Structure-biodistribution relation of neutral 99mTc(CO)3-complexes with tridentate N-substituted derivatives of aminoethylglycine and phenylenediamine

Article abstract of DOI:10.1016/j.tetlet.2006.04.136

Derivatives of ethylenediamine-N-acetic acid (EDAA = N-aminoethylglycine = AEG) and ortho-phenylenediamine-N-acetic acid (PDAA) with uncharged substituents on one or both of the amines form neutral complexes with a [^99mTc(CO)₃]⁺

Full text of DOI:10.1016/j.tetlet.2006.04.136

Tetrahedron Letters 47 (2006) 4641–4645
Structure–biodistribution relation of neutral 99mTc(CO)3-complexes
with tridentate N-substituted derivatives of aminoethylglycine
and phenylenediamine
Dirk Rattat, Jan Cleynhens, Christelle Terwinghe, An-Elisabeth De Greve
and Alfons Verbruggen*
Laboratory of Radiopharmaceutical Chemistry, Katholieke Universiteit Leuven, Herestraat 49/O&N2, PO Box 821,
B-3000 Leuven, Belgium
Received 28 March 2006; revised 21 April 2006; accepted 26 April 2006
Abstract—Derivatives of ethylenediamine-N-acetic acid (EDAA = N-aminoethylglycine = AEG) and ortho-phenylenedi-
amine-N-acetic acid (PDAA) with uncharged substituents on one or both of the amines form neutral complexes with a
[
99mTc(CO)3]+-moiety. We studied the influence of different modifications at the amines (e.g., with methyl, ethyl, butyl or benzyl
groups) on the behaviour of the 99mTc(CO)3-complexes in vivo in mice, with special focus on blood–brain barrier (BBB) passage.
The complexes have been characterised by reversed phase HPLC, logP, electrophoresis and some of them also by LC–MS. LogP
values of the 99mTc–tricarbonyl complexes varied from ꢀ0.52 (AEG) to 2.5 (N,N0-dibenzyl-EDAA). With increasing lipophilicity,
more of the activity was found in liver and intestines as compared to kidneys and urine for the more polar complexes. Brain uptake
was found for the 99mTc(CO)3-complexes with N,N0-dibutyl-ethylenediamine-N-acetic acid (0.34% of I.D. after 2 min) and ortho-
phenylenediamine-N-acetic acid (0.22% of I.D. after 2 min).
ꢀ 2006 Elsevier Ltd. All rights reserved.
Technetium–tricarbonyl complexes with
[
since Alberto et al. developed a convenient low pressure
a
fac-
charged (e.g., with ligands of N–N–N type such as dieth-
ylenetriamine) or negatively charged (e.g., with ligands
of O–N–O type such as iminodiacetic acid).5–7 In the
current study, we have investigated derivatives of ethyl-
enediamine-N-acetic acid (EDAA) and ortho-phenylene-
diamine-N-acetic acid (PDAA), which form neutral
complexes with the [99mTc(CO)3]+-moiety. The influence
of uncharged substituents on one or both of the amines
on the biodistribution of the resulting 99mTc–tricarbonyl
complexes in mice was investigated, with a special focus
on the ability of the 99mTc-tracers to pass the blood–
brain barrier (BBB). The clinical usefulness of, for
example, 99mTc-TRODAT-1 for diagnosis of Parkin-
son’s disease has shown that there is still a place for
new 99mTc-based radiopharmaceuticals, especially in
the field of neuroreceptor-targeting molecules.8,9 The
availability of 99mTc–tricarbonyl complexes with signifi-
cant brain uptake could result in a new generation of
99mTc-labelled agents for diagnosis and/or follow up
of neurological diseases.
