Synthesis of novel 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives for chemoselective attachment to unprotected polyfunctionalized compounds

Article abstract of DOI:10.1002/chem.200700231

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10- tetraazacyclodecane-l,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr³-octreotate by chemoselective oxime ligation and Cu^I-catalyzed azide-alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.

Full text of DOI:10.1002/chem.200700231

DOI: 10.1002/chem.200700231
Synthesis of Novel 1,4,7,10-Tetraazacyclodecane-1,4,7,10-Tetraacetic Acid
(DOTA) Derivatives for Chemoselective Attachment to Unprotected
Polyfunctionalized Compounds
Sebastian Knçr,[a] Armin Modlinger,[a] Thorsten Poethko,[b] Margret Schottelius,[b]
Hans-Jürgen Wester,[b] and Horst Kessler*[a]
In memoriam of Professor Miklos Bodanszky, a hero of peptide chemistry
Abstract: A convenient synthesis of
novel bifunctional poly(amino carbox-
ylate) chelating agents allowing chemo-
selective attachment to highly function-
alized biomolecules is described. Based
on the well known chelator 1,4,7,10-tet-
raazacyclodecane-1,4,7,10-tetraacetic
acid (DOTA), we synthesized novel bi-
functional chelating agents bearing ad-
ditional functional groups by alkylating
1,4,7,10-tetraazacyclododecane (cyclen)
with one equivalent of para-functional-
ized alkyl 2-bromophenyl-acetate and
three equivalents of tert-butyl 2-bro-
moacetate. The resulting compounds,
which contain an additional carbonyl
or alkyne functionality, allow site spe-
cific labeling of appropriately function-
alized unprotected biomolecules in a
rapid manner via click reactions. This
was demonstrated by the attachment of
our new DOTA derivatives to the so-
matostatin analogue Tyr3-octreotate by
chemoselective oxime ligation and CuI-
catalyzed azide–alkyne cycloaddition.
Initial biodistribution studies in mice
with the radiometalated compound
demonstrated the applicability of the
described DOTA conjugation.
Keywords: antitumor
agents
·
click reactions · isotopic labeling ·
oxime ligation
Introduction
DOTA-modified peptides are generally synthesized either
in solution[5] or on solid support attaching the DOTA resi-
due to a free amine of the resin bound peptide. For this, un-
protected[6] or more conveniently, protected DOTA deriva-
tives are used to overcome side reactions by polyactivation
of the four carboxylic groups of DOTA. Therefore, a
number of DOTA derivatives were developed allowing se-
lective formation of monoconjugates. For example, the tri-
protected and commercially available DOTA-tris(tert-butyl)
ester,[7] the corresponding benzyl protected analogues
For non-covalent binding of radionuclides, bifunctional che-
lating agents (BFCAs) are used to connect radioactive
markers and a targeting molecule. Among these BFCAs, the
1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic
acid
(DOTA) is a well-studied macrocyclic complex ligand which
has been shown to form extremely stable complexes of both
divalent and trivalent metals.[1,2] Radiopharmaceuticals con-
taining this ligand as metal chelator have found widespread
use in therapy and diagnostic imaging.[3,4]
DOTA-tris
ACHTREUNG
p-NCS-Bz-DOTA[9] or DOTAGA
AHCTREUNG
[a] S. Knçr, Dr. A. Modlinger, Prof. Dr. H. Kessler
Department Chemie, Lehrstuhl für Organische Chemie II
Technische Universität München, Lichtenbergstrasse 4
85747 Garching (Germany)
an additional unprotected carboxylic group, are widely used
BFCAs. In other approaches, derivatized amino acids con-
taining a DOTA moiety in the side chain were used[11] or
the DOTA moiety was synthesized stepwise on the N-termi-
nus of a resin bound peptide.[12] The main limitation of all
these methods is the introduction of the DOTA residue into
the bioconjugate via an electrophilic reaction. This proce-
dure tolerates no other N- or S-nucleophilic groups for a se-
lective reaction.
Fax : (+49)89-298-13210
E-mail: horst.kessler@ch.tum.de
[b] Dr. T. Poethko, Dr. M. Schottelius, Prof. Dr. H.-J. Wester
Department of Nuclear Medicine
Nuklearmedizinische Klinik und Poliklinik
Klinikum rechts der Isar, Technische Universität München
Ismaningerstrasse 22, 81675 München (Germany)
Chemoselective approaches allowing a site-selective func-
tionalization of polyfunctionalized compounds remain in
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
demand. Such a method would be a powerful tool for the
synthesis of DOTA-linked radiopharmaceuticals enabling
new synthetic strategies and the synthesis of complex struc-
tures. For example, we recently developed multimeric ami-
nooxy-functionalized RGD derivatives[13] which were 18F-la-
beled via oxime ligation using 4-[18F]fluorobenzaldehyde.[14]
A carbonyl functionalized DOTA derivative would allow a
selective conjugation with DOTA and subsequent labeling
with various radiometals. Very recently, Lin and co-workers
have demonstrated
a site-specific protein modification
Figure 1. Structures of 4-acetylphenyl-DOTA derivative 1 and ethynyl-
phenyl-DOTA derivative 2.
through CuI-catalyzed 1,2,3-triazole formation.[15] Applying
this methodology and using an alkyne functionalized DOTA
derivative would enable site specific DOTA labeling of pro-
teins. In a recent publication, Hovinen reported the synthe-
sis of an aminooxy-functionalized chelate by derivatization
of the tris tert-butyl ester of DOTA which was conjugated
with naltrexone and 2-deoxy-d-ribose.[16] However, the re-
ported procedure requires expensive starting material and
the applicability to polyfunctionalized compounds such as
peptides remains unclear.
easily synthesized by established synthetic methods. To
prove their applicability, Tyr3-octreotate, a somatostatin (sst)
analogue which is a well known targeting molecule for
tumor diagnostics and endoradiotherapeutic purposes,[40–45]
was chosen as a highly functionalized model compound pro-
viding a broad range of functionalities. Both classes of deriv-
atives proved to react selectively with appropriately modi-
fied unprotected Tyr3-octreotate. In addition, one of the ra-
dioligands was labeled with 68Ga and an initial biodistribu-
tion study in AR42J tumor bearing nude mice was per-
formed proving applicability of the modified linkage.
