Synthesis and metal complexation properties of Ph-DTPA and Ph-TTHA: Novel radionuclide chelating agents for use in nuclear medicine

Article abstract of DOI:10.1039/b413758b

We wish to report the synthesis and metal complexation properties of new radionuclide chelating agents for use in nuclear medicine. The strategy includes the facile preparation of rigid analogues of DTPA and TTHA possessing an aromatic ring. The aromatic

Full text of DOI:10.1039/b413758b

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A R T I C L E
Synthesis and metal complexation properties of Ph-DTPA
and Ph-TTHA: novel radionuclide chelating agents for use in
nuclear medicine
Se´bastien G. Gouin,a,b Jean-Franc¸ois Gestin,b Laurence Monrandeau,c
Fabienne Segat-Dioury,d Jean Claude Meslina and David Deniaud*a
a Laboratoire de Synthe`se Organique, UMR CNRS 6513, Faculte´ des Sciences et des
Techniques, 2, Rue de la Houssinie`re, BP 92208, 44072, Nantes Cedex, France.
E-mail: david.deniaud@chimie.univ-nantes.fr; Fax: 33 2 5112 5402; Tel: 33 2 5112
b INSERM U463, Chimie des Bioconjugue´s, 9, Quai Moncousu, 44093, Nantes, France
c Department of Medical Technology & Physics, Sir Charles Gairdner Hospital, Hospital Ave,
Nedlands, WA 6009, Australia
d Laboratoire de Chimie Organique, Conservatoire National des Arts et Metiers, ESA CNRS
7084, 2 Rue Conte´, 75003, Paris, France
Received 8th September 2004, Accepted 18th November 2004
First published as an Advance Article on the web 10th January 2005
We wish to report the synthesis and metal complexation properties of new radionuclide chelating agents for use in
nuclear medicine. The strategy includes the facile preparation of rigid analogues of DTPA and TTHA possessing an
aromatic ring. The aromatic structure used increased the stability of the complexes formed (pre-organization
concept) and they are easily functionalised for attaching to any support. The poly(amino)poly(carboxylic) acids,
Ph-DTPA (5a) and Ph-TTHA (5b) were obtained in five steps from phenylenediamine as the starting material with
overall yields of 42 and 20%, respectively. The key step in this synthetic process is the preparation of tri- and
tetra-amino compounds, 3a and 3b, respectively. In order to assess the ability of both ligands to complex with
different metals (111In, 153Sm, 90Y, 177Lu, 213Bi, 225Ac), along with their suitability for use in nuclear medicine, we used a
number of complementary tests. We were able to demonstrate the high complexation capacity of Ph-DTPA (5a) with
a broad range of radionuclides in a slightly acidic medium. In vitro stability studies show the high stability of
Ph-DTPA with 111In in human serum, a necessary condition for all medical applications. The protonation constant
(log KHi ) of Ph-DTPA (5a) was determined by potentiometric methods.
nitro group, the precursor of an isothiocyanate function, which
Introduction
enables chelating agents to be coupled to antibodies or haptens.42
Radioimmunotherapy (RIT) is a developing and promising
These considerations brought us to use a benzene ring for
technique for the treatment of small tumors.1 It consists of
the synthesis of novel rigid analogues of DTPA and TTHA,
injecting patients with a radiopharmaceutical able to target and
which were termed Ph-DTPA and Ph-TTHA, respectively.43,44
to selectively destroy tumor cells.2 The radionuclides usually
The complexation capacity of these compounds was tested on
a range of radionuclides used in nuclear medicine. Tests were
used are a or b− emitters with a short half-life, coupled to the
vector (an antibody or hapten) through a bifunctional chelating
also performed to determine the stability of these complexes in
agent.3–8 In order to deliver radionuclides to tumors whilst min-
human serum, an absolute requirement for potential use in IS
imizing irradiation of healthy tissue,9 the chelating agent must
and RIT. Furthermore, potentiometric titrations have been used
be able to form a stable complex in human serum. Classically
used ligands are polyaminopolycarboxylic acid compounds with
a sufficient number of O- and N-donor groups to satisfy the
to determine the protonation constants of Ph-DTPA (5a).
Results and discussion
high coordination number of the radionuclides, which generally
varies from 8 to 10.10–15 Diethylenetriaminepentaacetic acid
(DTPA) is particularly effective and this molecule is widely used
for radiodiagnostic purposes in immunoscintigraphy (IS).16–21
While not as frequently used,22 the homologous triethylenete-
traaminehexaacetic acid (TTHA) is nonetheless an interesting
ligand and forms chelates with high complexation constants with
a great number of lanthanides.23,24
It has been established that in order to improve the stability
of the complexes formed, it is necessary to use ligands with a
semi-rigid or rigid structure (pre-organization concept).25–27 This
is why nowadays many research projects actually focus on the
synthesis of chelating agents with cyclohexane, cyclopentane,
or macrocyclic structures.28–35 Few teams however work on the
development of ligands with a benzene skeleton.36–39 Mederos
et al. and Tanaka et al. have shown that aromatic analogues of
ethylenediaminetetraacetic acid (EDTA) are effective chelating
agents, notably in acidic conditions.40,41 Moreover, aromatic
rings have the property of being easily functionalized with a
Chemical synthesis
Ph-DTPA and Ph-TTHA were synthesized from ortho-
phenylenediamine according to similar protocols (Scheme 1).
