(3) 1.912(4), Re(1)–C(2)
1.919(4), Re(1)–O(5) 2.137(3), Re(1)–N(3) 2.156(3), Re(1)–N(4) 2.228(3); C(1)–
Re(1)–C(3) 86.32(17), C(1)–Re(1)–C(2) 92.87(16), C(3)–Re(1)–C(2) 86.72(17),
C(3)–Re(1)–O(5) 174.20(13), C(1)–Re(1)–N(3) 171.59(14), C(2)–Re(1)–N(4)
170.16(13). [Re(CO)3(L6)] Re(1)–C(3) 1.888(3), Re(1)–C(2) 1.912(3), Re(1)–C(1)
1.925(3), Re(1)–O(5) 2.1373(19), Re(1)–N(3) 2.157(2), Re(1)–N(4) 2.243(2); C(3)–
Re(1)–C(2) 88.29(12), C(3)–Re(1)–C(1) 87.00(13), C(2)–Re(1)–C(1) 88.12(13),
C(3)–Re(1)–O(5) 173.46(11), C(1)–Re(1)–N(3) 171.44(10), C(2)–Re(1)–N(4)
171.92(10).
Conjugation of various commercial or synthesized carboxylic
acid derivatives to the N(e) amino group of precursors 13 or
14 by standard amide coupling protocols was straightforward
(Scheme 3; see Experimental Section and the Supporting Infor-
mation for details). After successful coupling, the N(a)-Boc
group of the Lys moiety of intermediates was removed in
quantitative yield by treatment with TFA. In the case of
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
derivative 21, this also resulted in the cleavage of the tert-
butyl ester groups. For compounds 16 and 17, an additional
deprotection step was required to remove TBDMS and the
ethyl ester group, respectively. Thus, starting from either 13 or
14, alkyne substrates 15–22, ready for use in click conjuga-
tions, were obtained in 2–3 synthetic steps and in good yields
(60–90%). The alkyne derivatives described herein are already
interesting for the modification of (bio)molecules by CuAAC.
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Multifunctional 99mTc Radiopharmaceuticals
from precursor 20 could be used
for SPECT and optical (fluores-
cence) imaging whereas those
obtained from DOTA derivative
21 would enable combination
with other metals, such as para-
magnetic gadolinium, yielding a
potential MRI/SPECT probe. The
described approach for the prep-
aration of functionalized alkyne
pro-chelators for bioconjugation
by CuAAC is not limited to the
examples discussed above, but
is also applicable to virtually any
biochemical and chemical entity
of interest as well as combina-
tions thereof.
With the exception of the che-
lating system DOTA, the various
moieties conjugated to the N(a)-
propargyl Lys precursors 13 and
14 did not interfere with the
CuAAC and the subsequent che-
lation step with [M(CO)3]+. Thus,
compounds 15–20 were subject-
ed to CuAAC with benzyl azide
(7) under reaction conditions
similar to those described above
(Scheme 1). In all cases, ligand
systems L15–L20 were obtained
in good yields. Compound 21
was not a suitable substrate for
the copper-catalyzed cycloaddi-
tion because the copper catalyst
was sequestered by the macro-
cyclic chelator DOTA.[29] However,
Scheme 3. Synthesis of N(e)-functionalized, N(a)-propargyl Lys click precursors, CuAAC thereof with model com-
pound benzyl azide (7) and formation of the corresponding 99mTc and Re complexes. a) For 15: 14, NHS-m-dPEG15,
DMSO, RT, 24 h; for 16: 14, 1-O-methyl-b-d-glucuronic acid-2,3,4-tris-TBDMS ether,[16] HBTU, DIPEA, DMF, RT, 2 h;
for 17: 13, (R)-2-succinylamido-6-[4-(4-iodophenyl)butanamido]hexanoic acid ethyl ester,[16] HBTU, DIPEA, DMF, RT,
2 h; for 18: 14, biotin–NHS ester, DMSO, RT, 20 h; for 19: 13, acridine-9-carboxylic acid, HOBT, DCC, DMF, 808C,
18 h; for 20: 14, DY547–NHS ester, DMSO, RT, 18 h; for 21: 13, DOTA(tBu)3–NHS ester, CH2Cl2, reflux, 2 h;
b) CH2Cl2/TFA (4:1, 25 mLmmolꢀ1), RT, 18 h; c) for 16: TBAF, THF, RT, 18 h; for 17: MeOH, aqueous NaOH, RT, 18 h;
d) GdCl3, H2O, 908C, 1.5 h; e) for 15, 16, 18, 20: benzyl azide (7), tBuOH/H2O (1:1), excess Cu(0), RT, 20 h; for 17,
19, 22: benzyl azide (7), tBuOH/H2O (1:1), 20 mol% Cu(OAc)2, 40 mol% sodium ascorbate, RT, 18 h; f) for M=Re:
[Re(CO)3Br3][Et4N]2, alcohol or H2O, 608C, 0.5–4 h; for M=99mTc : [99mTc(CO)3(H2O)3]+, PBS pH ~7, 1008C, 30 min.
However, only when labeled with 99mTc is the in vivo tracking
and quantification of the conjugate possible.
CuAAC of the pre-formed[30,31] Gd3+ complex 22 provided the
desired product L22 cleanly, with quantitative conversion of
the substrates.[32] Metal labeling of the ligands L15, L20, and
L22 with [M(CO)3]+ (M=99mTc, Re) was performed according to
the procedure described for the amino acid model com-
pounds. Complexes [99mTc(CO)3(L)] (L=L15–L20 and L22) were
obtained as single products (HPLC), the identities of which
were again confirmed by HPLC comparison with the corre-
sponding fully characterized rhenium analogues [Re(CO)3(L)]
(L=L15–L19 and L22),[16] with the exception of [Re(CO)3(L20)]+,
which was available only in very small amounts. In addition,
we were able to show that our previously reported one-pot,
two-step protocol[9,13] can be applied not only to simple alkyne
precursors of ligand systems L2–L6 but also to functionalized
propargyl substrates 15, 18, and 20 (Figure 3). For example,
treatment of aliquots of the crude reaction solutions obtained
from the CuAAC of benzyl azide (7) and alkynes 15, 18, or 20
Compounds 15–17 contain various pharmacological modifi-
ers. It has been shown that PEGylation or glycosylation of pep-
tide-based radiotracers can improve their characteristics
in vivo.[21–23] Thus, derivatives 15 (PEG15) and 16 (glucuronic
acid) could be generally useful for the attachment of polar
modifiers to 99mTc(CO)3-based radiopharmaceuticals. Substruc-
ture 17 contains a recently published example of a small or-
ganic albumin-binding molecule (C) useful for prolonging the
circulation time of conjugates in the bloodstream (see
below).[24,25] Derivatives 18 and 19 carry biotin, an affinity tag
applicable for sequential bioconjugations (e.g., pre-targeting
approaches[26]) and acridine, a DNA intercalator, respectively.
Application of the latter to radiopharmaceuticals has been sug-
gested for the delivery of short-range Auger-electron-emitting
radioisotopes to the cell nucleus to enhance their therapeutic
efficacy.[27,28] Finally, alkyne precursors 20 and 21 functionalized
with an organic dye (DY547) or the macrocyclic chelator DOTA,
respectively, represent examples of substrates for the develop-
ment of dual-modality imaging agents. Conjugates derived
with
99mTc(CO)3(L)] (L=L15, L18, L20) directly in high purity (>
[
99mTc(CO)3(H2O)3]+ yielded final radioconjugates
[
90%), identical to those obtained with pre-synthesized and pu-
rified ligands. These examples represent unprecedentedly effi-
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T. L. Mindt, R. Schibli, et al.
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Figure 3. One-pot, two-step procedure (bottom) yields 99mTc-labeled conju-
gates, which are identical to those prepared by multistep synthesis involving
isolation and purification of intermediates (top).
cient examples of the combination a radionuclide chelate with
two different moieties without the use of protecting groups.
In vitro and in vivo evaluation
We previously reported a series of 99mTc-labeled derivatives of
bombesin (BBS),[22,33] a regulatory peptide that targets the gas-
trin-releasing peptide (GRP) receptor, which is overexpressed in
various tumor types such as prostate, breast, and small-cell
lung cancer.[34] Like other radiolabeled regulatory peptides po-
tentially useful as imaging probes for the detection of tumors
and metastasis,[35] radiotracers derived from BBS or its binding
sequence BBS(7–14) are rapidly degraded both extracellularly
and intracellularly by peptidases. The fast clearance of radiola-
beled peptides from the blood pool is generally appreciated
for diagnostic purposes. However, it also limits tumor uptake,
and thus may represent a potential limitation for their use as
therapeutic radiopharmaceuticals. Common strategies to
extend the serum half-life of peptide-based radiotracers in vivo
include PEGylation, the formation of multimers or structural
modifications for stabilization against enzymatic degrada-
tion.[23,36,37] Alternative approaches make use of albumin-bind-
ing molecules.[24,38] By noncovalent interaction with serum al-
bumin, this class of compounds has been shown to prolong
the in vivo circulatory half-life of conjugated probes (e.g., fluo-
rophores and MRI contrast agents) as well as antibody frag-
ments.[24,25] We reasoned that the fusion of a low-molecular-
weight albumin binder (Kd: micromolar range) to BBS(7–14) ra-
dioconjugates would result in a prolonged circulation time in
the blood without compromising the targeting capacity of the
high-affinity peptide (Kd: nanomolar range). In addition, associ-
ation of the radioconjugate to albumin could provide some
protection against enzymatic degradation.
Figure 4. Syntheses of a radiolabeled BBS(7–14) derivative A) functionalized
with an albumin binding moiety by click chemistry and B) reference com-
pound NaHisAc-BBS(7–14). a) EtOH/H2O (1:1), 10 mol% Cu(OAc)2, 20 mol%
sodium ascorbate, 608C, 4 h; b) 0.1 mm peptide in PBS (pH 6.5),
[
99mTc(CO)3]+ (200—500 MBq), 1 h at 758C. (Cha=Cyclohexylalanine, Nle=
Norleucine).
formation of a single radioactive species was confirmed in
each case by g-HPLC (radiochemical yield and purity >90%).
[
99mTc(CO)3(L24)] was assessed in vitro for its binding to albu-
min and GRP-receptor-overexpressing PC3 cells.[16] In brief, in-
cubation of [99mTc(CO)3(L24)] in mouse blood serum at 378C
for 15 min and analysis of samples by size-exclusion chroma-
tography indicated quantitative binding of the radioconjugate
to mouse serum albumin. Binding of [99mTc(CO)3(L24)] to intact
PC3 cells was also examined. Approximately 6% of total radio-
activity added was found bound to the cells after 1 h at 378C.
