Efficient synthesis of new tetradentate ligands with potential applications for 64Cu PET-imaging

Article abstract of DOI:10.1016/j.bmcl.2010.12.072

We wish to report the synthesis of new tetradentate ligands in less than 3 h in good to excellent yields from a commercially available compound using microwave-assisted technology. First tests of complexation showed a high ability of these ligands to chel

Full text of DOI:10.1016/j.bmcl.2010.12.072

Bioorganic & Medicinal Chemistry Letters 21 (2011) 924–927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of new tetradentate ligands with potential applications
for 64Cu PET-imaging
Ewen Bodio a, Karine Julienne a, Sébastien G. Gouin a, Alain Faivre-Chauvet b, David Deniaud a,
⇑
a CNRS, UMR 6230, Université de Nantes, Chimie et Interdisciplinarité: Synthèse Analyse Modélisation, UFR Sciences et Techniques, 2 Rue de la Houssinière, BP92208,
44322 Nantes Cedex 03, France
b Centre de Recherche en Cancérologie de Nantes-Angers, Inserm, Institut deBiologie, Université de Nantes, U892, 9 quai Moncousu, 44093 Nantes Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We wish to report the synthesis of new tetradentate ligands in less than 3 h in good to excellent yields
from a commercially available compound using microwave-assisted technology. First tests of complexa-
tion showed a high ability of these ligands to chelate 64Cu(II) in very diluted medium. These new systems
have the potential to be used for nuclear medicine and particularly for PET-imaging.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 October 2010
Revised 12 December 2010
Accepted 15 December 2010
Available online 19 December 2010
Keywords:
Chelating agent
Copper 64
PET
Thiazole
Microwave
In recent years, advances in imaging technology and more partic-
ularly in positron emission tomography (PET) have generated
increasing interest.1–5 PET has numerous advantages over other
imagery techniques such as good spatial resolution (better than
SPECT—Single Photon Emission Computed Tomography—) and excel-
lent sensitivity (better than all imaging technologies),2 versatility. . .
Its sensitivity is so high that it allows using only nanomolar to picom-
olar concentrations of radiopharmaceutical to get an image.6
Many positron-emitting radionuclides have been investigated.
18F is by far the most commonly used of the longer-lived PET radio-
nuclides (18F half-life = 110 min).7 The most famous example is
FDG ([18F]-fluorodeoxyglucose) which is widely used for tumor
imaging. However, when the pharmacokinetics of the radiophar-
maceutical are slow, the short half life of 18F can be inhibitory. In
these cases 64Cu (half-life = 761.9 min) seems an excellent alterna-
tive.8–10 Indeed, specific sites imaging can be reached by tethering
a copper chelating agent to biologically active molecules (sugar,
peptide, antibody. . .) that selectively bind to certain receptors in
vivo.1,2,11–13 The long half life of copper is particularly important
as several hours to several days may be necessary to get a signifi-
cant accumulation of the injected radionuclide in the tumor.8,9
Furthermore, 64Cu displays similarly good spatial resolution,
with respect to 18F (around 5.0 mm),14 some studies have shown
that it gives the highest effective dose and above all, that it can
be used in preliminary dosimetry studies for radioimmunotherapy
(use of radiolabelled antibodies to target and destroy tumor
cells).15–17 In fact, the main problem in radioimmunotherapy is
reproducibility in the measurement of be able to measure out
the amount of radioactivity bound to the tumor cells (it depends
on the kind of cancer, on the vector and even on the patient).
One promising solution is to use the dual emission nature of an ele-
ment such as copper which possess both a beta+ emitter (64Cu) and
a betaÀ emitter (67Cu). With 64Cu, radioactivity can be quantified
and as the same vector could be used to transport 67Cu to tumor
cells, the pharmacokinetics would be the same and the quantity
of 67Cu linked to tumor would be known.2,18
These considerations in combination with our groups expertise in
chelation chemistry and heterocyclic chemistry led us to explore the
design of new copper chelating agents. Copper complexes of bis(thi-
osemicarbazone) ligands represent an important class of com-
pounds.19–22 These ligands are particularly interesting due to their
rapid complexation kinetics, the ease and the efficiency of their syn-
thesis and their ability to form neutral complexes. Consequently, our
attention has been focused on these types of bis(thiosemicarbazone)
ligands. Moreover, our strategy allows one to tune the properties of
the ligands by varying R1 and R2 on the heterocyclic groups (Fig. 1).
By this way, we combine both our experience in the synthesis of che-
lating agents23–27 and our knowledge in heterocyclic chemistry.28–30
Our initial synthetic strategy hinged on of a biscoupling reaction
between a dicarbonyl compound, namely isophthalaldehyde, and
two thiazolohydrazines. Isophthalaldehyde is commercially avail-
able and the thiazolohydrazines were synthesized readily via adap-
tion of our recently reported thiazole synthesis (Scheme 1).28,29
⇑
Corresponding author.
E-mail address: david.deniaud@univ-nantes.fr (D. Deniaud).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.072