Thus, our goal was to design novel DOTA derivatives en-
abling the chemoselective attachment in presence of a wide
range of functional groups. Thereby, the focus was on struc-
tures readily accessible in few synthetic steps from cheap
commercial materials to make our method convenient and
even a general alternative to the widely used DOTA-tris-
(tert-butyl) ester. Furthermore, the accessibility of the corre-
spondingly polyfunctionalized compounds was an important
aspect in our consideration. Click reactions,[17] in particular
the oxime ligation[18,19] as well as the CuI-catalyzed azide–
alkyne cycloaddition[20,21] are well-studied and powerful re-
actions for chemoselective couplings which fulfil all require-
ments for our purpose. The oxime ligation denotes the
highly selective reaction between an aminooxy component
and aldehydes or methyl ketones[22] under formation of an
oxime bond, which is known to be stable both in vitro and
in vivo.[23] The reaction was shown to tolerate every free
amino acid side chain except an N-terminal cysteine and
found widespread use, for example, in the synthesis of tem-
plate-assembled synthetic proteins,[24,25] radioactive labeled
peptide conjugates,[23,26] cyclic peptides[27] and protein ana-
logues.[28,29] The CuI-catalyzed azide–alkyne cycloaddition is
a catalyzed variant of the chemoselective Huisgen 1,3-dipo-
lar cycloaddition[30–33] of an azide and an alkyne for the for-
mation of a triazole which has found application in various
developments in medicinal chemistry.[34–39] To provide chem-
ists with different methodologies for the synthesis of DOTA
conjugates, we focused on appropriate DOTA derivatives
for both reaction types described above.
Results
Synthesis of a carbonyl-substituted DOTA derivative for
chemoselective attachment to aminooxy-functionalized pep-
tides viaoxime ligation : Planning our synthetic strategy, we
decided to attach the carbonyl functionality to the DOTA
moiety. The aminooxy group can be readily implemented in
peptides as a aminooxy-functionalized building block (e.g.
(Boc-aminooxy)acetic acid, Boc-O-(Fmoc-amino)-serine
(Boc-Ams
ACHTREUNG
Fmoc-aminopropionic acid (Fmoc-Dpr
AHCTREUNG
carbonyl functionality, a methyl ketone was preferred over
an aldehyde due to its significant higher stability. This proce-
dure avoids additional protection and deprotection steps,
which would have been essential when working with an al-
dehyde, and makes the final compound storable for longer
periods.
Our synthesis started with 4-acetylphenylboronic acid (3)
which was reacted with tert-butyl 2-bromoacetate in a
Suzuki-type cross coupling reaction.[46] The resulting 2-(4-
acetylphenyl) substituted acetate 4 was obtained in 76%
yield (Scheme 1). The a-bromination using N-bromosuccini-
mide (NBS) in presence of catalytic amounts of bromine
and initiation with light gave the a-bromo ester 5 in high
yield (88%). The latter compound was slowly added to a
1.2-fold excess of cyclen in DMF in the presence of K2CO3
to afford the monoalkylated cyclen-adduct 6 (75% yield).
Subsequent peralkylation with tert-butyl 2-bromoacetate
then gave the desired tetraester 1 (72% yield).
Thus, in this report we present the synthesis of 2-[1-
(1,4,7,10-tetraazacyclodecane)-4,7,10-tris(tert-butylacetate)]-
(4-acetylphenyl) acetic acid tert-butyl ester (1) as carbonyl
component for oxime ligations with aminooxy-functional-
ized compounds and 2-[1-(1,4,7,10-tetraazacyclodecane)-
4,7,10-tris(tert-butylacetate)]acetic acid methyl ester (2) for
CuI-catalyzed azide–alkyne cycloadditions with azide func-
tionalized compounds (Figure 1). Both compounds can be
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H. Kessler et al.
rectly reacted with NBS. In this manner, 10 could be ob-
tained in 47% yield together with unreacted 9 which was re-
covered in 49% yield. This result is explained by the much
higher acidity of the a-bromo ester 10 to 9 leading to a
rapid deprotonation of the formed product with one equiva-
lent of the lithium enolate of 9 during the reaction. Howev-
er, based on recovered starting material 9, which could be
readily separated by flash chromatography, the yield was
almost quantitative. The monoalkylation of cyclen with a-
bromo ester 10 was performed in an analogous way as de-
scribed above to yield 11. After peralkylation with tert-butyl
2-bromoacetate and cleavage of the TMS group using tetra-
butylammonium fluoride (TBAF), the tetraester 2 was ob-
tained in 85% yield over two steps.
Scheme 1. Synthesis of carbonyl-substituted DOTA derivative 1. a)
BrCH2CO2tBu, Pd(OAc)2/P(o-Tol)3, K2CO3, THF/H2O, RT, 18 h ; 76 %;
A
ACHTREUNG
b) NBS, Br2, hn, CCl4, 608C, 1 h; 88%; c) cyclen, K2CO3, DMF, RT, 10h;
75%; d) BrCH2CO2tBu, K2CO3, DMF, RT, 4 h; 72%.
Synthesis of alkyne-substituted DOTA derivatives for che-
moselective attachment to azido functionalized peptides via
CuI-catalyzed azide–alkyne cycloaddition: To design the ap-
propriate DOTA derivative for attachment to peptides via
CuI-catalyzed azide–alkyne cycloaddition,[17] we decided to
introduce the alkyne functionality to the DOTA derivative,
as azido functionalized peptides are readily accessible, for
example, by introduction of azido acids[47–49] or by diazo
transfer on the solid phase.[50] After our positive results with
the synthesis of the carbonyl-substituted derivative, we
again chose 2-bromo-2-phenylacetic acid as the core residue
for the implementation of the alkyne, providing an analo-
gous synthetic route as described above.
