Synthesis of DOTA-conjugated multimeric [Tyr3]octreotide peptides via a combination of Cu(I)-Catalyzed Click cycloaddition and thio acid/Sulfonyl azide Sulfo-Click amidation and their in vivo evaluation

Article abstract of DOI:10.1021/jm100246m

Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr ³]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition (click reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation (sulfo-click reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr ³]octreotide conjugate displayed the highest binding affinity (IC₅₀ = 1.32 nM) followed by dimeric [Tyr³]octreotide (2.45 nM), [DOTA⁰,Tyr³]octreotide (2.45 nM), and tetrameric [Tyr³]octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the ¹¹¹In-labeled monomeric [Tyr³]octreotide conjugate had the highest tumor uptake (42.3 ± 2.8 %ID/g) at 2 h p.i., which was better than [¹¹¹In-DOTA⁰,Tyr³]octreotide (19.5 ± 4.8 %ID/g). The ¹¹¹In-labeled dimeric [Tyr ³]octreotide conjugate showed a long tumor retention (25.3 ± 5.9 %ID/g at 2 h p.i. and 12.1 ± 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications.

Full text of DOI:10.1021/jm100246m

3944 J. Med. Chem. 2010, 53, 3944–3953
DOI: 10.1021/jm100246m
Synthesis of DOTA-Conjugated Multimeric [Tyr3]Octreotide Peptides via a Combination
of Cu(I)-Catalyzed “Click” Cycloaddition and Thio Acid/Sulfonyl Azide “Sulfo-Click”
Amidation and Their in Vivo Evaluation
Cheng-Bin Yim,†,‡ Ingrid Dijkgraaf,‡ Remco Merkx,† Cees Versluis,§ Annemarie Eek,‡ Gwenn E. Mulder,†
Dirk T. S. Rijkers,† Otto C. Boerman,‡ and Rob M. J. Liskamp*,†
†Department of Pharmaceutical Sciences, Faculty of Science, Division of Medicinal Chemistry and Chemical Biology,
Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands,
‡Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, Geert Grooteplein Zuid 8, 6525 GA Nijmegen,
The Netherlands, and §Biomolecular Mass Spectrometry and Proteomics Group, Department of Pharmaceutical Sciences,
Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received November 30, 2009
Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated
monomeric, dimeric, and tetrameric [Tyr3]octreotide-based analogues as a tool for tumor imaging and/
or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cyclo-
addition (“click” reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent
metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation (“sulfo-click” reaction). In a
competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr3]octreotide
conjugate displayed the highest binding affinity (IC50 = 1.32 nM) followed by dimeric [Tyr3]octreotide
(2.45 nM), [DOTA0,Tyr3]octreotide (2.45 nM), and tetrameric [Tyr3]octreotide (14.0 nM). Biodistribu-
tion studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the 111In-labeled
monomeric [Tyr3]octreotide conjugate had the highest tumor uptake (42.3 ( 2.8 %ID/g) at 2 h p.i., which
was better than [111In-DOTA0,Tyr3]octreotide (19.5 ( 4.8 %ID/g). The 111In-labeled dimeric [Tyr3]-
octreotide conjugate showed a long tumor retention (25.3 ( 5.9 %ID/g at 2 h p.i. and 12.1 ( 1.3 %ID/g at
24 h p.i.). These promising results can be exploited for therapeutic applications.
Introduction
has an enhanced stability toward proteolytic degradation, lead-
ing to a prolonged inhibitory effect in somatostatin-responsive
tumors.8 Several radiopharmaceuticals based on [Tyr3]octreo-
tide, functionalized with a diethylenetriamine pentaacetic acid
(DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA), are applied for imaging and treatment of neuro-
endocrine tumors in cancer patients.9-13
There is a growing interest in the development of multi-
valent receptor targeting ligands because they have higher
binding affinities than monovalent analogues.14-19 A promis-
ing approach toward polyvalent systems is the use of dendri-
mers as a versatile scaffold. Unique features, such as the
stepwise synthetic control of branching, the well-defined
monodisperse structure, the possibility of specifically tailored
peripheral groups, and the efficient and reproducible syn-
thesis, make dendrimers ideal molecular scaffolds for the simul-
taneous presentation of biologically relevant ligands.20-26 We
have described a versatile procedure using amino acid based
dendrimers, with a considerable degree of molecular diversity in
the dendrimer synthetic strategy, to design and synthesize multi-
valent carbohydrates27-30 and peptides.31-34
Somatostatin is a cyclic tetradecapeptide secreted in several
locations in the gastrointestinal system and throughout the
central nervous system. It plays an important regulatory role
in neurotransmission and secretion, and the peptide hormone is
known to control cell proliferation in normal tissues and
tumors.1-4 It elicits its biological effects via high-affinity inter-
actions with a family of five different somatostatin receptors
(SSTR1-5).5-7 Pathologically up-regulated receptors, such as
overexpression of especially SSTR2a in neuroendocrine tumors,
can be exploited in functional imaging techniques, allowing
radionuclide imaging of tumor lesions. The short plasma half-
life and consequent limited therapeutic potential of somatosta-
tin have been overcome with the availability of [Tyr3]octreotide,
a long-acting, synthetic octapeptide analogue of the naturally
occurring hormone. This chemically engineered [Tyr3]octreotide
*To whom correspondence should be addressed. Phone: þ31 30 253
7396. Fax: þ31 30 253 6655. E-mail: r.m.j.liskamp@uu.nl.
aAbbreviations: SSTR2, somatostatin subtype 2 receptor; DOTA,
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Fmoc, 9H-
fluoren-9-ylmethoxycarbonyl; TMOB, 2,4,6-trimethoxybenzyl; EDCI,
ethyl-3-(3-dimethylaminopropyl)carbodiimide; tBu, tert-butyl; Cbz,
benzyloxycarbonyl; TFA, trifluoroacetic acid; BOP, benzotriazoly-
loxy-tris(dimethylamino)phosphonium hexafluorophosphate; DMEM,
Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin;
PBS, phosphate buffered saline; IC50, half maximal inhibitory concen-
tration; CI, confidence interval; DMF, dimethylformamide; DCM,
dichloromethane; DMAP, 4-dimethylaminopyridine.
An important issue is the complete and efficient attachment of
ligands to dendrimers. In addition to other chemoselective
conjugation reactions,35-40 the Cu(I)-catalyzed variant41-44 of
the Huisgen 1,3-dipolar cycloaddition45 has led to a plethora of
biomedical applications in drug discovery, drug delivery, and
nanomedicine research involving peptidomimetics, biomaterials,
r
pubs.acs.org/jmc
Published on Web 04/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3945
Scheme 1. Click Chemistry Reactionsa
Figure 1. Chemical structure of N3-Ahx-D-Phe-cyclo(Cys-Tyr-
D-Trp-Lys-Thr-Cys)-Threol 1.
DOTA-conjugated mono-, di-, and tetrameric [Tyr3]octreotide
peptides.
a (A) Cu(I)-catalyzed 1,3-dipolar cycloaddition of azides and terminal
alkynes, yielding 1,4-disubstituted 1,2,3-triazoles (“click chemistry”).
(B) Thio acid/sulfonyl azide amidation reaction via a thiatriazoline
intermediate, leading to amide, nitrogen, and sulfur (“sulfo-click
chemistry”).
