Comparison and systematic optimization of synthetic protocols for DOTA-hydrazide generation

Article abstract of DOI:10.1016/j.tetlet.2012.11.151

DOTA-based organometallic complexes are extensively used in functional and molecular imaging studies, as well as in radioimmunotherapy. DOTA forms thermodynamically stable and kinetically inert complexes with various different diagnostic and therapeutic metals, such as gadolinium, gallium, yttrium and indium. Here, we have compared and systematically optimized the two most commonly used synthetic protocols for generating DOTA-hydrazide complexes, identifying conditions which reduce overall reaction time and increase overall yield.

Full text of DOI:10.1016/j.tetlet.2012.11.151

Tetrahedron Letters 54 (2013) 918–920
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Comparison and systematic optimization of synthetic protocols for
DOTA–hydrazide generation
Felix Fuge a, Marek Weiler a, Jessica Gätjens a, Twan Lammers a,b,c, Fabian Kiessling a,
⇑
a Department of Experimental Molecular Imaging, Helmholtz Institute for Biomedical Engineering, RWTH–Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany
b Department of Targeted Therapeutic, MIRA Institute for Biomedical Technology and Technical Medicine University of Twente, Enschede, PO Box 217, 7500 AE Enschede,
The Netherlands
c Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
DOTA-based organometallic complexes are extensively used in functional and molecular imaging studies,
as well as in radioimmunotherapy. DOTA forms thermodynamically stable and kinetically inert com-
plexes with various different diagnostic and therapeutic metals, such as gadolinium, gallium, yttrium
and indium. Here, we have compared and systematically optimized the two most commonly used syn-
thetic protocols for generating DOTA–hydrazide complexes, identifying conditions which reduce overall
reaction time and increase overall yield
Received 28 September 2012
Revised 26 November 2012
Accepted 28 November 2012
Available online 19 December 2012
Keywords:
Cyclen
Ó 2012 Elsevier Ltd. All rights reserved.
Alkylation
Hydrazide
DOTA
Lanthanides
Molecular imaging plays an increasingly important role in (bio-)
medical research, enabling a better understanding of fundamental
molecular pathways in living organisms. Various different imaging
techniques are used in molecular imaging, such as positron emis-
sion tomography (PET), single photon emission tomography
(SPECT), magnetic resonance imaging (MRI) and optical imaging
(OI). Contrast media are crucial for biomedical imaging purposes,
to enhance the sensitivity for disease detection, and to gain impor-
tant information on disease-related cellular and molecular pro-
cesses. Common contrast agent generation techniques in this
area of research rely on the coupling of imaging probes to peptides,
proteins or antibodies.
since the synthesis and workup of DOTA are relatively complicated
and time-consuming.
Much effort has been invested in recent years to improve the
synthesis of DOTA complexes by selective N-alkylation.4–7
However, most methods are relatively laborious, they require long
reaction times and generally result in low yields. Selective N-deriv-
atization is accomplished either by protection-derivatization-
deprotection methods, or by direct derivatization.8 Alternatively,
also a bis-aminal strategy can be used for tetraazacylcododecane
synthesis and modification.9 Apart from methods involving tert-
butyloxycarbonyl, tosyl, formyl, sterically hindered reagents and
metal carbonyls for mono- and trialkylation, the direct derivatiza-
tion with chelating agents is the most efficient method, because of
less complexity and fewer reaction steps.5,10,11 Methods for bis-
alkylation are more scarce and often involve diprotection via tosyl,
methyl and phosphoryl groups, silicone intermediates and
oxamide groups. Because of the harsh conditions and low yields
of such approaches, the direct functionalization is often
preferred.8,12 Recent literature also suggest an approach via boc
and Cbz protection groups for bis-alkylation which eliminates
problems occurring in direct derivatization.8
DOTA
(1,4,7,10-tetraazacyclododecan-N,N0,N00,N000-tetraacetic
acid) is the most commonly used macrocyclic chelating agent for
the functionalization of peptides, proteins and antibodies with
contrast agents, forming thermodynamically stable and kinetically
inert complexes with most trivalent metal ions.1,2 To enable
efficient coupling to biomolecules, reactive groups need to be
introduced into the DOTA complex. To this end, three of the four
secondary amines of the cyclen are selectively alkylated with
tert-butylacetate, and the remaining amine is derivatized.3
Amine and hydrazide groups have been established as versatile
coupling sites in many different applications in the biomedical
research. However, more efficient chemical protocols are needed,
For the synthesis of cyclen (1,4,7,10-tetraazacyclododecane)
(1)–based DOTA derivatives with one reactive side arm as potential
coupling site for biomolecules by hydrazide groups, there are two
standard procedures. Both protocols are depicted in Scheme 1.
These methods have conceptual advantages and disadvantages
(such as good yields and high flexibility for method 1; and high
⇑
Corresponding author. Tel.: +49 (0) 241 8080116; fax: +49 (0) 241 803380116.
E-mail address: fkiessling@ukaachen.de (F. Kiessling).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.11.151