The binding could be blocked by the presence of excess
BBS(7–14) (1 mm), hence proving receptor specificity.
To study the effect of the albumin binder conjugated to ra-
diolabeled BBS derivatives in vivo, time-dependant biodistribu-
tion studies were performed with [99mTc(CO)3(L24)] and refer-
ence compound [99mTc(CO)3(NaHisAc-BBS(7–14))] in nude mice
bearing PC3 tumor xenografts.[16] Figure 5 shows selected data
of the biodistribution of the two radiotracers at 1 and 24 h
post injection (p.i.). Clearly, blood clearance was much slower
for [99mTc(CO)3(L24)] (14 and 6% of injected dose per gram
Stabilized BBS(7–14) derivative 23, containing a “clickable”
azide functionality attached to its N terminus, was prepared by
standard solid-phase peptide synthesis (SPPS) employing Fmoc
strategies in analogy to reported procedures (Figure 4).[12,33]
Alkyne 17 equipped with the albumin binding molecule C
(Scheme 3) was conjugated to azidopeptide 23 by CuAAC in a
mixture of water and ethanol at 608C to yield BBS(7–14) deriv-
ative L24. Reference compound NaHisAc-BBS(7–14), identical in
all respects but lacking the albumin binding moiety and func-
tionalized with the established N(a)-HisAc chelating system,
was prepared by SPPS.[39] Both peptides were readily radiola-
beled with [99mTc(CO)3]+ in PBS (pH 6.5) for 1 h at 758C. The
(IDgꢀ1) after
99mTc(CO)3(NaHisAc-BBS(7–14))] (0.23% at 1 h p.i., and com-
plete clearance after 24 h). The longer circulation time of
99mTc(CO)3(L24)] is likely the result of its binding to serum al-
1
and 24 h p.i., respectively) than for
[
[
bumin, which is also consistent with the higher uptake ob-
served in most organs and tissue 24 h p.i., in particular in the
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Multifunctional 99mTc Radiopharmaceuticals
tive, targeted radionuclide conjugates containing
pharmacological modifiers, affinity tags, or a second
imaging probe can be assembled in a few synthetic
steps with high yields using convenient and efficient
one-pot procedures. The extended “click-to-chelate”
approach was successfully applied to the preparation
of
a
novel 99mTc-labeled bombesin derivative
equipped with a low-molecular-weight albumin-bind-
ing molecule to extend the biological half-life of the
radioconjugate. Evaluation of the compound in vitro
and in vivo provided encouraging results in terms of
prolonged circulation time in the bloodstream as
well as receptor-specific targeting of GRP-receptor
positive tumors. The methodology presented herein
provides an attractive synthetic tool for the prepara-
tion of multifunctional 99mTc-based radiopharmaceuti-
cals with high potential for a multitude of applica-
tions.
Figure 5. Comparison of the biodistribution of [99mTc(CO)3(L24)] and [99mTc(CO)3(NaHisAc-
BBS(7–14))] in nude mice bearing PC3 xenografts 1 h and 24 h p.i. (n=3–4).
well-perfused heart, spleen, lung, liver, and kidney.[40] Initially,
Experimental Section
reference compound [99mTc(CO)3(NaHisAc-BBS(7–14))] was taken
up to a higher extent by GRP-receptor-positive tumors, colon,
and pancreas (1 h p.i.); however, accumulation of radioactivity
was significantly higher for [99mTc(CO)3(L24)] after 24 h. Biodis-
tribution studies performed after co-injection of natural bomb-
esin (0.1 mg per mouse; blocking experiments) revealed a sig-
nificant attenuation of the radioactivity in receptor-positive
colon and pancreas, while the uptake in non-targeted tissue
and organs (e.g., blood, liver, and kidney) remained un-
changed. These results confirm the specific, GRP-receptor-
mediated uptake of the radiotracer (see the Supporting Infor-
mation for details).
Only key intermediates and final products are described here. For
detailed information on experimental procedures, including gener-
al methods, equipment, solid-phase peptide synthesis (SPPS), in vi-
tro and in vivo experiments, and analytical data for all compounds
(1H and 13C NMR spectra, HPLC chromatograms of natRe and 99mTc
complexes), see the Supporting Information; CIF files of X-ray
structures are available at the CCDC (see below).
CAUTION: 99mTc is a g-emitter (140 keV) with a half-life of 6.01 h.
All reactions involving 99mTc were performed in a laboratory ap-
proved for the handling of radioisotopes, and appropriate safety
procedures were followed at all times to prevent contamination.
While the attachment of a low-molecular-weight albumin
binder to radiolabeled BBS derivatives resulted in the desired
prolongation of the circulation time of the conjugate in vivo,
the tumor-to-background ratio is not yet suitable for nuclear
imaging and therapy due to high background radioactivity. We
speculate that the interaction of the radioconjugate with albu-
min may be too strong, or alternatively, may restrict the acces-
sibility of the BBS moiety, both of which could influence the
binding to GRP receptors. Alternatively, the radiopeptide
bound to the albumin could be partly enzymatically degraded;
this could explain the observed high level of radioactivity cir-
culating in the blood after 24 h without an increase in uptake
by tumor tissue. Investigations employing analogues of the al-
bumin binder moiety C (Scheme 3) of lower affinity,[24] the use
of extended linkers between the different functional moieties
of the radioconjugate, and applications to other tumor-seeking
vectors are currently ongoing.
Chemical syntheses
General procedure A: Synthesis of N(a)-propargyl amino acid
derivatives. Method A1: Amino acid substrates were dissolved in
MeOH (4 mLmmolꢀ1) and K2CO3 (3 equiv), and propargyl bromide
(1; 80% in toluene, 2 equiv) was added. The mixture was stirred at
RT for 2 days, filtered and concentrated under reduced pressure.
The crude products were purified by flash chromatography on
silica gel. Method A2: Amino acid substrates were dissolved in THF
(15 mLmmolꢀ1) and DIPEA (2.5 equiv), and propargyl bromide (1;
80% in toluene, 2 equiv) was added. The mixture was stirred at
508C for 1–2 days and concentrated under reduced pressure. The
crude products were purified by flash chromatography on silica
gel.
General procedure B: CuAAC of alkynes with benzyl azide (7).
Method B1: N(a)-propargyl amino acid derivatives were dissolved
in tBuOH/H2O (1:1, 20–50 mLmmolꢀ1) and Cu(OAc)2 (0.1–0,2 equiv),
sodium ascorbate (0.2–0.4 equiv) and benzyl azide (7, 1.1 equiv)
were added. The mixture was stirred at RT for 18 h, diluted with
brine and extracted with EtOAc. The organic extracts were washed
with brine, dried over Na2SO4 and concentrated under reduced
pressure. The crude products were purified by semi-preparative
HPLC or flash chromatography on silica gel. Method B2: N(a)-prop-
argyl amino acid derivatives were dissolved in tBuOH/H2O (1:1, 50–
75 mLmmolꢀ1) and excess metallic Cu(0) (~40 mgmmolꢀ1) and
benzyl azide (7, 1.1 equiv) were added. The mixture was stirred at
RT for 20 h, filtered and concentrated under reduced pressure. The
Conclusions
In summary, we have shown that CuAAC can be used to com-
bine a (radio)metal chelating system, a targeting (bio)molecule,
and various secondary entities in a single step. Starting from
azide-functionalized (bio)molecules and suitable alkyne pro-
chelators derived from a common N(a)-propargyl Lys deriva-
ChemMedChem 2010, 5, 2026 – 2038
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2031

T. L. Mindt, R. Schibli, et al.
MED
crude products were purified by semi-preparative HPLC or flash
chromatography on silica gel.
N(a)-propargyl ValOMe (3). As per general procedure A1 from
HValOMe (HCl salt; 336 mg, 2.0 mmol) and purification by flash
chromatography with CH2Cl2/MeOH (25:1). Yellow oil (223 mg,
General procedure C: Removal of Boc protecting groups. Where
appropriate, the Boc group was cleaved by stirring intermediates
in CH2Cl2/TFA (4:1, ~50 mLmmolꢀ1) at RT for 18 h. Removal of the
volatile components under reduced pressure yielded the corre-
sponding amine products quantitatively as their TFA salts.
67%): IR (neat): n˜ =3311, 2958, 1727, 1465, 1201, 1144, 994 cmꢀ1
;
1H NMR (CDCl3): d=3.69 (s, 3H), 3.40 and 3.31 (each dd, each 1H,
J=16.7 and 2.6 Hz), 3.14 (d, 1H, J=5.8 Hz), 2.15 (t, 1H, J=2.6 Hz),
1.96–1.85 (m, 1H), 1.70 (bs, 1H), 0.91 and 0.89 (each d, each 3H,
J=6.7 Hz); 13C NMR (CDCl3): d=175.2, 81.8, 71.6, 66.2, 51.7, 37.7,
31.8, 19.3, 18.6 ppm; HRMS: [M+Na]+ =192.0966 (calcd for
C9H15NO2Na: 192.1001).
General procedure D: Synthesis of rhenium complexes.
[Re(CO)3Br3][Et4N]2 was prepared according to published proce-
dures. 1,2,3-Triazoles were dissolved in water, alcohol or mixtures
thereof (1:1; ~80 mLmmolꢀ1) and [Re(CO)3Br3][Et4N]2 (1 equiv) was
added. The solution was adjusted to pH ~7 by the addition of
dilute aqueous Et4NOH (~0.5%). The mixture was then stirred at
50–758C until HPLC analysis indicated complete conversion of the
substrates (0.5–4 h). The solution was concentrated under reduced
pressure, and the crude products were purified by semi-prepara-
tive HPLC or by using C18 Sep-Pakꢄ columns and mixtures of water
and methanol (0!50% MeOH). Occasionally, carbonyl ligands of
the complexes exhibited weak signals in the 13C NMR spectra. How-
ever, the presence of fac-Re(CO)3 could be confirmed in each case
by its characteristic and strong IR absorptions.[18]
N(a)-propargyl ProOMe (4). As per general procedure A1 from
HProOMe (HCl salt; 331 mg, 2.0 mmol) and purification by flash
chromatography with EtOAc/hexane (3:1). Yellow oil (250 mg,
75%): IR (neat): n˜ =3282, 2953, 2818, 1741, 1436, 1278, 1200, 1173,
1
1130, 905, 669 cmꢀ1; H NMR (CDCl3): d=3.70 (s, 3H), 3.57 (t, 2H,
J=2.5 Hz), 3.41 (dd, 1H, J=8.8 and 6.6 Hz), 3.06–3.00 (m, 1H),
2.73–2.67 (m, 1H), 2.17 (t, 1H, J=2.5 Hz), 2.16–2.07 (m, 1H), 2.00–
1.84 (m, 2H), 1.83–1.73 (m, 1H); 13C NMR (CDCl3): d=174.3, 78.6,
73.4, 62.8, 52.5, 52.2, 41.4, 29.8, 23.5 ppm; HRMS: [M+H]+
=
168.1019 (calcd for C9H14NO2: 168.1025).