Thus, we describe a two-stage ligation procedure that com-
bines the established Cu(I)-catalyzed “click” reaction between
dendrimeric alkynes and peptidic azides, and the subsequent
copper-free thio acid/azide “sulfo-click” amidation reaction
between the obtained dendrimeric peptide thio acids and a
DOTA-derived sulfonyl azide. The receptor binding charac-
teristics of the resulting mono-, di-, and tetrameric [Tyr3]-
octreotide conjugates were determined in a competitive bind-
ing assay using AR42J tumor cells and compared to native
[Tyr3]octreotide. Biodistribution of 111In-labeled compounds
was evaluated in an in vivo study with BALB/c nude mice with
subcutaneous AR42J tumors. The uptake of the 111In-labeled
DOTA-functionalized dimeric [Tyr3]octreotide conjugate was
higher at 6 and 24 h after administration when compared
to the tumor uptake of [111In-DOTA0,Tyr3]octreotide. These
findings are promising results for novel therapeutic applica-
tions of [Tyr3]octreotide-based conjugates.
oligonucleotides, and carbohydrates.46-55 This reaction,
which regiospecifically gives 1,4-disubstituted 1,2,3-triazoles
(Scheme 1A), is particularly suitable for chemoselective bio-
conjugation reactions with unprotected peptide ligands to
dendrimers.31-34
Recently, wereported the successful synthesis of multimeric
[Tyr3]octreotide conjugates using the Cu(I)-catalyzed click
reaction between dendrimeric alkynes and azido peptides.34
However, a disadvantage of this approach is that during the
click reaction, copper could be chelated by DOTA, as was
indeed observed previously.34 This will hamper efficient label-
ing of the DOTA moiety with radioactive metal ions and
hampers (radiolabeling and) in vivo applications. To avoid
copper contamination, alternative copper-free “click” ap-
proaches are particularly relevant.56-58 In the recent literature
interesting methodologies toward metal-free triazole forma-
tion are described that involve strained cyclooctyne deriva-
tives and electron-deficient alkyne or oxanorbornadiene
moieties.59-66 However, these conjugation procedures
lack regioselectivity, which prevents precise control over
the product that is formed, and often suffer from poor
conversion or relatively lengthy reaction times and/or
formation of (noxious) undesired side products.
As an alternative metal-free “click” reaction, the chemose-
lective amidation reaction between thio acids and sulfonyl
azides is receiving increased attention as an important cou-
pling method. The reaction between thio acids and (sulfonyl)
azides was first mentioned in the literature in 1980,67 and it
was assumed that the reaction took place via reduction of the
azide tothe correspondingaminepriortoacylation bythe thio
acid, a very rapid but otherwise conventional, nucleophilic
substitution.68,69 Thorough mechanistic studies by Williams
et al.70,71 revealed that this amidation reaction proceeded via a
thiatriazoline intermediate rather than via acetylation of the
free amine (Scheme 1B). The potentially chemical orthogon-
ality of this reaction has led to a renewed interest in the thio
acid/sulfonyl azide amidation reaction as a valuable synthetic
tool for the modification/coupling of unprotected peptides in
solution or on solid support72-75 and the chemoselective
modulation of bioactive proteins for biomedical research.76,77
Encouraged by these promising results, we extended the applica-
tion of the thio acid/sulfonyl azide amidation, which we denote
as “sulfo-click”,78 to the metal-free introduction of a DOTA-
sulfonyl azide in multimeric [Tyr3]octreotide thio acids. This
resulted in a highly efficient synthesis and radiometal-labeling of
Results and Discussion
Chemistry. Syntheses of Cyclic [Tyr3]Octreotide Azides
and Dendrimeric Alkyne-Based Thio Esters. Cyclic [Tyr3]-
octreotide 1 with an N-terminal azide functionality (Figure 1)
was prepared on the 2-chlorotrityl chloride resin via Fmoc/tBu
solid phase chemistry, as was described previously.34
The combination of two orthogonal conjugation methods,
as outlined in the Introduction, requires a bifunctional scaf-
fold with a peripheral acetylene moiety to facilitate the Cu(I)-
catalyzed click reaction and a thio acid functionality to
initiate the thio acid/sulfonyl azide amidation reaction. We
have developed a modular approach using easily accessible
building blocks, with a considerable degree of molecular
diversity in monomer structure, for the synthesis of bifunc-
tional amino acid based dendrimers. This flexible synthetic
strategy, based on the dihydroxybenzoic acid moiety, can be
easily modified with a variety of branching units, rigidity,
cavity size, and tunable surface functionalities.79 Alkyne-
derived benzoic acids 3-5 (Scheme 2) were prepared follow-
ing a procedure that was previously described.31,32,34 The
thio acid moiety can be readily prepared from a 2,4,6-tri-
methoxybenzyl (TMOB) thio ester precursor, according to
Vetter.80 Hence, mono-, di-, and tetrameric dendrimeric
alkynes 6-8 with TMOB-protected thio acids were synthe-
sized from the corresponding carboxylic acids 3-5 and
TMOB-thiol 2by EDCI-mediatedesterification, as is depicted
in Scheme 2.
Conjugation of [Tyr3]Octreotide to Dendrimers by the
Copper-Catalyzed Click Cycloaddition Reaction Procedure.
The Cu(I)-catalyzed coupling of azide 1 to TMOB-alkynes
6, 7, and 8 was performed as previously reported.31,32,34

3946 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yim et al.
Scheme 2. Synthesis of TMOB-Protected Mono-, Di-, and Tetrameric Alkyne-Derived Thio Esters 6, 7, and 8, Respectively
Scheme 3. Synthesis of Mono-, Di-, and Tetrameric [Tyr3]Octreotide Triazoles 9, 10, and 11, Respectively
The synthetic route toward the corresponding mono-, di-,
and tetrameric [Tyr3]octreotide conjugates 9, 10, and 11,
respectively, is outlined in Scheme 3. Following our proce-
dure for microwave-assisted cycloaddition chemistry, azide 1
was reacted with acetylene 6, 7, or 8 in the presence of CuSO4/
sodium ascorbate in THF/H2O (1:1 v/v) under microwave
irradiation at 100 °C. HPLC analysis showed complete con-
version within 5 min. Optimization studies showed that using

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3947
50 mol % CuSO4, accompanied by 2.5 equiv of sodium ascor-
bate, gave the best results for the examined click reactions. The
identity of the cycloadducts 9-11 was confirmed by mass
spectrometry.
times, and workup procedures were studied to improve yields
for the synthesis of compound 13; however, the yield obtained
with BOP reagent was the highest in this optimization series.
Next, N-acylsulfonamides 14-16 were prepared from thio
esters 9-11 in three steps according to previously established
protocols.70-72 Treatment with TFA of mono-, di-, or tetra-
meric [Tyr3]octreotide thio ester 9, 10, or 11 released the
corresponding thio acid intermediate which was immediately
reacted with DOTA(tBu)sulfonyl azide 13 in the presence of
2,6-lutidine as a base (Scheme 5). This amidation process was
monitored by HPLC and was found to be complete within
1 h. The obtained N-acylsulfonamides with a tBu-protected
DOTA moiety were treated with TFA to give, after purifica-
tion by HPLC, mono-, di-, and tetrameric DOTA-conju-
gated [Tyr3]octreotide sulfonamides 14, 15, and 16 in a
decent yield of 67% per step. Figure 2 shows the ESI-MS
spectrum of dimeric [Tyr3]octreotide sulfonamide 15 with no
indication of additional Cu-DOTA complexes. A clear ad-
vantage of both the applied cycloaddition chemistry and the
thio acid/sulfonyl azide amidation reaction is the orthogo-
nality with functional groups in this peptide. This study has
therefore clearly demonstrated the compatibility of these
chemoselective coupling methods using unprotected peptide
derivatives leading to no metal contamination of the DOTA
moiety.
Conjugation of DOTA to Mono-, Di-, and Tetrameric
[Tyr3]Octreotide Triazoles by the Thio Acid/Sulfonyl Azide
Amidation Ligation Method. For use in the thio acid/sulfonyl
azide amidation reaction, DOTA was derivatized with a
sulfonyl azide linker. For this purpose the Cbz-protected
taurylsulfonyl azide 12 was used.81 The N-terminal Cbz func-
tionality of 12 was removed by treatment with HBr/glacial
acetic acid, and the hygroscopic hydrobromide was trans-
formed into the hydrochloride using an ion-exchange resin.