Amide 1a was prepared by monoacylation of the starting
compound with DCC activated Cbz-glycine, according to the
method described by Bermejo et al.45 The carbobenzyloxy group
was then cleaved off by palladium-catalyzed hydrogenolysis
which generated diamine 2a with a 98% yield.46 The carbonyl
group was then effectively reduced by the BH3·DMS complex in
THF.47 The triamine thus obtained was isolated and purified as
its chlorohydrate 3a form through bubbling HCl gas in ethanol.48
In order to synthesize tetraamine 3b, Cbz-glycine was activated
into an acid chloride, then opposed to ortho-phenylenediamine
to obtain the original product 1c quantitatively, contrary to
classical coupling by DCC. It was not possible to deprotect
diamine 1c by hydrogenolysis in neutral medium with palladium
on charcoal as catalyst, probably due to the fact that 1c is highly
4 5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 5 4 – 4 6 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 1 Synthesis of polyamines 3a and 3b. Reagents and conditions: (i) Cbz-NHCH2CO2H, DCC, THF, 4 h, rt; (ii) Pd/C, H2, EtOH, 24 h,
rt; (iii) BH3-DMS, THF, 28 h, reflux; (iv) Fmoc-NHCH2COCl, (i-Pr)2EtN, THF, 1 h, rt for 1b; Cbz-NHCH2COCl, Et3N, CH2Cl2, 1.5 h, rt for 1c;
(v) from 1b, piperidine, DMF, 0.5 h, rt.
Scheme 2 Synthesis of Ph-DTPA (5a) and Ph-TTHA (5b). Reagents and conditions: (i) tert-buyl bromoacetate, KI, K2CO3, CH3CN, 80 ◦C, 72 h
for 4a and 96 h for 4b; (ii) NaOH 2 M, EtOH, 60 ◦C, 12 h then 6 M HCl.
insoluble in ethanol. This led us to change the nature of the
protective group on glycine. Compound 1b functionalized with
a Fmoc group that can be cleaved in basic medium, was syn-
thesized with a yield of 94%. Quantitative deprotection of both
nitrogens was achieved using piperidine in DMF. Reduction
of compound 2b was then carried out as previously described.
As opposed to triamine 3a, tetraamine 3b was not isolated
as its chlorohydrate form which is too hygroscopic to provide
convenient storage.
Alkylation of both compounds 3a and 3b was induced by
tert-butyl bromoacetate according to the method described by
Studer (KI, K2CO3). Products 4a and 4b were obtained with
respectively 65 and 41% yields (Scheme 2).49,50
The uncompleted cleavage of ester groups of compounds 4a
and 4b by using trifluoroacetic acid,51,52 led us to proceed the
deprotection step in basic medium in a mixture of ethanol and
2 M aqueous solution of sodium hydroxide. After acidification
with hydrochloric acid, Ph-DTPA (5a) and Ph-TTHA (5b) were
then isolated as their chlorohydrate forms with yields of 88 and
90%, respectively.
was conducted with UV-vis spectroscopy. The second test was
intended to estimate the rate of complexation in an extremely
diluted and slightly acidic medium. The last test would only be
performed if the first two tests proved conclusive and was aimed
at determining the stability of a metal complex in human serum,
a requirement for its use in RIT or IS.
The first test was performed with non-radioactive metal
cations and aimed at determining if ligands 5a and 5b were
effective chelates. As both ligands have a benzenic chromophore,
the corresponding absorption spectra exhibited an intense band
around 230 nm, which corresponds to a P → P* transition
(emax = 8500 dm3 mol−1 cm−1, kmax = 228.4 nm for 5a and
emax = 12000 dm3 mol−1 cm−1, kmax = 229 nm for 5b). Upon
metal complexation, an electronic perturbation is generated,
which usually translates into a modification of the absorption
spectrum. The procedure consists of dissolving the metal
trichloride in distilled water to a final concentration of 1 mM in
neutral medium (pH ca. 7). After 1 h incubation with constant
stirring at rt, a few ml of the solution are analyzed by UV-vis
spectroscopy (200–800 nm). Fig. 1 summarizes results obtained
with three metals (In, Y, and Sm) complexed with Ph-DTPA (5a)
and Ph-TTHA (5b).
Complexation tests
In these three examples, in the presence of an equimolar
amount of In(III), Y(III) or Sm(III), an important modification
of the UV absorption spectrum of Ph-DTPA (5a) and Ph-
TTHA (5b) was observed (except for Ph-TTHA with samarium).
The band around 230 nm disappears due to a hypochromic
In order to assess the ability of both ligands to complex different
metals along with their suitability for use in nuclear medicine,
three complementary tests were planned. The first test was
designed to evaluate the chelating efficiency of the ligands and
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radionuclides used in immunoscintigraphy (111In and 153Sm), and
b− and a emitters used in RIT (90Y, 177Lu, 213Bi and 225Ac).