N(a)-propargyl Gln(Et)OBn (5). As per general procedure A2 from
HGln(Et)OBn[16] ((90 mg, 0.34 mmol)) and purification by flash chro-
matography with CH2Cl2/MeOH (30:1). Brown oil (56 mg, 54%): IR
(neat): n˜ =3293, 2920, 1729, 1643, 1453, 1253, 1168, 964, 733, 697
General procedure F: Synthesis of 99mTc complexes. [Na][99mTcO4]
was eluted from a 99Mo/99mTc generator (Mallinckrodt-Tyco, Petten,
The Netherlands) with a 0.9% saline solution. The precursor
cmꢀ1 1H NMR (CDCl3): d=7.38–7.27 (m, 5H), 5.68 (bs, 1H), 5.16
;
[
99mTc(CO)3(H2O)3]+ was prepared according to published proce-
and 5.12 (each d, each 1H, J=12.0 Hz), 3.47–3.42 (m, 1H), 3.42 and
3.33 (each dd, each 1H, J=16.8 and 2.3 Hz), 3.21 (quint., 2H, J=
7.4 Hz), 2.26–2.15 (m, 2H), 2.12 (t, 1H, J=2.3 Hz), 1.92–1.80 (m,
3H), 1.07 (t, 3H, J=7.3 Hz); 13C NMR (CDCl3): d=174.4, 172.1,
135.7, 128.8, 128.6, 128.5, 81.7, 72.0, 66.9, 59.5, 37.0, 34.5, 32.8,
28.8, 15.0 ppm; HRMS: [M+Na]+ =325.1521 (calcd for
C17H22N2O3Na: 325.1528).
dures.[41] In brief, 1 mL [99mTcO4]ꢀ in 0.9% NaCl was added to the
IsoLinkTM kit (Mallinckrodt-Tyco, Petten, The Netherlands) via the
septum. The reaction was heated for 20 min at 1008C. The solution
was cooled and the adjusted to pH ~7 with a 1:1 mixture of 1m
phosphate buffer (pH 7.4) and 1m HCl. Aliquots of stock solutions
of the 1,2,3-triazole ligand systems in water (50 mL, 10ꢀ3–10ꢀ5 m)
were added to
a
solution of [
99mTc(CO)3(H2O)3]+ (100 mL;
~1 GBqmLꢀ1) and phosphate-buffered saline (PBS; 350 mL, pH 7.2)
N(a)-propargyl Lys(Ac)OMe (6). As per general procedure A2 from
HLys(Ac)OMe[16] (TFA salt; 144 mg, 0.44 mmol) and purification by
flash chromatography with CH2Cl2/MeOH (15:1). Yellow oil (100 mg,
73%): IR (neat): n˜ =3285, 2941, 1731, 1645, 1546, 1433, 1367, 1288,
was added to adjust the final concentration of the chelator (10ꢀ4
–
10ꢀ6 m). The reaction mixture was heated at 1008C for 30 min and
radiolabeling yields were determined by HPLC.
1
1198, 1171, 1139, 990 cmꢀ1; H NMR (CDCl3): d=6.20 (bs, 1H), 3.59
One-pot synthesis of 99mTc complexes. Aliquots (50 mL) of the fil-
tered solution of crude ligands (L15, L18, L20; ~15 mm in tBuOH/
H2O 1:1) were diluted with PBS (350 mL, pH 7) and [99mTc(CO)3-
(H2O)3]+ (100 mL; ~1 GBqmLꢀ1) was added. The reaction mixture
was heated at 1008C for 30 min and product formation was con-
firmed by g-HPLC.
(s, 3H), 3.32–3.19 (m, 3H), 3.07 (q, 2H, J=6.8 Hz), 2.11 (t, 1H, J=
2.2 Hz), 1.82 (s, 3H), 1.73 (bs, 1H), 1.60–1.43 (m, 2H), 1.42–1.33 (m,
2H), 1.31–1.20 (m, 2H); 13C NMR (CDCl3): d=175.1, 170.2, 81.3,
71.7, 59.7, 51.8, 39.2, 36.8, 32.7, 29.1, 23.1, 22.9 ppm; HRMS:
[M+Na]+ =263.1365 (calcd for C12H20N2O3Na: 263.1372).
Ligand L3. As per general procedure B1 from N(a)-propargyl
ValOMe (3; 40 mg, 0.24 mmol) and purification by flash chromatog-
raphy with EtOAc/hexane (3:1). Colorless oil (52 mg, 71%): IR
Solid-phase peptide synthesis (SPPS). The synthesis of peptides
23 and NaHisAc-BBS(7–14) was carried out on solid phase on a
Rink amide polystyrene resin (0.60 mmolgꢀ1) using standard Fmoc
strategy and procedures previously described.[12,16] After cleavage
from the resin and deprotection, the peptides were purified by
preparative HPLC and analyzed by LC–MS.
1
(neat): n˜ =2955, 1731, 1459, 1198, 1047, 721 cmꢀ1; H NMR (CDCl3):
d=7.32–7.26 (m, 3H), 7.20–7.17 (m, 2H), 5.43 (s, 2H), 3.85 and 3.66
(each d, each 1H, J=13.7 Hz), 3.61 (s, 3H), 2.98 (d, 2H, J=5.8 Hz),
1.88–1.80 (m, 2H), 0.83 and 0.81 (each d, each 3H, J=6.2 Hz);
1H NMR (CDCl3): d=175.4, 147.5, 134.9, 129.2, 128.8, 128.2, 127.8,
Peptide 99mTc labeling. Solutions of peptides L24 and NaHisAc-
BBS(7–14) (30–50 mL; 1 mm in PBS, pH ~6.5) were mixed with
500 mL of the [99mTc(CO)3(H2O)3]+ solution and heated at 758C for
1 h, after which the radiolabeled products were analyzed and puri-
fied prior to in vitro and in vivo experiments by HPLC.
66.8, 54.2, 51.6, 44.0, 31.7, 19.3, 18.7 ppm; HRMS: [M+Na]+
=
325.1633 (calcd for C16H22N4O2Na: 325.1640).
Ligand L4. As per general procedure B1 from N(a)-propargyl
ProOMe (4; 170 mg, 1.0 mmol) and purification by flash chroma-
tography with EtOAc/EtOH (25:1). Brown oil (285 mg, 95%): IR
(neat): n˜ =2955, 2826, 1735, 1455, 1433, 1205, 1186, 1126, 1047,
1
725 cmꢀ1; H NMR (CDCl3): d=7.36 (s, 1H), 7.32–7.26 (m, 3H), 7.21–
Characterization of compounds
7.18 (m, 2H), 5.45 and 5.40 (each d, each 1H, J=14.9 Hz), 3.91 and
3.73 (each d, each 1H, J=13.6 Hz), 3.56 (s, 3H), 3.22 (dd, 1H J=8.9
and 6.3 Hz), 3.06–3.00 (m, 1H), 2.47–2.41 (m, 1H), 2.09–1.99 (m,
1H), 1.91–1.64 (m, 3H); 13C NMR (CDCl3): d=174.5, 145.1, 134.7,
N(a)-propargyl GlyOMe (2).[13] As per general procedure A1 from
HGlyOMe (HCl salt; 250 mg, 2 mmol) and purification by flash chro-
matography with EtOAc/hexane (1:4). Yellow oil (235 mg, 72%).
2032
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ChemMedChem 2010, 5, 2026 – 2038

Multifunctional 99mTc Radiopharmaceuticals
129.1, 128.7, 128.1, 122.6, 64.9, 54.1, 53.4, 51.8, 48.8, 23.0,
14.2 ppm; HRMS: [M+H]+ =323.1477 (calcd for C16H20N4O2Na:
323.1484).
each 1H, J=15.7 Hz), 3.30–3.25 (m, 1H; overlapping with MeOD
signal; confirmed by COSY), 3.25–3.18 (m, 2H), 3.17–3.14 (m, 1H),
2.53–2.41 (m, 2H), 1.19–2.02 (m, 2H), 1.12 (t, 3H, J=7.3 Hz);
13C NMR (MeOD): d=198.3, 196.8, 196.6, 185.8, 174.8, 149.8, 135.5,
130.4, 130.2, 129.6, 124.4, 67.7, 56.5, 53.7, 35.5, 33.7, 29.8,
14.8 ppm; HRMS: [M+Na]+ =638.1019 (calcd for C20H22N5O6ReNa:
638.1025). Crystals suitable for X-ray analysis were obtained by
slow evaporation of a solution of [Re(CO)3(L5)] in CH2Cl2.
Ligand L5. As per general procedure B2 from N(a)-propargyl
Gln(Et)OBn (5; 25 mg, 0.08 mmol) and purification by flash chroma-
tography with CH2Cl2/MeOH (25:1). White solid (25 mg, 71%): mp
79–818C; IR (neat): n˜ =3301, 2970, 2934, 1731, 1652, 1543, 1453,
1
1213, 1167, 1050, 724, 699 cmꢀ1; H NMR (CDCl3): d=7.42–7.25 (m,
11H), 5.88 (bs, 1H), 5.49 (s, 2H), 5.18 and 5.15 (each d, each 2H,
J=12.2 Hz), 3.94 and 3.74 (each d, each 1H, J=13.6 Hz), 3.36 (dd,
1H, J=8.0 and 5.2 Hz), 3.26–3.14 (m, 2H), 2.31–2.13 (m, 2H), 2.11–
2.01 (m, 1H), 1.97 (bs, 1H), 1.93–1.83 (m, 1H), 1.07 (t, 3H, J=
7.3 Hz); 13C NMR (CDCl3) d174.6, 172.2, 147.0, 135.8, 134.8, 129.3,
128.9, 128.8, 128.6, 128.5, 128.3, 121.8, 66.9, 60.2, 54.3, 43.2, 34.4,
33.0, 29.0, 15.0 ppm; HRMS: [M+H]+ =436.2343 (calcd for
C24H30N5O3: 436.2349).
