Synthesis and evaluation of bombesin analogues conjugated to two different triazolyl-derived chelators for 99mTc labeling

Article abstract of DOI:10.1002/cmdc.201000191

Overexpression of the gastrin-releasing peptide receptor (GRPR) in a variety of human carcinomas has provided a means of diagnosis and treatment. Previously we reported a metabolically stable (NαHis)Ac-βAla- βAla-[Cha13,Nle¹⁴]BBS(7-14) analogue with high affinity for the GRPR. We have also shown that the biodistribution pattern of this fairly lipophilic, radiolabeled peptide can be enhanced by glycation, which is easily carried out by Cu^I-catalyzed cycloaddition. Herein, we further elaborate this "click approach" in the synthesis of a new series of triazole-based chelating systems as alternatives to the (NHis)Ac chelator for labeling with the ^99mTc(CO)₃ core. The bombesin analogues, containing these new chelating systems, were evaluated with regard to their synthesis and in vitro and in vivo properties, and were compared with their (NαHis)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor-to-background ratios. 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Full text of DOI:10.1002/cmdc.201000191

DOI: 10.1002/cmdc.201000191
Synthesis and Evaluation of Bombesin Analogues
Conjugated to Two Different Triazolyl-Derived Chelators
99m
for Tc Labeling
Luc Brans,[a] Elisa Garcꢀa-Garayoa,[b] Christian Schweinsberg,[b] Veronique Maes,[a]
Harriet Struthers,[c] Roger Schibli,[b, c] and Dirk Tourwꢁ*[a]
Overexpression of the gastrin-releasing peptide receptor
(GRPR) in a variety of human carcinomas has provided a
means of diagnosis and treatment. Previously we reported a
metabolically stable (NaHis)Ac-bAla-bAla-[Cha13,Nle14]BBS(7–14)
analogue with high affinity for the GRPR. We have also shown
that the biodistribution pattern of this fairly lipophilic, radiola-
beled peptide can be enhanced by glycation, which is easily
carried out by CuI-catalyzed cycloaddition. Herein, we further
elaborate this “click approach” in the synthesis of a new series
of triazole-based chelating systems as alternatives to the
(NaHis)Ac chelator for labeling with the 99mTc(CO)3 core. The
bombesin analogues, containing these new chelating systems,
were evaluated with regard to their synthesis and in vitro and
in vivo properties, and were compared with their (NaHis)Ac
counterparts. The influence of the chelator on biodistribution
properties was less than that of glycation, which clearly im-
proved the tumor-to-background ratios.
Introduction
Bombesin (BBS) is a 14 amino acid peptide (pGlu1-Gln2-Arg3-
Leu4-Gly5-Asn6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Met14-NH2)
that was isolated from the frog Bombina bombina by Anastasi
et al.[1] Since then, two bombesin-like peptides have been
found in mammals: gastrin-releasing peptide (GRP)[2] and neu-
romedin B (NMB).[3] These peptides contain 27 and 10 amino
acids, respectively, and have high homology with BBS. In mam-
mals, three BBS receptor subtypes have been characterized to
date which all belong to the family of G-protein-coupled re-
ceptors.[4] The NMBR, or BB1 receptor, subtype has high affinity
for NMB. The GRPR, or BB2 receptor, has high affinity for GRP
and an even higher affinity for BBS. The third subtype, BB3, is
an orphan receptor, as no high-affinity endogenous ligand has
been identified yet. The C-terminal nonapeptide sequence,
BBS(6–14), is the minimal fragment necessary to maintain full
GRPR affinity. Heimbrook et al. determined that acetylation of
the N-terminal amine in the octapeptide sequence Ac-BBS(7–
14) maintained affinity at nanomolar levels.[5,6]
To be useful for the diagnosis of abdominal lesions, a radio-
tracer must accumulate at the lowest possible levels in the ab-
domen, including in the liver and the intestines. Uptake at
these organs depends mainly on pharmacokinetic properties,
which are influenced by factors such as metabolic stability,[21]
polarity,[22,23] and charge.[24–26] Therefore, we evaluated different
glycation techniques for (NaHis)Ac-containing bombesin ana-
logues.[27] Bombesin analogue 2, which was glycated by CuI-
catalyzed alkyne/azide cycloaddition, exhibited an improved
biodistribution profile in PC-3 xenografts, resulting in nude
mice with enhanced tumor uptake, decreased hepatic activity,
and expedient excretion by the kidneys.[28] Glycation by CuI-
catalyzed cycloaddition also prevented glycation of the secon-
dary amine in the (NaHis)Ac chelator, which was encountered
during Maillard glycation.[27,29]
An alternative to the Na-carboxymethylhistidine [(NaHis)Ac]
chelator for 99mTc(CO)3, which requires multistep synthesis, is a
1,2,3-triazole-containing chelator. We recently reported the
single step synthesis of such a 1,2,3-triazole-containing chela-
tor by alkyne/azide cycloaddition.[30,31] Preliminary data regard-
In addition to the presence of BBS receptors in the periphery
and the central nervous system, evidence has been found that
these receptors are overexpressed on the membrane of several
tumor cells,[7] including prostate, breast, colon, and small-cell
lung carcinomas.[8–14] This provides an opportunity to use the
GRPR as a target for detection and treatment[15] of these can-
cers using radiolabeled BBS(7–14) ligands. Several 18F, 64Cu,
68Ga, 99mTc, 177Lu, 111In, and 188Re radiolabeled BBS(7–14) deriva-
tives have already been designed as tumor-selective tracers for
peptide receptor imaging and radiotherapy, but most of them
suffer from high uptake in the abdomen.[7,16–20] This is also the
[a] Dr. L. Brans, Dr. V. Maes, Prof. D. Tourwꢀ
Department of Organic Chemistry
Vrije Universiteit Brussel, 1050 Brussels (Belgium)
Fax: (+32)02-629-33-04
E-mail: datourwe@vub.ac.be
[b] Dr. E. Garcꢁa-Garayoa, Dr. C. Schweinsberg, Prof. R. Schibli
Center for Radiopharmaceutical Science
Paul Scherrer Institute, 5232 Villigen-PSI (Switzerland)
[c] Dr. H. Struthers, Prof. R. Schibli
Department of Chemistry and Applied Biosciences
ETH Zurich, 8093 Zurich (Switzerland)
case
for
the
99mTc(CO)3-labeled
(NaHis)Ac-bAla-bAla-
[Cha13,Nle14]BBS(7–14) (1; Figure 1) which we previously report-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000191.
ed.[21]
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D. Tourwꢀ et al.
