Facile synthesis of 1-(acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane: a reactive precursor chelating agent

Article abstract of DOI:10.1016/j.tetlet.2009.03.198

1-(Acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (DOTA-Tris-^tBu ester or 1) is a precursor chelating agent for lanthanide ions and can be efficiently labeled with various functional moieties or macromolecules to improve the targeting specificity, intracellular delivery, bio-compatibility, and pharmacokinetics of resulting contrast media used in molecular imaging. This compound is commercially available but the extremely high cost seriously limits its wide utilization. Thus, we sought a convenient and inexpensive synthesis of DOTA-Tris-^tBu ester that is readily adapted for use in any laboratory. The synthetic approach described here is straightforward and has an overall yield of 92%. Significantly, the product can be purified conveniently without using of a time- and labor-intensive column chromatography. Other advantages of this method, such as operational convenience, starting material availability, and atom efficiency make it very attractive to prepare DOTA-Tris-^tBu ester in large quantity with a reduced cost.

Full text of DOI:10.1016/j.tetlet.2009.03.198

Tetrahedron Letters 50 (2009) 2929–2931
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Facile synthesis of 1-(acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,
4,7,10-tetraazacyclododecane: a reactive precursor chelating agent
b
b,
Cong Li a,b, , Paul Winnard Jr. , Zaver M. Bhujwalla
*
*
a School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China
b JHU ICMIC Program, The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
1-(Acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (DOTA-Tris-tBu
ester or 1) is a precursor chelating agent for lanthanide ions and can be efficiently labeled with various
functional moieties or macromolecules to improve the targeting specificity, intracellular delivery, bio-
compatibility, and pharmacokinetics of resulting contrast media used in molecular imaging. This com-
pound is commercially available but the extremely high cost seriously limits its wide utilization. Thus,
we sought a convenient and inexpensive synthesis of DOTA-Tris-tBu ester that is readily adapted for
use in any laboratory. The synthetic approach described here is straightforward and has an overall yield
of 92%. Significantly, the product can be purified conveniently without using of a time- and labor-inten-
sive column chromatography. Other advantages of this method, such as operational convenience, starting
material availability, and atom efficiency make it very attractive to prepare DOTA-Tris-tBu ester in large
quantity with a reduced cost.
Article history:
Received 15 June 2008
Revised 27 March 2009
Accepted 27 March 2009
Available online 2 April 2009
Keywords:
DOTA
Cyclen
Contrast media
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
10
Real time non-invasive medical diagnosis in living subjects has
been possible with the development of diagnostic imaging tech-
niques such as X-ray computed tomography (CT), positron emis-
sion tomography (PET), magnetic resonance imaging (MRI) and
optical imaging. Contrast media play a crucial role in improving
the sensitivity and resolution of images, and achieving targeting
specificity at the molecular/cellular level. Among various parame-
ters to evaluate the performance of contrast media, low toxicity
tops the list. For example, lanthanide ions are widely used in
MRI contrast agents,1 radioactive tracers,2 and optical imaging
probes.3,4 However, free lanthanide ions usually show high toxicity
in vivo. To overcome this limitation, chelating agents that can coor-
dinate metal ions tightly are widely used to minimize toxicity. In
various chelating agents, polyazamacrocyclic 1,4,7,10-tetra-acetic
acid-1,4,7,10-tetraazacyclododecane (DOTA) demonstrated high
thermodynamic stability and kinetic inertness to a larger number
of transition and lanthanide metal ions.5 Nevertheless, small
molecular DOTA chelators have their own inherent shortcomings.
For example, commercial available MRI contrast agent Gd3+-DOTA
(DotaremTM) showed low relaxivity and extremely fast excretion
rate in vivo. To increase the relaxivity, Gd3+-DOTA complexes were
labeled to macromolecules, such as proteins,6 micellar aggregates,7
dendrimers,8 or liposomes9 by prolonging the rotational correla-
tion lifetime sR
.