F. Fuge et al. / Tetrahedron Letters 54 (2013) 918–920
919
Scheme 1. General overview of DOTA–hydrazide synthesis: reaction pathway 1 (reaction I, II, V–VI); reaction pathway 2 (reaction III–VI).
regio-selectivity and short reaction times for method 2), but they
have thus far never been compared. Here, we present the first di-
rect comparison and a systematic optimization of both synthetic
methods for DOTA–hydrazide generation.
To assess which of the two standard synthetic protocols for
DOTA–hydrazide generation is most efficient, we have primarily
focused on the first reaction step (see reaction I and III; Scheme 1),
since this is the most critical step in both pathways, and later
reactions are known to require relatively short reaction times,
and to result in good yields.
As exemplified by Scheme 1, the first step in pathway 1 begins
with a threefold alkylation of cyclen (1). This threefold alkylation
with tert-butylbromoacetate is a common method to generate
DO3A (2) by direct derivatization. This step is critical, because
the selective tris N-alkylation often leads to bis or quadruple alkyl-
ation as byproducts making workup complicated.
In recent years, many different methods and workup proce-
dures have been investigated to improve the regio-selectivity of
this reaction. Yields have ranged from ꢀ45% to 75%, and the best
results achieved thus far have been reported by Li and colleagues,
who obtained a yield of 77%, using a reaction time of 20 h at room
temperature.4,5 These results were obtained with MeCN and
3.1 equiv of tert-butylbromoacetate in the presence of triethyl-
amine (TEA) as a base.
We here elevated the reaction temperature to 60 °C, which en-
abled a reduction of the reaction time from 20 to 6 h, without a loss
in regio-selectivity, and with a concomitant gain in overall yield
(87%; Table 1). Purification was performed by HPLC. No significant
amounts of double or quadruple alkylated product were detected
by NMR or ESI-mass spectrometry. K2CO3 and KHCO3 as an alterna-
tive base had no influence on yield or purity of product (2).
Reaction II of pathway 1, that is, the alkylation of the free
secondary amine with ethyl bromoacetate (EBA; see Scheme 1),
afforded a good yield and purity (3). The reaction was performed
with K2CO3 in dry MeCN at 60 °C for 24 h, and the product was iso-
lated at a yield of 92% after HPLC purification.13
For introducing hydrazide moieties, the ethyl ester (4) was
brought to reaction with a 100-fold excess of hydrazine monohy-
drate in 10 mL ethanol and refluxed for 12 h. A yield of 86% was ob-
tained after 12 h of heating to reflux, comparable to 85% reported
in the literature.13 Reaction temperatures below 60 °C led to a sig-
nificantly lower yield or to no product.
The deprotection of the carboxylic acid (6) was achieved upon
reaction with concentrated HCl at room temperature for 5 h. The
resulting product (7) was evaporated to dryness, re-dissolved in
water, filtered (using a 0.2 lm syringe filter) and dried under
vacuum, according to previously established procedures.13 The
conversion was close to quantitative (96% after workup).
Using pathway 1, an overall yield of 67% was achieved for
DOTA–hydrazide synthesis compared to 55% in the literature.5,13,14
Furthermore, it was possible to reduce the overall reaction time by
15 h (from 62 to 47), and to increase the yield of the critical reac-
tion step I by 10% (from 77% to 87%).
Synthetic pathway 2 starts with a selective monoalkylation of
the secondary amines in cyclen (1) (see reaction III in Scheme 1).
As described above, the first reaction step in the generation of
DOTA–hydrazide is the most critical one, because a specific alkyl-
ation is needed, although in this case, the results are expected to
be better controllable than in the formation of trialkylated DO3A
(2). To investigate the regio-selectivity of reaction III, a series of
experiments were conducted with 0.8–1.3 equiv of the electrophile
EBA. The best results were achieved with 0.8 equivalents of EBA,
after a reaction time of 24 h at room temperature in CH2Cl2, result-
ing in a yield of 86% (as compared to 79% reported in the litera-
ture10). In the presence of excess cyclen, no multiple alkylation
products were detected by NMR and ESI-MS. Purification was again
performed using HPLC, and unreacted cyclen was retrieved and
used in further reactions. An alternative purification by column
chromatography was not performed, because decomposition in
the presence of silica gel has been reported in the literature.15
The reaction temperature was not elevated because of ring-closure
reactions at temperatures >40 °C.
Table 1
Overview of reaction conditions varied for reaction I (cf. Scheme 1)
For the synthesis of the orthogonally alkylated cyclen (5), the
monoalkylated product (4) was brought to reaction with tert-
butylbromoacetate. The best results were obtained with 3.1 equiv
in MeCN at room temperature after a reaction time of 4 h. Workup
conducted by HPLC resulted in a yield of 85%, comparable to 84%
reported in the literature.10
The formation of hydrazide (6) was performed according to the
protocol described in reaction V, rendering a yield of 82%. Depro-
tection of the carboxylic acid side arms was achieved within 4 h
Solvent
Electrophile (equiv)
Base
Time (h)
Temp (°C)
Yield (%)
MeCN
MeCN
MeCN
CHCl3
MeCN
CHCl3
MeCN
MeCN
3.5
3.3
3.3
3.3
3.3
3.5
3.1
3.5
K2CO3
TEA
K2CO3
K2CO3
K2CO3
TEA
48
48
48
24
24
24
6
25
25
25
25
25
25
60
60
47
79
71
29
38
66
87
52
TEA
TEA
6

