A straightforward and convenient pathway for the synthesis of functional bismacrocyclic ligands

Article abstract of DOI:10.1016/j.tetlet.2011.01.133

The synthesis of a novel DO3A-based bismacrocyclic ligand is reported. The synthetic pathway involved a series of simple and convenient steps, which can easily provide the desired product in larger quantities than produced in current synthetic procedures.

Full text of DOI:10.1016/j.tetlet.2011.01.133

Tetrahedron Letters 52 (2011) 1619–1622
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A straightforward and convenient pathway for the synthesis of functional
bismacrocyclic ligands
Pascal Kadjane a, Nikos K. Logothetis a,b, Goran Angelovski a,
⇑
a Physiology of Cognitive Processes, Max Planck Institute for Biological Cybernetics, 72076 Tübingen, Germany
b Imaging Science and Biomedical Engineering, University of Manchester, Manchester M13 9PL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel DO3A-based bismacrocyclic ligand is reported. The synthetic pathway involved a
series of simple and convenient steps, which can easily provide the desired product in larger quantities
than produced in current synthetic procedures. This method enables the facile preparation of binuclear
macrocyclic complexes which can be used in MRI, as well as other molecular imaging modalities.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 December 2010
Revised 20 January 2011
Accepted 26 January 2011
Available online 1 February 2011
Keywords:
Contrast agents
DO3A
Macrocyclic chelators
Magnetic resonance imaging
Synthesis
A number of imaging techniques in medicine, such as X-ray
computed tomography (CT), positron emission tomography (PET)
and magnetic resonance imaging (MRI), allow the non-invasive
diagnosis of disease or damage in living systems by providing
three-dimensional images of tissues. Among these, MRI remains
the most widely employed technique in the clinical field. Unlike
CT and PET, MRI does not require ionizing radiation, and exploits
the difference in water relaxation rates in tissues to generate spa-
tially resolved images. The contrast of such images may be consid-
erably enhanced by using paramagnetic contrast agents. Ever since
it was demonstrated that Gd-DTPA can increase the relaxation rate
(R1) of water protons,1 several Gd-based contrast agents (CAs) (e.g.,
Gd-DOTA, Gd-HPDO3A) have been developed to improve image
quality.2
acterization of the Gd3+ complexes indicated their considerable po-
tential to operate as SCAs. Interestingly, the use of responsive
bismacrocylic complexes proved to be significantly more effective
than their monomacrocyclic analogs. Specifically the binding of a
metal ion in a 1:1 stoichiometry, was actually found to affect
two Gd3+ in the SCA altering both their r1. Therefore the resulting
MR signal was twice as strong as the signal change produced from
an SCA that contains only one Gd3+ center. Unfortunately, however,
the synthesis of the bismacrocyclic SCAs is more challenging when
compared to their monomacrocyclic equivalents.
There are several possible strategies to couple the Ca2+-chelat-
ing moiety of the SCA to its MR reporting unit (Gd-DO3A). The
linking of BAPTA-, DTPA- or EDTA-modified Ca2+ chelators was
achieved by the reaction of a corresponding bis-anhydride with a
DO3A-alkylamine.10,11 The reaction yields were up to 55% and
purification by HPLC was required due to the high polarity of the
two resulting carboxylic acids. Alternatively, EGTA-derivatized
SCAs were made by coupling a DO3A-alkylamine with 2-bromo-
acetic acid, followed by an N-alkylation reaction to attach these
two DO3A-derivatives to the Ca2+ chelator. In this case, the isolated
yields of the bismacrocyclic derivatives were high (up to 81%),
however purification by size exclusion chromatography was re-
quired. Most significantly the purification methods needed to iso-
late the bismacrocyclic ligand precursors, or the final products, are
not always readily available in all laboratories.
Over the last decade, however, contrast agents have been also
developed for detecting and reporting specific biological events.3
Such bioactivated, smart contrast agents (SCAs) are capable of
responding to enzymatic activity, pH or biologically relevant metal
ions such as Zn2+, K+ or Ca2+, by changing the MRI signal induced by
a change in their microenvironment.4–8
Efforts to develop new Ca2+-responsive, MRI-detectable CAs,
have been particularly strong, as they can potentially revolutionize
neuroimaging by providing noninvasive functional markers.9 We
have recently synthesised several bismacrocyclic molecules that
contained various Ca2+-chelators.8,10,11 The physicochemical char-
Here we report a simple and convenient synthesis of a new
DO3A-based bismacrocyclic ligand. The practical advantage of this
synthetic methodology involves the facile purification of all the
⇑
Corresponding author. Tel.: +49 7071 601 916; fax: +49 7071 601 919.
E-mail address: goran.angelovski@tuebingen.mpg.de (G. Angelovski).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.01.133