Scheme 2. Synthesis of alkyne-functionalized DOTA derivatives. a)
The synthesis was started from commercially available 4-
iodophenylacetic acid (7). The free acid was protected as
methyl ester 8 by treating with thionyl chloride in methanol
in almost quantitative yield (93%) and high purity.
ꢀ
SOCl2, MeOH, 08C ! RT, 1 h; 93%; b) HC C-TMS, [Pd
A
NEt3, CH3CN, 08C ! RT, 3 h; 93%; c) 1) LDA, THF, ꢁ788C, 1 h; 2)
NBS, THF, ꢁ788C ! RT, 18 h; 47% (92% related to recovered 14); d)
cyclen, K2CO3, DMF, RT, 10h; 73%; e) 1) BrCH 2CO2tBu, K2CO3, DMF,
RT, 4 h; 2) TBAF ; THF, RT, 15 min; 85%.
Subsequently, 8 was coupled with trimethylsilyl (TMS)
acetylene in a Sonogashira reaction using [Pd(PPh3)4]/CuI as
G
a catalyst affording the 4-alkynylphenylacetate 9 in 93%
yield (Scheme 2).[51,52] The reaction was easy to handle and
proceeded cleanly. However, the a-bromination of 9 with
NBS failed with both, Br2/hn and 2,2’-azobisisobutyronitrile
(AIBN) as initiators. This is most likely due to side reactions
caused by the presence of a triple bond. Therefore, we cir-
cumvented this problem by introducing the bromide in an
electrophilic manner. For this purpose, we transformed ester
9 into the boron enolate in an analogous manner as de-
scribed by Evans et al.,[53] treating subsequently with lithium
diisopropyl amide (LDA) and Bu2BOTf. After addition of
N-bromosuccinimide as the electrophilic brominating re-
agent, the a-bromo ester 10 was isolated in rather poor
yield. In an attempt to further optimize the reaction, we
found that the conversion proceeds cleanly if the lithium
enolate of 9, obtained by deprotonation with LDA, was di-
With these novel functionalized chelators in hand, we
scrutinized chemoselective attachment to N-terminally ami-
nooxy and azide-functionalized Tyr3-octreotates 13 and 18.
Chemoselective synthesis of the DOTA-Tyr3-octreotate con-
jugate 14 via oxime ligation using the DOTA ketone deriva-
tive 1: In our initial experiments, the tert-butyl protected
DOTA ketone 1 was directly used for the oxime ligation.
However, the oxime bond of the resulting conjugate was un-
stable under the strong acidic conditions required for depro-
tection of the tert-butyl groups. Therefore the procedure was
switched and 1 was deprotected in situ prior to the ligation.
A quantitative cleavage was realized treating with 10n
aqueous HCl in dioxane 50:50 (Scheme 3). Alternative
methods applying formic acid,[54] 50% trifluoroacetic acid
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Chemoselective synthesis of the
DOTA-Tyr3-octreotate conju-
gate 19 via CuI-catalyzed azide–
alkyne
cycloaddition
using
DOTA alkyne derivative 2:
With respect to the azide func-
tionalization of Tyr3-octreotate,
we chose a simple N-terminal
elongation with 3-(3-azidopro-
pylcarbamoyl) propanoic acid
(16) on the solid support. Since
in several biological active pep-
tides it is of great importance
to use a spacer between BFCA
and the targeting moiety and
based on earlier experience in
our group, 16 was the linker of
choice. The two-step synthesis
of 16 starting from 1-bromo-3-
aminopropane is cheap, high
yielding, easy to scale up and
does not require any chroma-
tography (Scheme 4). Com-
pound 16 was attached to Tyr3-
octreotate following standard
peptide coupling protocols to
obtain the azido functionalized
Tyr3-octreotate 18 (see Support-
ing Information).
Scheme 3. Chemoselective oxime ligation reaction of DOTA derivative 1 and aminooxy functionalized Tyr3-oc-
treotate 13. a) 10n HCl/dioxane 1:1, RT, 18 h; b) CH3CN/H2O 1:1 (HPLC grade), pH 4 (TFA, HPLC grade),
RT, 18 h; 73% (two steps).
For the azide–alkyne cycload-
dition, a one-pot procedure was
worked out: Prior to the cycloaddition, methyl ester 2 was
(TFA)/water or a mixture of TFA, triisopropylsilane (TIPS)
and water (95:2.5:2.5)[55]—a standard deprotection mixture
used in Fmoc peptide chemistry—failed. The free chelator
12 thus obtained reacted cleanly with equimolar amounts of
aminooxy-functionalized Tyr3-octreotate 13 in an acetoni-
trile/water mixture at pH 4 (TFA) to give the desired conju-
gate 14 with high purity, as proven by HPLC analysis
(Figure 2).
saponified in situ using lithium hydroxide in THF/water.
It should be emphasized that any reaction where free ami-
nooxy functionalities occur, demand high solvent purities
(HPLC grade) in order to prevent side reactions with car-
bonyl functionalized impurities. The final product was fur-
ther purified by preparative HPLC to give 14 in 73% yield
and 97% purity.
Scheme 4. Synthesis of 3-(3-azidopropylcarbamoyl)propanoic acid (16).
a) NaN3, H2O, 80 8C, 24 h; 84%; b) succinic anhydride, NEt3, acetone,
RT, 15 h; 71%.
Surprisingly, this resulted in a
partial cleavage of tert-butyl
groups as well, which, however,
had no impact on the subse-
quent procedure. The resulting
mixture was further reacted
with the azido functionalized
Tyr3-octreotate 18 using Cu/
CuSO4 as catalyst system
(Scheme 5). After evaporation,
Figure 2. HPLC spectrum (UV 220nm) of crude DOTA conjugate 14 obtained by oxime ligation.
the crude mixture was directly
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H. Kessler et al.