E. Bodio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 924–927
925
provided 6 in 98% yield as a pure pale yellow powder. Compound 6
was converted to the bis(thiazadiene) 7 by a double condensation
reaction with commercially available N,N-dimethylformamide
dimethyl acetal (DMF-DMA) in ethanol. Compound 7 could be
isolated, but usually engaged directly in the double cyclization with
N
N
N
N
HN
NH
S
S
a-bromoketones. This strategy gave the expected ligands in 67–74%
overall yields (Scheme 2).
R1
R2
R2
R1
The scope of the reaction and especially the electronic effect of
the R group on the
a-bromoketones have been studied using vari-
O
R1
=
and R2 = H
ous -bromoketones (electron neutral: para-chlorobenzyl, electron
a
R
donating: tert-butyl and electron withdrawing: trifluoromethyl).
Ligand L1–L3 displayed poor water solubility, a very unfortunate
property for compounds aimed at biological applications. Conse-
quently the synthesis of the bis(ester) ligand L4 was targeted in or-
der to open easy access to the corresponding bis(carboxylate).
Moreover, modifications of ester functionalities are very versatile;
consequently such a ligand would offer a facile route to several
other chelating agents.
This approach allowed the three step synthesis of ligands L1–L4
in good yield.37,38 Furthermore, under microwave irradiation, the
overall synthesis was achieved in less than 3 h including all work-
ups and purifications (Table 1).
or R1 = H and R2 = R
Figure 1. Tetradentate ligands L1–L8.
Compound 1 was prepared in 78% yield by a monocondensation
of commercially available N,N-dimethylformamide dimethyl acetal
and thiourea in methanol, and was protected with a tert-butyloxy-
carbonyl group. Protected thiazadiene 2 was treated with an
a
-bromoketone, to give the corresponding S-alkyl salt which
underwent a spontaneous cyclization, followed by deamination
of the cycloadduct to afford the expected thiazole 3 in good yield.
Hydrazine 4 was synthesized by an electrophilic N-amination reac-
tion.31–33 The presence of the boc group enabled sodium hydride to
deprotonate the thiazadiene 2. The just formed amidure then re-
acted with O-(4-nitrobenzoyl)hydroxylamine to give the hydrazine
4 in 53% yield (the synthesis of O-(4-nitrobenzoyl)hydroxylamine
was previously reported by Carpino in a two step strategy).34 The
hydrazine was then deprotected in good yield.
To study the influence of the position of the R group on the hetero-
cycle, ligands with a substituent on position 5 of the thiazole rings
were synthesized. These chelating agents L5–L8 were efficiently pre-
pared from bis(thiosemicarbazone) 6 and the different previously
used a-bromoketones(Scheme3). S-Alkylintermediate8, underwent
in situ cyclization via condensation of the generated imines with the
ketones. The ligands L5–L8 were obtained in good to excellent overall
yields (70–96% from isophthalaldehyde) (Table 2), the difference of
yield being due to a more or less poor solubility in organic solvent.
For these kinds of chelating agents the ability to use various
Despite a successful test reaction with benzaldehyde, attempts
to obtain ligand L1, by condensation of hydrazines 4 or 5 with
isophthalaldehyde were unsuccessful.
a-bromoketones with electronically different R was important. As
We, thus, decided to change our approach. Instead of coupling the
heterocycles at the last step, we chose to start from isophthalalde-
hyde, and to synthesize in parallel, step by step, the two heterocycles.
The first step was a double condensation reaction of commercially
available thiosemicarbazide with isophthalaldehyde. The reaction
could be conducted using ‘classical heating’ to afford 6 in good yield
in 10 h. A highly efficient process was essential as purification was
challenging due to the poor solubility of the thiosemicarbazide and
products. Under microwave irradiation,35,36 the reaction time was
reduced to 15 min and the reaction was cleaner: a simple filtration
the R groups are directly bound to the heterocyclic rings of the
ligand, they should influence the electron density located on chelat-
ing atoms. Thus, the chelation properties of the ligand may be tuned
by changing of the
a-bromoketone.
Preliminary complexation tests were performed with L1 using
64CuCl2. To form the 64Cu-chelated complexes, 1
lL of an aqueous
ammonium acetate solution (2.5 M) was added to a 64CuCl2
solution (10À7 M) in aqueous hydrochloric acid (0.1 M) providing
a
64Cu(OAc)2 solution. To this medium were added 3
lL of an
O
R
S
S
1)
, DCM, r.t., 1 h
O
Boc2O, NaH
THF, 0 °C r.t., 12 h
Br
S
NHBoc
H2N
N
N
BocHN
N
N
R
N
3
2) TEA, r.t., 20 h
R = p-Cl-C6H4
1
2
quantit.
71%
1) NaH, THF, reflux, 1 h
O
NH2
O
2)
O2N
, THF, r.t., 12 h
O
O
H
N
Boc
N
NH2
1) TFA, DCM 0 °C
2) NaHCO3, water
r.t., 5 h
S
S
R
NH2
R
N
N
5
4
90%
53%
Scheme 1. The synthesis of thiaziolohydrazines 5.