For each radionuclide studied, a series of samples at con-
centrations ranging from 10−6 to 10−10 M were prepared in
a buffered aqueous solution (pH 5.5). For these tests, we
used a slightly acidic medium to avoid precipitation of metal
hydroxides. Increasing amounts of both ligands, Ph-DTPA (5a)
and Ph-TTHA (5b) were then added and the resulting samples
were incubated for 1 h at 37 ◦C. Thin-layer chromatography
(TLC) associated with a judicious choice of eluent enabled
the separation of the free form of the radionuclide from the
complexed one, as the former does not migrate in the established
conditions. Radioactivity on TLC plates was quantified using a
phosphorimager. These experiments were also done with DTPA,
Cy-DTPA, CHX-DTPA or DOTA (Fig. 2) as references for their
similar number and nature of coordinating atoms as well as
for their known high kinetics of complexation with the studied
radionuclides.53
This study was done with two c emitters used in IS, indium
(
111In, T1/2 = 2.83 d) and samarium (153Sm, T1/2 = 1.95 d),
which also has a b− component.54 Both types of emission of this
radionuclide could allow imaging and therapy to be combined.
Because no perturbation of the UV absorption spectrum had
been observed when samarium cation was incubated with Ph-
TTHA ligand (Fig. 1), this ligand was not tested with the
radioactive isotope (153Sm).
A representative TLC plate with Ph-DTPA–indium and Ph-
TTHA–indium complexes (ligand–indium ratio of 1 : 1) is shown
in Fig. 3. As staining intensity is a function of the locally emitted
radioactivity, the rate of complexation can be measured. Results
are summarized in Table 1.
Fig. 1 UV-vis spectra of 5a and 5b and their complexes with indium,
yttrium and samarium. Blue curve: 5a or 5b; red curve: Y complex; green
curve: In complex; black curve: Sm complex.
effect, that is indicative of a perturbation produced by the
complexation of the metal ion near the aromatic chromophore.
As a consequence, Ph-DTPA should be an effective ligand to
complex indium, yttrium, and samarium cations as well as the
homologous compound Ph-TTHA for indium and yttrium.
However, with the assumption that, for a given ligand, the
conformation of the complexed species is comparable for In(III),
Y(III), Sm(III) and thus should induce similar modification of
the UV absorption spectrum (as is the case for Ph-DTPA), Ph-
TTHA proved to be ineffective to chelate samarium cations.
At last, similar experiments enable us to demonstrate effective
chelation of both ligands with lutetium and bismuth. As non-
radioactive actinium trichloride was not available, analysis by
UV spectroscopy could not be performed.
Fig. 3 Thin-layer chromatography analysis of 5a-111In and 5b-111In
complexes.
Results showed that, under our experimental conditions, both
chelating agents complexed indium cations quantitatively and
proved to be as effective as the well known DTPA ligand. The
strong chelating capacity of Ph-DTPA (5a) was also observed
for samarium as the observed rates of complexation, 73% with a
stoichiometric amount of ligand, and 95% with a two-fold molar
excess, are comparable to those obtained with DTPA.
After having verified the ability of ligands 5a and 5b to
trap a given metal we assessed complexation effectiveness by
determining the rate of complexation. In order to do so in
labelling conditions close to those used in nuclear medicine,
Ph-DTPA and Ph-TTHA were tested with photon emitting
Fig. 2 Structure of polyaminocarboxylate ligands used as references.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 5 4 – 4 6 1
4 5 6

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Table 1 Complexation capacity of 5a and 5b with two photon emitters (111In and 153Sm)a
111In chelated/%
153Sm chelated/%
Molar equivalent of ligand DTPA
Ph-DTPA (5a)
Ph-TTHA (5b)
DTPA
Ph-DTPA (5a)
Ph-TTHA (5b)
1
2
5
100
100
—
98
100
—
100
100
—
76
100
100
73
95
100
—
—
—
153Sm] = 0.74 lmol dm−3; [111In] = 1.34 nmol dm−3; incubation = 1 h; pH = 5.5–5.8 (0.1 M sodium acetate buffer); temperature = 37 ◦C; each point
a
[
is the average of the percentage amount from three replicates.
Table 2 Complexation capacity of 5a and 5b with two b− particle emitters (90Y and 177Lu)a
90Y chelated/%
177Lu chelated/%
Molar equivalent of ligand
DTPA
Ph-DTPA (5a)
Ph-TTHA (5b)
DOTA
Ph-DTPA (5a)
Ph-TTHA (5b)
1
2
10
102
103
9
—
75
97
97
23
—
93
97
100
6
—
37
43
56
87
100
—
100
—
55
100
—
100
—
0
0
—
55
—
a [90Y] = 44 nmol dm−3; [177Lu] = 200 lmol dm−3; incubation = 1 h; pH = 5.5–5.8 (0.1 M sodium acetate buffer); temperature = 37 ◦C; each point is
the average of the percentage amount from three replicates.
Table 3 Complexation capacity of 5a and 5b with two a particle emitters (213Bi and 225Ac)a
213Bi chelated/%
Molar equivalent of ligand Cy-DTPA Ph-DTPA (5a)
225Ac chelated/%
CHX-DTPA
Ph-TTHA (5b)
Ph-DTPA (5a)
Ph-TTHA (5b)
102
103
104
105
106
0
0
69
86
—
17
17
67
70
88
—
—
—
—
0
—
—
—
0
—
—
—
0
49
71
73
—
23
0
0
213Bi] = 0.67 nmol dm−3; [225Ac] = 0.25 nmol dm−3; incubation = 1 h; pH = 5.5–5.8 (0.1 M sodium acetate buffer); temperature = 37 ◦C; each point
a
[
is the average of the percentage amount from three replicates.