1620
P. Kadjane et al. / Tetrahedron Letters 52 (2011) 1619–1622
Chart 1. The structure of the synthesized bismacrocycle L.
intermediates throughout the synthetic route. The bismacrocyclic
ligand L (Chart 1) was synthesized in eight steps starting from
the commercially available 1,13-diamino-4,7,10-trioxatridecan 1
(Scheme 1). The primary amines of 1 were easily converted to sec-
ondary N-benzyl amines by reductive amination using NaBH4,
leading to 2 in a 63% yield. In parallel, the tris-Boc-protected cyclen
3 was prepared as previously described12 and then reacted with
bromoacetonitrile in CH3CN in the presence of K2CO3 to provide
4 in a high yield (93%). Thereafter the nitrile functionality was
readily reduced to the amine 5 by hydrogenation at room temper-
ature, employing the Raney-nickel catalyst and a solution of 2 N
sodium hydroxide. This was transformed into the corresponding
amide 6 by the use of chloroacetic anhydride in CH2Cl2. Note that
6 can also be obtained using chloroacetyl chloride under the same
conditions yielding the same amount of product (81%). The con-
densation of 2.4 equiv of 6 with 1.0 equiv of the bis-amine 2 in
the presence of KI resulted in the formation of the bismacrocyclic
compound 7. The presence of the Boc protecting groups on the cy-
clen nitrogens apparently reduces the polarity of this whole mole-
cule when compared to the analogous tert-butyl ester derivative of
DO3A. Thus 7 is easily purified by column chromatography over
silica gel, resulting in a 75% yield. The removal of the benzyl groups
to give 8 was achieved by hydrogenolysis in EtOH using Pd/C as a
catalyst at 60 °C. It is important to note that no trace of product
could be observed when the hydrogenolysis was attempted at
room temperature, possibly because of the bulkiness of the Boc
protective groups. Following the successful removal of the benzyl
groups, 8 was treated with a mixture of TFA/CH2Cl2 to cleave the
Boc protecting groups. The excess TFA was removed by evapora-
tion under reduced pressure and the resulting crude compound
was alkylated with tert-butyl bromoacetate in basic conditions
producing the ligand precursor 9. Finally, the complete deprotec-
tion of the tert-butyl esters to obtain the desired ligand L, was
accomplished with formic acid at 60 °C.13
Scheme 1. Synthesis of L. Reagents and conditions: (a) benzaldehyde, NaBH4, CH3OH, 63%; (b) bromoacetonitrile, K2CO3, CH3CN, 70 °C, 93%; (c) Raney-nickel, 2 N NaOH,
EtOH, rt; (d) chloroacetic anhydride, Et3N, CH2Cl2, 0 °C, 81%; (e) chloroacetyl chloride, Et3N, CH2Cl2, 0 °C, 81%; (f) KI, K2CO3, CH3CN, 60 °C, 75%; (g) H2, Pd/C, EtOH, 60 °C; (h)
TFA/CH2Cl2, rt; (i) tert-butyl bromoacetate, K2CO3, CH3CN, 70 °C, 59% over three steps; (j) formic acid, 60 °C, 97%.