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containing bombesin analogues
was compared with the corre-
sponding (NaHis)Ac analogues.
Two varieties of triazole-based
chelators were constructed on a
resin-bound
bombesin
se-
quence. The first type, seen in 8
(Scheme 1), is obtained by “click-
ing” the commercially available
Boc-propargylglycine (Boc-Pra-
OH; 7) to the azidoacetylated
peptide (6).[30] The cycloaddition
reaction went to completion
upon shaking the solid-support-
ed azidopeptide (6; 1.0 equiv)
overnight with Boc-Pra-OH (7;
2.0 equiv), N,N-diisopropylethyla-
Figure 1. Overview of the bombesin analogues.
ing 99mTc(CO)3-labeled bombesin analogue 3 have been previ-
ously reported.[30]
We report herein the detailed synthesis of bombesin ana-
logues conjugated to two types of triazole-based chelators for
labeling with the readily available 99mTc(CO)3 core. These tria-
zole-based chelators (3, 4a/b, and 5) do not contain the reac-
tive secondary amine, thus permitting selective derivatization
of the peptide sequence, which can be carried out after chela-
tor conjugation. This is in contrast to peptides containing the
(NaHis)Ac chelator (1 and 2) in which selective glycation by
Maillard reaction was hampered by the secondary amine of
the (NaHis)Ac chelator.[29] The labeling and in vitro and in vivo
properties of analogues 3–5, (Figure 1) are compared with
(NaHis)Ac-containing counterparts 1 and 2. Therefore, the pre-
viously published data for 1 and 2 is presented for compari-
son.
Scheme 1. Synthesis and radiolabeling of b1TA-containing bombesin ana-
logues. a) 7 (2.0 equiv), CuI (0.2 equiv), DIEA (2.0 equiv), DMF, 16 h; b) 10%
TA/EDT (7:3) in TFA, 3 h; c) [99mTc(CO)3(H2O)3]+.
mine (DIEA; 2.0 equiv), and CuI (0.2 equiv) in N,N-dimethylfor-
mamide (DMF). Cleavage of bound peptide 8 from the resin
with simultaneous side chain deprotection afforded bombesin
analogue 3. The resulting chelator, b-(1-peptidyl-1H-[1,2,3]tria-
zol-4-yl)Ala, is denoted as b1TA, where the number 1 indicates
the position to which the peptide chain is attached. This no-
menclature is unambiguous, considering the 1,4-regiospecifici-
ty of CuI-catalyzed alkyne/azide cycloaddition.
The [Cha13,Nle14]BBS(7–14) sequence was used for all ana-
logues.[38] Both substitutions stabilized the binding sequence
while retaining binding affinity. Either one or two bAla residues
were used as a spacer between the chelator and the binding
sequence to minimize the influence of the bulky radiometal
complex on binding affinity for the GRPR. It was also shown
that the (bAla)2 spacer in (NaHis)Ac-bAla-bAla-[Cha13,Nle14]BBS-
(7–14) (1) improved the biodistribution profile by improving
in vivo tumor uptake.[21]
Results and Discussion
Synthesis
The discovery by Tornøe[32] that the slow cycloaddition of an
azide and an alkyne can be accelerated by CuI catalysis, which
was primarily developed further by the Sharpless group,[33,34]
led to addition of an important new member to the family of
“click” reactions. In fact, the popularity of this reaction and the
constant use of the term “click” in this regard has led to the
common misuse of “click” to denote only the CuI-catalyzed re-
action between an azide and an alkyne.
We applied the click reaction of an azide and an alkyne to
synthesize two chelators as alternatives for the histidine-like
chelators (NaHis)Ac and (NeHis)Ac, which were previously devel-
oped by our research group and by Alberto et al., respective-
ly.[35,36] The resulting triazole ring is known to be a good ligand
for various transition metals.[37] We evaluated the synthesis of
these triazole-based chelators and their capacity for labeling
with the 99mTc(CO)3 core. The biodistribution of these triazole-
The second type of triazole chelator is obtained by CuI-cata-
lyzed alkyne/azide cycloaddition of Boc-bN3Ala-OH (16) and an
alkynoic acid (10 or 11), coupled to the resin-supported pep-
tide 9 (Scheme 2). This provides the b-(4-peptidyl-1H-[1,2,3]tria-
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Comparing Triazolyl-Based with (NaHis)Ac 99mTc Chelators
Scheme 2. Synthesis and radiolabeling of b4TA-containing bombesin analogues. a) 10 (3.0 equiv), DIC (3.0 equiv), HOBt (3.0 equiv), DMF, 2 h; b) 11 (3.0 equiv),
DIC (3.0 equiv), HOBt (3.0 equiv), DMF, 2 h; c) 16 (2.0 equiv), CuI (0.2 equiv), DIEA (2.0 equiv), DMF, 16 h; d) 10% TA/EDT (7:3) in TFA, 3 h; e) [99mTc(CO)3(H2O)3]+.
zol-1-yl)Ala chelator (4a/b), abbreviated as b4TA, due to the at-
tachment of the peptide chain to position four of the five-
membered ring. Boc-bN3Ala-OH (16), required for the synthesis
of the b4TA chelating system, was successfully prepared from
Boc-Dap-OH as described in literature.[39] A customized proce-
dure to remove the sulfonamide by-product by buffered ex-
traction was described by Lundquist et al.[40] for the purification
of a-azido acids, and was successfully applied to the synthesis
of Boc-bN3Ala-OH.