Subsequently, DOTA-Tris(tBu) was coupled using BOP to give
tBu-protected DOTA-derived sulfonyl azide 13 in 20% yield
(Scheme 4), after column chromatography. Several attempts
using different coupling reagents, concentrations, reaction
Scheme 4. Synthesis of tBu-Protected DOTA-Derived Sulfonyl
Azide 13
Radiolabeling. 111In-Labeling of the DOTA-conjugated
mono-, di-, and tetrameric [Tyr3]octreotide peptides 14-16
was performed in NH4OAc buffer (pH 5.5) with 111InCl3 by
Scheme 5. Synthesis of Mono-, Di-, and Tetrameric DOTA-Conjugated [Tyr3]Octreotide Sulfonamides 14, 15, and 16, Respectively

3948 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yim et al.
heating the reaction mixture for 10-15 min at 95 °C, and the
labeling process was monitored by radio-HPLC. The radio-
chemical yield was more than 98% with a specific activity of
7.4 MBq/μg for both 111In-labeled 14 (corresponding to 13.6
GBq/μmol) and 15 (22.7 GBq/μmol) and was 80% for 111In-
labeled 16 (43.3 GBq/μmol). These efficient labeling yields
are evidently the result of the current approach, which
comprises a significant synthetic improvement toward
DOTA-conjugated mono- and multimeric [Tyr3]octreotide
biomolecules without contamination by Cu-DOTA adducts
resulting from the remaining copper ions in a Cu(I)-cata-
lyzed “click” cycloaddition.34
AR42J tumor cells, using [111In-DOTA0,Tyr3]octreotide as a
radiotracer. The IC50 values were calculated from fitted
sigmoidal displacement curves as depicted in Figure 3A and
are summarized in Table 1. As a reference, commercially
available [DOTA0,Tyr3]octreotide was included as one of
the competing ligands in the same in vitro assay. The obtained
IC50 values for the AR42J cell line were in the low nanomolar
range, indicating the high binding affinity of the synthesized
compounds.
Both the monomeric [Tyr3]octreotide conjugate 14 and the
dimeric [Tyr3]octreotide conjugate 15 had IC50 values of 1.32
and 2.45 nM, respectively, comparable to that of the refer-
ence compound [DOTA0,Tyr3]octreotide (IC50 = 2.45 nM).
When the valency was increased from two to four [Tyr3]-
octreotide moieties (compound 16), a 5.7-fold decrease in
affinity (IC50 = 14.0 nM) was observed. The displacement
curve of conjugate 16 (Figure 3A) did not show a sigmoidal
shape, suggesting that the compound also bound aspecifi-
cally. The hypothesized increase in receptor affinity due to
multimerization was not observed, especially in the case of
[Tyr3]octreotide tetramer 16, which may be attributed to the
limited water solubility of these [Tyr3]octreotide conjugates.
To assess the binding properties of In-containing DOTA-
complexes, an additional binding experiment was performed
with 115In-labeled DOTA-conjugated compounds 14-16
using similar protocols. Overall, the IC50 values for all
115In-labeled compounds improved by a factor of 1.4-2.9
(Figure 3B and Table 1). This improvement in binding
strength was of importance and justified the use of these
compounds in in vivo experiments.
Lipophilicity Studies. The log(D) values of 111In-labeled
DOTA-conjugated mono-, di-, and tetrameric [Tyr3]octreotide
sulfonamides 14, 15, and 16 were -2.20 ( 0.17, -1.79 ( 0.11,
and -0.71 ( 0.01, respectively, and obtained from n-octanol/
PBS partition coefficient measurements. Compared to [111In-
DOTA0,Tyr3]octreotide (log(D) = -3.59 ( 0.14), 111In-labeled
compound 14 was more lipophilic reflected by the log D value
which was 25-fold higher. This could be attributed to the
aromatic character of the dendrimer-derived linker moiety.
Receptor Binding Studies. Binding affinities for the somato-
statin receptor of the DOTA-conjugated mono-, di-, and
tetrameric [Tyr3]octreotide peptides 14, 15, and 16, respec-
tively, were determined using a competitive binding assay with
Biodistribution Study. The in vivo characteristics and
tumor targeting of 111In-labeled 14-16 were investigated in
BALB/c nude mice with subcutaneous SSTR2 expressing
AR42J tumors. 111In-labeled [DOTA0,Tyr3]octreotide was
taken as the reference compound (see Supporting Informa-
tion Figure SI 11A and Table SI 11B), with tumor uptake of
19.5 ( 4.84 %ID/g at 2 h p.i. and 7.47 ( 0.78 %ID/g at 24 h
p.i. The organ distribution of 111In-labeled 14 and 15 after 2,
6, and 24 h p.i. is summarized in Figure 4 and in Table 2.
111In-Labeled compound 14 (Figure 4A) had a rapid and
high tumor uptake of 42.33 ( 2.80 %ID/g at 2 h p.i., and this
could be blocked by co-injection of an excess of octreotide, an
indication that tumor accumulation was a receptor-mediated
Figure 2. ESI-MS spectrum of dimeric [Tyr3]octreotide sulfonam-
ide 15: m/z found, 3070.0; calcd, 3069.3 (isotopic)/3071.6 (average).
Figure 3. (A) Competitive binding assay of mono-, di-, and tetrameric DOTA-conjugated systems of [Tyr3]octreotide 14 (0), 15 (4), and 16
(3), respectively, displacing [111In-DOTA0,Tyr3]octreotide on AR42J tumor cells (n = 2 for each data point). [DOTA0,Tyr3]octreotide (O) was
used as reference compound. (B) Competitive binding assay of mono-, di-, and tetrameric 115In-DOTA-conjugated systems of [Tyr3]octreotide
14 (9), 15 (2), and 16 (1), respectively, displacing [111In-DOTA0,Tyr3]octreotide on AR42J tumor cells. [115In-DOTA0,Tyr3]octreotide (b) was
used as reference compound. The error bars indicate the range of the 95% confidence interval.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3949
Table 1. IC50 Values for Competitive Binding Assay of Mono-, Di-, and
Tetrameric [0]DOTA- and [115In]DOTA-Conjugated Systems of [Tyr3]-
Octreotide Displacing [111In-DOTA0,Tyr3]Octreotide with AR42J Tumor
Cellsa
Table 2. Biodistribution of [111In]DOTA-Conjugated [Tyr3]Octreotide
Analogue 14 and [111In]DOTA-ConjugatedDimeric [Tyr3]Octreotide 15
in AR42J Tumor-Bearing BALB/c Nude Mice after 2, 6, and 24 h p.i.a
time after injection
competing ligand
IC50 (nM)
95% CI (nM)
2 h
6 h
24 h
2 h þ cold
DOTA
monomer 14
[Tyr3]Octreotide Monomer 14
1.32
2.45
14.0
2.45
1.10 - 1.57
2.14 - 2.81
4.30 - 45.5
2.26 - 2.65
dimer 15
tetramer 16
blood
tumor
0.27 ( 0.03
0.13 ( 0.06 0.04 ( 0.01 3.46 ( 1.28
42.33 ( 2.80 21.32 ( 6.85 10.75 ( 2.98 4.82 ( 0.87
[DOTA0,Tyr3]octreotide
pancreas 2.92 ( 0.43
adrenals 1.86 ( 0.58
0.63 ( 0.21 0.17 ( 0.03 1.07 ( 0.27
0.78 ( 0.41 0.63 ( 0.27 2.38 ( 0.53
[
115In]DOTA
monomer 14
dimer 15
tetramer 16
0.950
1.16
4.75
1.45
0.845 - 1.07
1.05 - 1.28
3.49 - 6.45
1.26 - 1.68
kidney
liver
54.19 ( 7.44 10.96 ( 4.62 2.53 ( 0.67 62.74 ( 15.28
0.52 ( 0.03
0.22 ( 0.06 0.09 ( 0.01 3.61 ( 1.21
6.11 ( 1.89 2.83 ( 0.76 2.16 ( 0.34
stomach 7.54 ( 2.22
[
115In-DOTA0,Tyr3]octreotide
[Tyr3]Octreotide Dimer 15
a n = 2 for each data point.
blood
tumor
1.52 ( 0.36
0.86 ( 0.11 0.17 ( 0.01 2.31 ( 0.30
25.31 ( 5.86 30.91 ( 6.16 12.14 ( 1.28 5.55 ( 0.43
pancreas 2.96 ( 0.09
adrenals 2.69 ( 0.37
1.69 ( 0.35 0.36 ( 0.02 1.18 ( 0.18
2.49 ( 0.43 1.27 ( 0.50 1.56 ( 0.50
kidney
liver
92.20 ( 13.08 65.61 ( 9.71 11.74 ( 3.31 57.78 ( 3.79
18.67 ( 1.66 14.92 ( 2.13 5.54 ( 0.90 22.67 ( 1.86
stomach 10.21 ( 2.26 7.78 ( 2.44 2.97 ( 0.35 3.46 ( 0.24
a Each value represents the mean % injected dose per gram of tissue (
SD of five animals (2 h þ cold: three animals). Mean tumor weight was
0.24 g.
blood clearance was very fast (0.27 ( 0.03 %ID/g at 2 h p.i. and
0.04 ( 0.01 %ID/g at 24 h p.i.) with the tumor/blood ratios
increasing from 156 ( 18.9 at 2 h p.i. to 268 ( 104 at 24 h p.i.