P. Kadjane et al. / Tetrahedron Letters 52 (2011) 1619–1622
1621
Scheme 2. Reagents and conditions: (a) tert-butyl bromoacetate, K2CO3, 80 °C, 59%; (b) H2, Pd/C, CH3OH, rt; (c) benzyl bromoacetate, K2CO3, CH3CN, 80 °C, 50%; d) H2, Pd/C,
CH3OH, rt; e) tris-t-Bu-DO3A-EA, EDCI/NMM/HOBt or HBTU or carbonyldiimidazole, DMF, 60 °C; f) formic acid, 60 °C.
The effectiveness of this synthetic strategy was demonstrated
when comparing it to those more frequently used. This includes
the synthesis of the bismacrocyclic ligand L via the condensation
of the biscarboxylic acid 13 with tris-tert-Bu-DO3A-ethylamine11
by using common coupling reagents for peptide synthesis14 to
optimize the reaction yield (Scheme 2).
sponding lanthanide chloride salt. The r1 of the Gd2L was
3.9 mMꢀ1sꢀ1 in HEPES buffer (pH 7.4), 25 °C and 300 MHz. The de-
cay of the luminescent intensity of the Eu2L was measured in buf-
fered H2O and D2O, yielding with the lifetime values
sH2O = 0.73 ms
and D2O = 1.43 ms. Using the empirical equation for the estimation
s
of the hydration number,18 q was determined to be 0.4. Both the r1
and q are in the range typically observed for bismacrocyclic com-
pounds of this type.8 However, the relaxometric titration experi-
ments, performed in the absence and presence of Ca2+ and Mg2+
exhibited only moderate r1 changes upon the addition of up to
20 equiv. of either Ca2+ or Mg2+. The maximum r1 value reached
4.3 mMꢀ1sꢀ1, corresponding to a 10% increase from the initial r1
value. The similar nature of the results obtained from these two
titrations showed that the complex responds poorly to both of
these ions, despite possessing a Ca2+-chelator which exhibits a
selective interaction towards alkaline earth metal ions when incor-
porated in a responsive fluorescence dye.19
The biscarboxylic acid 13 was prepared initially from 2, so that
orthogonal protecting groups could be attached to both of the car-
boxylates of the desired product. The secondary amine 2 was alkyl-
ated with tert-butyl bromoacetate to yield 10. The benzyl groups
were removed by hydrogenation and subsequent alkylation of 11
with benzyl bromoacetate yielded the tetraester 12 which contains
two different protecting groups that can be removed selectively
under acidic (tert-Bu esters) or reductive (benzyl esters) condi-
tions. The benzyl esters were converted to acids by hydrogenation
using identical reductive conditions described previously for 13.
The first condensation reactions utilized the conventional re-
agents EDCI, NMM and HOBt. However, only the trace amounts
of the desired product 9 could be isolated (Table 1). The use of
HBTU provided 9 in a moderate yield after column chromatogra-
phy purification over silica gel. The tert-butyl esters were removed
successfully using formic acid, however during the Gd3+-complex
In summary, we have developed an easy and efficient procedure
to obtain a new DO3A-based bismacrocyclic ligand. This simplified
route involves a few facile synthetic steps requiring relatively easy
purification procedures to obtain bismacrocyclic products. The
effectiveness of this route was demonstrated by the synthesis of
the molecule which can serve as potential SCA. The procedure
can be applied to the future synthesis of a versatile range of bis-
macrocyclic products and can be used for preparation of various
functional ligands that can find application in several molecular
imaging methods.
formation and the subsequent relaxometric titration with Ca2+
,
the formation of the precipitate was observed. This can be ex-
plained by the contamination of 9 with the hexafluorophosphate
ðPF6ꢀÞ anions, which are then hydrolyzed to phosphoric acid dur-
ing the acid mediated deprotection of the tert-butyl esters.15 Ulti-
mately, it is the presence of these phosphate anions in the
solution that form insoluble salts in the presence of cations such
Acknowledgments
as Gd3+ and Ca2+ 16,17
Finally, the reaction involving the third cou-
.
pling reagent, carbonyldiimidazole, gave 9 in a similar yield when
compared to HBTU. However, in this case the complete cleavage of
the tert-butyl esters using formic, trifluoroacetic or hydrochloric
acid could not be achieved.
We are thankful to Dr. M. Placidi and Dr. I. Mamedov for helpful
discussions. Financial support of the Max-Planck Society and the
German Research Foundation (DFG, grant AN 716/2-1) is gratefully
acknowledged. The European COST Action D38 ‘Metal-Based Sys-
tems for Molecular Imaging Applications’ is also acknowledged.
Finally Gd3+ and Eu3+ complexes of the novel ligand L were pre-
pared and their physicochemical properties were investigated. The
complexation was performed in H2O at neutral pH from corre-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.01.133.
Table 1
Conditions for the coupling reaction of 13 and tris-t-Bu-DO3A-ethylamine to yield 9
Coupling agent
Solvent and temperature
Yield%
References
EDCI/NMM/HOBt
HBTU
Carbonylimidazole
DMF, rt or 60 °C
DMF, 60 °C
DMF, 60 °C
—
32
33
1. Felix, R.; Schorner, W.; Laniado, M.; Niendorf, H. P.; Claussen, C.; Fiegler, W.;
Speck, U. Radiology 1985, 156, 681–688.