Meanwhile, to image specific cellular/molecular
evens in vivo, Gd3+-DOTA was conjugated to targeting domains
such as antibodies,11 peptides,12 and biotin/avidin.13 Additionally,
Gd3+-DOTA was also functionalized with polyethyl glycols (PEGs)14
or crown ethers15 to increase its biocompatibility and circulation
lifetime in circulation system.
To efficiently label the DOTA chelate to various molecules, a
reactive group for covalent binding must be introduced into this
chelator. Since amine groups are prevalent in biomolecules, and
the conversion of a pendant carboxylic acid of DOTA into a carbox-
amide does not significantly affect the stability of the labeling me-
tal complex,
a facile approach is the conjugation of DOTA
derivatives with biomolecules though a physiologically stable
amide bond. In this respect, regio-selective activation of carboxylic
acid in DOTA to react with amines under a mild reaction condition
is a feasible way to achieve this goal.
1-(Acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-
tetraazacyclododecane (DOTA-tris-tBu ester or 1) as a reactive che-
lating agent has been widely used to label DOTA chelator to various
biomolecules. In this molecule, three of the four secondary amines
in the cyclen ring were regioselectively alkylated with tert-butyl
acetate, and the remaining one was functionalized with an acetic
acid. In this way, this compound can be directly and specifically
conjugated with biomolecules by forming an amide bond without
the potential intermolecular cross-linking.16 Additionally, three
pendant tert-butyl acetates can be easily hydrolyzed to acetic acids
that are ready for complexation with metal ions.
* Corresponding authors. Tel.: +86 21 6404 1911; fax: +86 21 6404 2268 (C.L.);
tel.: +1 410 955 6864; fax: +1 410 614 1948 (Z.M.B.).
E-mail addresses: congli@mri.jhu.edu (C. Li), zaver@mri.jhu.edu (Z.M. Bhujwalla).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.198

2930
C. Li et al. / Tetrahedron Letters 50 (2009) 2929–2931
Despite the wide application potential of compound 1 in medi-
ous solution of KOH (0.17 M, final concentration), and a product
with higher polarity was detected after stirring for 4 h at 50 °C.
To increase the yield, longer reaction time (up to 24 h) and higher
concentration of NaOH (up to 0.5 M, final concentration) were ap-
plied. However, no obvious improvement in yield was achieved. To
clarify this experimental result, a similar reaction was conducted
in MeOH/H2O. Disappointingly, an identical phenomenon was ob-
served and the yield of product was still low. MS analysis revealed
a new product 1-(methyl acetate)-4,7,10-tris(tert-butoxycarbon-
ylmethyl)-1,4,7,10-tetraazacyclododecane 4, which assumes the
base-catalyzed trans-esterification in the methanol. To prevent
the re-esterification, different solvent systems including CH3CN/
H2O, dioxane/H2O, and DMF/H2O (v:v = 2:1) were tested in the
presence of NaOH (0.4 M, final concentration) at 50 °C (Table 1).
The experimental results showed that dioxane/H2O was the sol-
vent of choice, which gave the highest yield (>90%) of product 1
and no other by-products were detected by either TCL or MS. Com-
pared with CH3CN and DMF, the high stability of dioxane in basic
solution and its excellent solubility to compound 3 are the main
reasons for its optimal performance in this reaction.
In order to investigate the relationship between base concentra-
tion and the yields of 1, comparative studies were performed in the
presence of various concentrations of NaOH at 50 °C (Fig. 1). The
yield of 1 was proportional to the concentrations of NaOH added
in the range of 0–0.4 M, and the maximal value of 95% was ob-
tained after a 4-h reaction in dioxane/H2O with 0.4 M NaOH. Fur-
ther increase of base did not improve the yield. The yield
decreased slightly after NaOH concentration exceeded 1.0 M. It is
also noteworthy that no product with higher polarity than 1 was
detected even at the highest concentration of NaOH tested, which
indicates that the tert butyl-acetate remained intact even under
highly basic surroundings.