920
F. Fuge et al. / Tetrahedron Letters 54 (2013) 918–920
after HCl treatment, according to the method described earlier for
reaction VI of synthetic pathway 1.
isterium für Bildung und Forschung (BMBF), project number
0315415C.
Synthetic pathway 2 resulted in an overall yield of 59%, com-
pared to 53% suggested by the literature with an overall reaction
time of 46 h.10,13,14
In pathway 1, the overall yield exceeds the results of pathway 2
and the literature values by more than 10%. Reducing the reaction
time by 14 h furthermore partially overcomes an important limita-
tion of this method (i.e. its relatively long reaction time), together
indicating that pathway 1 is preferable over pathway 2 for obtain-
ing high yields of DOTA–hydrazide within an acceptable time
frame.
References and notes
1. Liu, W.; Hajibeigi, A.; Lin, M.; Rostollan, C. L.; Kovacs, Z.; Oz, O. K.; Sun, X. Bioorg.
Med. Chem. Lett. 2008, 18, 4789–4793.
2. Kramer-Marek, G.; Shenoy, N.; Seidel, J.; Griffiths, G. L.; Choyke, P.; Capala, J.
Eur. J. Nucl. Med. Mol. Imaging 2011, 38, 1967–1976.
3. Li, C.; Winnard, P., Jr.; Bhujwalla, Z. M. Tetrahedron Lett. 2009, 50, 2929–2931.
4. Dadabhoy, A.; Faulkner, S.; Sammes, P. J. Chem. Soc., Perkin. Trans. 2 2002, 348–
357.
5. Li, Cong; Wong, W.-T. Tetrahedron 2004, 60, 5595–5601.
6. Wängler, C.; Wängler, B.; Eisenhut, M.; Haberkorn, U.; Mier, W. Bioorg. Med.
Chem. 2008, 16, 2606–2616.
In conclusion, a comparative analysis and systematic optimiza-
tion of the two most commonly used synthetic protocols for
generating DOTA–hydrazide was performed, improving overall
yields and reducing reaction times, without a loss in regio-selectiv-
ity. The higher flexibility of pathway 1 with regard to coupling site-
containing side chains and its higher overall yield (at a comparable
reaction time) exemplify that this synthetic strategy should be
preferred for DOTA–hydrazide synthesis.
7. Yoo, B.; Sheth, V. R.; Pagel, M. D. Tetrahedron Lett. 2009, 50, 4459–4462.
8. De Leon-Rodriguez, L. M.; Kovacs, Z.; Esqueda-Olivia, A. C.; Miranda-Olvera, A.
D. Tetrahedron Lett. 2006, 47, 6937–6940.
9. Develay, S.; Tripier, R.; Chuburu, F.; Le Baccon, M.; Handel, H. Eur. J. Org. Chem.
2003, 3047–3050.
10. Andre, J. P.; Geraldes, C. F.; Martins, J. A.; Merbach, A. E.; Prata, M. I.; Santos, A.
C.; de Lima, J. J.; Toth, E. Chem. Eur. J. 2004, 10, 5804–5816.
11. Massue, J.; Plush, S. E.; Bonnet, C. S.; Moore, D. A.; Gunnlaugsson, T. Tetrahedron
Lett. 2007, 48, 8052–8055.
12. Li, C.; Wong, W. T. J. Org. Chem. 2003, 68, 2956–2959.
13. Frullano, L.; Tejerina, B.; Meade, T. J. Inorg. Chem. 2006, 45, 8489–8491.
14. Bernhard, C.; Goze, C.; Rousselin, Y.; Denat, F. Chem. Commun. 2010, 46, 8267–
8269.
Acknowledgments
15. Wängler, B.; Beck, C.; Wagner-Utermann, U.; Schirrmacher, E.; Bauer, C.; Rösch,
F.; Schirrmacher, R.; Eisenhut, M. Tetrahedron Lett. 2006, 47, 5985–5988.
The authors gratefully acknowledge the financial support by the
German Ministry for Education and Research (BMBF)/Bundesmin-