1622
P. Kadjane et al. / Tetrahedron Letters 52 (2011) 1619–1622
2. Reichert, D. E.; Lewis, J. S.; Anderson, C. J. Coord. Chem. Rev. 1999, 184, 3–66.
3. Que, E. L.; Chang, C. J. Chem. Soc. Rev. 2010, 39, 51–60.
13. Analytical data for the ligand L: 1H NMR (300 MHz, C. d.-m., 76H), 1.83–1.47
(m, 4H). 13C {1H} NMR (75 MHz, CDCl3): d 175.3, 175.1, 170.9, 170.6, 170.0,
165.8, 69.7, 69.6, 69.4, 69.3, 69.1, 68.9, 68.8, 68.2, 56.9, 56.6, 56.4, 55.4, 55.3,
54.6, 54.1, 52.5, 51.6, 51.4, 51.0, 50.7, 50.3, 49.6, 48.4, 48.1, 48.0, 47.6, 36.6,
34.2, 25.2, 23.6. ESI-HRMS: for C50H89N12O21ꢀ: calcd 1193.62707 [MꢀH]ꢀ,
found 1193.62714.
14. Han, S. Y.; Kim, Y. A. Tetrahedron 2004, 60, 2447–2467.
15. Deifel, N. P.; Holman, K. T.; Cahill, C. L. Chem. Commun. 2008, 6037–
6038.
16. Pan, H. B.; Darvell, B. W. Cryst. Growth Des. 2009, 9, 639–645.
17. Laurent, S.; Vander Elst, L.; Henoumont, C.; Muller, R. N. Contrast Media Mol.
Imaging 2010, 5, 305–308.
18. Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle, L.; de
Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin Trans.2 1999, 493–
503.
4. Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats, R.; Jacobs, R. E.;
Fraser, S. E.; Meade, T. J. Nat. Biotechnol. 2000, 18, 321–325.
5. Zhang, S. R.; Wu, K. C.; Sherry, A. D. Angew. Chem., Int. Ed. 1999, 38, 3192–3194.
6. Major, J. L.; Boiteau, R. M.; Meade, T. J. Inorg. Chem. 2008, 47, 10788–10795.
7. Hifumi, H.; Tanimoto, A.; Citterio, D.; Komatsu, H.; Suzuki, K. Analyst 2007, 132,
1153–1160.
8. Angelovski, G.; Fouskova, P.; Mamedov, I.; Canals, S.; Toth, E.; Logothetis, N. K.
Chem.BioChem. 2008, 9, 1729–1734.
9. Jasanoff, A. Curr. Opin. Neurobiol. 2007, 17, 593–600.
10. Mishra, A.; Fouskova, P.; Angelovski, G.; Balogh, E.; Mishra, A. K.; Logothetis, N.
K.; Toth, E. Inorg. Chem. 2008, 47, 1370–1381.
11. Dhingra, K.; Fouskova, P.; Angelovski, G.; Maier, M. E.; Logothetis, N. K.; Toth, E.
J. Biol. Inorg. Chem. 2008, 13, 35–46.
12. Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997, 119, 3068–3076.
19. Kakizawa, Y.; Akita, T.; Nakamura, H. Chem. Lett. 1993, 1671–1674.