cal diagnostic imaging, to purchase the needed quantities is extre-
mely costly, which severely limits its usage. Several synthetic
procedures to prepare compound 1 were reported, however, the
shortcomings include the requirement of expensive reactive re-
sin17 or catalyst,18 and relatively low total yield17,18 making them
difficult to prepare 1 within a reasonable cost. tert-Butyl ester as
a protecting group of carboxylic acids is widely used in synthetic
chemistry because it is easily removed in acidic conditions, while
demonstrating stability in basic surroundings. Meanwhile, alkyl
esters, such as ethyl acetate, can be hydrolyzed efficiently in both
acidic and basic surroundings. In this work, we present a novel
synthetic method to prepare chelator 1 efficiently by taking advan-
tage of selective hydrolysis of pedant ethyl acetate but not tert-bu-
tyl acetates under basic conditions. Compared with the previous
methods, the advantages that include high yield, convenient puri-
fication process, ease in handling, and starting material concentra-
tion-independent yield confer the preparation of compound 1 with
a reasonable price.
In a typical synthetic procedure (Scheme 1), 1.0 equiv ethyl bro-
moacetate in 5 mL CH3CN was added dropwise under N2 into a
mixture of 1,4,7-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaz-
acyclododecane 219 and anhydrous K2CO3 (1.5 equiv) in 30 mL
CH3CN at 55–60 °C. The reaction was monitored by TLC until all
starting material was consumed. At the end of reaction, K2CO3
was filtered and the solvent was removed to give 1-(ethoxycarbon-
ylmethyl)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetra-
azacyclododecane 3 with a yield of 98%.20
The selective hydrolysis of ethyl acetate in compound 3 was
tested in a series of hydrolytic reaction conditions as shown in Ta-
ble 1, and TLC and MS were used to monitor the reaction process.
First, compound 3 was dissolved in the mixture of EtOH and aque-
The functions of both temperature and reaction time to the
yield of compound 1 under the reaction condition of dioxane/
H2O/0.4 M NaOH were also studied (Fig. 2). The hydrolytic rate of
N-alkylated ethyl acetate accelerated substantially at high temper-
ature. At room temperature, the yield of 95% was achieved after 8 h
of reaction, while this yield was attained in less than 4 h at 45–
50 °C. However, by-products with higher polarity than 1 were de-
tected by TLC after 4 h of reaction when the temperature was
above 80 °C. Mass spectrum analysis demonstrated that tert-butyl
acetate began to hydrolyze at the temperature above 80 °C, and
that the by-products were proved to be chelators with two or three
N-alkylated acetic acids.
tBuOOC
tBuOOC
tBuOOC
tBuOOC
COOEt
N
N
N
N
N
N
HN
N
BrCH2COOEt
CH3CN/K2CO3
COOtBu
COOtBu
tBuOOC
2
3
COOH
N
N
N
N
Dioxane/NaOH
50 °C, 4 h
tBuOOC
COOtBu
1
Scheme 1. Synthetic procedure to prepare compound 1.
100
80
Table 1
Effect of solvents and auxiliary bases to the yield of compound 1
Entry
Cond.a
Base
Yieldb (%)
1
4
CH3ONac
NaOHd
CH3ONac
NaOHd
NaOHd
NaOHd
NaOHd
21
56
15
28
68
73
96
n.d.e
n.d.e
33
60
1
2
3
4
5
6
7
EtOH
EtOH
MeOH
MeOH
DMF
MeCN
Dioxane
34
n.d.e
n.d.e
n.d.e
40
a
Starting material 3 with the concentration of 15 mM in selected solvent sys-
tems, 4–6 h, 50 °C.
20
b
0.0
0.2
0.4
0.6
0.8
1.0
Yield of compound 1 or 4 is calculated by comparing the proton integration of –
OCH2 in 3 or –OCH3 in 4 with CCH3 in tert-butyl group from 1H NMR spectra.
Conc. of NaOH (M)
c
CH3ONa was added with catalytic dosage.
NaOH was added as the aqueous solution with a final concentration of 0.4 M.
Not detected.
d
Figure 1. Plot of the yield of compound 1 as a function of concentrations of NaOH in
dioxane/H2O after reaction for 4 h at 50 °C.
e

C. Li et al. / Tetrahedron Letters 50 (2009) 2929–2931
2931
and starting material availability, all of which are very helpful in
reducing the production cost of this compound.