The higher tumor uptake of 111In-labeled compound 14 com-
pared to [111In-DOTA0,Tyr3]octreotide emphasizes the impor-
tance of the implemented sulfonamide linker and dendrimer-
derived core for tumor targeting.
111In-Labeled dimer [Tyr3]octreotide 15 showed a high
and specific tumor uptake of 25.31 ( 5.86 %ID/g at 2 h p.i.
(Figure 4B, Table 2). Interestingly, the activity in the tumor
remained high at 6 h p.i. (30.91 ( 6.16 %ID/g), which was an
indication for a prolonged retention of this compound in the
tumor. The tumoruptakeof111In-labeled 15 at6 h was similar
to the tumor uptake of the 111In-labeled [Tyr3]octreotide
monomer 14 (21.32 ( 6.85 %ID/g at 6 h p.i.) (P = 0.063).
Apart from the undesirable kidney accumulation, radi-
olabeled 15 had a high liver uptake (18.67 ( 1.66 %ID/g at
2 h p.i. to 5.54 ( 0.90 %ID/g at 24 h p.i.), which was
completely absent for radiolabeled 14 (0.52 ( 0.03 %ID/g
at 2 h p.i. to 0.09 ( 0.01 %ID/g at 24 h p.i.). However, from
the results of the octanol/PBS partition coefficient studies it
was expected that the increased lipophilic character of the
dimer 15 could have implications for liver uptake and kidney
accumulation.
Also, the in vivo tumor uptake of 111In-labeled dimeric
[Tyr3]octreotide conjugate 15 was significantly higher than
[
111In-DOTA0,Tyr3]octreotide at 6 and 24 h after administra-
tion (P < 0.05). From this, the dimeric [Tyr3]octreotide con-
jugate 15 showed interesting properties for radionuclide therapy
studies because of its long tumor retention. The receptor binding
affinity may be improved, and the undesirable kidney and liver
accumulation may be limited, by further optimizing the spacer
length and hydrophilic character.28,30 These considerations are
currently addressed in a study in which the effects of different
spacers are investigated.
Figure 4. Biodistribution of [111In]DOTA-conjugated [Tyr3]octreotide
analogue 14 (A) and [111In]DOTA-conjugated dimeric octreotide 15 (B)
in BALB/c nude mice bearing subcutaneous AR42J expressing tumors
in the right flank. Values are given as percentage of the injected dose per
gram of tissue (n = 5 mice/group). Blocking was performed by co-
injection of, respectively, 900 and 1500 molar excess of octreotide. Mice
were dissected at 2, 6, and 24 h p.i.
The biodistribution of 111In-labeled [Tyr3]octreotide tetra-
mer 16 indicated that this compound behaved quite differently
compared to its monomeric and dimeric counterparts, 14 and
15, respectively. Besides the high liver uptake, biodistribution
process. Lower, but specific, uptake was observed in the pan-
creas, stomach, and colon. Apart from the kidney, the tumor
was the tissue with the highest radioactivity concentration. Its

3950 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yim et al.
data of 111In-labeled 16 showed a high spleen uptake (Suppor-
ting Information Figure SI 12A and Table SI 12B), which could
be attributed to the rather lipophilic character and thus reduced
water solubility of 111In-labeled 16. Unfortunately, 111In-la-
beled [Tyr3]octreotide tetramer 16 showed negligible tumor
accumulation. On the basis of the octanol/PBS partition
coefficient, receptor binding properties, and tissue distribution
data, it became clear that tetramerization of [Tyr3]octreotide
had profound implications on its lipophilic character and
consequently on receptor affinity and tumor uptake.
on Merck precoated silica gel 60 F-254 (0.25 mm) plates. Spots
were visualized with UV light, ninhydrin, or Cl2/N,N,N0,N0-tetra-
methyl-4,40-diaminodiphenylmethane (TDM).83 Column chroma-
˚
tography was performed with Silicycle silica gel 60 A (particlesizeof
€
41-63 μm). Melting points were measured on a Buchi Schmelz-
punktbestimmungsapparat (according to Dr. Tottoli) and are
uncorrected. Microwave assisted reactions were carried out in a
Biotage initiator. H NMR spectra were recorded on a Varian
1
G-300 (300 MHz) spectrometer, and chemical shifts are given in
ppm (δ) relative to TMS (0.00 ppm). 13C NMR spectra were
recorded on a Varian G-300 (75.5 MHz) spectrometer, and chemi-
cal shifts are given in ppm (δ) relativetoCDCl3 (77.0 ppm). The13
C
NMR spectra were recorded using the attached proton test (APT)
sequence. (Semi)Preparative HPLC runs were performed on a
Kratos HPLC workstation, and analytical HPLC runs were carried
out on a Shimadzu HPLC system. Two buffer systems were used:
(I) buffer A consisting of 0.1% trifluoroacetic acid (TFA) in
CH3CN/H2O, 5:95 v/v, and buffer B consisting of 0.1% TFA in
CH3CN/H2O, 95:5 v/v, and (II) buffer C consisting of 0.1% TFA
in CH3OH/H2O, 20:80 v/v, and buffer D consisting of 0.1% TFA in
CH3OH/H2O, 95:5 v/v. Preparative HPLC runs were performed on
an Alltech Adsorbosphere XL C8 column (250 mm ꢀ 22 mm, pore
Conclusions
In the present study, we have demonstrated that the
combined approach using the Cu(I)-catalyzed 1,3-dipolar
cycloaddition between dendrimeric alkynes and peptidic
azides and subsequent copper-free thio acid/sulfonyl azide
amidation reaction with DOTA-derived sulfonyl azides is
a powerful method for the synthesis of DOTA-conjugated
mono- and multimeric [Tyr3]octreotide biomolecules.
Since the DOTA moiety was not contaminated with resi-
dual Cuþ/Cu2þ, radiolabeling with 111In3þ of the final com-
pounds was highly efficient, resulting in a specific activity of
up to 43 GBq/μmol.
In a competitive binding assay using AR42J tumor cells it
was shown that the IC50 values of mono- and multimeric
[Tyr3]octreotide-based conjugates were all in the low nanomo-
lar range. The monomeric [Tyr3]octreotide conjugate 14 dis-
played the highest binding affinity (IC50=1.32 nM) followed
by dimeric [Tyr3]octreotide 15 (2.45 nM), and tetrameric
[Tyr3]octreotide (14.0 nM). The effect of [Tyr3]octreotide
multimerization on its receptor-binding affinity was less than
expected, which may be attributed to the decreased water
solubility, especially in the case of tetramer 16. Thus, the high
lipophilic character of tetrameric [Tyr3]octreotide conjugate 16
could have compromised the receptor binding affinity of this
compound.
˚
size of 90 A, particlesizeof10μm) usingalineargradientofbufferB
(0-100% in 120 min) in buffer A at a flow rate of 12.0 mL/min, and
semipreparative HPLC runs were performed on an Alltech Alltima
˚
C8 column (250 mm ꢀ 10 mm, pore size of 90 A, particle size of
10 μm) using a linear gradient of buffer B (0-100% in 120 min) in
buffer A at a flow rate of 4.8 mL/min. Analytical runs were
performed on an Alltech Alltima C8 column (250 mm ꢀ 4.6 mm,
˚
pore size of 90 A, particle size of 5 μm) using a linear gradient
of buffer B (0-100% in 20 min) in buffer A at a flow rate of
1.0 mL/min or a linear gradient of buffer D (0-100% in 40 min) in
buffer C at a flow rate of 0.5 mL/min. The purity of all newly
synthesized compounds was judged by analytical HPLC and
was found to be at least 98%. Characterization of the com-
pounds was performed with electrospray ionization (ESI) mass
spectrometry on a Micromass LCT mass spectrometer calib-
rated with CsI using nano-ESI at 1200 V capillary voltage and
50 V at the sample cone.
Chemistry. The syntheses of N3-Ahx-D-Phe-cyclo(Cys-Tyr-D-
Trp-Lys-Thr-Cys)-Threol (1), 3-(prop-2-ynyloxy)benzoic acid
(3), 3,5-bis(prop-2-ynyloxy)benzoic acid (4), and 3,5-bis(2-(3,5-
bis(prop-2-ynyloxy)benzamido)ethoxy)benzoic acid (5) have
been described previously.34 The synthesis of 2,4,6-trimethox-
ybenzyl thiol (2) was performed as described by Vetter.80 Details
of the synthesis and characterization of compounds 7, 8, 10, 11,
and 16 are described in the Supporting Information.