[Re(CO)3(L6)]. As per general procedure D from ligand L6 (25 mg,
0.07 mmol) in methanol/water (1:1) and purification by trituration
in water (twice). White solid (35 mg, 83%; purity according to
HPLC>95%): mp 138–1428C; [a]2D0 =+19.8 (c=1 in MeOH); IR
(neat): n˜ =3165, 2939, 2022 and 1885 (strong), 1631, 1454, 1366,
1
722 cmꢀ1; H NMR (MeOD): d=8.10 (s, 1H), 7.45–7.32 (m, 5H), 6.63
(bd, 1H, J=5.0 Hz; exchanges with MeOD after 18 h), 5.67 (s, 2H),
4.35–4.19 (m, 2H; becomes 2 doublets (4.30 and 4.22, each 1H, J=
15.0 Hz) after completed H/D exchange), 3.31–3.24 (m, 2H), 3.13 (t,
1H, J=6.4 Hz), 1.94 (s, 3H), 1.91–1.82 (m, 2H), 1.60–1.52 (m, 4H);
13C NMR (MeOD): d=198.5, 196.8, 195.6, 186.3, 173.5, 149.6, 135.5,
130.5, 130.2, 129.6, 124.5, 67.7, 56.5, 53.8, 40.2, 33.9, 30.2, 24.5,
22.6 ppm; HRMS: [M+Na]+ =652.1181 (calcd for C21H24N5O6ReNa:
652.1182). Crystals suitable for X-ray analysis were obtained by
slow diffusion of CH2Cl2 into a solution of [Re(CO)3(L6)] in MeOH.
Ligand L6. As per general procedure B2 from N(a)-propargyl Ly-
s(Ac)OMe (6; 80 mg, 0.3 mmol) and purification by flash chroma-
tography with CH2Cl2/MeOH (15:1). Yellow oil (100 mg, 81%): IR
(neat): n˜ =3294, 2932, 1731, 1652, 1548, 1453, 1433, 1286, 1200,
1
1169, 1137, 1047, 719 cmꢀ1; H NMR (CDCl3): d=7.41–7.35 (m, 4H),
7.31–7.26 (m, 2H), 5.72 (bs, 1H), 5.52 (s, 2H), 3.92 and 3.76 (each d,
each 1H, J=14.0 Hz), 3.71 (s, 3H), 3.31 (t, 1H, J=6.4 Hz), 3.22 (q,
2H, J=6.5 Hz), 1.97 (s, 3H), 1.93 (bs, 1H), 1.74–1.55 (m, 2H), 1.52–
1.43 (m, 2H), 1.42–1.32 (m, 2H); 13C NMR (CDCl3): d=175.4, 170.1,
146.9, 134.7, 129.1, 128.7, 128.1, 121.6, 60.5, 54.1, 51.8, 43.2, 39.3,
32.7, 29.1, 23.3, 22.9 ppm; HRMS: [M+H]+ =374.2188 (calcd for
C19H28N5O3: 374.2192).
Intermediate 9. To a solution of BocLysOMe·AcOH (8; 942 mg,
68%, 2.0 mmol) in THF (15 mL) and DIPEA (1.4 mL, 8.0 mmol) was
added dropwise at 08C trifluoroacetic acid (TFA) anhydride (834 mL,
6.0 mmol). The solution was warmed to RT and stirred for 5 h. The
mixture was diluted with water and extracted with EtOAc. The or-
ganic layers were washed with water and then combined, dried
over Na2SO4 and concentrated under reduced pressure. Purification
of the crude product by flash chromatography on silica gel with
EtOAc/hexane (1:1.5) gave product 9 as a yellow oil (0.7 g, 98%):
[a]2D0 =ꢀ15.2 (c=1 in MeOH); IR (neat): n˜ =3324, 2943, 1706, 1518,
[Re(CO)3(L3)]. As per general procedure D from ligand L3 (7 mg,
0.03 mmol) in ethanol and purification by Sep-Pakꢄ (1 g). White
solid (20 mg, 83%; purity according to HPLC>95%): mp>2408C
(dec.); [a]2D0 =+21.3 (c=1 in MeOH); IR (neat): n˜ =2963, 2019 and
1
1892 (strong), 1630, 1047, 1022, 932 cmꢀ1; H NMR ([D4]MeOH): d=
1
1367, 1206, 1161, 720 cmꢀ1; H NMR (CDCl3): d=6.67 (bs, 1H), 5.08
8.10 (s, 1H), 7.43–7.34 (m, 5H), 6.42 (bs, 1H), 5.71 and 5.67 (each d,
each 1H, J=14.9 Hz), 4.84–4.80 (shoulder of HDO signal, m, 1H),
4.29–4.21 (m, 2H), 2.34–2.26 (m, 1H), 1.16 and 1.11 (each d, each
3H, each J=7.1 Hz); 13C NMR ([D4]MeOH): d=196.9, 195.4, 195.2,
184.5, 148.0, 134.0, 128.9, 128.7, 128.8, 123.0, 70.6, 55.0, 53.2, 31.6,
17.8, 17.2 ppm; HRMS: [M+Na]+ =581.0814 (calcd for
C18H19N4O5ReNa: 581.0811).
(bd, 1H, J=8.5 Hz), 4.30–4.21 (m, 1H), 3.71 (s, 3H), 3.33 (q, 2H, J=
6.7 Hz), 1.85–1.74 (m, 1H), 1.67–1.55 (m, 3H), 1.45–1.31 (m, 2H),
1.41 (s, 9H); 13C NMR (CDCl3): d=173.3, 157.6 (q, J=38.3 Hz), 155.7,
116.1 (q, J=287.3 Hz), 80.3, 53.2, 52.6, 39.8, 32.8, 28.5, 28.4,
22.7 ppm; HRMS: [M+Na]+ =379.1453 (calcd for C14H23N2O5F3Na:
379.1457).
Intermediate 10. As per general procedure C from intermediate 9
(680 mg, 1.9 mmol). Yellow oil (700 mg, quantitative): [a]2D0 =+10.4
(c=1 in MeOH); IR (neat): n˜ =3088, 2952, 1701, 1670, 1557, 1441,
[Re(CO)3(L4)]. As per general procedure D from ligand L4 (28 mg,
0.1 mmol) in ethanol and purification by Sep-Pakꢄ (1 g). White
solid (30 mg, 55%; purity according to HPLC >95%): mp>2408C
(dec.); [a]2D0 =+8.2 (c=1 in MeOH); IR (neat): n˜ =2955, 2021 and
1
1183, 1156, 1132, 722 cmꢀ1; H NMR ([D4]MeOH with TMS): d=4.00
1877 (strong), 1638, 1355 cmꢀ1 1H NMR (CDCl3): d=7.72 (s, 1H),
;
(t, 1H, J=6.8 Hz), 3.80 (s, 3H), 3.30–3.25 (m, 2H, obscured by the
MeOD signal), 1.99–1.85 (m, 2H), 1.59 (quint., 2H, J=7.5 Hz), 1.52–
1.32 (m, 2H); 13C NMR ([D4]MeOH with TMS): d=170.9, 161.6 and
159.2 (each q, each J=37.0 Hz), 117.6 and 116.6 (each q, each J=
7.44–7.36 (m, 5H), 5.75 and 5.58 (each d, each 1H, J=15.2 Hz),
4.14 and 4.09 (each d, each 1H, J=14.7 Hz), 3.99–3.95 (m, 1H),
3.41–3.30 (m, 1H), 3.27 (dd, 1H, J=10.6 and 6.6 Hz), 2.48–2.40 (m,
1H), 2.35–2.26 (m, 1H), 2.21–2.15 (m, 1H), 2.10–2.03 (m, 1H);
13C NMR (CDCl3): d=197.5, 196.7, 195.5, 183.3, 146.8, 133.3, 129.6,
129.5, 128.8, 123.5, 71.0, 68.3, 60.6, 56.3, 31.8, 24.7 ppm; HRMS:
[M+Na]+ =579.0652 (calcd for C18H20N4O5ReNa: 579.0654). Crystals
suitable for X-ray analysis were obtained by slow diffusion of
hexane into a solution of [Re(CO)3(L4)] in THF.
287.0 Hz), 53.8, 53.6, 40.1, 31.1, 29.3, 23.1 ppm; HRMS: [M+H]+
=
257.1110 (calcd for C9H16N2O3F3: 257.1113).
Intermediate 11. As per general procedure A2 from intermediate
10 (TFA salt; 667 mg, 1.8 mmol) at RT for 2 days and purification
by flash chromatography with EtOAc/hexane (1:1). Yellow oil
(360 mg, 68%): [a]2D0 =ꢀ10.0 (c=1 in MeOH); IR (neat): n˜ =3306,
[Re(CO)3(L5)]. As per general procedure D from ligand L5 (15 mg,
0.03 mmol) in ethanol/water (5:1) and purification by Sep-Pakꢄ
(2 g). White solid (13 mg, 62%; purity according to HPLC>94%):
mp>2008C (dec.); [a]2D0 =+15.9 (c=0.5 in MeOH); IR (neat): n˜ =
2943, 1706, 1554, 1437, 1201, 1172, 1156, 722 cmꢀ1 1H NMR
;
(CDCl3): d=6.56 (bs, 1H), 3.72 (s, 3H), 3.46–3.31 (m, 5H), 2.18 (t,
1H, J=2.5 Hz), 1.74 (bs, 1H), 1.72–1.64 (m, 1H), 1.61–1.54 (m, 3H),
1.46–1.36 (m, 2H); 13C NMR (CDCl3): d=175.3, 157.5 (q, J=38.2 Hz),
116.1 (q, J=287.9 Hz), 81.4, 72.1, 59.9, 52.2, 39.8, 37.2, 32.5, 28.6,
22.9 ppm; HRMS: [M+H]+ =295.1263 (calcd for C12H18N2O3F3:
295.1270).