In order to check our proce-
dure, we repeated the reaction
with the azido functionalized,
but non-cysteine containing
sample peptide 3-(3-azidopro-
pylcarbamoyl)propanoyl-Tyr-
Glu-Trp-Lys (20, see Supporting
Information). As expected, the
reaction sequence proceeded
without side reactions. Depro-
tection of the tert-butyl esters
gave the crude product with
high purity (see Supporting In-
formation). After HPLC purifi-
cation the corresponding conju-
gate 21 was obtained in 51%
yield (four steps).
Biodistribution studies: The
DOTA–Tyr3-octreotate conju-
gate 14 was chosen for biodis-
tribution studies to verify the
applicability of the new DOTA
derivatives. 68Ga labeling was
performed using
generator to give [68Ga]14 with
specific activity of
a
68Ge/68Ga
570Cimmol ꢁ1 at the time point
of injection into mice. [68Ga]14
was obtained in 55.7% radio-
chemical yield and 91.4% radi-
ochemical purity. The biodistri-
bution data for [68Ga]14 in
Scheme 5. Chemoselective CuI-catalyzed azide–alkyne cycloaddition of DOTA derivative 17 and azido func-
tionalized Tyr3-octreotate 18. a) LiOH, THF/H2O, RT, 18 h; b) 1) Cu/CuSO4, THF/H2O, RT, 18 h; 2) TFA/
TIPS/H2O 95:5:5, RT, 2 h; 3) Na2S, THF/H2O, RT; 4) DMSO, NH3, CH3CN/H2O, RT, 24 h; 37% (five steps).
deprotected by adding TFA/TIPS/water 95:2.5:2.5[55] to
afford the crude product 19 with high purity, as shown by
HPLC analysis (Figure 3). ESI mass spectroscopy showed
that 19 was obtained as a copper complex. Thus, the copper
ions were removed from the solution and from the chelator
by precipitation with sodium sulfide to give the pure free
conjugate. During the four-step procedure, no side reactions
with functional groups were observed, except of a reductive
opening of the intramolecular disulfide bond in the last step
which was easily reformed in quantitative yield by treating
with DMSO in acetonitrile/water for 24 h. After HPLC pu-
rification, 19 was obtained in 37% yield and 97% purity
from 18 (five steps).
AR42J tumor bearing nude mice 30and 60min p.i. are dis-
played in Figure 4.
At both time points, blood concentration of the radioli-
gand was comparably high (2.37ꢂ0.35 and 1.71ꢂ0.17%
iDgꢁ1 at 30and 60min p.i., respectively). While kidney ac-
cumulation rapidly decreased within the observation period
(20.0ꢂ2.4 to 11.9ꢂ1.9% iDgꢁ1), indicating renal clearance
of [68Ga]14, the tracer accumulation in the other excretion
organs, that is, liver and intestine, decreased only slowly
over time, probably due to nonspecific accumulation that is
not associated with excretion. In the sst-expressing tissues, a
strong divergence between the tumor and the other sst-posi-
tive organs was observed.
While tracer accumulation in
pancreas, adrenals and stomach
significantly decreased between
30and 60min p.i., tumor accu-
mulation remained almost con-
stant within this period (7.73ꢂ
1.52 and 7.46ꢂ1.30at 30and
60min p.i., respectively). This
and the overall decrease in
non-specific tracer accumula-
tion in the other organs lead to
Figure 3. HPLC spectrum (UV 220nm) of crude DOTA conjugate 19 obtained after click reaction and subse-
quent deprotection (step 2, Scheme 5).
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Novel DOTA Derivatives
FULL PAPER
the a-bromo esters 5 and 10
were straightforward and the
products were obtained in good
yields.
For the CuI-catalyzed azide–
alkyne
cycloaddition[17,20,34–39]
we used 1.2 equivalents of
copper sulfate together with of
an excess of copper metal,
which leads to in situ genera-
tion of the catalytically active
CuI species. Although there are
reports[56] where only 1 mol%
of CuII salts are used, we had to
use an excess, given the fact
that one equivalent of the CuII
Figure 4. Biodistribution of [68Ga]14 in AR42 J tumor bearing nude mice 30and 60min p.i. ( n=5; n=3 for
the competition study). Data are given in% injected dose per gram tissue (% iDgꢁ1) and are meansꢂSD.
increasing tumor/organ ratios between 30and 60min p.i.
(see Supporting Information). That the tumor accumulation
is mainly receptor mediated was demonstrated in a competi-
tion study (60min p.i.) by co-injection of an excess of unla-
beled competitor (20 mg Tyr3-octreotate per mouse). Under
these conditions, tumor accumulation was reduced 2.68ꢂ
0.36% iDgꢁ1.
is immediately complexed with the DOTA residue. Howev-
er, in the literature there are a plethora of different proce-
dures[56] and for our purposes, optimization of the catalyst
loading was not essential as dissolved copper ions could be
precipitated and filtered off by addition of sodium sulfide.
Using Tyr3-octreotate as a model compound, the conjugation
by azide–alkyne cycloaddition was shown to be applicable
with a broad range of functionalities. As the only side reac-
tion, an opening of disulfide bridge was found. However,
this causes no problem, as the reduced DOTA conjugate is
smoothly recyclized in quantitative yield. Nevertheless, this
side reaction should be kept in mind when planning to syn-
thesize a disulfide bridged conjugate. Of course, it can be
avoided if the linear precursor peptide is used for conjuga-
tion and the disulfide bridge is formed afterwards. Hence,
our alkyne derivatized chelator offers a promising alterna-
tive for attachment of BFCAs in a highly chemoselective
manner as demonstrated by reaction with model peptides 18
and 20. During the reviewing process we became aware of
an alternative approach for DOTA conjugation via azide–
alkyne cycloaddition.[57]
The oxime ligation of our new methyl ketone functional-
ized chelator 12 with unprotected N-terminally aminooxy-
functionalized Tyr3-octreotate proceeded smoothly and with-
out by-products, thereby adding another example of the che-
moselective condensation of a methyl ketone and a hydrox-
ylamine. Hence, we have successfully shown the chemoselec-
tive attachment of a novel bench stable DOTA derivative
suitable for the ligation with aminooxy-functionalized bio-
molecules. In comparison to the recently presented amino-
oxy-functionalized DOTA derivatives,[16] our inverse ap-
proach allows the introduction of our chelator at any posi-
tion in a peptide sequence, since various appropriate amino-
oxy functionalized building blocks for SPPS are commercial-
Discussion
DOTA and its derivatives emerged as an important class of
chelators for imaging technologies in medicine due to their
ability to form very stable complexes with a variety of di-
and trivalent metal ions. For the convenient synthesis of che-
lator conjugated targeting biomolecules, different suitable
prochelators bearing orthogonally protected carboxy groups
have been described. However, so far there is only one
report published in the literature on the synthesis of proche-
lators enabling connections other than through amine or car-
boxyl functionalities within the targeting molecule.[16] Our
approach in developing BFCAs which allow chemoselective
attachment via click reaction resulted in the synthesis of
novel chelators 1 and 2.