S-2,4,6-Trimethoxybenzyl 3-(Prop-2-ynyloxy)benzothioate (6).
To a solution of 3 (100 mg, 568 μmol, 1.1 equiv) in dry DCM
(10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI) (149 mg, 775 μmol, 1.5 equiv) and 4-dimethyla-
minopyridine (DMAP) (4.0 mg, 33 μmol, 0.05 equiv) followed by
2 (112 mg, 516 μmol, 1.0 equiv) were added. After being stirred for
72 h at room temperature under an N2 atmosphere, the reaction
mixture was concentrated in vacuo. The yellow oily residue was
dissolved in DCM (50 mL) and washed with 1 N KHSO4, brine,
dried (Na2SO4), and concentrated in vacuo. The crude product
was purified by column chromatography and isolated as a white
solid in 39% yield (210 μmol, 75 mg). Rf(EtOAc/hexane, 2:8 v/v):
0.32. Mp: 84 °C. 1H NMR (300 MHz, CDCl3): δ = 7.61 (m, 1H),
7.55 (m, 1H), 7.33 (m, 1H), 7.14 (m, 1H), 6.13 (s, 2H), 4.72 (m, 2H),
4.40 (s, 2H), 3.81 (m, 9H), 2.53 (m, 1H). 13C NMR (75 MHz,
CDCl3): δ = 192.8, 161.2, 159.6, 157.8, 139.0, 129.8, 120.9, 120.4,
112.9, 104.8, 90.8, 78.3, 76.1, 56.2, 56.1, 55.6, 22.8.
111In-Labeled [Tyr3]octreotide conjugate 14 showed the
highest tumor uptake (42.3 ( 2.8 %ID/g at 2 h p.i.) in
BALB/c nude mice inoculated with sc AR42J tumors, which
was considerably higher than that for [111In-DOTA0,
Tyr3]octreotide (19.5 ( 4.8 %ID/g at 2 h p.i.). Radiolabeled
[Tyr3]octreotide dimer 15 exhibited a long tumor retention,
probably due to the bivalency effect, which implied that
dimeric [Tyr3]octreotide conjugate 15 could have interesting
properties for therapeutic and clinical applications.
Experimental Section
General Procedures. Materials and Analysis Methods. Unless
stated otherwise, chemicals were obtained from commercial
sources and used without purification. Peptide-grade solvents
were purchased from Biosolve and used directly except for
dimethylformamide (DMF), N-methylpyrrolidone (NMP),
˚
and dichloromethane (DCM), which were dried on 4 A mole-
cular sieves prior to (solid phase) synthesis. 2-Chlorotrityl chloride
resin was purchased from Tianjin Nankai Hecheng Science &
Technology Company Ltd., and NR-fluorenylmethyloxycarbonyl
(Fmoc) amino acids were purchased from Advanced ChemTech,
Alexis, or Novabiochem. Commercially available (Novartis)
octreotide (Sandostatin) was used as a receptor-selective ligand.
Solid phase synthesis was carried out in plastic syringes with a
polyethylene frit (20 μm) obtained from Applied Separations Inc.
The resin loading was determined by measuring the UV absorbance
of the piperidine-dibenzofulvene adduct (λmax = 300 nm).82 Thin
layer chromatography (TLC) and Rf values were determined
Monomeric [Tyr3]Octreotide Peptide Thio Ester (9). Alkyne
6 (3.0 mg, 7.9 μmol) and azido peptide 1 (9.3 mg, 7.9 μmol,
1.0 equiv) were dissolved in THF/H2O (1:1, v/v, 0.4 mL). To this
solution, aqueous CuSO4 (16 μL, 0.1 M, 1.6 μmol, 0.2 equiv) and
sodium ascorbate (16 μL, 0.5 M, 7.9 μmol, 1.0 equiv) were
added. The obtained reaction mixture was stirred for 5 min

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3951
under microwave irradiation (100 °C). Then the volatiles were
removed under reduced pressure and the residue was taken up in
CH3CN/H2O (1:1, v/v, 2.0 mL) and lyophilized. After purifica-
tion by semipreparative HPLC (C8) compound 9 was obtained
in 47% yield (3.7 μmol, 5.8 mg). tR(C8, buffer system I): 22.6
min. ESI-MS: m/z = 1546.63 [M þ H]þ, 1569.61 [M þ Na]þ,
773.83 [M þ 2H]2þ (C75H95N13O17S3, M = 1545.61).
gradient of buffer B (5-100% in 20 min) in buffer A (buffer A
consisting of 0.1% TFA in water; buffer B consisting of 0.1% TFA
in ethanol). Retention times were 17.3, 18.3, and 18.4 min for 14, 15,
and 16, respectively.
Determination of the Octanol-PBS Partition Coefficients. A
total of approximately 148 kBq (4 μCi, 3.7 MBq/μg) of the 111In-
labeled compounds 14, 15, and 16 in 500 μL of PBS (pH 7.4) was
mixed with 500 μL of n-octanol. The resulting biphasic system
was shaken vigorously for 2 min. The two layers were subse-
quently separated by centrifugation (5 min, 250g), and the
counts in 100 μL aliquots (n = 3) of both the organic and the
aqueous layers were measured in a γ-counter (Perkin-Elmer).
The partition coefficient (log(D)) was determined from at least
two independent experiments and calculated from the formula
log(D) = log10[(counts in octanol layer)/(counts in aqueous
layer)].
Receptor Binding Studies. The half maximal inhibitory con-
centration (IC50) of compounds 14, 15, and 16 for the SSTR2 on
AR42J tumor cells was determined in a competitive binding
assay, using [111In-DOTA0,Tyr3]octreotide as a tracer. Radi-
olabeling was performed as described above. AR42J cells were
seeded into six-well plates at 8 ꢀ 105 cells/well and cultured until
confluent. Cells were washed twice with binding buffer (DMEM
with 0.1% w/v bovine serum albumin). Subsequently, binding
buffer (1.5 mL), unlabeled peptide 14, 15, or 16 in a range from
0.1 to 300 nM, and 111In-labeled [Tyr3]octreotide (40 000 dpm)
were added to each well. After incubation at 37 °C for 3 h, the
medium was removed and cells were washed twice with binding
buffer and extracted from the wells, and cell-associated radio-
activity was determined in a γ-counter. For the “cold” labeling
of monomeric (14), dimeric (15), and tetrameric (16) [Tyr3]-
octreotide conjugates with 115In3þ, each of the peptides was
dissolved in an aqueous solution (25 μL, 10 mM NH4OAc).
Subsequently, a 3 molar excess of InCl3 (Aldrich Chemical Co.)
was added. The In(III) complexation was performed at 40 °C for
2 h.84 GraphPad Prism software (version 4.00 for Windows,
GraphPad Software, Inc.) was used to calculate IC50 values and
to determine statistical significance at the 95% confidence
interval with a P value of <0.05 being considered significantly
different.