99mTc(CO)3]-moiety have been the focus of interest
synthesis in aqueous media for the precursor fac-
[
99mTc(CO)3(H2O)3]+.1,2 A recently developed kit for
preparation of this precursor complex (IsoLinkTM,
Tyco-Mallinckrodt, Petten, The Netherlands), which
contains the solid sodium boranocarbonate as both
source of CO and reducing agent, made its use even
more attractive.3 Major characteristics of this Tc–tricar-
bonyl complex are the presence of three stable CO-
groups and three labile water molecules. The latter can
be replaced by a variety of donating ligands.4 Especially
tridentate amine containing ligands form complexes of
high stability when labelled with a [99mTc(CO)3]+-moi-
ety. Depending on the type of ligand and heteroatoms,
the resulting complexes can be neutral (e.g., with ligands
of N–N–O type such as aminoethylglycine), positively
Keywords: Technetium; Tricarbonyl; Imaging agents; Biodistribution;
Brain uptake; LC–MS.
*
The model compound AEG offers a primary amine,
a secondary amine and a carboxylic acid group for
Corresponding author. Tel.: +32 16 330446; e-mail: Alfons.
Verbruggen@pharm.kuleuven.be
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.04.136

4642
D. Rattat et al. / Tetrahedron Letters 47 (2006) 4641–4645
R1 R2
R1
R2
1. methanol,
N
N
N
N
N
N
N
N
N
N,N-diisopropylethylamine,
tertiary-butyl bromoacetate,
4 °C, overnight
HO
O
HO
O
HO
O
OH
N
2. trifluoroacetic acid
145.18
160.22
N,N,N'-TM-EDAA
173.24
N,N-DE-EDAA
H
R3
R3
N,N-DM-EDAA
O
(R1, R2, R3 = methyl, ethyl, butyl, benzyl or a proton)
N
N
N
N
Scheme 1. Preparation of N-alkyl substituted derivatives of amino-
ethylglycine. N-alkylated derivatives of ethylenediamine reacted with
tert-butyl bromoacetate to tridentate N–N–O type ligands. Deprotec-
tion of the resulting tertiary butyl esters was achieved with trifluoro-
acetic acid.
HO
O
HO
O
229.34
297.38
N,N'-DBu-EDAA
N,N'-DBn-EDAA
complex formation with the [99mTc(CO)3]+-moiety,
resulting in the neutral complex [99mTc(CO)3(AEG)].
Previous studies have shown excellent complex forming
properties of AEG as tridentate ligand, but no brain up-
take of the formed 99mTc-complex.10 A logP value of
ꢀ0.52 further characterised the complex as probably
too hydrophilic to cross the blood brain barrier.
NH2
NH
N
N
OH
O
OH
O
163.16
PDAA
178.19
MPDAA
Figure 1. Tridentate N–N–O type ligands for labelling with
99mTc(CO)3]+and their molecular masses (EDAA = ethylenediamine-
N-acetic acid; PDAA = ortho-phenylenediamine-N-acetic acid).
In the present study, we have investigated the prepara-
tion and biological characteristics of a series of more
lipophilic derivatives of [99mTc(CO)3(AEG)], modified
with respect to AEG by a gradually increasing number
of methyl, ethyl or benzyl substituents on one or both
of the amines. In addition, two derivatives of phenylene-
diamine have been included. The ligands were prepared
by reacting the bidentate N-alkylated starting materials
N,N-dimethyl-ethylenediamine, N,N,N0-trimethyl-ethyl-
enediamine, N,N-diethyl-ethylenediamine, N,N0-dibutyl-
ethylenediamine and N,N0-dibenzyl-ethylenediamine
(starting materials commercially available from Fluka
or Aldrich) with tert-butyl protected bromoacetic
acid, to introduce a single acetic acid substituent at
one of the amines (Scheme 1). In the same way we mod-
ified ortho-phenylenediamine and N-methyl-ortho-
phenylenediamine.
[
(PDAA) after deprotection. The identity of the depro-
tected ligands was confirmed by high-resolution mass
spectrometry.