For the synthesis of both compounds, a monoalkylation of
cyclen had to be accomplished to obtain 6 and 11. In the lit-
erature, there are several examples of similar monoalkyla-
tions where high yields of monoalkylated product were ach-
ieved by applying cyclen in great excess (e.g. 2 equiv[7] or
5 equiv[8]) and where the yields are calculated based on the
amount of alkylating agents. As a result of cyclen being the
most expensive reagent in the sequence, in our synthesis we
used almost equimolar amounts of the alkylating agent and
obtained satisfying yields of 75 and 73% for compounds 6
and 11. Although the crude product could be directly fur-
ther alkylated, purification of this intermediate was carried
out via flash chromatography at this stage, as it was easier
to separate the monoalkylated product from higher alkylat-
ed products and unreacted cyclen, rather than separation
after peralkylation, where the resulting compounds only
differ in the nature of the alkyl substituent. The syntheses of
ly available (e.g. Boc-Ams
Aoa)-OH).
N
N
As a first proof of principle, we used the 68Ga-labeled de-
rivative of the oxime linked DOTA conjugate 14 in an in
vivo experiment. AR42J tumor bearing nude mice were
treated with 68Ga labeled 14 per mouse and investigated for
tumor accumulation at 30and 60min p.i. The results exhibit
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H. Kessler et al.
18 h at room temperature, the mixture was filtered and the solvent re-
moved under reduced pressure. The residue was taken up in EtOAc
(250mL) and subsequently washed with saturated aqueous NH 4Cl
(150mL), saturated aqueous NaHCO 3 (150mL) and brine. After drying
over MgSO4, the organic layer was filtered through silica gel and concen-
trated. Flash chromatography on silica gel (EtOAc/hexane 1:8) yielded 4
(8.30g, 76%) as a pale yellow solid. Rf =0.27 (EtOAc/hexane 1:4); m.p.
50–528C; 1H NMR (360MHz, CDCl 3): d=7.92 (d, J=8.2 Hz, 2H), 7.37
(d, J=8.1 Hz, 2H), 3.58 (s, 2H), 2.59 (s, 3H), 1.43 (s, 9H); 13C NMR
(90MHz, CDCl 3): d=197.7, 170.0, 140.1, 135.8, 129.4 (2C), 128.5 (2C),
81.2, 42.6, 28.0, 26.5; HRMS (EI): m/z: calcd for C14H18O3: 234.12559;
found 234.12532 [M]+.
good contrast and high accumulation in the tumor (see
Figure 4).
Conclusion
The novel alkyne- and keto-functionalized DOTA deriva-
tives described in this article allow a facile and chemoselec-
tive conjugation with polyfunctionalized compounds. With
regard to the synthesis of our new modified chelators, we
have developed economical straightforward procedures
avoiding complicated protection group chemistry consider-
ing the final application of the BFCA. This allows easy
access to labeled compounds in few synthetic steps. Both
the alkyne as well as the methyl ketone functionalized
DOTA derivatives proved to react selectively in the corre-
sponding conjugation with N-terminally modified Tyr3-oc-
treotate. Furthermore, the pharmacokinetics of [68Ga]14 in
AR42J tumor bearing nude mice demonstrate the suitability
of the chemoselective BFC conjugation approach for the
synthesis of new radiometalated peptide ligands for diagnos-
tic and therapeutic in vivo applications.
A
mide (NBS) (2.58 g, 14.5 mmol, 1.2 equiv) and Br2 (2 drops) were added
to a solution of 4 (2.84 g, 12.1 mmol, 1.0equiv) in dry CCl 4 (250mL).
The mixture was heated to reflux, illuminated with a 500 W halogen
lamp for 10min and stirred for further 50min under reflux. After cooling
to room temperature, the reaction solution was filtered, the solvent con-
centrated and the crude product purified by flash chromatography on
silica gel (EtOAc/hexane 1:10) to give 5 (3.34 g, 88%) as a pale yellow
solid. Rf =0.27 (EtOAc/hexane 1:4); m.p. 54–568C; 1H NMR (360MHz,
CDCl3): d=7.93 (d, J=8.4 Hz, 2H), 7.62 (d, J=8.3 Hz, 2H), 5.27 (s,
1H), 2.59 (s, 3H), 1.45 (s, 9H); 13C NMR (90MHz, CDCl 3): d=197.2,
166.6, 141.1, 137.3, 128.8 (2C), 128.6 (2C), 83.5, 47.1, 27.6, 26.6; MS (EI):
m/z (%): 314 (<1) [M
(81Br)ꢁOtBu]+, 239 (8) [M
C10H881BrO2: 240.96872; found 240.96833 [M
[M
(79Br)ꢁOtBu]+.