Two b− emitters potentially used in RIT were then studied:
yttrium (90Y, T1/2 = 2.67 d), which is currently used in clinical
applications,12,55 and lutetium (177Lu, T1/2 = 6.71 d). Results are
presented in Table 2 and show important differences in the
affinity of Ph-DTPA and Ph-TTHA for these radionuclides.
When 93% of yttrium cation is complexed in a presence of
ten-fold molar excess of ligand Ph-DTPA (5a), Ph-TTHA (5b)
appeared to be less effective as only 37% of the radionuclide is
chelated in the same conditions. Furthermore, even a 103-fold
molar excess of ligand 5b only chelates 56% of the yttrium cation.
It should be noted that, in these experiments, the measured
rate of complexation is not directly proportional to the number
of equivalents of ligand as the solution of the metallic cation
contains a small part of non-radioactive isotopes which are
undetected by the radioactivity counter. However, it remains
a method of choice for estimating the rate of complexation of
radionuclides in high dilute medium.
with the radionuclide, a high excess of ligand has been used to
ensure the total complexation of the cationic species. Results
obtained with bismuth-213 showed the superior complexation
ability of both Ph-DTPA (5a) and Ph-TTHA (5b) over Cy-DTPA
(Table 3), which is currently considered the best chelating agent
for bismuth.13,57,58 Indeed, as opposed to 5a and 5b, a greater
excess of Cy-DTPA must be used to observe complexation with
bismuth. Conversely, Ph-DTPA (5a) and Ph-TTHA (5b) were
ineffective at chelating actinium; no complexation was observed,
even with high excess of ligand (106 mol equiv.). However, in such
conditions, CHX-DTPA achieves complexation of actinium-
225 with a rate of 23%. The poor results obtained with this
radionuclide are in accordance with the reported results that
showed improved complexation rates with macrocyclic ligands
rather than with their linear analogues.59
In conclusion, these tests revealed the promising complexation
properties of Ph-DTPA (5a) for the radionuclides studied. This
ligand proved to be more effective than DTPA and Cy-DTPA,
to complex yttrium-90 and bismuth-213, respectively. Results
obtained with Ph-TTHA (5b) were not as clear cut. While
being rather ineffective at complexing yttrium-90, lutetium-177,
and actinium-225, this chelating agent was shown to chelate
indium and bismuth effectively, as reflected by the high rates
of complexation obtained. We thus envisaged the evaluation of
the stability of these complexes in human serum, an essential
condition for their use in IS and RIT.
Results obtained with lutetium (177Lu), and DOTA as refer-
ence compound, showed even more marked differences between
5a and 5b.56 With a two-fold molar excess of ligand, Ph-
DTPA proved to be competitive to DOTA with a complete
complexation of lutetium, as opposed to Ph-TTHA, which only
complexed 55% of lutetium cation with a ligand–metal ratio of
100 : 1.
Experiments conducted with bismuth (213Bi, T1/2 = 45.6 min)
and actinium (225Ac, T1/2 = 10 d)—both of them are being tested
as a emitters in phase I and II of clinical trials—were carried out
using solutions with even lower radionuclide concentrations. As
radioactive sources and buffered solutions contain unknown and
varying amounts of non-radioactive isotopes which compete
A number of serum proteins and enzymes have the ability to
trans-chelate radionuclides from metal complexes or to cleave
off radiopharmaceuticals. In order to be used in IS and RIT,
these decomplexation processes must be avoided as much as
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Fig. 4 Stability of 111In–Ph-TTHA and 111In–Ph-DTPA complexes in human serum.
Table 4 Comparison of the protonation constants of Ph-DTPA (5a)
possible. Very few novel chelating agents have been shown to
form complexes with a good stability in blood. The kinetic
inertness of a complex in serum appears to be a major property
to evaluate this stability,60 and we thus decided to study our
complexes in such conditions.
and Ph-DTA with the constants of linear analogues (DTPA and EDTA)
Protonation
constants
Ph-DTPA 5aa
DTPAb
Ph-DTAc
EDTAd
After incubation of buffered solutions of radionuclides and
ligand in human serum, the separation of the complexed forms
of radionuclide (serum protein-bound versus ligand-bound)
was realized by size exclusion chromatography and quantified
by radioactivity measurements. Typical profiles obtained are
presented in Fig. 4 where the blue curve corresponds to serum
protein-bound metal, and the other curves reflect radioactivity
emitted by metal complexes as a function of time of incubation.
This study was realized only for metal chelates having a
quantitative rate of complexation (Ph-DTPA–In, –Sm, –Y, –Lu,
and Ph-TTHA–In).