file,[28] we also prepared the glycated counterpart of b1TA ana-
logue 3. For this analogue, both chelator conjugation and gly-
cation were carried out by CuI-catalyzed alkyne/azide cycload-
dition (Scheme 3). Peptide 17, containing an alkyne, was Fmoc
deprotected and then “clicked” to N3-b-d-glucose(OAc)4 (18;
8.0 equiv), in the presence of DIEA (4.0 equiv) and CuI
(0.4 equiv), to provide compound 19. Subsequently, azidoacetic
acid was coupled to the peptide, yielding compound 20,
which was then “clicked” to Boc-Pra-OH (7). Compound 21 was
then cleaved from the resin and acetates on the glucose
moiety were removed by aminolysis in aqueous ammonia/
methanol to provide bombesin analogue 5 (see Table 1). These
“click” examples further illustrate the general potential for the
azide/alkyne cycloaddition reaction in bioconjugation applica-
tions.[41]
After coupling the simplest alkynoic acid, propynoic acid
(10), to the solid-supported peptide, yielding compound 12,
CuI-catalyzed cycloaddition was carried out successfully over-
night with Boc-bN3Ala-OH (16; 2.0 equiv), DIEA (2.0 equiv), and
CuI (0.2 equiv) in DMF. Cleavage of peptide 14 from the resin
yielded peptide 4a. However, the bombesin analogue contain-
ing the b4TA chelator 4a could only be labeled with the
99mTc(CO)3 core in ~60% yield. Moreover, the 99mTc(CO)3 com-
plex of 4a appeared to be unstable, likely because of the
lower electron density at the coordinating N2 of the triazole
ring, which is enhanced by the fact that the carbonyl group
forms a conjugated system with the triazole, rendering the
ring even more electron deficient.
In vitro and in vivo assays
Radiolabeling
The radiolabeling yields for [99mTc(CO)3]BBS analogues 3 and 5,
which contain the b1TA chelator, were higher than 95%, which
is similar to the labeling yield for the (NaHis)Ac chelator.[35] The
physicochemical properties of the labeled and unlabeled pep-
To increase the distance between the carbonyl and the tria-
zole ring, 4-pentynoic acid (11) was coupled to the resin
bound peptide 9 (Scheme 2). To
maintain a similar spacer length,
only one bAla residue was incor-
porated in this analogue. Subse-
quently, Boc-bN3Ala-OH (16;
2.0 equiv) was “clicked” to resin-
bound peptide 13 (1.0 equiv),
using catalytic quantities of CuI
(0.2 equiv) and DIEA (2.0 equiv)
in DMF. Despite our efforts, we
were not able to label com-
pound 4b in a high yield or
obtain the stable 99mTc(CO)3 com-
plex.
Because the glycated counter-
Scheme 3. Synthesis of (b1TA)Ac-Ala(NTG)-bAla-bAla[Cha13,Nle14]BBS(7–14): a) 20% piperidine/DMF, 2ꢃ10 min;
b) N3-b-d-glucose(OAc)4 (8.0 equiv), CuI (0.4 equiv), DIEA (4.0 equiv), DMF, 16 h; c) N3CH2COOH (3.0 equiv), DIC
(3.0 equiv), HOBt (3.0 equiv), DMF, 2 h; d) Boc-Pra-OH (2.0 equiv), CuI (0.2 equiv), DIEA (2.0 equiv), DMF, 16 h;
part 2 of (NaHis)Ac-bAla-bAla-
BBS(7–14)-NH2 (1) displayed an
improved biodistribution pro- e) 10% TA/EDT (7:3) in TFA, 3 h; f) 25% aqueous NH3/MeOH (1:1), [peptide]=2 mm, 16 h.
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D. Tourwꢀ et al.
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range. The binding affinity (KD) of analogues 1, 2, 3, and 5 is in
the sub-nanomolar range, indicating that replacement of the
(NaHis)Ac chelator with the b1TA chelator has no influence on
binding affinity. The affinities of radiolabeled compounds 4a
and 4b could not be determined, likely due to rapid decompo-
sition of the [99mTc(CO)3]b4TA complex in the in vitro assay con-
ditions used.[30] Therefore, no additional in vitro and in vivo
tests were carried out for these compounds. The finding that
the labeled analogues exhibited higher binding affinities than
their non-labeled counterparts is in agreement with previous
observations for bombesin and neurotensin analogues.[21,42,43]
Table 1. Analytical data for bombesin analogues 1–5.
Compd
Formula
Yield
[%][a]
Mr calcd
[Da]
ESIMS(+)
[M+H]+
HPLC
tR [min]
Purity
[%][b]
1
2
3
4a
4b
5
C61H90N18O14
C72H106N22O20
C57H84N18O13
C59H87N19O14
C58H86N18O13
C71H105N23O20
47
42
39
23
41
30
1298.7
1598.8
1228.6
1285.7
1242.7
1599.8
1299.4
1599.4
1229.2
1286.6
1243.4
1600.3
13.2
13.1
13.4
13.4
13.4
12.0
>98
>98
>98
>98
>98
>98
[a] After purification. [b] After preparative HPLC.
tides were sufficiently different to allow simple purification by
preparative HPLC. The [Re(CO)3] complexes of 3 and 5 were
prepared, and their identities were confirmed by mass spec-
trometry. In contrast, the labeling yields of analogues 4a and
4b, containing the isomeric b4TA, were lower than 60%. More-
over, these complexes were shown to be unstable (vide infra).
The lower labeling yields and stability of Tc(CO)3 complexes in-
volving the b4TA chelator (Scheme 2), compared with those
containing the b1TA chelator, have been previously reported
(see “inverse click” ligands in reference [30]). This has been at-
tributed to lower electron density at the coordinating N2 rela-
tive to that at the coordinating triazole N3 in the b1TA chela-
tor.[30] The electron density at N2 is even lower for 4a, due to
conjugation of the carbonyl group.