926
E. Bodio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 924–927
S
NH2
H2N
N
H
DMF-DMA
N
S
N
S
HN
NH
N
S
N
S
EtOH, MW (120 °C), 15 min
HN
H2N
NH
NH2
DMF, MW (50 °C), 10 min
O
O
N
N
N
N
6
7
98%
O
1)
, DMF
Br
R
N
N
N
HN
NH
2) Et3N, DMF, MW (50 °C), 10 min
S
N
S
O
O
R
R
L1 - L4
68-76% (2 steps)
Scheme 2. The synthesis of ligands L1–L4.
Table 1
Table 2
Yields of ligands L1–L4
Yields of ligands L5–L8
Entry
R
Overall yield (%)
Entry
R
Overall yield (%)
L1
L2
L3
L4
p-Cl-C6H4-
tBu-
F3C-
69
67
74
71
L5
L6
L7
L8
p-Cl-C6H4-
tBu-
F3C-
96
97
70
93
EtO2C-
EtO2C-
aqueous sodium acetate buffer and 4
1 lL of a solution of L1 in DMF (1 or 10 equiv) was finally added.
l
L of dimethylformamide.
of 1:1. The different results show that is not a binuclear complex like
is classically described in the literature.40–44 We obtained a single
copper centre with four donor atoms from the chelator.
The solution was incubated at room temperature for 30 min. The
chelation yield was measured on a phosphorimager apparatus
after TLC on silica gel plates.
The experimental results revealed a high chelation rate of 70%
when one equivalent of L1 was used, and more than 80% when
10 equiv were used. These results compare in comparison with
currently used chelation agents.2 These results are very exciting
as they suggest that the complexation kinetics are very fast even
in hostile conditions: room temperature, acid buffer and in very
diluted medium (10À9 M).
The composition of the synthesized complex CuL1 was estab-
lished by IR, UV, mass spectrometry (MALDI) and elemental analysis
with stable isotope of copper.39 The NMR spectrum was presented
broad peaks due to this paramagnetic configuration. Energy-disper-
sive X-ray spectroscopy (EDXS) indicated a copper to ligand L1 ratio
In conclusion, we have designed new tetradentate ligands and
more particularly, we have developed two fast and efficient origi-
nal methods for their microwaved assisted synthesis. In two or
three steps, these methods furnished quite sophisticated ligands
in less than 3 h and in good to excellent yields (from 67% to
97%). Furthermore, the versatility of our two synthetic methods
should allow us to study the influence that the nature and position
of substituents position on the heterocyclic rings has on the chela-
tion properties. A preliminary complexation test using radioactive
copper demonstrated the ability of L1 to chelate this metal very
efficiently both kinetically and thermodynamically. These results
encourage us to envision the synthesis of functionalized analogues
of ligands L1–L8 giving chelates that could be grafted to chemical
or biological vectors.
O
Br
R
N
S
N
S
N
N
N
N
N
N
HN
H2N
NH
NH2
HN
NH
HN
NH
DMF, rt, 2 h
.2HBr
S
NH
O
HN
O
S
S
S
R
R
R
R
L5 L6
-
6
8
71-99%
Scheme 3. The synthesis of ligands L5–L8.

E. Bodio et al. / Bioorg. Med. Chem. Lett. 21 (2011) 924–927
31. Erdik, E.; Ay, M. Chem. Rev. 1989, 89, 1947.
927
Acknowledgments
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33. Shen, Y.; Friestad, G. K. J. Org. Chem. 2002, 67, 6236.
The authors are grateful to the Région Pays de la Loire, to the
French Ministry of Education and to the CNRS for financial support.
34. Carpino, L. A.; Giza, C. A.; Carpino, B. A. J. Am. Chem. Soc. 1959, 81, 955.
35. Loupy, A. Spectra Anal. 1993, 22, 33.
36. Kremsner, J. M.; Kappe, C. O. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.)
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37. General procedure for ligands L1–L4: Thiosemicarbazide (2.19 mmol, 2 equiv)
and isophtalaldehyde (1.10 mmol, 1 equiv) were suspended in 4 mL of ethanol
in a 10 mL-microwave reactor. The reactor was placed in the microwave oven.
The reactor was heated from room temperature to 120 °C (OF) on 2 min by
monomode microwaves irradiation (power: 150 W, stirring: 50%, ventilation:
1/3), maintained at 120 °C for 15 min (power: 50 W, stirring: 50%, ventilation:
1/3) and let cooled down to room temperature (power: 0 W, stirring: 50%,
ventilation: 3/3). The mixture was filtered and washed with ethanol to give 6.
Compound 6 (0.71 mmol, 1 equiv) and DMF-DMA (1.78 mmol, 2.5 equiv) were
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