−log KH1
−log KH2
−log KH3
−log KH4
−log KH5
−R log KHi
10.29
6.33
4.05
3.02
—
10.06
8.32
4.13
2.50
2.29
27.30
6.64
4.76
3.60
2.88
0.60
17.88
9.79
6.21
2.45
1.95
1.50
21.90
23.69
a 0.1 M KCl, 25 ◦C. b Ref.63, 1 M KCl, 25 ◦C. c Ref.64, 0.1 M KCl, 25 ◦C.
d Ref.65, 1 M KCl, 25 ◦C.
peralkylation of compounds 3a and 3b. We demonstrated the
high complexation capacity of 5a, the rigid analogue of DTPA
with a broad range of radionuclides (111In, 153Sm, 90Y, 213Bi, 177Lu)
in slightly acidic medium (pH = 5.5). Chelation rates obtained
with this ligand proved to be of the same order of magnitude, and
sometimes even greater, than those obtained with DTPA. We
also noted an important difference in the chelation ability of
both ligands with Sm, Y and Lu as Ph-DTPA (5a), with 8
coordination centers, usually has a higher complexation capacity
than Ph-TTHA 5b, with 10 coordination centers. This result is
in accordance with observations made when comparing DTPA
and Cy-DTPA complexes with the homologous TTHA and Cy-
TTHA complexes, which are usually less stable. Indeed, when the
metal coordination number is exceeded, an excessive increase in
the number of chelating functions of a ligand would often would
lead to the formation of more labile complexes.66
Moreover, Ph-DTPA (5a) was shown to form stable complexes
with indium in human serum, a necessary condition for all
medical applications. We report here the acidity constants of
this ligand determined by potentiometric titration.
These preliminary results encouraged us to envision the syn-
thesis of functionalized analogues of Ph-DTPA (5a) in order to
develop a ligand that could be grafted to chemical or biological
vectors. As Ph-DTPA (5a) is able to complex different metals,
it may be used for applications other than nuclear medicine
when functionalized. For instance, binding of this molecule to
substrates such as silica gels may be considered in solid–liquid
extraction systems used for the treatment of effluents.67,68
The 111In complex formed with Ph-TTHA (5b) exhibited poor
stability in human serum as after 4 h of incubation (green curve),
trans-complexation of more of 50% of 111In from Ph-TTHA
to human serum proteins had occurred. This phenomenon
is amplified after 24 h incubation at 37 ◦C, as most of the
radioactivity is detected on human serum proteins. Similar
results were found for Ph-DTPA–Y, Ph-DTPA–Lu, and Ph-
DTPA–Sm complexes as after 15 h of incubation, more than
50% of the radionuclide was bound to serum proteins.
However, with Ph-DTPA, the trans-complexation of 111In did
not occur and after 145 h of incubation (6 d) only 6% of the
radionuclide (pink curve) had been displaced, which suggests
that this complex is a good candidate for IS.
As a consequence, the evaluation of the stability constant of
the complex formed with Ph-DTPA (5a) and 111In should be
fundamental for medical applications.
In order to do so, the determination of the acidity constants
of the ligand is usually needed, so we decided to conduct
potentiometric titrations of the fully protonated ligand (5a) as
reported for other polyaminopolycarboxylate ligands.61 In our
conditions,62 four stepwise protonation constants were found
(Table 4).
By comparison with the aliphatic analogue DTPA, the first
and the third protonation constants are very similar. The second
one is lower than that of DTPA and is responsible of an overall
decrease of basicity of the ligand. This result is in accordance
with the decrease of basicity observed by rigidification of EDTA
with a benzenic ring (Ph-DTA, Fig. 2).
Experimental
Conclusion
Chemistry
We have developed two original structures Ph-DTPA (5a) and
Ph-TTHA (5b) that mimic the heteroatom linking of DTPA and
TTHA. Both of them were synthesized in five steps with respec-
tive overall yields of 42 and 20%, the rate-limiting step being the
All reagents were purchased from Acros Organics and Aldrich.
All chemicals were reagent grade and used without further
purification, and all solvents were freshly distilled before use.
The CNRS Analysis Laboratory (Vernaison) performed the
4 5 8
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elemental analyses. Column chromatography was conducted
over silica gel 60 (40–63 lm), available from E. Merck. Melting
points measured using a Reichert microscope are uncorrected.
The 13C and 1H-NMR spectra were recorded at rt using a Bruker
AC200 operating at 50 and 200 MHz, respectively. Chemical
shifts (d) are given in ppm downfield from tetramethylsilane as
internal standard. Mass spectra were determined with a Hewlett
Packard 5989 spectrometer. The IR spectra were obtained using
a Bruker Vector 22 spectrometer.
was repeated three times to afford product 2b as a colorless oil.
(200.7 mg, 100%): IR (KBr): 3258, 1669, 1598, 1525, 1296 cm−1;
dH (CD3OD) 3.48 (s, 4H, 2CH2), 7.23 (s, 2H, 2CaH), 7.60 (s, 2H,
2CbH); dC (CD3OD) 45.4 (2CH2), 126.0 (2CH), 127.1 (2CH),
131.6 (2CarN), 173.9 (2CO); MS m/z: 223 (100, [M + H]+), 180.