Biodistribution. All animal experiments were conducted in
compliance with the animal welfare committee requirements of
our institution and performed according to national regula-
tions. The rat pancreatic AR42J tumor was grown in the right
flank of male BALB/c nude mice weighing 20-25 g (6-8 weeks)
by subcutaneous injection of 5 ꢀ 106 cells (0.2 mL) from cell
culture. After approximately 2 weeks, when tumors had a
diameter of 4-6 mm, mice were randomly divided into groups
of five mice each. Each group received an intravenous injection
via the tail vein with 0.37 MBq (0.1 μg) 111In-DOTA-labeled
[Tyr3]octreotide conjugate 14, 15, or 16. At 2, 6, or 24 h
postinjection (p.i.) the animals were euthanized by CO2/O2
asphyxiation. Blood, tumor, and tissues of interest were dis-
sected, weighed, and counted in a γ-counter along with three 100
μL aliquots of the diluted standard representing 1% of the
injected activity. From this the percentage injected dose per
gram (%ID/g) for each tissue was calculated. Additional groups
of three mice were co-injected intravenously with 50 μg of
octreotide (Sandostatin) and sacrificed after 2 h to determine
nonspecific binding of the radiotracers. Data are expressed as
the mean ( SD, calculated in Microsoft Excel. Statistical
analysis of biodistribution data used the unpaired t test (SPSS
Inc. software, version 16.0.01).
tBu-Protected DOTA-Derived Sulfonyl Azide (13). HCl H-
3
Tau-N3 (prepared from Cbz-Tau-N3 as described by Brouwer
et al.81) (205 mg, 1.11 mmol, 1.8 equiv) was dissolved in DCM
(5 mL). To this solution, DOTA(Tris-tBu) (354 mg, 618 μmol, 1.0
equiv) and benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) (331 mg, 749 μmol, 1.2 equiv) followed
by N,N-diisopropylethylamine (DIPEA) (305 μL, 1.75 mmol, 2.8
equiv) were added. The reaction mixture was stirred at room
temperature overnight and subsequently diluted with DCM (50
mL). This solution was washed with 5% NaHCO3 (3 ꢀ 50 mL) and
brine (50 mL), dried (Na2SO4), and concentrated in vacuo. Purifica-
tion of compound 13 by column chromatography required two runs
(DCM/MeOH, 95:5 v/v). Compound 13 was isolated in 20% yield
(124 μmol, 87 mg). Rf(DCM/MeOH, 95:5 v/v): 0.29. Mp: 86 °C. 1H
NMR (300 MHz, CDCl3): δ = 6.73 (m, 1H), 3.75 (m, 2H), 3.58 (m,
2H), 3.55-1.90 (broad m, 24H), 1.47 (s, 27H). 13C NMR (75 MHz,
CDCl3): δ = 172.8, 82.3, 82.2, 55.9, 54.2, 34.1, 28.2.
Monomeric [Tyr3]Octreotide DOTA-Conjugated Sulfonamide
(14). Triazole 9 (2.5 mg, 1.6 μmol) was treated with TFA/
triisopropylsilane (TIS) (95:5 v/v, 400 μL) for 3 h at room
temperature. After concentration in vacuo, the residue was
dissolved in dry DMF (120 μL) and sulfonyl azide 13 (1.1 mg,
1.6 μmol, 1.0 equiv) followed by 2,6-lutidine (1.0 μL, 8.5 μmol,
5.2 equiv) were added. The reaction mixture was stirred for 1 h at
room temperature and subsequently concentrated in vacuo. This
residue was treated with TFA/TIS/H2O (95:2.5:2.5, v/v/v) for 3 h at
room temperature. Compound 14 was obtained in 25% yield
(0.40 μmol, 0.76 mg) after purification by semipreparative HPLC
(C8). tR(C8, buffer system I): 18.28 min. ESI-MS: m/z = 921.87
[M þ 2H]2þ, 614.90 [M þ 3H]3þ (C83H115N19O23S3, M= 1841.76).
Dimeric [Tyr3]Octreotide DOTA-Conjugated Sulfonamide
(15). The synthesis of compound 15 was performed as described
for compound 14 starting from triazole 10 (3.9 mg, 1.4 μmol)
and sulfonyl azide 13 (1.0 mg, 1.4 μmol, 1.0 equiv). Compound 15
was obtained in 28% yield (0.40 μmol, 1.2 mg) after purification
by semipreparative HPLC (C8). tR(C8, buffer system I):18.53 min.
ESI-MS: m/z = 1536.31 [M þ 2H]2þ, 1024.00 [M þ 3H]3þ, 768.51
[M þ 4H]4þ (C141H192N32O36S5, M = 3071.55).
Cell Culture. The AR42J cell line (rat tumor of the exocrine
pancreas) was a generous gift from Erasmus University Rotter-
dam, The Netherlands. These cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco) supplemented with
10% fetal calf serum, 1% penicillin/streptomycin, and 1%
glutamine. Cells were grown in a humidified atmosphere with
5% CO2 at 37 °C.
Radiolabeling. DOTA-conjugated compounds 14, 15, and 16
were labeled with 111InCl3 (Covidien) in 0.25 M NH4OAc
solution (pH 5.5) for 15 min at 95 °C. Labeling reactions were
performed in acid-washed Protein LoBind safe-lock tubes
(Eppendorf GmbH). Radiochemical purity was determined by
instant thin-layer chromatography (ITLC) on silica gel (Tec-
Control chromatography strips, Biodex Medical Systems,
Inc.). The ITLC analysis was performed using two different mobile
phases: 0.1 M EDTA/0.1 M NH4OAc (1:1, v/v; Rf(bound 111In) =
0; Rf(unbound 111In) = 1) and THF/0.25 M NH4OAc (1:1, v/v;
Rf(colloid) = 0; Rf(unbound 111In or 111In-labeled compound) >
0.5). Radiochemically pure (>99%) tracers were obtained by
preparative RP-HPLC prior to use for partition coefficient mea-
surements (log(D)) and animal experiments. Preparative isolation
of 111In-labeled compounds were carried out on an Agilent HPLC
system with an in-line NaI radiodetector (Raytest GmbH) using a
Acknowledgment. The authors thank Bianca Lemmers
and Kitty Lemmens for animal handling/care during in vivo
experiments. Professor Marion de Jong (Erasmus Medical
Center Rotterdam, The Netherlands) is acknowledged for
providing the rat pancreatic AR42J tumor cell line, and Lieke
˚
Zorbax Eclipse C8 column (150 mm ꢀ 4.6 mm, pore size: of 300 A,
particle size of 5 μm) at a flow rate of 1.0 mL/min using a linear

3952 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Yim et al.
Joosten is thanked for technical assistance throughout the in
vitro experiments.
(23) Wolinsky, J. B.; Grinstaff, M. W. Therapeutic and diagnostic
applications of dendrimers for cancer treatment. Adv. Drug Deli-
very Rev. 2008, 60, 1037–1055.
(24) Villalonga-Barber, C.; Micha-Screttas, M.; Steele, B. R.; Georgo-
poulos, A.; Demetzos, C. Dendrimers as biopharmaceuticals:
synthesis and properties. Curr. Top. Med. Chem. 2008, 8, 1294–
1309.
Supporting Information Available: Synthesis and character-
1
ization of compounds 7, 8, 10, 11, and 16; H and 13C NMR
spectra of TMOB-thio esters 6-8 and DOTA-derived sulfonyl
azide 13; HPLC and high resolution ESI-MS analyses of
triazoles 9-11 and DOTA-conjugated [Tyr3]octreotide com-
pounds 14-16; biodistribution data of 111In-labeled tetrameric
[Tyr3]octreotide 16 and [111In-DOTA0,Tyr3]octreotide. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(25) Tekade, R. K.; Kumar, P. V.; Jain, N. K. Dendrimers in oncology:
an expanding horizon. Chem. Rev. 2009, 109, 49–87.
(26) Medina, S. H.; El-Sayed, M. E. H. Dendrimers as carriers for
delivery of chemotherapeutic agents. Chem. Rev. 2009, 109, 3141–
3157.
(27) Autar, R.; Khan, A. S.; Schad, M.; Hacker, J.; Liskamp, R. M. J.;
Pieters, R. J. Adhesion inhibition of F1C-fimbriated Escherichia
coli and Pseudomonas aeruginosa PAK and PAO by multivalent
carbohydrate ligands. ChemBioChem 2003, 4, 1317–1325.
(28) Joosten, J. A. F.; Loimaranta, V.; Appeldoorn, C. C. M.; Haataja,
S.; Ait El Maate, F.; Liskamp, R. M. J.; Finne, J.; Pieters, R. J.
Inhibition of Streptococcus suis adhesion by dendritic galabiose
compounds at low nanomolar concentration. J. Med. Chem. 2004,
47, 6499–6508.
(29) Joosten, J. A. F.; Tholen, N. T. H.; Ait El Maate, F.; Brouwer,
A. J.; van Esse, G. W.; Rijkers, D. T. S.; Liskamp, R. M. J.; Pieters,
R. J. High-yielding microwave-assisted synthesis of triazole-linked
glycodendrimers by copper-catalyzed [3 þ 2] cycloaddition. Eur. J.
Org. Chem. 2005, 3182–3185.
(30) Branderhorst, H. M.; Liskamp, R. M. J.; Visser, G. M.; Pieters,
R. J. Strong inhibition of cholera toxin binding by galactose
dendrimers. Chem. Commun. 2007, 5043–5045.