The Tc–tricarbonyl precursor [99mTc(CO)3(H2O)3]+ was
prepared by addition of a solution of [99mTcO4]ꢀ (eluate
from a commercially available Ultratechnekow FM
99mTc-generator; Tyco-Mallinckrodt, Petten, The Neth-
erlands; activities up to 1850 MBq in 1 ml saline) to a
vial containing a mixture of NaBH4 (25 mg), Na2CO3
(4.5 mg) and K–Na–tartrate (20 mg) under an
atmosphere of CO and heating for 20 min at 70 ꢁC.1
For labelling of the ligands, 0.3 ml of the [99mTc(CO)3-
(H2O)3]+ solution was added to a solution of the
HPLC-isolated ligands, the mixture was then adjusted
to pH 10 and heated at 70 ꢁC for 10 min. The reaction
mixtures were then analysed by RP-HPLC. All tested
aminoethylglycine derivatives afforded a main radiola-
belled reaction product in high yield (80–98%), although
N,N0-dibutyl-EDAA and N,N0-dibenzyl-EDAA showed
lower labelling yields than the other ligands. Labelling
of PDAA with a 99mTc–tricarbonyl moiety was also rea-
lised with an excellent labelling yield (>90%), while the
additional alkyl group in MPDAA significantly de-
creased the yield (30–40%). For some of the ligands
the formation of diastereomers was observed, namely
for N,N0-dibutyl-EDAA, N,N0-dibenzyl-EDAA and
MPDAA. For each of these ligands, the diastereomer
eluting last during HPLC analysis on a reversed phase
column was formed in a predominant way (70–90%).
For example, [99mTc(CO)3(DBu)] showed two peaks
on HPLC (tR = 18.57 min and tR = 19.08 min) in a rel-
ative ratio of 30:70. LC–MS measurements confirmed
that both labelled compounds had the same mass, corre-
sponding to the assumed 99mTc(CO)3-complex. Biodis-
The reaction products with only one acetic acid group
substituent were isolated by HPLC using a mobile phase
containing 0.1% trifluoroacetic acid. This concentration
of trifluoroacetic acid was sufficient for a complete
removal of the tertiary butyl protective group after incu-
bation at room temperature for 10 min (see footnote ).
Figure 1 shows the structures of the obtained tridentate
N–N–O type derivatives of ethylenediamine-N-acetic
acid (EDAA) and o-phenylenediamine-N-acetic acid
Typical procedure: equimolar amounts (1 mmol) of N,N-diisopropyl-
N-ethylamine (DIEA), tert-butyl-bromoacetate and an ethylenedia-
mine derivative, for example, N,N,N0-trimethyl-ethylenediamine,
were dissolved in methanol (3 ml) and kept at 4 ꢁC overnight. The
resulting protected ethylenediamine-N-acetic acid derivatives were
isolated by HPLC (X-Terra RP-18 column; 4.6 mm · 250 mm;
Waters, Brussels, Belgium; gradient elution from 0.1% trifluoroacetic
acid in water to 0.1% trifluoroacetic acid in acetonitrile in 20 min;
flow rate 1 ml/min). Incubation of the collected HPLC-eluate
fractures containing the desired product for 10 min at room temper-
ature afforded the fully deprotected ligands under these conditions
(by the trifluoroacetic acid in the mobile phase).

D. Rattat et al. / Tetrahedron Letters 47 (2006) 4641–4645
4643
tribution experiments have always been performed with
the predominant compound. To elucidate which confor-
mation was present in the predominant isomer, crystal-
lisation of the corresponding rhenium complex followed
by X-ray crystallography would be necessary, but this
was not part of the study.
All 99mTc-complexes were isolated by HPLC and ana-
lysed for charge by paper electrophoresis and for lipo-
philicity by logP determination. Paper electrophoresis
of the 99mTc-complexes was performed on Whatman 4
paper strips (13 cm · 4 cm) using 0.05 M ammonium
acetate buffer pH 7.4 as electrolyte solution and a volt-
age of 400 V for 20 min. Pertechnetate was used as ref-
erence compound to prove migration of the charged
99mTc-complexes. As expected, none of the tested com-
pounds showed any migration during electrophoresis,
indicating that the complexes are neutral. LogP values
have been determined according to a literature proce-
dure.11,12 For the 99mTc(CO)3-complexes with the
following ligands they were found to be 0.15 for N,N-di-
methyl-EDAA, 0.21 for N,N,N0-trimethyl-EDAA, 0.98
for N,N-diethyl-EDAA, 1.65 for N,N0-dibutyl-EDAA,
2.5 for N,N0-dibenzyl-EDAA and 0.87 for ortho-PDAA.