N-(2-Aminophenyl)ethylenediamine trichloride (3a)69
Compound 2a (400 mg, 2.47 m◦mol) was dissolved in anhydrous
THF (10 cm3) and cooled to 0 C before addition of BH3·DMS
(6.2 cm3, 12.2 mmol) 2 M in THF. The solution was stirred at
reflux for 28 h under nitrogen. The solution was evaporated to
dryness and the residue, cooled in an ice bath. Methanol was
added (25 cm3◦) and the solution was saturated with HCl gas
for 0.5 h at 0 C. The solution was evaporated to dryness and
the residue was redissolved in absolute ethanol (10 cm3). The
solution was saturated with HCl gas 0.5 h at 0 ◦C. The resulting
pink precipitate was filtered on a frit under argon and vacuum
o-Phenylene-N,N-bis-[2-(fluoren-9-
yl)methyloxycarbonylaminoethanamide] (1b)
N-(9-Fluorenylmethoxycarbonyl)glycine (1 g, 3.36 mmol) and
oxalyl chloride (457 mg, 3.60 mmol) were added to anhydrous
THF (15 cm3) at 0 ◦C under inert atmosphere. A catalytic
quantity of DMF (24 mg, 0.33 mmol) was added and the
mixture was stirred for 45 min at rt. The solution was injected
under inert atmosphere into a mixture of o-phenylenediamine
(151 mg, 1.40 mmol), diisopropylethylamine (1.37 g, 10.6 mmol)
in anhydrous THF (15 cm3). The resulting solution was stirred
for 1 h at rt. After evaporation under reduced pressure, the resi-
due was taken up in dichloromethane (50 cm3) and the organic
phase is washed with water (40 cm3). The organic phase
precipitate was filtered on a frit and rinsed with dichloromethane
(10 cm3) to afford compound 1b as a white solid. (898 mg, 94%):
mp: 168–170 ◦C; IR (KBr): 3316, 1708, 1676, 1526, 1253 cm−1;
dH (DMSO) 3.88 (s, 4H, 2CH2), 4.24 (s, 2H, 2CH), 4.32 (s, 4H,
2CH2), 7.10–7.90 (m, 20H, 20CarH), 9.36 (s, 2H, 2Car NHCO);
dC (DMSO) 44.2 (2CH), 46.7 (2CH2), 65.9 (2CH2), 120.1, 121.4,
◦
dried. (579 mg, 91%): mp: 210–212 C; IR (KBr): 3429, 2879,
1507, 1326 cm−1; dH (CD3OD) 3.27 (t, 2H, CH2NH2, J = 5.8 Hz),
3.58 (t, 2H, NHCH2, J = 5.8 Hz), 7.00 (m, 2H, 2CarH), 7.40
(m, 2H, 2CarH); dC (CD3OD) d 39.7 (CH2NH2), 41.8 (NHCH2),
115.7 (CarH), 119.4 (Car), 121.0 + 125.5 + 132.0 (3CarH), 142.9
(Car). L-SIMS− m/z 256.6 ([M − H]+, 100), 403, 331.
o-Phenylene-N,N-bis(ethylenediamine) (3b)
Compound 2b (680 mg, 3.05 mmol) and a 2 M solution of
complex BH3·DMS in THF (7.5 cm3, 15.0 mmol) were added
to anhydrous THF (25 cm3) at 0 ◦C. The mixture was heated
at reflux under nitrogen atmosphere for 23 h. After evaporation
under reduced pressure, the residue was dissolved in methanol
(25 cm3). Bubbling of hydrochloric acid gas into the solution
was carried out for 30 min at 0 ◦C. The solution was evaporated
under reduced pressure and the residue was dissolved in absolute
ethanol (18 cm3). Bubbling of hydrochloric acid gas into the solu-
tion was repeated as previously described. The precipitate was fil-
tered on a frit, dried under reduced pressure and added to a 5 M
NaOH solution (80 cm3). The product was extracted from the
aqueous phase with dichloromethane (3 × 100 cm3). The organic
phase was dried over magnesium sulfate, filtered, and evaporated
under reduced pressure to afford compound 3b as a colorless
oil. (343 mg, 58%): IR (KBr): 3345, 2936, 2861, 1598 cm−1; dH
(CDCl3) 2.43 (br s, 4H, 2NH2), 2.97 (t, 4H, 2CH2, J = 5.2 Hz),
3.16 (t, 4H, 2CH2, J = 5.2 Hz), 6.65 (m, 2H, 2CH), 6.78 (m,
2H, 2CH); dC (CDCl3) 41.2 (2CH2), 46.9 (2CH2), 111.6 (2CH),
119.1 (2CH), 137.4 (2CN); L-SIMS+ m/z 195.0 ([M + H]+, 100).