(R/S)-tert-Butyl 2-[1-(1,4,7,10-tetraazacyclodecane)]-2-(4-acetylphenyl)-
A
N
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
AHCTREUNG
acetate (6): A solution of 5 (500 mg, 1.60 mmol, 1.0 equiv) in DMF
(50mL) was added dropwise at room temperature over 10h to a suspen-
sion of 1,4,7,10-tetraazacyclodecane (331 mg, 1.92 mmol, 1.2 equiv) and
K2CO3 (552 mg, 3.99 mmol, 2.5 equiv) in DMF (20mL). The mixture was
filtered and concentrated under reduced pressure. Flash chromatography
on silica gel (MeOH/CHCl3 1:7!7:1, 1% NEt3) yielded 6 (488 mg, 75%)
as a pale yellow solid. Rf =0.10 (MeOH/CHCl3 3:1, 1% NEt3); m.p. 33–
368C; 1H NMR (360MHz, CDCl 3): d=7.91 (d, J=8.4 Hz, 2H), 7.43 (d,
J=8.2 Hz, 2H), 4.62 (s, 1H), 2.90–2.67 (m, 11H), 2.64–2.44 (m, 10H),
1.47 (s, 9H); 13C NMR (90MHz, CDCl 3): d=197.4, 170.7, 142.3, 136.4,
129.3, 128.3, 81.9, 67.8, 49.3, 47.7, 45.9, 45.8, 28.1, 26.5; HRMS (EI): m/z:
calcd for C22H36N4O3: 404.27875; found 404.27951 [M]+.
Experimental Section
General methods: Tritylchloride polystyrol (TCP) resin (0.94 mmolgꢁ1
)
was purchased from PepChem (Tübingen, Germany). Coupling reagents
and amino acid derivatives were purchased from Novabiochem, Neosys-
tem, and IRIS Biotech GmbH, Merck Biosciences, Perseptive Biosystems
GmbH and Neosystem. Dry solvents were purchased from Fluka. All
other reagents and solvents were purchased from Merck, Aldrich and
Fluka and were used as received. Standard syringe techniques were ap-
plied for transferring dry solvents. Thin-layer chromatography (TLC) was
performed on aluminium-backed plates Merck silica gel 60F 254. Com-
pounds were visualized by UV absorption at 254 nm or coloration with
cerium ammonium molybdate (CAM). Flash chromatography was per-
formed on silica gel 60(Merck, 230–400mesh) (ca. 50g for 1 g of materi-
al to be separated) with the indicated eluent. Solvents for chromatogra-
phy were distilled prior to use. Chromatographic elution solvent systems
are reported as volume/volume ratios. RP-HPLC analyses was performed
using an Omnicrom YMC column (4.6 mm”250mm, 5 mm C18,
1 mLminꢁ1) and detection at 254 nm for non-peptidic compounds and
220nm for peptidic compounds. The eluent was a linear gradient from
water (0.1% TFA) to acetonitrile (0.1% TFA) over 30 minutes. The re-
tention time (tR) of the analytical RP-HPLC is given in minutes with the
gradient in percentage of acetonitrile. NMR: Bruker AC-250, AV-360,
AV-500 and DMX500. 1H and 13C NMR spectra were recorded at ambi-
ent temperature. Spectra were calibrated to their respective solvent sig-
nals (CDCl3: 1H 7.26 ppm, 13C 77.0ppm; [D 4]MeOH: 1H 3.31 ppm, 13C
49.05 ppm;). Chemical shifts (d) are reported in parts per million (ppm)
and coupling constants (J values) are given in Hertz (Hz). The following
abbreviations were used to explain the multiplicities: s, single; d, doublet;
t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; m,
multiplet; b, broad. MS: Finnigan MAT 8200 (EI), Finnigan LCQ (ESI).
Peptide sequence analysis was performed on a Bruker Ultraflex TOF/
TOF.
A
acetate)]-2-(4-acetylphenyl)acetate (1): A solution of tert-butyl 2-bro-
moacetate (0.84 mL, 5.71 mmol, 3.3 equiv) in DMF (20 mL) was added at
room temperature over 30min to a suspension of 6 (700 mg, 1.73 mmol,
1.0equiv) and K 2CO3 (1.08 mg, 7.79 mmol, 4.5 equiv) in DMF (50 mL).
After stirring for 4 h, the mixture was filtrated and concentrated under
reduced pressure. Flash chromatography on silica gel (MeOH/CHCl3 9:1,
1% TEA) yielded 1 (930mg, 72%) as a pale yellow solid. 99% purity;
RP-HPLC (10!100%) tR =22.0min; Rf =0.83 (MeOH/CHCl3 3:1, 1%
NEt3); m.p. 70–718C; 1H NMR (360MHz, CDCl 3): d=7.94 (d, J=8.3 Hz,
2H), 7.11 (d, J=8.2 Hz, 2H), 4.66 (s, 1H), 3.60(d, J=17.3 Hz, 1H), 3.45
(d, J=17.6 Hz, 1H), 3.38 (d, J=17.5 Hz, 1H), 3.22–3.05 (m, 3H), 2.98–
2.70(m, 6H), 2.61 (s, 3H), 2.61–2.48 (m, 2H), 2.46–2.38 (m, 1H), 2.36–
2.24 (m, 2H), 2.22–2.04 (m, 5H), 1.49 (s, 9H), 1.48 (s, 9H), 1.46 (s, 9H),
1.39 (s, 9H); 13C NMR (90MHz, CDCl 3): d=197.6, 173.4, 173.1, 173.0,
172.9, 137.2, 136.6, 130.3 (2C), 128.1 (2C), 82.9, 82.2, 82.1, 82.0, 65.4,
56.0, 55.8, 55.5, 52.7, 52.4, 52.1, 48.5, 48.1, 48.0, 47.9, 44.5, 27.9 (3C), 27.8
(6C), 27.7 (3C), 26.6; MS (EI): m/z (%): 746.0(9) [ M]+, 645.1 (47)
[MꢁCO2tBu]+; HRMS (EI): m/z: calcd for C35H57N4O7: 645.42273; found
645.42257 [MꢁCO2tBu]+.