Dischino et al. suggested a log P range of 1–2.5 as ideal
for a potential brain uptake.13
To support the supposed structures of tridentate
99mTc(CO)3-complexes with the studied ligands, we per-
formed LC–MS measurements at the example of
[
99mTc(CO)3(DBu)], which turned out to be the key
compound of the study (DBu = N,N0-dibutyl-EDA-N-
acetic acid). Mass spectra were recorded using LC–MS
(Waters separation module, XTerra MS C18 column
50 mm · 2.1 mm, 3-in. NaI(Tl) radiation detector,
Micromass LCT mass spectrometer and MassLynx soft-
ware, Waters-Micromass, Manchester, UK). The col-
umn was eluted at a flow rate of 300 ll/min with
gradient mixtures of water (containing 0.01% formic
acid) and acetonitrile (linear gradient from 0% acetoni-
trile at 0 min to 50% at 20 min, then kept at 50% till
30 min). A solution of 0.01% kryptofix 2.2.2 in
CH3CN–H2O (50:50) was added to the mobile phase
at a flow rate of 1 ll/min and served as lock mass
(377.26 Da) for accurate mass determination. Generator
eluate with a high relative content of 99Tc was used for a
carrier-added synthesis of the complexes to enhance the
signal in mass spectrometry. The solution with the com-
plex was concentrated to obtain a detectable signal, but
this remained close to the detection limit of the mass
spectrometer. An alternative procedure was the addition
of 99Tc to the 99mTc generator eluate for the carbonyl-
ation reaction (200–300 ll of an aqueous 99Tc solution,
containing 15 lg/ml NH4TcO4). Mass spectrometry
supported the supposed structure of [99mTc(CO)3(D-
Bu)]. In Figure 2, the single ion mass chromatogram
shows the complex at a retention time of 21.41 min
and the detected mass of 414.98 Da corresponds with
the theoretical value of 415.11 Da [M+H+]+ for this
complex.
Figure 2. LC–MS analysis of the Tc–tricarbonyl complex
[
99/99mTc(CO)3(DBu)]ꢁ: (a) calculated mass spectrum, (b) detected
mass spectrum and (c) single ion mass chromatogram.
male NMRI mice (body mass 28–42 g). A 99mTc-activity
of approximately 40 kBq/mouse was injected via a tail
vein. Four mice were sacrificed at 2 min p.i., the other
four at 60 min p.i. and the organs and body parts were
dissected and weighed. The activity in the dissected
organs and body parts was measured using a Wallac
1480 WIZARD 300 automatic gamma counter (Perkin–
Elmer, Boston, USA). For calculation of total blood
radioactivity, blood mass was assumed to be 7% of the
body mass.14 Table 1 shows the results of the biodistri-
bution experiments for selected organs as percentage of
injected dose (% of I.D.). As a general tendency we
found an increasing liver uptake and a decreasing renal
excretion with increasing lipophilicity (and increasing
logP) of the compounds. Heart, lungs, stomach and
spleen did not show any significant uptake.
For
[
99mTc(CO)3(DBu)] and
[
99mTc(CO)3(PDAA)],
brain uptake was found at 2 min after injection, namely
0.34% of I.D. and 0.22% of I.D., respectively.
[
99mTc(CO)3(MPDAA)] was not yet fully characterised,
but the additional methyl group did not change the
brain uptake significantly (0.19% of I.D. after 2 min)
as compared to [99mTc(CO)3(PDAA)].
Biodistribution experiments of each of the isolated
99mTc(CO)3-complexes was determined in eight normal

4644
D. Rattat et al. / Tetrahedron Letters 47 (2006) 4641–4645
Table 1. Results of biodistribution experimets of 99mTc-labelled compounds in mice (activity in % of injected dose; n = 4 at each time point)
99mTc(CO)3-complexes with
DM–EDAA
TM–EDAA
DE–EDAA
DBu–EDAA
DBn–EDAA
PDAA
% I.D. 2 min p.i.