124.7, 125.3, 127.1, 127.6, 128.9, 130.3, 140.7, 143.8 (10Car
+
20CarH), 156.8 (2NHCO), 168.5 (2NHCO); L-SIMS+ m/z 683
([M + H]+, 100); Anal. calcd. for C40H34N4O6: C, 72.12; H, 5.61;
N, 8.21. Found: C, 72.27; H, 5.28; N, 8.10.
o-Phenylene-N,N-bis-(2-benzoxycarbonylaminoethanamide)
(1c)
A round-bottom flask equipped with stirring bar and two
addition funnels, containing N-carbobenzyloxyglycine (1 g,
4.78 mmol) in anhydrous dichloromethane (15 cm3) was placed
under nitrogen. The flask was submerged in an ice-water
bath at 0 ◦C, and oxalyl chloride (669 mg, 4.9 mmol) and
dimethylformamide (22 mg, 0.3 mmol) were added. The ice-
water bath was removed but stirring continued for 90 min at
rt. A mixture of o-phenylenediamine (258 mg, 2.39 mmol) and
triethylamine (911 mg, 9 mmol) in anhydrous dichloromethane
(20 cm3) was added dropwise to the solution. The solution was
stirred at rt for 1.5 h under nitrogen atmosphere, and then treated
with 2 M hydrochloric acid (60 cm3). The organic phase was
separated and shaken with water (60 cm3), dried over MgSO4
and concentrated to afford 1c as a white solid. (1.17 g, 100%):
mp: 179–181 ◦C; IR (KBr): 3421, 3268, 1705, 1681, 1543 cm−1;
dH (DMSO) 3.84 (d, 4H, 2CH2, J = 5.6 Hz), 5.06 (s, 4H, 2CH2),
7.14–7.19 (m, 2H, 2CH), 7.35 (s, 10H, 10CarH), 7.52–7.63 (m,
4H, 2CH + 2CH2NH), 9.35 (br s, 2H, 2CarNH); dC (DMSO) 44.2
(2CH2), 65.7 (2CH2), 124.7 (2CH2), 125.1 (2CH2), 127.8 (6CH),
128.4 (4CH), 130.3 (2CarN), 136.9 (2C), 156.7 (2NHCOO),
168.4 (2NHCO); MS m/z: 152, 126 (100), 108; Anal. calcd.
for C26H26N4O6: C, 63.66; H, 5.34; N, 11.42. Found: C, 63.60;
H, 5.38; N, 11.42.
N,N,Nꢀ-Tri-tert-butoxycarbonylmethyl-Nꢀ-tert-
butoxycarbonylmethyl-Nꢀ-[2-di(tert-
butoxycarbonylmethyl)aminophenyl]ethylenediamine (4a)
Potassium carbonate (3.21 g, 23 mmol) and potassium iodide
(78 mg, 0.4 mmol) were added to a solution of 3a (600 mg,
2.3 mmol) in freshly distilled acetonitrile (40 cm3). The mixture
was stirred at 80 ◦C, and tert-butylbromoacetate (4.48 g, 23
mmol) was added dropwise. The reaction mixture was kept at
this temperature over a period of 72 h under argon, and then
cooled to rt, filtered, and concentrated under reduced pressure.
Dichloromethane was added (50 cm3) and the solution washed
with water (80 cm3), dried (MgSO4), and evaporated. The residue
was purified by flash chromatography on silica using hexane–
triethylamine (49 : 1) as eluent. This procedure was repeated
with dichloromethane–ethyl acetate (19 : 1) as eluent to give 4a
as a colorless oil. (1.1 g, 65%): IR (KBr): 2978, 2931, 1740, 1367,
1149 cm−1; dH (CDCl3) 1.37 (s, 27H, 9CH3), 1.44 (s, 18H, 6CH3),
2.83 (t, 2H, CH2, J = 5.8 Hz), 3.41 (m, 6H, 3CH2), 4.10 (s, 4H,
2CH2), 4.15 (s, 2H, CH2), 6.90–7.10 (m, 4H, CarH); dC (CDCl3)
28.2 (15CH3), 48.9 (CH2), 51.9 (CH2), 53.5 (2CH2), 53.6 (CH2),
56.1 (2CH2), 80.6 (2C), 81.0 (3C), 120 + 121.5 + 122.3 + 122.5
(4CarH), 141.3 + 141.8 (2CarN), 170.3 (2CO), 170.7 (2CO), 171.0
o-Phenylene-N,N-bis(2-aminoethanamide) (2b)
Compound 1b (600 mg, 0.90 mmol) and piperidine (4 cm3) were
mixed in anhydrous DMF (20 cm3). The solution was stirred at
reflux 30 min under nitrogen. The solution was evaporated to
dryness and the residue was dissolved in diethyl ether (10 cm3).
The solution was washed with water (60 cm3), and the aqueous
phase was evaporated to dryness. The residue was redissolved
in water and the solution evaporated to dryness. This procedure
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 5 4 – 4 6 1
4 5 9

View Article Online
(CO); MS m/z: 722 (100, M+), 608, 450; HRMS (DCI) m/z
trichloride (2 mg, ca. 5.10−5 M) in deionized water (10 cm3). The
final metal concentration of each solution ranged from 5.10−4 to
10−3 M in a final volume of 15 cm3. Solutions were stirred at rt
for 1 h. Absorbance intensity was monitored between 200 and
800 nm with a SHIMADZU-UV 2501 PC spectrophotometer.
calcd. for C38H63N3O10 [M]+ 722.4592, found 722.4593.