(31) Rijkers, D. T. S.; van Esse, G. W.; Merkx, R.; Brouwer, A. J.;
Jacobs, H. J. F.; Pieters, R. J.; Liskamp, R. M. J. Efficient
microwave-assisted synthesis of multivalent dendrimeric peptides
using cycloaddition reaction (click) chemistry. Chem. Commun.
2005, 4581–4583.
(32) Dijkgraaf, I.; Rijnders, A. Y.; Soede, A.; Dechesne, A. C.; van Esse,
G. W.; Brouwer, A. J.; Corstens, F. H. M.; Boerman, O. C.;
Rijkers, D. T. S.; Liskamp, R. M. J. Synthesis of DOTA-conju-
gated multivalent cyclic-RGD peptide dendrimers via 1,3-dipolar
cycloaddition and their biological evaluation: implications for
tumor targeting and tumor imaging purposes. Org. Biomol. Chem.
2007, 5, 935–944.
(33) Arnusch, C. J.; Branderhorst, H. M.; de Kruijff, B.; Liskamp,
R. M. J.; Breukink, E.; Pieters, R. J. Enhanced membrane pore
formation by multimeric/oligomeric antimicrobial peptides. Bio-
chemistry 2007, 46, 13437–13442.
(34) Yim, C.-B.; Boerman, O. C.; de Visser, M.; de Jong, M.; Dechesne,
A. C.; Rijkers, D. T. S.; Liskamp, R. M. J. Versatile conjugation of
octreotide to dendrimers by cycloaddition (“click”) chemistry to
yield high-affinity multivalent cyclic peptide dendrimers. Biocon-
jugate Chem. 2009, 20, 1323–1331.
(35) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Synthesis of proteins by native chemical ligation. Science 1994,
266, 776–779.
(36) Dawson, P. E.; Kent, S. B. H. Synthesis of native proteins by
chemical ligation. Annu. Rev. Biochem. 2000, 69, 923–960.
(37) Saxon, E.; Bertozzi, C. R. Cell surface engineering by a modified
Staudinger reaction. Science 2000, 287, 2007–2010.
(38) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. A “traceless” Staudin-
ger ligation for the chemoselective synthesis of amide bonds. Org.
Lett. 2000, 2, 2141–2143.
(39) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E. Nucleo-
philic catalysis of hydrazone formation and transimination: im-
plications for dynamic covalent chemistry. J. Am. Chem. Soc. 2006,
128, 15602–15603.
(40) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Nucleophilic catalysis
of oxime ligation. Angew. Chem., Int. Ed. 2006, 45, 7581–7584.
(41) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed
1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org.
Chem. 2002, 67, 3057–3064.
References
(1) Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Butcher, M.; Rivier, J.;
Guillemin, R. Hypothalamic polypeptide that inhibits the secretion
of immunoreactive pituitary growth hormone. Science 1973, 179,
77–79.
(2) Reichlin, S. Somatostatin. N. Engl. J. Med. 1983, 309, 1495–1501.
(3) Reichlin, S. Somatostatin (second of two parts). N. Engl. J. Med.
1983, 309, 1556–1563.
(4) Weckbecker, G.; Lewis, I.; Albert, R.; Schmid, H. A.; Hoyer, D.;
Bruns, C. Opportunities in somatostatin research: biological,
chemical and therapeutic aspects. Nat. Rev. Drug Discovery 2003,
2, 999–1017.
(5) Yamada, Y.; Post, S. R.; Wang, K.; Tager, H. S.; Bell, G. I.; Seino,
S. Cloning and functional characterization of a family of human
and mouse somatostatin receptors expressed in brain, gastrointest-
inal tract, and kidney. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 251–
255.
(6) Meyerhof, W. The elucidation of somatostatin receptor functions:
a current view. Rev. Physiol. Biochem. Pharmacol. 1998, 133, 55–
108.
(7) Patel, Y. C. Somatostatin and its receptor family. Front. Neuroen-
docrinol. 1999, 20, 157–198.
(8) Bauer, W.; Briner, U.; Doepfner, W.; Haller, R.; Huguenin, R.;
Marbach, P.; Petcher, T. J.; Pless, J. SMS 201-995: a very potent
and selective octapeptide analogue of somatostatin with prolonged
action. Life Sci. 1982, 31, 1133–1140.
(9) Lamberts, S. W. J.; Krenning, E. P.; Reubi, J.-C. The role of
somatostatin and its analogs in the diagnosis and treatment of
tumors. Endocr. Rev. 1991, 12, 450–482.
(10) Froidevaux, S.; Eberle, A. N. Somatostatin analogs and radio-
peptides in cancer therapy. Biopolymers (Pept. Sci.) 2002, 66, 161–
183.
(11) Kaltsas, G. A.; Papadogias, D.; Makras, P.; Grossman, A. B.
Treatment of advanced neuroendocrine tumours with radiola-
belled somatostatin analogues. Endocr.-Relat. Cancer 2005, 12,
683–699.
(12) Forrer, F.; Valkema, R.; Kwekkeboom, D. J.; de Jong, M.;
Krenning, E. P. Peptide receptor radionuclide therapy. Best Pract.
Res., Clin. Endocrinol. Metabol. 2007, 21, 111–129.
(13) de Jong, M.; Breeman, W. A. P.; Kwekkeboom, D. J.; Valkema, R.;
Krenning, E. P. Tumor imaging and therapy using radiolabeled
somatostatin analogues. Acc. Chem. Res. 2009, 42, 873–880.
(14) Varki, A. Biological roles of oligosaccharides: all of the theories are
correct. Glycobiology 1993, 3, 97–130.
(15) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Polyvalent interac-
tions in biological systems: implications for design and use of
multivalent ligands and inhibitors. Angew. Chem., Int. Ed. 1998,
37, 2754–2794.
(16) Wester, H.-J.; Kessler, H. Molecular targeting with peptides or
peptide-polymer conjugates: just a question of size. J. Nucl. Med.
2005, 46, 1940–1945.
(17) Tweedle, M. F. Adventures in multivalency: the Harry S. Fischer
memorial lecture. Contrast Media Mol. Imaging 2006, 1, 2–9.
~
(18) Martos, V.; Castreno, P.; Valero, J.; de Mendoza, J. Binding to
(42) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed regio-
selective “ligation” of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 2002, 41, 2596–2599.
protein surfaces by supramolecular multivalent scaffolds. Curr.
Opin. Chem. Biol. 2008, 12, 698–706.
(19) Pieters, R. J. Maximising multivalency effects in protein-
carbohydrate interactions. Org. Biomol. Chem. 2009, 7, 2013–
2025.
(43) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. CuI-catalyzed
alkyne-azide “click” cycloadditions from a mechanistic and syn-
thetic perspective. Eur. J. Org. Chem. 2006, 51–68.
(20) Boas, U.; Heegaard, P. M. H. Dendrimers in drug research. Chem.
Soc. Rev. 2004, 33, 43–63.
(21) Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka, F. C. Designing
(44) Meldal, M.; Tornøe, C. W. Cu-catalyzed azidealkyne cycloaddi-
tion. Chem. Rev. 2008, 108, 2952–3015.
(45) Huisgen, R. 1,3-Dipolar Cycloadditions. Introduction, Survey,
Mechanism. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A.,
Ed.; Wiley: New York, 1984; Vol. 1, pp 1-176.
ꢀ
dendrimers for biological applications. Nat. Biotechnol. 2005, 23,
1517–1526.
(22) Yang, H.; Kao, W. J. Dendrimers for pharmaceutical and biome-
dical applications. J. Biomater. Sci., Polym. Ed. 2006, 17, 3–19.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3953
(46) Kolb, H. C.; Sharpless, K. B. The growing impact of click
chemistry on drug discovery. Drug Discovery Today 2003, 8,
1128–1137.
(47) Dedola, S.; Nepogodiev, S. A.; Field, R. A. Recent applications of
the CuI-catalysed Huisgen azide-alkyne 1,3-dipolar cycloaddition
reaction in carbohydrate chemistry. Org. Biomol. Chem. 2007, 5,
1006–1017.
Metal-free triazole formation as a tool for bioconjugation. Chem-
BioChem 2007, 8, 1504–1508.