Internalization and efflux
Based on the internalization and externalization patterns
(Figure 2), there are no crucial differences between the ana-
logues. In general, all analogues rapidly internalized, reaching
a plateau after 60 min with ~25–35% of the applied dose
found inside the cells. From the internalization curves of
(NaHis)Ac-conjugated bombesin analogues 1 and 2, it can be
concluded that glycation has a positive influence on internali-
zation. The same trend was not observed for b1TA-containing
bombesin analogues 3 and 5. Blockade experiments using an
excess of native BBS(1–14) also showed that binding and inter-
nalization were very specific. In regard to externalization, no
profound differences were observed for analogues 1, 2, and 3;
~30–40% of the internalized activity remained in the cells after
5 h. The b1TA-containing, glycated analogue (5) had longer
cellular retention than the other analogues, with 60% of the
activity remaining in the cells 5 h after maximal internalization.
Metabolic stability studies
The stability of the new radiolabeled bombesin analogues was
determined in human plasma as described in the experimental
part, and was in agreement with the stability of our other
[Cha13,Nle14]BBS(7–14) peptide analogue.[21] The half-life was
determined to be 16 h.
Binding tests
The unlabeled bombesin analogues show high binding affinity
for GRP receptors, as shown in Table 2. All of the analogues
were able to displace the binding of 99mTc(CO)3-labeled
(NaHis)Ac-BBS(7–14) to GRP receptors in human prostate ade-
nocarcinoma PC-3 cells, exhibiting IC50 values in the nanomolar
Table 2. Binding to the GRPR in PC-3 cells of the labeled (KD) and unla-
beled (IC50) [Cha13,Nle14]BBS(7–14) analogues.
Compd
IC50 [nm]
KD [nm]
1[a]
2[b]
3
4a
4b
5
5.1Æ1.7
3.2Æ1.2
1.9Æ1.1
ND
4.2Æ0.7
5.2Æ0.1
0.18Æ0.12
0.29Æ0.16
0.19Æ0.06
ND[c]
ND[c]
0.60Æ0.27
[a] Data from Ref. [21], reported here as a reference value. [b] Data from
Ref. [27,28], reported here as a reference value. [c] Could not be deter-
mined due to instability of the complex.
Figure 2. Time course of a) internalization and b) externalization after maxi-
mal internalization for 99mTc(CO)3-labeled bombesin analogues 1, 2, 3, and 5
in PC-3 cells after incubation at 378C (n=2–3 in triplicate).
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Comparing Triazolyl-Based with (NaHis)Ac 99mTc Chelators
Biodistribution studies
Liver uptake was similar for analogues 1 and 3 at 0.5 h p.i.,
although analogue 3 had a slightly faster clearance rate. Liver
uptake for the glycated analogues was also very similar and
was very low at all time points. When the liver uptake for 2
and 5 is compared with unglycated analogues 1 and 3, it is
evident that glycation results in a significant decrease in liver
accumulation. The increase in hydrophilicity after glycation de-
creases hepatobiliary excretion, which explains the observed
decrease in liver uptake.
Animal experiments were performed in nude mice bearing PC-
3 tumor xenografts with bombesin analogues 3 and 5. The col-
lected data were compared with reference analogues 1 and 2
(Table 3). At 0.5 h post-injection (p.i.), blood activity was about
threefold higher for bombesin analogues 2, 3, and 5 than for
analogue 1, although these analogues had a faster rate of
clearance. For the unglycated analogues, kidney uptake was
higher for analogue 3 at 0.5 h p.i. alone. At later time points,
both analogues showed similar renal uptake, due to a faster
excretion of compound 3. For glycated analogues 2 and 5,
uptake in the kidneys was nearly identical for all time points,
with a relatively high early uptake followed by fast excretion.
Kidney uptake was higher for the glycated analogues than for
their unglycated counterparts at all time points, which can be
attributed to their higher hydrophilicity and correspondingly
lower hepatobiliary excretion. Nevertheless, kidney uptake of
all analogues decreased significantly over time, indicating that
the radioligand was not trapped in the kidneys.
Colon uptake and, more notably, pancreatic uptake, were
significant for all analogues. This was expected, as these
organs are known to be GRPR-positive in rodents.[4] Uptake in
both organs was successfully blocked by co-injection with
BBS(1–14) at 1.5 h p.i., demonstrating the high specificity of
in vivo uptake. Glycated analogues 2 and 5 were characterized
by higher uptake in receptor-positive organs. The presence of
the b1TA-containing chelator resulted in slower clearance.
Tumor uptake was similar for compounds 2 and 5, but
higher than for compounds 1 and 3 at all time points. The
b1TA glycated analogue (3) exhibited a higher tumor uptake
than compound 1, particularly at the earliest p.i. time point.
This would indicate that carbohydration has a greater impact
on tumor uptake than chelation.
Table 3. Biodistribution of [99mTc(CO)3]BBS analogues 1, 2, 3, and 5 in
nude mice bearing PC-3 xenografts.[a]
Compd Organs
Blood
0.5 h
1.5 h
5.0 h
1.5 h blocked
0.58Æ0.21 0.36Æ0.66 0.23Æ0.11
0.47Æ0.07
1.23Æ0.08
0.76Æ0.19
2.13Æ0.36
6.48Æ2.88
1.19Æ0.07
1.11Æ0.17
0.37Æ0.09
Stomach 1.37Æ0.62 0.73Æ0.47 0.60Æ0.33
Kidney 1.82Æ0.51 0.79Æ0.35 0.39Æ0.20
Tumor-to-tissue ratios
Tumor-to-blood ratios indicate the overall radioactive back-
ground due to circulation of the radiolabeled compound in
the blood, and are a valuable tool to compare the applicability
of various radiopharmaceuticals in tumor diagnosis. The
tumor-to-blood ratio for analogue 1 remained low at all p.i.
time points. In comparison, there was a remarkable increase in
the tumor-to-blood ratio for b1TA-containing bombesin coun-
terpart 3 during the same time. Glycated bombesins 2 and 5
had similar tumor-to-blood ratios, except at 1.5 h p.i., and ex-
hibited higher tumor-to-blood ratios relative to the unglycated
bombesins. This difference can be explained by the faster
blood clearance and higher tumor uptake resulting from glyca-
tion (Figure 3).