3306, 2925, 2013 and 1889 (strong), 1640, 1452, 1353, 1045 cmꢀ1
;
1H NMR (MeOD; recorded after amide proton has exchanged): d=
8.09 (s, 1H), 7.42–7.34 (m, 5H), 5.68 (s, 2H), 4.33 and 4.24 (each d,
ChemMedChem 2010, 5, 2026 – 2038
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2033

T. L. Mindt, R. Schibli, et al.
MED
Intermediate 12. Intermediate 11 (295 mg, 1.0 mmol) was dis-
solved in THF (10 mL) and DIPEA (420 mL, 2.5 mmol) and BOC2O
(327 mg, 1.5 mmol) was added. The solution was stirred at 558C
for 2 days and concentrated under reduced pressure. Purification
of the crude product by flash chromatography on silica gel with
EtOAc/hexane (1:2) gave compound 12 as a yellow oil (382 mg,
97%): [a]2D0 =ꢀ34.1 (c=1 in MeOH); IR (neat): n˜ =3306, 2949, 1706,
was deprotected according to procedure C to yield compound 15
as a colorless oil (60 mg, quantitative; purity according to HPLC>
95%): HRMS-ESI: [M+Na]+ =953.5400 (calcd for C43H82N2O19Na:
953.5409); NMR spectra were inconclusive presumably because of
micelle formation.
Compound 16. TBDMS-protected glucuronic acid[16] (100 mg,
0.18 mmol) was dissolved in DMF (3 mL) and HBTU (65 mg,
0.17 mmol) and DIPEA (65 mL, 0.36 mmol) were added. The solution
was stirred at RT for 20 min and then added dropwise at RT to a
suspension of precursor 14 (TFA salt; 72 mg, 0.18 mmol) in DMF
(3 mL) containing DIPEA (140 mL, 0.72 mmol). The mixture was
stirred at RT for 2 h and the resulting solution was diluted with
aqueous citric acid (1m, pH 3–4) and extracted with EtOAc. The or-
ganic phases were washed with aqueous citric acid (0.1m) and
brine and then combined, dried over Na2SO4 and concentrated
under reduced pressure. Purification of the crude product by flash
chromatography on silica gel with CH2Cl2/MeOH gave the fully pro-
tected intermediate as a white solidified foam (94 mg, 64%):
HRMS-ESI [M+Na]+ =839.4709 (calcd for C39H76N2NaO10Si3:
839.4705). The intermediate was deprotected in two steps. 1) Re-
moval of the TBDMS ether groups: TBAF (124 mg, 3.3 equiv) in THF
(3 mL) at RT for 18 h followed by purification using Sep-Pakꢄ (5 g;
0!50% MeOH, rest: water). White amorphous solid (38 mg, 78%):
HRMS [M+Na]+ =497.2115 (calcd for C21H34N2NaO10: 497.2111).
2) Removal of the N(a)-Boc group according to general procedure
C yielded TFA salt of compound 16 as colorless oil (39 mg, quanti-
tative): IR (neat): n˜ =3298, 2949, 2864, 1734, 1660, 1554, 1443,
1
1554, 1441, 1364, 1206, 1161, 866 cmꢀ1; H NMR ([D6]DMSO, 758C):
d=9.11 (bs, 1H), 4.36 (bs, 1H), 4.02 (dd, 1H, J=18.3 and 2.8 Hz),
3.97 (bd, 1H, J=18.3 Hz), 3.65 (s, 3H), 3.20 (t, 2H, J=6.9 Hz), 2.95
(t, 1H, J=2.5 Hz), 1.98–1.89 (m, 1H), 1.86–1.78 (m, 1H), 1.61–1.49
(m, 2H), 1.41 (s, 9H), 1.42–1.32 (m, 2H); 13C NMR ([D6]DMSO, 758C):
d=170.9, 156.9 (q, J=36.2 Hz), 153.8, 115.7 (q, J=289.5 Hz), 79.9,
79.8, 72.9, 58.2, 51.3, 38.7, 35.0, 28.5, 27.52, 27.47, 22.7 ppm; HRMS:
[M+Na]+ =417.1596 (calcd for C17H25N2O5F3Na: 417.1613).
Precursor 13. Intermediate 12 (150 mg, 0.35 mmol) was dissolved
in MeOH (0.5 mL) and NH4HCO3 (5 mL; 1m in H2O/MeOH 1:1) were
added. The solution was stirred at 558C for 3 days, diluted with
brine and extracted with EtOAc. The organic phases were washed
once with brine and then combined, dried over Na2SO4 and con-
centrated under reduced pressure. The crude product was purified
by chromatography on silica gel at ambient pressure with CH2Cl2/
MeOH (10:1, containing 0.5% Et3N) to yield product 13 as a color-
less oil (68 mg, 60%): [a]2D0 =ꢀ25.3 (c=1 in MeOH); IR (neat): n˜ =
3270, 2925, 1742, 1695, 1364, 1247, 1163, 772 cmꢀ1 1H NMR
;
([D6]DMSO with one drop D2O, 758C; despite the elevated temper-
ature there are still multiple signals observed): d=4.36 (bs, 1H),
4.05–3.95 (m, 2H), 3.65 (s, 3H), 3.07–2.86 (several multiplets, total
3H), 2.59–2.54 (m, 2H), 1.95–1.74 (several multiplets, total 4H),
1.42 and 1.40 (each s (intensity ~1:2), total 9H), 1.35–1.25 (m, 2H);
1H NMR ([D6]DMSO with one drop D2O, 758C): d=171.0, 170.9,
80.0, 79.7, 72.9, 72.0, 58.3, 51.2, 40.8, 35.0, 32.1, 28.7, 28.5, 27.6,
27.5, 22.8 ppm; HRMS: [M+H]+ =299.1966 (calcd for C15H27N2O4:
299.1971).
1
1190, 1144, 1083, 1026, 723 cmꢀ1; H NMR ([D4]MeOH): d=4.24 (d,
1H, J=7.5 Hz), 4.10 (dd, 1H, J=5.0 and 6.5 Hz), 4.04 and 3.99
(each dd, each 1H, each J=2.6 and 16.6 Hz), 3.71 (d, 1H, J=
9.4 Hz), 3.54 (s, 3H), 3.47 (t, 1H, J=9.2 Hz), 3.42 (t, 1H, J=9.0 Hz),
3.30–3.23 (m, 3H), 3.22 (t, 1H, J=8.2 Hz), 2.09–1.92 (m, 2H), 1.65–
1.57 (m, 2H), 1.56–1.38 (m, 2H); 13C NMR ([D4]MeOH): d=172.1,
171.0, 105.8, 79.8, 77.7, 76.5, 74.7, 74.4, 73.7, 60.0, 57.8, 39.5, 36.6,
30.0, 29.9, 23.0 ppm (signals of the TFA counter ion are not listed);
HRMS [M+Na]+ =397.1586 (calcd for C16H26N2NaO8: 397.1587).
Precursor 14. Intermediate 12 (660 mg, 1.67 mmol) was dissolved
in MeOH (10 mL) and aqueous K2CO3 (0.5m, 10 mL) was added.
The mixture was stirred at 758C for 3 h after which TLC indicated
completed conversion of the substrate. The solution was adjusted
to pH 6 by addition of aqueous TFA (1m) and the volatile compo-
nents were removed under reduced pressure. The residue was
stirred several times in EtOH, centrifuged (3 min, 16000 rpm) and
the decanted solutions were concentrated under reduced pressure
to yield the TFA salt of product 14 as a white solid (540 mg, quan-
titative): [a]2D0 =ꢀ30.4 (c=0.5 in MeOH); IR (neat): n˜ =3401, 3297,
Compound 17. (R)-2-succinylamido-6-(4-(4-iodophenyl)butanami-
do)-hexanoic acid ethyl ester[16] (25 mg, 0.045 mmol) was dissolved
in DMF (2 mL) and HBTU (17 mg, 0.045 mmol) and DIPEA (16 mL,
0.09 mmol) were added. The solution was stirred at RT for 20 min
and then added dropwise at RT to a solution of precursor 13
(14 mg, 0.045 mmol) in DMF (2 mL) containing DIPEA (16 mL,
0.09 mmol). The mixture was stirred at RT for 2 h and the resulting
solution was diluted with aqueous citric acid (1m) and extracted
with EtOAc. The organic phases were washed with aqueous citric
acid (0.1m) and brine and then combined, dried over Na2SO4 and
concentrated under reduced pressure. Purification of the crude
product by flash chromatography on silica gel with CH2Cl2/MeOH
gave the protected intermediate as a colorless oil (30 mg, 81%):
HRMS-ESI [M+Na]+ =849.2906 (calcd for C37H55IN4NaO9: 849.2911).
The intermediate was deprotected in two steps. 1) Removal of the
N(a)-Boc group according to general procedure C gave a yellow oil
(25 mg, quantitative): HRMS [M+Na]+ =749.2393 (calcd for
C32H47IN4NaO7: 749.2382). 2) Saponification of the ethyl ester by
stirring the isolated compound in MeOH (1 mL) and aqueous
NaOH 1m (1 mL) at RT for 18 h. The pH was adjusted to pH 4–5 by
addition of aqueous HCl 1m and concentrated under reduced
pressure. The residue was stirred several times in EtOH (2 mL), cen-
trifuged (3 min, 13000 rpm) and the solution was decanted and
concentrated under reduced pressure to yield compound 17 as a
yellow oil (25 mg, quantitative): IR (neat): n˜ =3278, 2931, 2861,
2973, 1672, 1581, 1387, 1203, 1167, 1136, 719 cmꢀ1 1H NMR
;
([D6]DMSO with one drop D2O, 758C): d=4.13 (bs, 1H), 4.02 and
3.89 (each dd, each 1H, J=18.1 and 3.0 Hz), 2.71 (t, 1H, J=2.5 Hz),
2.66 (t, 2H, J=6.6 Hz), 1.93–1.85 (m, 1H), 1.63–1.53 (m, 1H), 1.53–
1.45 (m, 2H), 1.39 (s, 9H), 1.37–1.29 (m, 2H); 13C NMR ([D6]DMSO
with one drop D2O, 758C): d=173.7, 158.4 (q, J=37.5 Hz), 155.2,
117.2 (q, J=303 Hz), 82.7, 82.3, 82.2, 78.7, 61.0, 33.7, 30.2, 29.3,
28.1, 23.7 ppm; HRMS: [M+H]+ =285.1812 (calcd for C14H25N2O4:
285.1814).
Compound 15. Precursor 14 (TFA salt; 40 mg, 0.10 mmol) was sus-
pended in dry DMSO (MS 4 ꢃ, 2 mL) and NHS-m-dPEG15 (BioCon-
nect, 86 mg, 0.10 mmol) was added. The resulting solution was
stirred at RT for 24 h and quenched by addition of water (1 mL).