Methyl 2-(4-(2-(trimethylsilyl)ethynyl)phenyl)acetate (9): [Pd(PPh3)4]
A
(3.6 g, 3.15 mmol, 0.07 equiv) and CuI (6.00 g, 31.5 mmol, 0.7 equiv) was
added under argon at 08C to a solution of methyl (4-iodophenyl)acetate
(8) (12.8 g, 45 mmol, 1.0equiv), trimethylsilylacetylene (9.30mL,
67.5 mmol, 1.5 equiv) and triethylamine (TEA) (14.9 mL, 108 mmol,
2.4 equiv) in dry CH3CN (120mL). After stirring for 30min at 0 8C and
3 h at room temperature, the mixture was filtered through a short
tert-Butyl 2-(4-acetylphenyl)acetate (4): A solution of 4-acetylphenylbor-
onic acid (3) (9.84 g, 60.0 mmol, 1.3 equiv) in THF/H2O (160mL/2.2 mL)
was added dropwise under argon over 1 h to a suspension of tert-butyl 2-
bromoacetate (6.80mL, 46.4 mmol, 1.0equiv), Pd
1.50mmol, 3 mol%), (o-tolyl)3 (1.36 g, 4.46 mmol, 10mol%) and
K2CO3 (34.6 g, 0.25 mmol, 5.4 equiv) in THF (160 mL). After stirring for
A
P
ACHTREUNG
6088
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6082 – 6090

Novel DOTA Derivatives
FULL PAPER
column of silica gel using EtOAc/hexane 1:1 as eluent. The solvent was
removed under reduced pressure and the residue purified by flash chro-
matography on silica gel (EtOAc/hexane 1:80!1:20) to yield 9 (10.3 g,
93%) as pale yellow crystals. Rf =0.27 (EtOAc/hexane 1:10); m.p. 55–
588C; 1H NMR (360MHz, CDCl 3): d=7.42 (d, J=8.4 Hz, 2H), 7.21 (d,
J=8.5 Hz, 2H), 3.68 (s, 3H), 3.61 (s, 2H), 0.25 (s, 9H); 13C NMR
(90MHz, CDCl 3): d=171.4, 134.3, 132.0 (2C), 129.1 (2C), 122.0, 104.7,
94.2, 52.0, 41.0, ꢁ0.0 (3C); HRMS (EI): m/z: calcd for C14H18O2Si:
246.10761; found 246.10744 [M]+.
13 (6.1 mg, 4.5 mmol, 1.0equiv) was added. After stirring for 18 h, the sol-
vent was concentrated and the crude product was directly purified by
semipreparative RP-HPLC (20!50%, 30min) to yield 14 (6.1 mg, 73%)
as a colorless powder after lyophilization. 97% purity; RP-HPLC (10!
60%); tR =18.1 min; MS (ESI): m/z: calcd for C75H99N15O22S2: 1625.7;
found 1626.6 [M+H]+, 1664.6 [M+K]+.
Chemoselective click reaction—Synthesis of DOTA-Tyr3-octreotate de-
rivative 19: A solution of LiOH (0.33 mg, 14 mmol, 3.4 equiv) in H2O
(30 mL) was added at room temperature to a solution of 2 (3.1 mg,
4.1 mmol, 1.0equiv) in THF (0.2 mL) and the mixture stirred for 18 h.
Subsequently, H2O (0.2 mL), peptide 18 (5.5 mg, 4.1 mmol, 1.0equiv),
0.1m aqueous CuSO4 (49 mL, 4.9 mmol, 1.2 equiv) and copper powder
(10mg) were added and the mixture stirred for 18 h. After this time, the
copper powder was filtered off, the solvent removed under reduced pres-
sure and the tert-butyl esters were cleaved by treating with a mixture of
TFA/TIPS/H2O 95:5:5 (1 mL) for 2 h. The solvent was again removed
and the residue was taken up THF/H2O 1:1 (1 mL) to precipitate the
copper salts by addition of Na2S·9H2O (12 mg, 49 mmol, 12.0equiv). The
mixture was filtrated and the crude product directly purified by semipre-
parative RP-HPLC (20!50%, 30min) to yield linear 19 (3.0mg, 37%)
as a colorless powder after lyophilization. The linear peptide was recycl-
ized in quantitative yield by stirring in CH3CN/H2O/DMSO 1:1:0.2
(4 mL) at pH 8 (25% NH3 solution) for 24 h. After evaporation and lyo-
philization from CH3CN/H2O 1:2 (10mL, pH 1–3 (TFA)), 19 (3.0mg,
37% from 18) was isolated as a white powder. 97% purity; RP-HPLC
(10!60%) tR =16.1 min; MS (ESI): m/z: calcd for C80H106N18O22S2:
1734.7; found 868.8 [(M+2H)/2]+, 1735.5 [M+H]+.
A
2-bromo-2-(4-(2-(trimethylsilyl)ethynyl)phenyl)acetate
(10): Lithium diisopropylamide (2m solution in THF/n-heptane/ethylben-
zene, 5.04 mL, 10.1 mmol, 1.2 equiv) was added at ꢁ788C to a solution of
9 (2.07 g, 8.40 mmol, 1.0 equiv) in dry THF (20 mL) and the solution
stirred for 1 h. After this time, a suspension of NBS (1.79 g, 10.1 mmol,
1.2 equiv) in dry THF (20mL) was added and the mixture warmed to
room temperature over 18 h. The solvent was removed under reduced
pressure, the residue suspended in CCl4 (30mL), filtered and evaporated.