Urine + kidneys
Liver
Intestines
Stomach
Spleen
10.0
22.1
10.6
1.4
7.5
22.9
9.3
1.3
1.4
6.6
36.8
14.3
1.1
23.9
33.5
9.4
0.9
0.9
8.7
39.7
11.7
1.8
9.3
24.4
9.0
1.2
0.9
1.1
0.9
1.9
Lungs
1.5
1.2
0.6
2.5
1.2
1.6
Heart
0.5
0.9
0.5
0.9
0.9
0.6
Blood
Brain
23.0
0.0
17.9
0.01
8.6
0.0
18.0
0.34
8.1
0.0
23.0
0.23
% I.D. 60 min p.i.
Urine + kidneys
Liver
Intestines
Stomach
Spleen
26.4
17.3
15.1
0.7
20.9
36.7
13.3
0.6
15.3
36.4
24.0
0.9
19.4
41.1
14.3
0.8
4.7
26.9
56.8
0.3
24.8
20.6
15.4
1.1
0.4
0.2
0.5
1.0
0.2
0.7
Lungs
0.5
0.4
0.4
0.9
0.3
0.7
Heart
0.3
0.2
0.2
0.7
0.1
0.4
Blood
Brain
5.5
0.0
7.0
0.0
4.0
0.0
3.4
0.22
3.0
0.0
13.0
0.12
Increased liver uptake, enhanced intestinal excretion and
reduced renal excretion can be expected in case of increas-
ing lipophilicity of (similar) compounds.15 None of the
tested compounds showed a significant activity in the
stomach, indicating stable 99mTc–tricarbonyl complexes
and no re-oxidation to pertechnetate. From the
99mTc(CO)3-labelled complexes with N,N-dimethyl-
EDAA (DME), N,N,N0-trimethyl-EDAA (TME), N,N-
diethyl-EDAA (DET), N,N0-dibutyl-EDAA (DBu) and
N,N0-dibenzyl-EDAA (DBZ) only [99mTc(CO)3(DBu)]
showed a clear brain uptake. This seems to support the
theory of an ‘ideal logP range’ of 1–2.5 for potential brain
uptake of radiopharmaceuticals suggested in the litera-
ture.13 The logP of [99mTc(CO)3(DE)] (0.98) and
Of the 99mTc(CO)3-complexes investigated in this study,
99mTc(CO)3(AEG)], [99mTc(CO)3(DM)], [99mTc(CO)3-
(TM)],
99mTc(CO)3(DE)] and 99mTc(CO)3(DBn)]
[
[
[
showed no brain uptake in mice. On the other hand,
the dibutyl-aminoethylglycine derivative [99mTc(CO)3-
(DBu)] and the phenylenediamine derivative [99mTc-
(CO)3(PDAA)] are able to pass the blood–brain barrier
and showed a clear brain uptake. With these compounds
as examples, 99mTc–tricarbonyl complexes with triden-
tate ligands may also be considered as potential brain
imaging agents. By further altering chain length or nat-
ure of the N-substituents of ethylenediamine and phenyl-
enediamine, derivatives with an optimised uptake will be
explored in future experiments.
[
99mTc(CO)3(DBn)] (2.5) slightly falls outside this range
and these complexes do not show brain uptake. Other
effects seem to play a role in case of the phenylenediamine
complex [99mTc(CO)3(PDAA)], as it showed brain
uptake, but had a logP of only 0.87.
References and notes
1. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P.; Abram,
U.; Kaden, T. A. J. Am. Chem. Soc. 1998, 120, 7987–
7988.
Brain uptake of the tested compounds is lower as com-
pared to that of classic brain perfusion agents such
as 99mTc–HMPAO, which showed a brain uptake in
mice of 2.9% of I.D. for the d,l-isomer and 1.2% of
I.D for the meso isomer after 1 min.16 Other examples,
for example, 99mTc–(SNS/S) mixed ligand complexes
as described by Tsoukalas et al.,17 are with 0.41% of
I.D. in brain after 5 min in the same order of magnitude
as the complex [99mTc(CO)3(DBu)] presented here.
2. Alberto, R.; Schibli, R.; Waibel, R.; Abram, U.; Schubi-
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