Purification of the mixture by semi-preparative HPLC (5%!80%
CH3CN within 14 min, rest: 0.1% aqueous TFA; flow: 3.5 mLminꢀ1
)
yielded N(a)-Boc protected intermediate as a colorless oil (60 mg,
58%; purity according to HPLC>95%): HRMS [M+Na]+
1053.5944 (calcd for C48H90N2O21Na: 1053.5934). The intermediate
=
1
1723, 1634, 1460, 1217, 1005, 735 cmꢀ1; H NMR (MeOD): d=7.61
2034
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ChemMedChem 2010, 5, 2026 – 2038

Multifunctional 99mTc Radiopharmaceuticals
and 6.99 (each d, each 2H, J=8.2 Hz), 4.35 (dd, 1H, J=9.0 and
4.9 Hz), 4.05–3.94 (m, 3H), 3.22 (t, 1H, J=2.5 Hz), 3.21–3.13 (m,
4H), 2.61–2.53 (m, 4H), 2.50–2.44 (m, 2H), 2.20 (t, 2H, J=7.8 Hz),
2.03–1.86 (m, 5H), 1.77–1.66 (m, 1H), 1.61–1.41 (m, 6H), 1.45–1.36
(m, 2H); 13C NMR (MeOD): d=175.9, 175.7, 175.0, 174.8, 171.7,
143.0, 138.7, 132.0, 91.6, 79.6, 74.7, 60.9, 53.8, 40.2, 39.8, 36.7, 36.6,
35.9, 32.4, 32.3, 32.2, 30.2, 30.1, 30.0, 28.7, 24.4, 23.1 ppm; HRMS
[M+Na]+ =707.1902 (calcd for C29H41IN4NaO7: 707.1912).
883.3616). The intermediate was deprotected according to proce-
dure C to yield compound 20 as a red solid (50% yield for 2 steps
as determined by UV/Vis: l=557 nm, d=1 cm, e=
150000mꢀ1 cmꢀ1; purity according to HPLC ꢁ 95%): HRMS [M]+
=
783.3104 (calcd for C39H51N4O9S2 783.3092).
Compound 21. A solution of precursor 13 (45 mg, 0.15 mmol),
DOTA(tBu)3-NHS ester (100 mg, 0.15 mmol) and DIPEA (52 mL,
0.3 mmol) in CH2Cl2 (5 mL) was stirred at reflux for 2 h. The solution
was diluted with CH2Cl2 and washed twice with aqueous citric acid
(0.1m). The organic extracts were dried over Na2SO4 and concen-
trated under reduced pressure to yield the fully protected inter-
mediate as a solidified white foam (110 mg, 86%): LR-MS [M+Na]+
=876.2 (calcd for C43H76N6NaO11: 876.1). The intermediate was de-
protected according to procedure C to yield compound 21 as a
yellow oil (penta-TFA salt, 90 mg, quantitative): IR (neat): n˜ =3093,
Compound 18. Precursor 14 (TFA salt; 40 mg, 0.1 mmol) was sus-
pended in dry DMSO (MS 4 ꢃ, 2 mL) and biotin–NHS ester (34 mg,
0.1 mmol) was added. The resulting mixture was stirred at RT for
20 h and quenched by addition of water (1 mL). Purification of the
mixture by semi-preparative HPLC (5%!95% CH3CN within
15 min, rest: 0.1% aqueous TFA; flow: 3.5 mLminꢀ1) yielded the
N(a)-Boc protected intermediate as a white solid (40 mg, 78%,
purity according to HPLC>95%): HRMS [M+Na]+ =533.2402
(calcd for C24H38N4NaO6S: 533.2410). The intermediate was depro-
tected according to procedure C to yield compound 18 as a color-
less oil (41 mg, quantitative; purity according HPLC>95%): IR
(neat): n˜ =3293, 2929, 1672, 1642, 1561, 1461, 1190, 1136, 720
1
2861, 1730, 1665, 1190, 1136, 839 cmꢀ1; H NMR (MeOD): d=4.17
(dd, 1H, J=7.3 and 4.6 Hz), 4.10–3.65 (several broad signals, 8H),
4.07 and 3.99 (each d, 1H, J=15.3 Hz), 3.87 (s, 3H), 3.50–3.15 (bs,
18H), 3.24 (t, 1H, J=2.1 Hz), 2.09–1.89 (m, 2H), 1.63–1.52 (m, 2H),
1.43–1.31 (m, 2H) (13C NMR is not conclusive due to similar chemi-
cal shifts of signals of the DOTA moiety); HRMS [M+H]+ =607.3059
(calcd for C26H44N6NaO9: 607.3067).
1
cmꢀ1; H NMR ([D4]MeOH with TMS): d=4.50 (dd, 1H, J=4.5 and
7.4 Hz), 4.31 (dd, 1H, J=4.6 and 7.4 Hz), 4.10 (t, 1H, J=6.1 Hz),
4.04 and 3.99 (each dd, each 1H, each J=2.0 and 17.2 Hz), 3.25–
3.18 (m, 4H), 2.93 (dd, 1H, J=4.9 and 12.7 Hz), 2.71 (d, 1H, J=
12.7 Hz), 2.21 (t, 2H, J=7.3 Hz), 2.06–1.96 (m, 2H), 1.78–1.35 (m,
10H); 13C NMR ([D4]MeOH with TMS): d=176.4, 171.0, 166.3, 79.8,
74.4, 63.6, 61.8, 60.1, 57.1, 41.2, 39.8, 36.9, 36.6, 30.1, 30.0, 29.9,
29.6, 27.0, 23.2 ppm (signal of TFA not observed); HRMS [M+Na]+
=433.1878 (calcd for C19H30N4NaO4S: 433.1885).
Compound 22. Compound 21 (penta-TFA salt; 34 mg, 0.03 mmol)
was dissolved in water (4 mL) and GdCl3 (hexahydrate; 11 mg,
0.03 mmol) was added. The solution was stirred at 908C for 1.5 h.
Purification by semi-preparative HPLC (isocratic 5% CH3CN, rest:
0.1% aqueous TFA; flow: 3 mLminꢀ1) gave the TFA salt of Gd-com-
plex 22 as a white solid (15 mg, 60%; purity according to HPLC>
95%): HRMS [M+H]+ =740.2246 (calcd for C26H42GdN6O9:
740.2254).
Compound 19. Acridine-9-carboxylic acid (67 mg, 0.3 mmol), pre-
cursor 13 (90 mg, 0.3 mmol) and HOBT (45 mg, 0.33 mmol) were
dissolved in DMF (4 mL) and DCC (68 mg, 0.33 mmol) was added.
The reaction mixture was stirred at 808C for 18 h, diluted with
water and extracted with CH2Cl2. The organic phases were washed
with water and then combined, dried over Na2SO4 and concentrat-
ed under reduced pressure. Purification of the crude product by
flash chromatography on silica gel with mixtures of EtOAc/hexane
gave the N(a)-Boc protected intermediate as a yellow oil (106 mg,
Ligand L15. As per general procedure B2 from compound 15
(19 mg, 0.02 mmol). The mixture was filtered through Celite and
treated with QuadraPureTM; IDA resin (~0.25 g) for 18 h. Filtration
and removal of volatile components under reduced pressure yield-
ed 1,2,3-triazole X as a yellow oil (18 mg, 84%, purity according to
HPLC>90%): HRMS [M+H]+ =1064.6226 (calcd for C50H90N5O19:
1064.6230).
70%; purity according to HPLC>95%): HRMS-ESI [M+H]+
=
Ligand L16. As per general procedure B2 from compound 16 (TFA
salt; 24 mg, 0.05 mmol) and purification by semi-preparative HPLC
(5%!90% CH3CN within 14 min, rest: 0.1% aqueous TFA; flow:
3 mLminꢀ1). White solid (50 mg, 60%; purity according to HPLC>
95%): mp>1508C (dec.); IR (neat): n˜ =3318, 2929, 2964, 1646,
504.2502 (calcd for C29H34N3O5: 504.2498). The intermediate was
dissolved in TFA/CH2Cl2 (1:4, 5 mL) and kept at RT overnight. The
solution was diluted with CH2Cl2 and washed with aqueous NaOH
(0.1m). The organic phase was dried over Na2SO4 and concentrated
under reduced pressure to yield compound 19 as a yellow oil
(80 mg, 94%): IR (neat): n˜ =3260, 2929, 1730, 1640, 1545, 1436,
1
1554, 1455, 1192, 1127, 1057, 1027, 719 cmꢀ1; H NMR ([D4]MeOH):
d=8.14 (s, 1H), 7.42–7.32 (m, 5H), 5.64 (s, 2H), 4.41 and 4.38 (each
d, each 1H, J=14.8 Hz), 4.27 (d, 1H, J=7.7 Hz), 4.01 (t, 1H, J=
5.8 Hz), 3.72 (d, 1H, J=9.3 Hz), 3.53 (s, 3H), 3.48 (t, 1H, J=8.6 Hz),
3.43 (t, 1H, J=8.7 Hz), 3.29–3.23 (m, 3H), 2.08–1.93 (m, 2H), 1.63–
1.54 (m, 2H), 1.53–1.46 (m, 1H), 1.45–1.35 (m, 1H); 13C NMR
([D4]MeOH): d=172.0, 171.4, 139.7, 136.4, 130.3, 130.0, 129.4,
127.1, 105.6, 77.5, 76.5, 74.6, 73.5, 60.6, 57.9, 55.3, 41.9, 39.6, 30.0,
29.9, 23.0 ppm; HRMS [M+H]+ =508.2404 (calcd for C23H34N5O8:
508.2407).
1
1212, 752 cmꢀ1; H NMR (CDCl3): d=8.04 (d, 2H, J=9.0 Hz), 7.90 (d,
2H, J=8.7 Hz), 7.67 (t, 2H, J=8.1 Hz), 7.45 (t, 2H, J=7.5 Hz), 7.03
(bs, 1H; exchanges with D2O), 3.71 (s, 3H), 3.71–3.65 (m, 2H), 3.45
(t, 1H, J=6.8 Hz), 3.42 and 3.35 (each dd, each 1H, each J=2.4
and 16.8 Hz), 2.18 (t, 1H, J=2.4 Hz), 1.88–1.77 (m, 4H; one proton
exchanges with D2O), 1.75–1.68 (m, 1H), 1.65–1.54 (m, 2H);
13C NMR (CDCl3): d=175.1, 167.0, 148.3, 141.1, 130.3, 129.4, 126.7,
125.3, 122.0, 81.3, 71.8, 59.8, 51.9, 40.0, 36.9, 32.7, 29.5, 23.2 ppm;
HRMS [M+H]+ =404.1976 (calcd for C24H26N3O3: 404.1974).