Liver
5.26Æ1.26 2.36Æ0.87 1.83Æ0.75
1[b]
Small Int 5.00Æ0.69 1.83Æ0.57 0.32Æ0.19
Pancreas 19.00Æ7.60 6.16Æ1.80 0.69Æ0.53
Colon
2.65Æ0.30 4.36Æ3.60 2.55Æ1.77
Tumor 1.53Æ0.26 0.80Æ0.35 0.41Æ0.27
Blood
1.54Æ0.30 0.18Æ0.01 0.06Æ0.01
0.19Æ0.08
0.28Æ0.10
0.84Æ0.40
1.59Æ0.31
0.87Æ0.13
0.80Æ0.27
0.40Æ0.15
1.02Æ0.44
Stomach 2.11Æ0.50 0.92Æ0.26 0.36Æ0.01
Kidney 4.86Æ1.10 1.00Æ0.11 0.35Æ0.07
Liver
4.05Æ0.81 1.15Æ0.17 0.38Æ0.06
3
Small Int 10.05Æ3.90 7.29Æ1.99 7.41Æ3.04
Pancreas 29.52Æ6.82 12.46Æ2.91 4.20Æ0.28
Colon
6.99Æ1.92 2.67Æ1.01 3.27Æ1.25
Tumor 3.76Æ1.47 1.20Æ0.75 0.80Æ0.44
Blood
1.77Æ0.16 0.34Æ0.11 0.07Æ0.01
0.49Æ0.15
0.47Æ0.11
2.06Æ0.71
0.98Æ0.80
3.64Æ1.15
3.33Æ0.28
0.74Æ0.07
1.01Æ0.09
Stomach 4.18Æ0.20 1.82Æ0.33 0.54Æ0.12
Kidney 7.48Æ0.53 2.65Æ0.80 0.95Æ0.05
Tumor-to-kidney ratios were generally lower than the other
ratios, due to the constant clearance of radiopharmaceuticals
through the renal pathway. Because of the higher tumor
uptake for analogues 2, 3, and 5 relative to bombesin 1, the
tumor-to-kidney values for the former are higher (Tu/Kiꢀ2 or
3) than the latter (Tu/Kiꢀ1) at 5.0 h p.i. (Figure 3).
Liver
1.74Æ0.07 0.64Æ0.33 0.22Æ0.01
2[c]
Small Int 8.51Æ1.49 4.55Æ1.24 1.77Æ0.46
Pancreas 63.17Æ8.23 26.96Æ2.53 3.70Æ1.15
Colon 12.93Æ1.88 7.31Æ1.22 6.43Æ0.79
Tumor 5.64Æ2.06 3.62Æ1.09 2.49Æ1.32
High tumor-to-liver ratios are preferable, as high uptake in
the liver may hamper imaging of abdominal lesions and meta-
stases. For compounds 1 and 3, tumor-to-liver ratios were
fairly low and similar (~1) at early time points, with a small in-
crease for compound 3 observed at 5.0 h p.i. The ratios for 2
and 5 were markedly higher at all time points than those for
unglycated analogues 1 and 3, and increased with time, likely
the result of increased hydrophilicity. This higher hydrophilicity
after glycation resulted in decreased hepatobiliary excretion
and, in turn, lower liver uptake and higher tumor uptake
(Figure 3).
Blood
1.40Æ0.39 0.19Æ0.06 0.08Æ0.01
0.16Æ0.03
0.04Æ0.96
1.63Æ0.20
0.77Æ0.08
1.32Æ0.32
Stomach 4.67Æ2.06 1.46Æ0.21 0.96Æ0.33
Kidney 6.36Æ0.81 2.20Æ0.60 0.89Æ0.15
Liver
0.96Æ0.09 0.31Æ0.05 0.23Æ0.07
5
Small Int 7.21Æ2.47 3.34Æ0.61 0.66Æ0.14
Pancreas 51.99Æ5.52 31.40Æ2.81 17.65Æ2.00 1.91Æ0.25
Colon
9.21Æ2.07 6.14Æ0.78 4.65Æ1.75
0.36Æ0.14
0.68Æ0.22
Tumor 4.97Æ1.14 4.41Æ0.55 2.61Æ0.81
[a] Dose: 3.7 MBq per mouse i.v.; animals in the blocked study received
an i.v. co-injection of 0.1 mg native BBS(1–14). Data are expressed as a
percentage of injected dose per gram of tissue ÆSD; n=3–4. [b] Data
from Ref. [21]. [c] Data from Ref. [28].
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D. Tourwꢀ et al.
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SPECT/CT images were acquired for compounds 1 (ungly-
cated, NaHis chelator), 2 (glycated, NaHis chelator), and 5 (gly-
cated, bT1A chelator) at 1.5 h p.i. The tumor xenografts were
clearly delineated for glycated analogues 2 and 5, (Figure 4b
and 4c), which all showed much lower accumulation in the
liver and gastrointestinal tract, due to their higher hydrophilici-
ty and decreased hepatobiliary excretion. In contrast, com-
pound 1 exhibited higher uptake in the abdominal area and
lower tumor uptake (Figure 4a), as observed in the biodistribu-
tion study. Similar imaging results were obtained for both gly-
cated analogues, indicating that the chelator has no influence
on pharmacokinetic properties.
Conclusions
The b-[1-(peptidyl)-1H-1,2,3-triazol-4-yl]Ala chelator, termed
b1TA, can be easily conjugated to a solid-supported peptide
sequence before labeling in high yield with the 99mTc(CO)3
core. Compared with the (NaHis)Ac chelator, the b1TA chelator
has a major advantage in that it does not contain a reactive
secondary amine, which may cause a selectivity problem
during the conjugation of other molecules to the peptide,
such as carbohydrates or fluorophores. In contrast, the second
type of triazole chelator, the b-[4-(peptidyl)-1H-1,2,3-triazol-1-
yl]Ala, or b4TA, chelator,b4TA although equally straightforward
to synthesize, did not form stable complexes with the
99mTc(CO)3 core and is therefore not suitable for radiolabeling.