Purification by flash chromatography on silica gel (gradient EtOAc/
hexane 1:80!1:20, 1% NEt3) gave 10 (1.28 g, 47%; 92% related to re-
covered 9 (1.01 g, 49%)). Rf =0.42 (EtOAc/hexane, 1:10); m.p. 82–848C;
1H NMR (360MHz, CDCl 3): d=7.47 (d, J=8.7 Hz, 2H), 7.44 (d, J=
8.7 Hz, 2H), 5.32 (s, 1H), 3.77 (s, 3H), 0.25 (s, 9H); 13C NMR (90MHz,
CDCl3): d=168.3, 135.7, 132.2 (2C), 128.5 (2C), 124.2, 104.1, 95.8, 53.3,
45.8, 45.8, ꢁ0.1 (3C); HRMS (EI): m/z: calcd for C14H1781BrO2Si:
326.01608; found 326.01622 [M
(R/S)-Methyl 2-[1-(1,4,7,10-tetraazacyclodecane)]-2-(4-(2-(trimethylsilyl)-
ethynyl)-phenyl)acetate (11): solution of 10 (316 mg, 0.97 mmol,
1.0equiv) in DMF (50mL) was added dropwise at room temperature
over 10h to suspension of 1,4,7,10-tetraazacyclodecane (cyclen)
A
ACHTREUNG
A
Animal experiments: Due to its high sst2-somatostatin receptor expres-
sion, the rat pancreatic tumor cell line AR42J was used as a tumor
model.[58] To establish tumor growth, cells were detached from the sur-
face of the culture flasks using 1 mm EDTA in PBS, centrifuged and re-
suspended in serum-free culture medium (RPMI-1640, Biochrom, Berlin,
Germany). Concentration of the cell suspension was 3.7”106 cells per
100 mL serum. Nude mice (female, 6–8 weeks) were injected 100 mL of
the cell suspension subcutaneously into the flank. Ten days after tumor
transplantation all mice showed solid palpable tumor masses (tumor
weight 0.7–1.4 g) and were used for the experiments. For biodistribution
studies, mice were intravenously injected 38 mCi [68Ga]14 (corresponding
to 0.15 mg of peptide) in 100 mL PBS into the tail vein. Non-specific
tissue accumulation of the radioligand was determined by coinjection of
an excess of cold competitor (20 mg Tyr3-octreotide per mouse). At differ-
ent time points after radioligand injection (30and 60min p.i.) mice ( n=5
per time point; n=3 for blocking study at 60min p.i.) were sacrificed
and dissected. The organs of interest were removed, weighed and count-
ed in a g counter (Wallach, Turku, Finland). Data are expressed as per-
cent injected dose per gram tissue (% iDgꢁ1).
a
(200 mg, 1.16 mmol, 1.2 equiv) and K2CO3 (160mg, 1.16 mmol, 1.2 equiv)
in DMF (10mL). The mixture was filtered and concentrated under re-
duced pressure. Flash chromatography on silica gel (MeOH/CHCl3 1:1!
9:1, 0.5% NEt3) yielded 11 (295 mg, 73%) as a pale yellow solid. Rf =
1
0.10 (MeOH/CHCl3 9:1, 0.5% NEt3); m.p. 70–758C; H NMR (500 MHz,
[D4]MeOH): d=7.49 (d, J=8.2 Hz, 2H), 7.30(d, J=8.3 Hz, 2H), 4.90(s,
1H), 3.78 (s, 3H), 3.21–3.01 (m, 6H), 3.01–2.82 (m, 8H), 2.71–2.62 (m,
2H), 0.23 (s, 9H); 13C NMR (125 MHz, [D4]MeOH): d=174.2, 135.6,
133.2 (2C), 130.8 (2C), 124.9, 105.4, 95.9, 67.2, 53.0, 48.4 (2C), 47.0 (2C),
44.9 (2C), 44.4 (2C), ꢁ0.0 (3C); MS (ESI): m/z: calcd for C22H36N4O2Si:
416.3; found 417.4 [M+H]+, 439.4 [M+Na]+.
(R/S)-Methyl 2-[1-(1,4,7,10-tetraazacyclodecane)-4,7,10-tris(tert-butylace-
tate)]-2-(4-ethynyl)phenyl)acetate (2): A solution of tert-butyl 2-bromoa-
cetate (615 mL, 4.19 mmol, 3.3 equiv) in DMF (20mL) was added at
room temperature over 30min to a suspension of 11 (530mg, 1.27 mmol,
1.0equiv) and K 2CO3 (634 mg, 4.57 mmol, 3.6 equiv) in DMF (50mL).
After stirring for 4 h, the mixture was filtered, concentrated under re-
duced pressure and the residue dissolved in THF (20mL). Then, tetrabu-
tylammonium fluoride (TBAF) (481 mg, 1.52 mmol, 1.2 equiv) was
added, and after stirring for 15 min, the solvent was removed and the
crude product purified by flash chromatography on silica gel (MeOH/
CHCl3 1:10, 1% NEt3) to yield 2 (508 mg, 85%) as a pale yellow solid.
99% purity; RP-HPLC (10!100%) tR =20.0 min; Rf =0.28 (MeOH/
CHCl3 1:9, 1% NEt3); m.p. 63–688C; 1H NMR (500 MHz, [D4]MeOH):
d=7.48 (d, J=8.2 Hz, 2H), 7.20(d, J=8.0Hz, 2H), 4.83 (s, 1H), 3.75 (d,
J=17.2 Hz, 1H), 3.74 (s, 3H), 3.55 (s, 1H), 3.54 (d, J=17.5 Hz, 1H), 3.51
(d, J=17.6 Hz, 1H), 3.26–3.21 (m, 1H), 3.18–3.05 (m, 4H), 2.99–2.92 (m,
3H), 2.89 (d, J=17.6 Hz, 1H), 2.87 (d, J=17.6 Hz, 1H), 2.74–2.63 (m,
2H), 2.33 (d, J=11.5 Hz, 1H), 2.28–2.12 (m, 4H), 2.12–2.05 (m, 2H),
1.54 (s, 9H), 1.52 (s, 18H); 13C NMR (125 MHz, [D4]MeOH): d=176.3,
175.4, 175.1, 174.7, 134.1, 132.8, 131.7, 123.8, 83.8, 83.5, 83.1, 79.7, 66.4,
59.61, 59.59, 59.57, 57.1, 56.8, 56.7, 54.2, 53.9, 53.7, 53.2, 45.9, 28.5, 28.4,
28.3, 24.8; HRMS (EI): m/z: calcd for C37H58N4O8: 686.42546; found
686.42532 [M]+.
Acknowledgements
The authors thank Mona Wolff, Burghard Cordes and Helmut Krause for
their technical assistance. Financial support by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie is gratefully ac-
knowledged.
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Received: February 8, 2007
Published online: May 14, 2007
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