Ligand L17. As per general procedure B2 from compound 17
(35 mg, 0.05 mmol) and purification using a C-18 Sep-Pakꢄ column
and mixtures of water and methanol (0% ! 100% MeOH). White
solid (20 mg, 50%; purity according to HPLC>95%): IR (neat): n˜ =
3302, 2929, 2860, 1737, 1634, 1599, 1549, 1484, 1456, 1395, 1381,
1355, 1231, 1203, 1173, 1122, 1053, 1027, 1007, 823, 792, 719, 693,
Compound 20. Precursor 14 (TFA salt; 0.54 mg, 1.35 mmol) was dis-
solved in DMSO (100 mL) and DIPEA (5.5 mmol) and DY547-NHS
(1.0 mg, 1.35 mmol) were added. The solution was stirred at RT in
the dark for 18 h and then diluted with water (0.4 mL). Purification
of the mixture by analytical HPLC (5%!90% CH3CN within 13 min,
rest: 0.1% aqueous TFA; flow 1 mLminꢀ1) gave the N(a)-Boc inter-
mediate as a red solid (~0.6 mg, 50%; purity according to HPLC>
95%): HRMS-ESI [M]+ =883.3609 (calcd for C44H59N4O11S2:
665 cmꢀ1 1H NMR ([D6]DMSO): d=8.06 (s, 1H), 8.03 (d, 1H, J=
;
7.7 Hz), 7.73 (dd, 2H, J=9.8, 5.3 Hz), 7.62 (d, 2H, J=8.3 Hz), 7.41–
7.27 (m, 5H), 7.00 (d, 2H, J=8.3 Hz), 5.59 (s, 2H), 4.11 (m, 1H), 3.93
ChemMedChem 2010, 5, 2026 – 2038
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2035

T. L. Mindt, R. Schibli, et al.
MED
(d, 1H, J=13.9 Hz), 3.80 (d, 1H, J=13.9 Hz), 3.16 (t, 2H, J=6.3 Hz),
2.97 (m, 4H), 2.48 (m, 2H, obscured by DMSO signal), 2.35 (t, 2H,
J=7.8 Hz), 2.26 (t, 2H, J=7.8 Hz), 2.04 (t, 2H, J=7.4 Hz), 1.75 (m,
2H, J=7.4 Hz), 1.70–1.46 (m, 4H), 1.42–1.20 (m, 8H); 13C NMR
([D6]DMSO): d=173.8, 173.2, 171.5 (two C=O), 171.0, 143.7, 141.6,
137.0, 136.0, 130.8, 128.7, 128.1, 127.9, 123.7, 91.2, 60.4, 52.8, 51.9,
41.8, 39.6, 38.3, 38.2, 34.7, 34.0, 31.3, 30.9, 30.8, 30.7, 28.9, 28.7,
26.7, 22.8, 22.6 ppm; HRMS [M+H]+ =818.2698 (calcd for
C36H49IN7O7: 818.2738).
labeled in situ without further purification in analogy to previously
reported procedures.[9,13]
Peptide NaHisAc-BBS(7–14). Prepared by SPPS. White solid
(7.8 mg, 34%; purity>95%): LC–MS: [M]+ =1428, 715 (calcd for
C66H97N19O17: 1428.594).
[Re(CO)3(L15)]. As per general procedure D from ligand L15
(11 mg, 0.01 mmol) in MeOH/H2O (1:1) and purification by semi-
preparative HPLC (5!95% CH3CN within 14 min, rest: 0.1% aque-
ous TFA; flow: 3 mLminꢀ1). Colorless oil (8 mg, 60%, purity accord-
ing to HPLC>95%): [a]2D0 =+23.6 (c=0.2 in MeOH); IR (neat): n˜ =
Ligand L18. As per general procedure B2 from compound 18
(26 mg, 0.05 mmol) and purification by semi-preparative HPLC
(5%!95% CH3CN within 15 min, rest: 0.1% aqueous TFA; flow:
3.5 mLminꢀ1). White solid (18 mg, 66%): mp 145–1488C; IR (neat):
2870, 2023 and 1887 (strong), 1638, 1450, 1346, 1102, 946 cmꢀ1
HRMS (calcd for C53H88N5O22NaRe:
[M+Na]+ =1356.5382
1356.5376).
;
n˜ =3320, 2929, 1665, 1638, 1457, 1190, 1129, 715 cmꢀ1 1H NMR
;
([D6]DMSO with one drop D2O): d=8.21 (s, 1H), 7.41–7.29 (m, 5H),
5.62 (s, 2H), 4.31 dd, 1H, J=4.9 and 7.8 Hz), 4.26 (s, 2H), 4.13 (dd,
1H, J=4.4 and 7.8 Hz), 3.93 (dd, 1H, J=4.8 and 6.6 Hz), 3.12–3.05
(m, 1H), 2.99 (t, 2H, J=6.1 Hz), 2.80 (dd, 1H, J=4.9 and 12.5 Hz),
2.57 (d, 1H, J=12.5 Hz), 2.03 (t, 2H, J=7.1 Hz), 1.92–1.76 (m, 2H),
1.65–1.55 (m, 1H), 1.52–1.15 (m, 10H); 13C NMR ([D6]DMSO with
one drop D2O): d=172.4, 169.8, 162.9, 138.0, 135.5, 128.8, 128.3,
128.0, 125.7, 61.0, 59.1, 58.2, 55.3, 53.0, 40.0, 39.7, 37.8, 35.1, 28.5,
28.2, 27.9, 25.2, 21.6 ppm (some signals are obscured by the sol-
vent peak but confirmed by HSQC); HRMS [M+H]+ =544.2707
(calcd for C26H38N7O4S: 544.2706).
[Re(CO)3(L16)]. As per general procedure D from ligand L16
(25 mg, 0.05 mmol) in MeOH/H2O (1:1) and purification by semi-
preparative HPLC (5%!90% CH3CN within 13 min, rest: 0.1%
aqueous TFA; flow 3 mLminꢀ1). White amorphous solid (29 mg,
75%; purity according to HPLC>95%): mp>1508C (dec.); [a]D20
ꢀ12.0 (c=1 in MeOH); IR (neat): n˜ =3281, 2932, 2026 and 1905
(strong), 1670, 1441, 1198, 1141, 1030, 723 cmꢀ1 1H NMR
=
;
([D4]MeOH): d=8.10 (s, 1H), 7.44–7.34 (m, 5H), 5.68 (s, 2H), 4.31–
4.20 (m, 3H), 3.71 (d, 1H, J=9.8 Hz), 3.53 (s, 3H), 3.49 (t, 1H, J=
9.8 Hz), 3.42–3.32 (m, 1H), 3.39 (t, 1H, J=9.6 Hz), 3.27–3.19 (m,
2H), 3.11 (t, 1H, J=6.8 Hz), 1.92–1.84 (m, 2H), 1.65–1.53 (m, 4H);
13C NMR ([D4]MeOH): d=198.5, 197.0, 196.6, 186.3, 172.2, 149.7,
135.6, 130.4, 130.3, 129.7, 124.4, 105.8, 77.7, 76.6, 74.7, 73.7, 67.8,
Ligand L19. As per general procedure B1 from compound 19
(40 mg, 0.1 mmol) and purification by flash chromatography with
CH2Cl2/MeOH (25:1). Yellow oil (50 mg, 93%): IR (neat): n˜ =3260,
57.8, 56.5, 53.8, 39.6, 33.8, 30.2, 24.4 ppm; HRMS [M+Na]+
=
800.1553 (calcd for C26H32N5O11ReNa: 800.1554).
3061, 2938, 1730, 1649, 1543, 1515, 1457, 1439, 1203, 758 cmꢀ1
;
1H NMR (CDCl3): d=8.07 (d, 2H, J=9.3 Hz), 7.93 (d, 2H, J=7.7 Hz),
7.68 (t, 2H, J=8.3 Hz), 7.46 (t, 2H, J=7.6 Hz), 7.34–7.25 (m, 3H),
7.27 (s, 1H), 7.21–7.15 (m, 2H), 7.05 (bs, 1H), 5.38 and 5.35 (each d,
each 1H, J=16.8 Hz), 3.84 (d, 1H, J=14.0 Hz), 3.73–3.62 (m, 3H),
3.68 (s, 3H), 3.34 (t, 1H, J=6.3 Hz), 2.00 (bs, 1H), 1.84–1.66 (m, 4H),
1.62–1.51 (m, 2H); 13C NMR (CDCl3): d=175.3, 167.0, 148.4, 146.8,
141.2, 134.6, 130.3, 129.4, 129.0, 128.7, 128.0, 126.7, 125.3, 122.1,
121.5, 60.5, 54.0, 51.9, 43.1, 39.9, 32.7, 29.3, 23.2 ppm; HRMS
[M+H]+ =537.2613 (calcd for C31H33N6O3: 537.2614).
[Re(CO)3(L17)]. As per general procedure D from ligand L17
(10 mg, 0.01 mmol) in MeOH/H2O (1:1). The complex was purified
using a Sep-Pak column and eluting with a mixture of MeOH
(80%) and water. White solid (5 mg, 38%; purity according to
HPLC>95%): IR (neat): n˜ =3286, 2933, 2020 and 1897 (strong),
1
1639, 1545, 1202, 1144, 723 cmꢀ1; H NMR ([D4]MeOH): d=7.60 (d,
2H, J=8.3 Hz), 7.44–7.31 (m, 6H), 6.99 (d, 2H, J=8.3 Hz), 5.67 (d,
2H J=8.7 Hz,), 4.34–4.20 (m, 2H), 3.20–3.13 (m, 3H), 2.58 (t, 2H),
2.52–2.48 (m, 3H), 2.18 (t, J=7.5 Hz, 2H), 1.95–1.78 (m, 4H), 1.75–
1.64 (m, 2H), 1.59–1.29 (m, 12H); 13C NMR ([D4]MeOH): d=174.4,
173.3, 149.1, 148.2, 141.5, 137.2, 134.0, 130.41, 128.9, 128.7, 128.1,
128.1, 122.9, 122.5, 90.3, 66.2, 62.1, 61.4, 55.0, 54.9, 38.7, 38.5, 35.1,
35.0, 34.3, 31.5, 31.0, 30.9, 30.8, 28.8, 28.7, 28.5, 28.3, 27.1, 26.0,
23.0, 23.0, 22.8, 22.3, 21.7 ppm. HRMS-ESI [M+H]+ =1088.2061
(calcd for C39H48IN7O10Re: 1088.2065).
Ligand L20. As per general procedure B2 from compound 19
(0.49 mg, 0.6 mmol) and purification by analytical HPLC (5%!90%
CH3CN within 13 min, rest: 0.1% aqueous TFA; flow 1 mLminꢀ1).