No notable differences were observed in the in vitro proper-
ties of the studied analogues, except for the slightly lower ex-
ternalization of compound 5 despite a lower internalization.
With regard to biodistribution, only a few differences were
found for the b1TA-containing compounds relative to their
(NaHis)Ac-conjugated counterparts. More remarkable differen-
ces were observed after glycation, with both the b1TA-contain-
ing glycated analogue (5) and the (NaHis)Ac glycated analogue
(2) exhibiting better biodistribution, with higher tumor uptake
and lower liver uptake, as a result of the shift from hepatobili-
Figure 3. a) Tumor-to-blood, b) tumor-to-kidney, and c) tumor-to-liver ratios
at various p.i. time points for glycated and unglycated 99mTc(CO)3-labeled
bombesin analogues 1, 2, 3, and 5 in nude mice bearing PC-3 xenografts.
Figure 4. SPECT/CT imaging of nude mice with PC-3 tumor xenografts 1.5 h after injection of the radiolabeled BBS analogues (20 MBq, i.v.). The images corre-
spond to the coronal (left) and sagittal (right) sections for a) compound 1, b) compound 2, and c) compound 5.
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ChemMedChem 2010, 5, 1717 – 1725

Comparing Triazolyl-Based with (NaHis)Ac 99mTc Chelators
Synthesis of b1TA-CH2CO-bAla-[Cha13,Nle14]BBS(7–14)-NH2 (3).
The solid-supported bAlaGln(Trt)Trp(Boc)AlaValGlyHis(Trt)ChaNle
peptide sequence was synthesized as described in the solid-phase
peptide synthesis section. Azidoacetic acid (3.0 equiv)[44] was subse-
quently coupled to this peptide using DIC (3.0 equiv) and HOBt
(3.0 equiv) in DMF. After 2.0 h, the solvent and reagents were re-
moved by filtration, and the resin was washed with DMF, iPrOH,
and then DMF again. A solution of Boc-Pra-OH (2.0 equiv), CuI
(0.2 equiv), and DIEA (2.0 equiv) in DMF was added to the resin,
and the reaction mixture was allowed to shake for 16 h at 258C.
The solvent and reagents were removed by filtration, and the resin
was washed with DMF, iPrOH and Et2O and dried under reduced
pressure. Finally, peptide 3 was obtained following resin cleavage
and purification as described in the general procedure above.
ary to renal excretion. This led to bombesin analogues with
greater potential for future targeting of bombesin receptor-
positive tumors.
Experimental Section
The Supporting Information contains information on chemical sup-
pliers and equipment, an overview of methods used for compound
analysis and purification, and HPLC and MS data for the prepared
BBS analogues. Azidoacetic acid and trifluoromethanesulfonyl
azide were prepared as described.[40,44] Reference peptides 1 and 2
were prepared as previously described by our research group.[21,27]
Synthesis of b4TA-CO-bAla-bAla-[Cha13,Nle14]BBS(7–14)-NH2 (4a).
General protocol for peptide synthesis and purification
The
solid-supported
bAlabAlaGln(Trt)Trp(Boc)AlaValGlyHis-
(Trt)ChaNle peptide sequence was synthesized as described in the
solid-phase peptide synthesis section. Propynoic acid (3.0 equiv)
was subsequently coupled to this peptide using DIC (3.0 equiv)
and HOBt (3.0 equiv) in DMF. After 2.0 h, the solvent and reagents
were removed by filtration, and the resin was washed with DMF,
iPrOH, and then DMF again. Next, Boc-bN3Ala-OH (2.0 equiv), CuI
(0.2 equiv), and DIEA (2.0 equiv) in DMF were added to the resin.
The reaction mixture was allowed to shake for 16 h at 258C. The
solvent and reagents were removed by filtration, and the resin was
washed with DMF, iPrOH and Et2O and dried under reduced pres-
sure. Peptide 4a was obtained following resin cleavage and purifi-
cation as described in the general procedure above.
The peptide analogues were synthesized manually in plastic syring-
es (2.5–5 mL) with a PE frit (MultiSynTech GmbH, Germany) on Rink
amide polystyrene resin, according to the Fmoc peptide synthesis
protocol. The resin (0.60 mmolgÀ1) was weighed and swollen in
CH2Cl2 for 10 min. The solvent was removed by filtration, and the
resin was washed with DMF. The entire peptide sequence was syn-
thesized by consecutive Fmoc deprotections and amino acid cou-
plings. After each Fmoc deprotection and coupling step, solvent
and reagents were removed by filtration and by thorough washing
of the resin with DMF, iPrOH, and then DMF again. Fmoc deprotec-
tions were carried out in a 20% solution of piperidine/DMF (2ꢃ
10 min). Each amino acid coupling was performed by adding the
Fmoc protected amino acid (3.0 equiv), with DIC (3.0 equiv) and
HOBt (3.0 equiv), to the resin in DMF ([Fmoc-AA-OH]ꢀ0.5m). The
mixture was allowed to shake for 2 h. Coupling of Fmoc-Nle-OH to
the resin was performed twice. Completeness of the other cou-
plings was assessed by the ninhydrin test, and coupling was only
repeated for those reactions with positive test results.[45]
Synthesis of b4TA-CH2CH2CO-bAla-[Cha13,Nle14]BBS(7–14)-NH2
(4b). The synthesis of compound 4b differed only in the spacer
from the preparation of compound 4a. The spacer of compound
4b contains a pentynoic acid and only one bAla residue.