100
80
60
40
20
0
Acknowledgments
This work was supported by NIH P50 CA103175 (JHU ICMIC
Program) and R21 CA128957.
References and notes
25 oC
50 oC
80 oC
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Valtancoli, B.; Virtuani, M. J. Chem. Soc., Dalton Trans. 2000, 697.
6. Li, C.; Winnard, P. T., Jr.; Takagi, T.; Artemov, D.; Bhujwalla, Z. M. J. Am. Chem.
Soc. 2006, 128, 15072.
0
2
4
6
8
10
Reaction time (h)
7. Nicolle, G. M.; Toth, E.; Eisenwiener, K. P.; Macke, H. R.; Merbach, A. E. J. Biol.
Inorg. Chem. 2002, 7, 757.
Figure 2. Plot of the yield of compound 1 as a function of temperature and reaction
time in dioxane/H2O/NaOH (0.4 M).
8. Jaszberenyi, Z.; Moriggi, L.; Schmidt, P.; Weidensteiner, C.; Kneuer, R.; Merbach,
A. E.; Helm, L.; Toth, E. J. Biol. Inorg. Chem. 2007, 12, 406.
9. Kamaly, N.; Kalber, T.; Ahmad, A.; Oliver, M. H.; So, P. W.; Herlihy, A. H.; Bell, J.
D.; Jorgensen, M. R.; Miller, A. D. Bioconjugate Chem. 2008, 19, 118.
10. Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Chem. Soc. Rev. 1998, 27, 19.
11. Lipinski, M. J.; Amirbekian, V.; Frias, J. C.; Aguinaldo, J. G.; Mani, V.; Briley-
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2006, 56, 601.
12. Ke, T.; Jeong, E. K.; Wang, X.; Feng, Y.; Parker, D. L.; Lu, Z. R. Int. J. Nanomed.
2007, 2, 191.
13. Artemov, D.; Mori, N.; Ravi, R.; Bhujwalla, Z. M. Cancer Res. 2003, 63, 2723.
14. Mohs, A. M.; Wang, X.; Goodrich, K. C.; Zong, Y.; Parker, D. L.; Lu, Z. R.
Bioconjugate Chem. 2004, 15, 1424.
15. Li, C.; Li, Y. X.; Law, G. L.; Man, K.; Wong, W. T.; Lei, H. Bioconjugate Chem. 2006,
17, 571.
16. Lewis, M. R.; Raubitschek, A.; Shively, J. E. Bioconjugate Chem. 1994, 5, 565.
17. Oliver, M.; Jorgensen, M. R.; Miller, A. D. Synlett 2004, 3, 453.
18. Heppeler, A.; Froidevaux, S.; Mäcke, H. R.; Jermann, E.; Béhé, M.; Powell, P.;
Hennig, M. Chem. Eur. J. 1999, 5, 1974.
1.00
0.95
0.90
0.85
19. Li, C.; Wong, W. T. Tetrahedron 2004, 60, 5595.
20. Preparation of the 1-(ethyl acetate)-4,7,10-tris(tert-butoxycarbonylmethyl)-
1,4,7,10-tetraazacyclododecane
1,4,7,10-tetraazacyclododecane
3.
2
1,4,7-Tris(tert-butoxycarbonyl-methyl)-
(1.0 g, 1.9 mmol) was dissolved in a
0
20
40
60
80
100
mixture of 50 mL anhydrous acetonitrile and K2CO3 (540 mg, 3.8 mmol,
2 equiv). Then ethyl bromoacetate (317 mg, 1.9 mmol, 1.0 equiv) in 5 mL
acetonitrile was added. This suspension was allowed to stir for 12 h under N2
at 70 °C. The reaction was monitored by TLC plates. After all the starting
material was consumed, crude product (light-yellow oil) was purified by flash
chromatography on silica gel (dichloromethane/methane = 10/1 (v/v),
Conc. of starting material 3 (mM)
Figure 3. Plot of the yield of compound 1 as a function of the concentration of
starting material 3 in dioxane/H2O/NaOH (0.4 M) after 4 h of reaction at 50 °C.