(66) van Berkel, S. S.; Dirks, A. J.; Meeuwissen, S. A.; Pingen, D. L. L.;
Boerman, O. C.; Laverman, P.; Delft, F. L.; Cornelissen, J. J. L. M.;
Rutjes, F. P. J. T. Application of metal-free triazole formation in
the synthesis of cyclic RGD-DTPA conjugates. ChemBioChem
2008, 9, 1805–1815.
(48) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba,
G.; Genazzani, A. A. Click chemistry reactions in medicinal
chemistry: applications of the 1,3-dipolar cycloaddition between
azides and alkynes. Med. Res. Rev. 2008, 28, 278–308.
(49) Moorhouse, A. D.; Moses, J. E. Click chemistry and medicinal
chemistry: a case of “cyclo-addiction. ChemMedChem 2008, 3,
715–723.
(50) Lutz, J.-F.; Zarafshani, Z. Efficient construction of therapeutics,
bioconjugates, biomaterials and bioactive surfaces using azi-
de-alkyne “click” chemistry. Adv. Drug Delivery Rev. 2008, 60,
958–970.
(51) Hein, C. D.; Liu, X.-M.; Wang, D. Click chemistry, a powerful tool
for pharmaceutical sciences. Pharm. Res. 2008, 25, 2216–2230.
(52) Nwe, K.; Brechbiel, M. W. Growing applications of “click chem-
istry” for bioconjugation in contemporary biomedical research.
Cancer Biother. Radiopharm. 2009, 24, 289–302.
(53) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.;
Finn, M. G.; Fokin, V. V.; Sharpless, K. B.; Hawker, C. J. Multi-
valent, bifunctional dendrimers prepared by click chemistry. Chem.
Commun. 2005, 5775–5777.
(54) Gopin, A.; Ebner, S.; Attali, B.; Shabat, D. Enzymatic activation of
second-generation dendritic prodrugs: conjugation of self-immo-
lative dendrimers with poly(ethylene glycol) via click chemistry.
Bioconjugate Chem. 2006, 17, 1432–1440.
(55) Carlmark, A.; Hawker, C.; Hult, A.; Malkoch, M. New methodol-
ogies in the construction of dendritic materials. Chem. Soc. Rev.
2009, 38, 352–362.
(56) Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Click chemistry
beyond metal-catalyzed cycloaddition. Angew. Chem., Int. Ed.
2009, 48, 4900–4908.
(57) Singh, I.; Vyle, J. S.; Heaney, F. Fast, copper-free click chemistry: a
convenient solid-phase approach to oligonucleotide conjugation.
Chem. Commun. 2009, 3276–3278.
(67) Hakimelahi, G. H.; Just, G. A simple synthesis of 2,2-disubstituted
tetrahydrothiophenes. Tetrahedron Lett. 1980, 21, 2119–2122.
(68) Rosen, T.; Lico, I. M.; Chu, D. T. W. A convenient and highly
chemoselective method for the reductive acetylation of azides.
J. Org. Chem. 1988, 53, 1580–1582.
ꢀ
(69) Rakommanomana, N.; Lacombe, J.-M.; Pavia, A. A. Reduction-
ꢀ
ꢀ
ꢀ
acetylation selective des azido-sucres par le melange acide thioace-
ꢀ
tique-thioacetate de potassium. Carbohydr. Res. 1990, 197, 318–
323.
(70) Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J.
The reaction of thio acids with azides: a new mechanism and new
synthetic applications. J. Am. Chem. Soc. 2003, 125, 7754–7755.
(71) Kolakowski, R. V.; Shangguan, N.; Sauers, R. R.; Williams, L. J.
Mechanism of thio acid/azide amidation. J. Am. Chem. Soc. 2006,
128, 5695–5702.
(72) Merkx, R.; Brouwer, A. J.; Rijkers, D. T. S.; Liskamp, R. M. J.
Highly efficient coupling of β-substituted aminoethane sulfonyla-
zides with thioacids, towards a new chemical ligation reaction. Org.
Lett. 2005, 7, 1125–1128.
(73) Barlett, K. N.; Kolakowski, R. V.; Katukojvala, S.; Williams, L. J.
Thio acid/azide amidation: an improved route to N-acyl sulfona-
mides. Org. Lett. 2006, 8, 823–826.
(74) Merkx, R.; van Haren, M. J.; Rijkers, D. T. S.; Liskamp, R. M. J.
Resin-bound sulfonyl azides: efficient loading and activation strat-
egy for the preparation of the N-acyl sulfonamide linker. J. Org.
Chem. 2007, 72, 4574–4577.
(75) Zhang, X.; Lu, X.-W.; Liu, C.-F. Solid-phase synthesis of peptide
thio acids through hydrothiolysis of resin-bound peptide thioe-
sters. Tetrahedron Lett. 2008, 49, 6122–6125.
(76) Hu, X.; Sun, J.; Wang, H.-G.; Manetsch, R. Bcl-XL-templated
assembly of its own protein-protein interaction modulator from
fragments decorated with thio acids and sulfonyl azides. J. Am.
Chem. Soc. 2008, 130, 13820–13821.
(58) Singh, I.; Zarafshani, Z.; Lutz, J.-F.; Heaney, F. Metal-free “click”
chemistry: efficient polymer modification via 1,3-dipolar cycload-
dition of nitrile oxides and alkynes. Macromolecules 2009, 42,
5411–5413.
(59) Li, Z.; Seo, T. K.; Ju, J. 1,3-Dipolar cycloaddition of azides with
electron-deficient alkynes under mild condition in water. Tetrahe-
dron Lett. 2004, 45, 3143–3146.
(77) Zhang, X.; Li, F.; Lu, X.-W.; Liu, C.-F. Protein C-terminal
modification through thio acid/azide amidation. Bioconjugate
Chem. 2009, 20, 197–200.
(78) Rijkers, D. T. S.; Merkx, R.; Yim, C.-B.; Brouwer, A. J.; Liskamp,
R. M. J. “Sulfo-click” for ligation as well as for site-specific
conjugation with peptides, fluorophores and metal-chelators.
J. Pept. Sci. 2010, 16, 1–5.
(60) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A strain-promoted [3
þ 2] azide-alkyne cycloaddition for covalent modification of
biomolecules in living systems. J. Am. Chem. Soc. 2004, 126,
15046–15047.
(61) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang,
P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-
free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 16793–16797.
(79) Brouwer, A. J.; Mulders, S. J. E.; Liskamp, R. M. J. Convergent
synthesis and diversity of amino acid based dendrimers. Eur. J.
Org. Chem. 2001, 1903–1915.
(80) Vetter, S. Direct synthesis of di- and trimethoxybenzyl thiols from
the corresponding alcohol. Synth. Commun. 1998, 28, 3219–3223.
(81) Brouwer, A. J.; Merkx, R.; Dabrowska, K.; Rijkers, D. T. S.;
Liskamp, R. M. J. Synthesis and applications of β-aminoethane-
sulfonyl azides. Synthesis 2006, 455–460.
(62) Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. Second-
generation difluorinated cyclooctynes for copper-free click chem-
istry. J. Am. Chem. Soc. 2008, 130, 11486–11493.
(63) Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons, G.-J.;
Popik, V. V. Selective labeling of living cells by a photo-triggered
click reaction. J. Am. Chem. Soc. 2009, 131, 15769–15776.
(64) Ning, X. H.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Visualizing
metabolically labeled glycoconjugates of living cells by copper-free
and fast Huisgen cycloadditions. Angew. Chem., Int. Ed. 2008, 47,
2253–2255.
(82) Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Chang, C.-D. Solid phase synthesis without
repetitive acidolysis. Preparation of leucyl-alanyl-glycylvaline
using 9-fluorenylmethyloxycarbonylamino acids. Int. J. Pept. Pro-
tein Res. 1979, 13, 35–42.
(83) von Arx, E.; Faupel, M.; Brugger, M. Das 4,40-tetramethyldiami-
no-diphenylmethan reagens (TDM): eine modification der chlor-o-
€
€
tolidin farbereaktion fur die dunnschichtchromatographie.
J. Chromatogr. 1976, 120, 224–228.
(84) Cooper, M. S.; Sabbah, E.; Mather, S. J. Conjugation of chelating
agents to proteins and radiolabeling with trivalent metallic iso-
topes. Nat. Protoc. 2006, 1, 314–317.
(65) van Berkel, S. S.; Dirks, A. J.; Debets, M. F.; van Delft,
F. L.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; Rutjes, F. P. J. T.