Synthesis of b1TA-CH2CO-Ala(NTG)-bAla-bAla-[Cha13,Nle14]BBS(7–
14)-NH2 (5). The solid-supported PrabAlabAlaGln(Trt)Trp-
(Boc)AlaValGlyHis(Trt)ChaNle peptide sequence was synthesized as
described in the solid-phase peptide synthesis section. Next, N3-b-
d-glucose(OAc)4 (8.0 equiv), CuI (0.4 equiv), and DIEA (4.0 equiv) in
DMF were added to the resin. The reaction mixture was allowed to
shake for 16 h at 258C, then the solvent and reagents were re-
moved by filtration, and the resin was washed with DMF, iPrOH,
and then DMF again. Azidoacetic acid (3.0 equiv) was subsequently
coupled to the peptide using DIC (3.0 equiv) and HOBt (3.0 equiv)
in DMF. After 2.0 h, the solvent and reagents were removed by fil-
tration, and the resin was washed with DMF, iPrOH, and then DMF
again. Then a solution of Boc-Pra-OH (2.0 equiv), CuI (0.2 equiv),
and DIEA (2.0 equiv) in DMF were added to the resin. The reaction
mixture was allowed to shake for 16 h at 258C. The solvent and re-
agents were removed by filtration, and the resin was washed with
DMF, iPrOH, and Et2O and dried under reduced pressure. After si-
multaneous deprotection of functional groups and cleavage of the
peptide from the resin, as described in the general procedure
above, the crude peptide was dissolved in a 25% aqueous NH3
(pH 9)/MeOH (1:1) solution and stirred for 24 h at 258C. The crude
reaction mixture was purified by preparative HPLC.
Peptides were cleaved from the Rink amide resin by the addition
of a TFA/thioanisole/ethanedithiol (90:7:3) mixture to the resin in
the plastic syringe (~0.3 mL per 100 mg resin). The mixture was al-
lowed to shake for 3 h at 258C before the resin was filtered. The fil-
trate was collected in a Falcon tube containing 10 mL cold Et2O,
which caused the peptide to precipitate. The tube was stored at
48C for 30 min, then the peptide was isolated by centrifugation
and decanting of the Et2O. Et2O was again added to the peptide in
the tube and the centrifugation and decantation processes were
repeated twice more. The crude peptide was then purified by
preparative HPLC.
Synthesis of Boc-bN3Ala-OH (17). TfN3 (2.0 equiv) was added drop-
wise to a solution of 213 mg Boc-Dap-OH (1.0 equiv), 216 mg
K2CO3 (1.5 equiv), and 2.6 mg CuSO4·5H2O (0.01 equiv) in 20 mL
H2O/MeOH (1:2). The reaction mixture stirred for 16 h, then the or-
ganic solvents were removed in vacuo and the remaining aqueous
solution was diluted with 50 mL H2O. The solution was acidified
with 37% HCl(aq) to pH 6. Next, 50 mL of 0.25m phosphate buffer
(pH 6.2, 6.8 g KH2PO4 and 0.88 g K2HPO4 in 200 mL H2O) were
added and the aqueous phase was extracted with EtOAc (4ꢃ
15 mL). The aqueous phase was acidified to pH 2 with 37% HCl(aq)
and extracted with CH2Cl2 (4ꢃ50 mL). The combined CH2Cl2 phases
were dried over MgSO4, filtered, and concentrated under reduced
pressure to obtain a colorless oil in 61% yield (147 mg). Rf =0.1
(EtOAc); 1H NMR (250 MHz, CDCl3): d=1.47 (s, 9H), 3.58–3.99 (m,
2H), 4.50–4.65 (m, 1H), 5.45 (d, 3JHH =7.1 Hz, 1H); 13C NMR
(63 MHz, CDCl3): d=28.2, 52.4, 53.3, 81.0, 155.4, 173.9; MS (ESI+)
m/z [M+H]+: 231.
Labeling procedures
99mTc(CO)3 labeling
Approximately 1 mL of a Na[99mTcO4] (~5 GBq) was eluted from a
generator and was added to the Isolink mixture, containing Na2-
(H3BCO2) (4.5 mg), borax (2.9 mg), K-Na tartrate tetrahydrate
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D. Tourwꢀ et al.
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(9 mg), and Na2CO3 (7–8 mg). This mixture was heated for 20 min
at 1008C and subsequently adjusted to pH 6.5 with a mixture of
HCl and phosphate buffer; 500 mL were withdrawn and combined
with 15–30 mL of the peptide solution (1 mm), following by heating
of the mixture at 758C for 1 h to enable labeling.
solubilized in 1n NaOH (2ꢃ400 mL). Radioactivity was measured by
g counter, and experiments were performed 2–3 times in triplicate.
Internalization
PC-3 cells at confluence were placed in 6-well plates. 24 h later,
they were incubated with labeled analogues (2–4 kBq) in culture
medium for 5, 15, 30, 60 and 120 min at 378C. To determine non-
specific binding, 1 mm natural BBS was added. After incubation, the
supernatant was collected and the cells were washed twice with
cold PBS. Surface-bound activity was removed by acid wash
(50 mm glycine/HCl, 100 mm NaCl, pH 2.8, 2ꢃ600 mL for 5 min) and
collected. Internalized activity was measured by lysing the cells
with 1n NaOH (2ꢃ600 mL). Radioactivity of all collected fractions
was measured by g counter. Results are expressed as percentage
of total added radioactivity per mg protein. Experiments were per-
formed 2–3 times in triplicate.
Re(CO)3 labeling
[ReBr3(CO)3][Et4N]2 (10À3 m in H2O, 200 mL) was added to a solution
of bombesin analogue 3 or 5 in H2O (10À3 m, 100 mL), and the solu-
tion was heated at 1008C for 1 h. HPLC analysis indicated complete
conversion of the starting material. The complexes were character-
ized by ESIMS: [Re(CO)3(3)]: C60H83N18O16Re; calcd m/z [M+H]+
=
1499.6; [M+2H]+/2=750.3, found 1499.7 and 750.3. [Re(CO)3(5)]:
C74H103N23O23Re; calcd m/z [M+H]+ =1868.7; [M+H]+/2=935.9,
found 935.9.