o-Phenylene-N,N-bis(ethylenediamine-N,N,Nꢀ-tri(tert-
butyl)triacetate) (4b)
Compound 3b (143 mg, 0.73 mmol), potassium carbonate
(1 g, 7.33 mmol), potassium iodide (24.9 g, 0.15 mmol) and
tert-butylbromoacetate (1.43 g, 7.33 mmol) were dissolved in
anhydrous acetonitrile (15 cm3). The mixture was stirred for 96 h
at 80 ◦C under argon. After evaporation under reduced pressure,
the residue was taken up in dichloromethane (40 cm3) and the
organic phase was washed with water (20 cm3) and dried over
magnesium sulfate. The solution was filtered and evaporated un-
der reduced pressure. The residue was purified by chromatogra-
phy with petroleum ether–dichloromethane–triethylamine (93 :
5 : 2) to afford compound 4b as a colorless oil. (264 mg, 41%):
IR (KBr): 2978, 2933, 1742, 1148 cm−1; dH (CDCl3) 1.36 (s, 18H,
6CH3), 1.44 (s, 36H, 12CH3), 2.81 (t, 4H, 2CH2, J = 7.0 Hz),
3.40 (m, 12H, 6CH2), 4.08 (s, 4H, 2CH2), 6.88 (m, 2H, 2CH),
7.06 (m, 2H, 2CH); dC (CDCl3) 28.3 (18CH3), 49.1 (2CH2), 52.1
(2CH2), 53.6 (2CH2), 56.1 (4CH2), 80.6 (2C), 81.0 (4C), 121.4 +
122.2 (4CH), 141.7 (2CarN), 170.7 (4CO), 170.9 (2CO); MS
m/z: 879 [M + H]+, 607, 362, 336 (100); HRMS [M + Na+] m/z
calcd. for C46H78N4O12 [M]+ 901.5514, found 901.5512.
Radiolabelling
Stock solutions of each chelate (2, 0.2, 0.02 and 0.002 mg.cm−3)
were prepared in 0.1 M sodium acetate, pH 5.5. Radionuclide–
chelating agent complexes were formed by adding a defined
volume of the appropriate radionuclide stock solution to
increasing amounts of each chelating agent [DTPA, Cy-DTPA,
DOTA, CHX-DTPA, Ph-DTPA (5a), and Ph-TTHA (5b)]. The
final volume was adjusted to 500 lL with a 0.1 M solution of
sodium acetate. The final pH of each solution ranged from 5.5
to 5.8 with a radionuclide concentration ranging from 10−6 to
10−10 M. Solutions were incubated at 37 ◦C for 1 h, 1 lL of
each sample was taken, and complexation was measured with
a PhosphorImager 445SI after thin-layer chromatography on
silica plates (MERCK Art. 5714) by elution with 0.1 M sodium
acetate (pH 5.5).
Radionuclides were produced by Cis-Bio International for
153Sm and 111In, by MDS Nordion for 90Y, by A n s t o fo r 177Lu
and by Transuraniens Institute for 213Bi and 225Ac.
N-(2-N,N-Dicarboxymethylaminophenyl)ethylenediamine-
N,Nꢀ,Nꢀ-triacetic acid trichloride (5a)
Serum stability
40 to 120 lL of previously prepared buffered solutions of
radionuclide-Ph-DTPA (5a) and radionuclide-Ph-TTHA (5b)
complexes (pH 5.5) were added to fresh human serum (4 cm3).
Serum samples were incubated at 37 ◦C. Portions of the serum
solution (500 lL) were periodically removed and analyzed on
a PD10 column (size exclusion chromatography). The column
was eluted with phosphate buffer solution at a flow rate of
1 cm3 min−1. The amount of radioactivity in each sample
was determined using an automatic PerkinElmer WALLAC
WIZARD 3 counter.
A mixture of 4a (190 mg, 0.26 mmol), ethanol (12 cm3) and
2 M aqueous solution of NaOH (12 cm3) was stirred 12 h
at 60 ◦C. The solution was evaporated to dryness, and the
residue was dissolved in cool water (12 cm3). Three molar
hydrochloric acid was then added until pH < 7 and the solution
had evaporated to dryness. Acetone was added (40 cm3) and
the solution was stirred for 10 min at rt. NaCl was filtered on a
frit and the filtrate, evaporated to dryness to give acid 5a as a
white powdery solid, which was vacuum dried and kept under
nitrogen. Acid 5a is an hygroscopic compound. (127 mg, 88%):
mp: 125–127 ◦C; IR (KBr): 3328, 3005, 1734, 1419, 1218 cm−1;
dH (D2O) 3.47 (br s, 2H, CH2), 3.52 (br s, 2H, CH2), 4.07 (s, 2H,
CH2), 4.20 + 4.26 (2s, 8H, 4CH2), 7.09–7.17 (m, 4H, 4CarH); dC
(D2O) 51.3 (CH2), 56.6 (CH2), 56.7 (2CH2), 58.2 (4CH2), 125.1 +
126.3 + 128.2 + 129.1 (4CarH), 144.3 + 146.1 (2CarN), 172.3
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0
◦C. 6 M HCl was added until the solution became acidic.
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4 6 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 5 4 – 4 6 1

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66 A. E. Martell and R. M. Smith, Critical stability constants, volume 4:
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67 G. Dubois, R. Tripier, S. Brande`s, F. Denat and R. Guilard, J. Mater.
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68 F. Denat, G. Dubois, R. Tripier, G. Guilard and B. Roux-Fouillet,
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69 Compound 3a is now commercially available (Interchim Intermedi-
ates).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 4 5 4 – 4 6 1
4 6 1