Red solid (50% yield as determined by UV/Vis: l=557 nm, d=
1 cm, e=150000mꢀ1 cmꢀ1; purity according to HPLC
HRMS-ESI [M]+ =916.3737 (calcd for C46H58N7O9S2: 916.3732).
ꢁ
95%):
[Re(CO)3(L18)]. As per general procedure D from ligand L18
(22 mg, 0.04 mmol) in MeOH/H2O (1:1) and purification by semi-
preparative HPLC (15%!95% CH3CN within 15 min, rest: 0.1%
aqueous TFA; flow 3 mLminꢀ1). White solid (20 mg, 62%; purity ac-
cording to HPLC>95%): mp 165–1688C; [a]2D0 =+31.5 (c=1 in
MeOH); IR (neat): n˜ =2938, 2018 and 1889 (strong), 1631, 1439
Ligand L22. As per general procedure B1 from compound 22 (TFA
salt; 13 mg, 0.015 mmol) and purification by semi-preparative
HPLC (5%!95% CH3CN within 14 min, rest: 0.1% aqueous TFA;
flow: 3 mLminꢀ1). White solid (68%; purity according to HPLC>
95%): mp 110–1158C; IR (neat): n˜ =2957, 2870, 1744, 1674, 1622,
1196, 1137, 718 cmꢀ1
C33H49GdN9O9: 873.2894).
;
HRMS [M+H]+ =873.2899 (calcd for
cmꢀ1; H NMR ([D4]MeOH): d=8.10 (s, 1H), 7.44–7.34 (m, 5H), 5.68
1
(s, 2H), 4.48 (dd, 1H, J=7.8 and 4.8 Hz), 4.32–4.20 (m, 3H), 3.28–
3.13 (m, 3H), 3.11 (t, 1H, J=6.7 Hz), 2.92 (dd, 1H, J=12.6 and
5.0 Hz), 2.71 (d, 1H, J=12.6 Hz), 2.21 (t, 2H, J=7.2 Hz), 1.92–1.84
(m, 2H), 1.78–1.59 (m, 4H), 1.58–1.53 (m, 4H), 1.48–1.39 (m, 2H);
13C NMR ([D4]MeOH): d=198.6, 197.0, 196.7, 186.1, 176.4, 166.3,
149.7, 135.6, 130.4, 130.2, 129.6, 124.4, 67.7, 63.5, 61.8, 57.1, 56.5,
53.8, 41.2, 40.0, 37.0, 33.9, 30.3, 29.9, 29.6, 27.0, 24.5 ppm; HRMS
[M+H]+ =836.1843 (calcd for C29H36N7NaO7ReS: 836.1852).
Peptide 23. Prepared by SPPS. White solid (7.4 mg, 40%; purity>
95%): LC–MS: [M]+ =1316, 659 (calcd for C60H89N19O15: 1316.467).
Peptide L24. 50 mL of peptide 23 (10 mm in EtOH), 62.5 mL of com-
pound 17 (10 mm in 1:1 EtOH/H2O), 12.5 mL of Cu(OAc)2 (10 mm in
H2O) and 25 mL of sodium ascorbate (10 mm n in H2O) were stirred
at 608C for 4 h. Formation of the product was monitored by ana-
lytical HPLC and the reaction mixture analyzed by MS: [M+2H]2+
=
[Re(CO)3(L19)]. As per general procedure D from ligand L19
1001.62 (calcd for C89H130IN23O22: 2001.03). The solution was radio-
(16 mg, 0.03 mmol) in MeOH/H2O (1:1). Adjustment of the pH of
2036
www.chemmedchem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 2026 – 2038

Multifunctional 99mTc Radiopharmaceuticals
the reaction mixture to pH 8 by addition of aqueous NaOH (0.1m)
resulted in precipitation of the product. Centrifugation (2 min,
13200 rpm, 16.1 g), decanting the solution and drying of the solid
provided [Re(CO)3(L19)] as a yellow powder (18 mg, 75%; purity
according to HPLC>95%; 1H NMR indicates the presence of 2%
(w/w) Et4NBr): mp>2508C (dec.); [a]2D0 =+25.5 (c=1.0 in MeOH);
In vitro Experiments
Mouse serum albumin binding. Evaluation of the binding of ra-
diolabeled peptides to mouse albumin was performed after incu-
bation of the radiolabeled conjugate (~100 kBq) with 500 mL of
mouse serum (Sigma–Aldrich) for 15 min at 378C. Samples were
analyzed by FPLC (column: Superose 12, Pharmacia Biotech) using
IR (neat): n˜ =2939, 2018 and 1894 (strong), 1642, 1260, 756 cmꢀ1
;
PBS as eluent at a flow rate of 0.5 mLminꢀ1
.
1H NMR ([D4]MeOH with one drop of 1% DCl; recorded after com-
plete H/D exchange): d=8.38 (d, 2H, J=8.0 Hz), 8.35–8.28 (m, 4H),
8.14 (s, 1H), 8.02–7.98 (m, 2H), 7.43–7.35 (m, 5H), 5.71 and 5.68
(each d, each 1H, each J=15.4 Hz), 4.36 (d, 1H, J=16.1 Hz), 4.26
(dd, 1H, J=1.0 and 16.1 Hz), 3.75 (t, 2H, J=6.9 Hz), 3.22 (t, 1H, J=
6.7 Hz), 2.04–1.99 (m, 2H), 1.93–1.85 (m, 2H), 1.84–1.68 (m, 2H);
13C NMR ([D4]MeOH with one drop of 1% DCl): d=198.6, 196.8,
196.6, 186.1, 166.4, 153.4, 149.6, 142.6, 138.7, 135.5, 130.4, 130.2,
129.6, 128.3, 128.2, 124.5, 124.1, 122.1, 67.6, 56.5, 53.8, 41.1, 33.9,
30.1, 24.7 ppm; HRMS [M+H]+ =793. 1776 (calcd for C33H30N6O6Re:
793.1784).
Binding to PC-3 cells. The binding to GRP receptors was tested in
intact PC-3 cells. Cells were placed in 6-well plates one day prior to
the assay (106 cells per well). Cells were incubated in triplicate with
the radiolabeled conjugate (20 kBq) for 1 h at 378C. Then the cells
were washed twice with PBS to remove unbound radiotracers and
recovered with NaOH (1m). Bound radioactivity was measured in a
g counter. Nonspecific binding was determined in the presence of
1 mm natural bombesin.
Biodistribution studies
[Re(CO)3(L20)]. As per general procedure D from ligand L20
(~0.1 mg, 0.1 mmol) in EtOH/H2O (1:1) and purification by analytical
HPLC (5%!90% CH3CN within 13 min, rest: 0.1% aqueous TFA;
flow 1 mLminꢀ1) yielded a red solution of Re complex X (HPLC:
quantitative reaction; purity>95%): IR (neat): n˜ =3415, 2920, 2023
and 1909 (strong), 1681, 1441, 1199, 1136, 1031, 722 cmꢀ1; HRMS
[M]+ =1186.3050 (calcd for C49H57N7O12ReS2: 1186.3059).
Animal experiments were conducted in compliance with the Swiss
animal protection laws and the ethical principles and guidelines
for scientific animal trials of the Swiss Academy of Medical Sciences
and the Swiss Academy of Natural Sciences. Female CD-1 nu/nu
mice (6–8-week-old) were injected s.c. with 8ꢅ106 cells in 150 mL
culture medium without supplements. Three weeks after tumor im-
plantation the mice were injected i.v. with ~1 MBq of the radiola-
beled peptides (3–4 animals per group). At different p.i. times (1
and 24 h), the animals were sacrificed by cervical dislocation. The
organs and blood were collected and weighed. The radioactivity
was measured in a g counter. To determine specificity of the
in vivo uptake, one group of mice received a co-injection of
100 mg of unlabeled natural BBS and the radiolabeled analogue
and sacrificed 1.5 h p.i. Results are presented as percentage of in-
jected dose per gram of tissue (% IDgꢀ1).
[Re(CO)3(L22)]. As per general procedure D from ligand L22 (5 mg,
5 mmol) in water and purification by semi-preparative HPLC (5%!
95% CH3CN within 14 min, rest: 0.1% aqueous TFA; flow:
3 mLminꢀ1). White solid (3 mg, 54%; purity according to HPLC>
95%): mp>2008C (dec.); [a]2D0 =+12.3 (c=0.3 in MeOH); IR (neat):
n˜ =3249, 2938, 2020 and 1901 (strong), 1623, 1403, 1198, 1140,
1083, 717 cmꢀ1
;
HRMS [M+H]+ =1127.2033 (calcd for
C35H46GdN9O12Re: 1129.2064).
Acknowledgements
We thank Professor Dario Neri (ETH) for providing the albumin
binder, Dr. Bernhard Pfeiffer (ETH) for assistance with NMR spec-
troscopic measurements, Dr. Cristina Mꢂller, Alain Blanc, Olga
Gasser (PSI) and Philipp Ansorge for technical assistance, and
Covidien, Petten (The Netherlands) for financial support.
Crystal data
Crystallographic data were collected at 183(2) K on an Oxford Dif-
fraction Xcalibur system with a Ruby detector using MoKa radiation
(l=0.7107 ꢃ) that was graphite-monochromated. Suitable crystals
were covered with oil (Infineum V8512, formerly known as Para-
tone N), mounted on top of a glass fiber and immediately trans-
ferred to the diffractometer. The program suite CrysAlisPro was used
for data collection, semi-empirical absorption correction and data
reduction.[42] The structures of [Re(CO)3(L6)] and [Re(CO)3(L5)] were
solved with direct methods using SIR97[43] whereas the one of
[Re(CO)3(L4)] was solved with the help of SIR2004.[44] All structures
were refined by full-matrix least-squares methods on F2 with
SHELXL-97[45] and checked for higher symmetry using the program
Platon.[46] Crystal data and structure refinement can be found in
table S3 of the Supporting Information. In addition, CCDC-781592,
-781593, and -781594 contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via http://
www.ccdc.cam.ac.uk. The structure of [Re(CO)3(L4)] contains seven
complexes, two and a half molecules of THF as well as half a mole-
cule of water in the asymmetric unit. Six out of the seven com-
plexes overlay almost perfectly if the phenyl groups are omitted.
Only in one case is the whole benzyl group oriented differently.
Keywords: bombesin · click chemistry · imaging agents ·
multifunctional conjugates · technetium
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2037

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Received: August 11, 2010
Revised: September 6, 2010
Published online on October 4, 2010
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