In vitro biological evaluation of peptides
Efflux
Cell line
For externalization studies, PC-3 cells at confluence were incubated
with labeled analogues (2–4 kBqwellÀ1) in culture medium for 1 h
at 378C to allow binding and maximal internalization. The superna-
tant was discarded, and the cells were washed twice with cold
PBS. New medium (2 mL) was added, and the cells were incubated
again at 378C for various times (30, 60, 150 and 300 min). The su-
pernatant was collected, and the cells were washed twice with
cold PBS. Internalized radioactivity was measured by lysing the
cells with 1n NaOH (2ꢃ600 mL). Radioactivity of the supernatant
(externalized activity) and of the lysate (internalized activity) was
determined by g counter. Results are expressed as a percentage of
the total activity associated with the cells. Experiments were per-
formed 2–3 times in triplicate.
Cells were maintained in DMEM with GLUTAMAX-I, supplemented
with 10% FCS, 100 IUmLÀ1 penicillin G sodium, 100 mgmLÀ1 strep-
tomycin sulfate, and 0.25 mgmLÀ1 amphotericin B. They were incu-
bated at 378C in an atmosphere containing 5% CO2 and were sub-
cultured twice weekly after detaching the cells with trypsin-EDTA.
Metabolic stability studies
The metabolic stability of the radiolabeled analogues was deter-
mined in human plasma. Each analogue (3–4 MBqmLÀ1, 1.5–
2 pmolmLÀ1) was incubated for different time periods (from 0–
24 h) at 378C. Following incubation, proteins were precipitated
with CH3CN/EtOH (1:1) and TFA (0.01%) and were centrifuged at
48C (10 min, 20000 g). The supernatant was filtered and analyzed
by RP-HPLC. Different peaks corresponding to the intact peptide
and the degradation products were observed in the radioactivity
chromatograms. The percentage of intact peptide was determined
for each incubation time.
In vivo biological evaluation of peptides
Biodistribution
All animal experiments were conducted in compliance with the
Swiss animal protection laws and with the ethical principles and
guidelines for scientific animal trials established by the Swiss Acad-
emy of Medical Sciences and the Swiss Academy of Natural Scien-
ces. Female CD-1 nu/nu mice 6–8 weeks old (Charles River Labora-
tories, Sulzfeld, Germany) were subcutaneously injected with 8ꢃ
106 PC-3 cells in 150 mL culture medium without supplements. Ap-
proximately three weeks after tumor implantation, the mice (3–6
per group) were injected i.v. with 3–4 MBq of the radiolabeled pep-
tide (0.2 pmol per mouse). At 0.5, 1.5, and 5 h p.i., the animals
were sacrificed by cervical dislocation. The organs (heart, lung,
spleen, kidneys, pancreas, stomach, small intestine, large intestine,
liver, muscle, bone), tumor xenografts, and blood were collected
and weighed, and the radioactivity was measured by g counter. To
determine the specificity of in vivo uptake, one group of mice re-
ceived a co-injection of 100 mg unlabeled natural BBS(1–14) and
the radiolabeled analogue and were sacrificed 1.5 h p.i. Results are
expressed as a percentage of injected dose per gram of tissue.
Binding assays
Inhibition studies
Intact human prostate adenocarcinoma PC-3 cells at confluence
were placed in 48-well plates. After 24 h, the cells were incubated
with increasing concentrations of BBS analogues (0–30000 nm), in
the presence of 4 kBq of 99mTc(CO)3-(NaHis)Ac-BBS(7–14) for 1 h at
378C. A specific binding buffer was used that includes protease in-
hibitors (50 mm HEPES, 125 mm NaCl, 7.5 mm KCl, 5.5 mm
MgCl·6H2O, 1 mm EGTA, 5 gLÀ1 BSA, 2 mgLÀ1 chymostatin,
100 mgLÀ1 soybean trypsin inhibitor, 50 mgLÀ1 bacitracin, pH 7.4).
Next, the cells were washed twice with cold PBS. Bound radioactiv-
ity was recovered by solubilizing the cells in 1n NaOH (2ꢃ400 mL)
at 378C, with radioactivity measured in a g counter. Experiments
were performed 2–3 times in triplicate.
Saturation studies
Imaging
Cells were placed in 48-well plates and, after 24 h, were incubated
with increasing concentrations of the labeled BBS analogues (0.01–
2 nm) for 1 h at 378C in the binding buffer described above. Non-
specific binding was determined by co-incubation with 1 mm natu-
ral BBS. After 1 h, the cells were washed twice with cold PBS and
Single-photon emission computed tomography/X-ray computed
tomography (SPECT/CT) images were collected postmortem, 1.5 h
after i.v. injection of the 99mTc(CO)3-labeled BBS (20 MBq). Images
were obtained on
a X-SPECT system (Gamma Medica, Inc.),
equipped with a single head SPECT device and a CT device. SPECT
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Comparing Triazolyl-Based with (NaHis)Ac 99mTc Chelators
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List of abbreviations
(NaHis)AcNa-carboxymethylhistidine
b1TA
b4TA
BBS
b-[1-(peptidyl)-1H-1,2,3-triazol-4-yl]Ala
b-[4-(peptidyl)-1H-1,2,3-triazol-1-yl]Ala
bombesin
Boc
tert-butyloxycarbonyl
cyclohexylalanine
Cha
Dap
DIC
2,3-diaminopropionic acid
diisopropylcarbodiimide
N,N-diisopropylethylamine
dimethylformamide
DIEA
DMF
EDT
Fmoc
GRP
HOBt
Nle
ethanedithiol
9-fluorenylmethyloxycarbonyl
gastrin-releasing peptide
1-hydroxybenzotriazole
norleucine
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NMB
PC-3
Pra
neuromedin B
human prostate adenocarcinoma cell line
propargylglycine
TA
thioanisole
TFA
trifluoroacetic acid
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TfN3
trifluoromethanesulfonyl azide
Acknowledgements
L.B. thanks the Institute for the Promotion of Innovation through
Science and Technology in Flanders (IWT-Vlaanderen) for grant
support. V.M. is a postdoctoral researcher for the Fund for Scien-
tific Research (Flanders). This work was funded by FWO grant
G.0036.04 and by Swiss Cancer League grant KLS 02040-02-2007.
Keywords: bombesin
radiochemistry · triazolyl · tumor targeting
·
chelators
·
click chemistry
·
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