Tf = 0.35) to give
3 as a
light yellow foam (1.1 g, yield: 98%). 1H NMR
(400 MHz, CDCl3): d 4.04 (q, J = 7.4 Hz, 2H), 3.78–1.65 (a set of very broad and
multiple peaks with an integration corresponding to 24H), 1.36 (s, 27H), 1.16
(t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 175.7 (C), 172.4 (2 Â C), 172.2
(C), 82.0 (2 Â C), 81.9 (C), 59.2 (CH2), 57.4 (CH2), 56.2 (2 Â CH2), 55.7 (CH2),
53.0–50.8 (4 Â CH2, a set of broad peaks), 50.4-47.4 (4 Â CH2, a set of broad
peaks), 28.2 (6 Â CH3), 28.1 (3 Â CH3), 13.6 (CH3); FAB+-MS m/z 623 (M+Na)+;
HRFAB+-MS calcd for C30H56N4O8Na (M+Na)+ 623.3996, found 623.3986.
21. Preparation of the 1-(acetic acid)-4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,
10-tetraazacyclododecane 1. 1-(Ethyl acetate)-4,7,10-tris(tert-butoxycarbonylm-
ethyl)-1,4,7,10-tetraazacyclododecane 3 (400 mg, 0.67 mmol, crude product
without purification) was dissolved in the mixture (15 mL) of dioxane and NaOH
withtheratio of3:1 (v:v). This solutionwasstirred vigorously forabout 4 hunder
N2 at 50 °C. Dioxane was removed in vacuo and water (20 mL) was added. The
mixture was extracted 3Â with CH2Cl2 (3 Â 30 mL). The organic phases were
combined and further washed 2Â with brine. The organic solution was pre-dried
and the solvent was removed to leave a colorless glass-like solid product. This
material was re-dissolved in 20 mL diethyl ether and allowed to re-crystallize by
cooling the solution at 4 °C for overnight. The final product 3 was obtained as a
white solid with the yield of 94%. 1H NMR (400 MHz, CDCl3): d 3.65–1.68 (a set of
very broad and multiple peaks with an integration corresponding to 24H), 1.39
(s, 27H); 13C NMR (100 MHz, CDCl3): d 175.9 (C), 172.4 (2 Â), 172.1 (C), 82.0
(2 Â C), 81.9 (C), 57.3 (CH2), 56.3 (2 Â CH2), 55.6 (CH2), 53.2–50.9 (4 Â CH2, a set
of broad peaks), 50.5–47.7 (4 Â CH2, a set of broad peaks), 28.3 (6 Â CH3), 28.1
(3 Â CH3); FAB+-MS m/z 595 (M+Na)+; HRFAB+-MS calcd for C28H52N4O8Na
(M+Na)+ 595.3683, found 595.3668.
We further investigated the relationship between the concen-
trations of starting material 3 and the yields of hydrolytic product
1 in dioxane/H2O/0.4 M NaOH at 50 °C. After 4 h of reaction, the
yield of 1 remained above 92% while the concentration of 3 in-
creased from 5 to 100 mM (Fig. 3). Significantly, product 1 can be
purified conveniently without the time- and labor-costly column
chromatography. Simple extraction steps offered the purified com-
pound 1 that was well characterized by 1H, 13C NMR and MS.21 The
solvent-friendly synthetic procedure and convenient purification
strongly indicate that production of compound 1 in large scale
and at reduced cost is possible.
In this study, we developed an efficient two-step synthetic pro-
cedure to prepare 1-(acetic acid)-4,7,10-tris(tert-butoxycarbon-
ylmethyl)-1,4,7,10-tetraazacyclododecane (1), a reactive chelating
agent that is widely used in medical diagnostic and therapeutic
applications. Compared with previous works, our method not only
gives the highest yield, but also offers the attractive features such
as operational convenience, solvent-friendliness, easy purification,