Triaza-based amphiphilic chelators: Synthetic route, in vitro characterization and in vivo studies of their Ga(III) and Al(III) chelates

Article abstract of DOI:10.1016/j.jinorgbio.2010.06.002

Radiogallium chelates are important for diagnostic imaging in nuclear medicine (PET (positron emission tomography) and γ-scintigraphy). Micelles are adequate colloidal vehicles for the delivery of therapeutic and diagnostic agents to organs and tissues. I

Full text of DOI:10.1016/j.jinorgbio.2010.06.002

Journal of Inorganic Biochemistry 104 (2010) 1051–1062
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Triaza-based amphiphilic chelators: Synthetic route, in vitro characterization and in
vivo studies of their Ga(III) and Al(III) chelates
Arsénio de Sá a, M. Isabel M. Prata b, Carlos F.G.C. Geraldes c, João P. André a,
⁎
a
Centro de Química, Campus de Gualtar, Universidade do Minho, 4710-057 Braga, Portugal
IBILI, Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal
b
c
Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, e Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3001-401 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Radiogallium chelates are important for diagnostic imaging in nuclear medicine (PET (positron emission
tomography) and γ-scintigraphy). Micelles are adequate colloidal vehicles for the delivery of therapeutic and
diagnostic agents to organs and tissues. In this paper we describe the synthesis and in vitro and in vivo
studies of a series of micelles-forming Ga(III) chelates targeted for the liver. The amphiphilic ligands are
based on NOTA (NOTA=1,4,7-triazacyclonoane-N,N′N″-triacetic acid) and bear a α-alkyl chain in one of the
pendant acetate arms (the size of the chain changes from four to fourteen carbon atoms). A multinuclear
NMR study (1H, 13C, 27Al and 71Ga) gave some insights into the structure and dynamics of the metal chelates
in solution, consistent with their rigidity and octahedral or pseudo-octahedral geometry. The critical micellar
concentration of the chelates was determined using a fluorescence method and 27Al NMR spectroscopy (Al
(III) was used as a surrogate of Ga(III)), both showing similar results and suggesting that the chelates of
NOTAC6 form pre-micellar aggregates. The logP (octanol–water) determination showed enhancement of the
lipophilic character of the Ga(III) chelates with the increase of the number of carbons in the α-alkyl chain.
Biodistribution and γ-scintigraphic studies of the 67Ga(III) labeled chelates were performed on Wistar rats,
showing higher liver uptake for [67Ga](NOTAC8) in comparison to [67Ga](NOTAC6), consistent with a longer
α-alkyl chain and a higher lipophilicity. After 24 h both chelates were completely cleared off from the tissues
and organs with no deposition in the bones and liver/spleen. [67Ga](NOTAC8) showed high kinetic stability
in blood serum.
Received 29 January 2010
Received in revised form 8 June 2010
Accepted 11 June 2010
Available online 3 July 2010
Keywords:
Triaza
Orthogonal protection
Amphiphilic
Gallium
Aluminum
Radiopharmacy
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
respectively, when hexacoordinated), charge and coordination chem-
istry. Transferrin has two binding sites for Fe(III), that have also high
Radiogallium chelates are of great interest in the field of medical
imaging. 67Ga (t1/2 =3.25 days), a γ emitter, is useful for γ-
scintigraphy, while 68Ga (t1/2 =68 min), a β+ emitter, is adequate
for positron emission tomography (PET). 67/68Ga(III), with a very well
known coordination chemistry, allows easy radiopharmaceutical
preparation, as the radiometal can be very rapidly inserted in
adequate molecules, contrarily to the covalently bound PET radio-
isotopes 18F or 11C, leading to a minimum loss of activity [1].
Ga(III) is a hard Lewis acid that forms thermodynamically stable
chelates with ligands that are hard Lewis bases, often with a
coordination number of six. The main requirements for a Ga(III)-
based radiopharmaceutical agent are the thermodynamic stability
towards hydrolysis and the kinetic inertness during the period of
clinical use in order to avoid ligand exchange with the blood serum
proteins, such as transferrin (Tf). Ga(III) is quite similar to the high
spin Fe(III) ion in what concerns their ionic radii (62 pm and 65 pm,
affinity for Ga(III) (log K(Ga–Tf) =20.3 [2]), and is present in high
concentrations in plasma, 2.5×10−3 M. Thus, when 67Ga(III) is
injected in the form of gallium citrate (or another low stability
complex) more than 90% of this metal is complexed by transferrin.
Several chelators for Ga(III) have been proposed, the majority of
them being hexadentate [1,3–11]. Among the chelators, triaza
macrocycles display high conformational and size selectivity towards
Ga(III) ions. The high thermodynamic stability of the Ga(III) chelates
of nine-membered triaza ligands is due to the adequate fit of the
relatively small cation in the macrocyclic cavity. The high thermody-
namic stabilities of Ga(III) chelates of NOTA-like ligands are illustrated
respectively by log KGa(NOTA) =30.98 and log KGa(NODASA) =30.9(0.2)
(NOTA = 1,4,7-triazacyclonoane-N,N′N″-triacetic acid;
NODASA= 1,4,7-triazacyclonoane-N-succinic acid-N′N″-diacetic
acid) [9,12]. Their high resistance against exchange of Ga(III) in
blood serum and acid-catalyzed dissociation has been demonstrated
[9].
Some of the ligands currently used for the preparation of
radiopharmaceuticals of Ga(III) are bifunctional, meaning that they
present a functionality that allows covalent coupling to a targeting
⁎
Corresponding author. Tel.: +351 253 604 385; fax: +351 253 604 382.
E-mail address: jandre@quimica.uminho.pt (J.P. André).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.06.002

1052
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
vector (e.g. peptides), besides binding the metal ion. The coupling to
the targeting moiety requires that the pro-chelator, often bearing
carboxylic acid groups, has the possibility of orthogonal protection
[13].
of [Ga(H2O)6]3+ at 0 ppm as reference. pH measurements were
performed on a pH meter Crison micro TT 2050 with an electrode
Mettler Toledo InLab 422. Mass spectra (ESI+) were performed on a VG
Autospec M spectrometer or on a Finnigan LXQ MS Detector.
Micelles are colloidal aggregates that have been extensively used
as drug carriers to improve pharmacokinetic properties or the
bioavailability of the drug, to increase the target-to-background
ratio of the drug or to deliver hydrophobic drugs [14–17]. These self-
assembly constructs accumulate in macrophage-rich tissues, such as
liver and spleen, undergoing endocytosis/phagocytosis [14,18].
Micelles loaded with suitable reporter groups can have application
in medical imaging. Gd(III)-containing micelles are of interest as MRI
contrast agents due to the possibility of delivering high payloads of
paramagnetic ion to the biological receptors and to the expected
increase in their relaxivity due to the slowing down of the rotational
dynamics of the chelates upon aggregation [19,20]. By their turn,
radio-labeled (125I, 111In, 153Sm) micelles have been employed in γ-
scintigraphic studies for the visualization of macrophage-rich tissues
such as lymph nodes, liver and spleen [21–24].
2.2. Synthetic procedures
2.2.1. Diphenyldiazomethane (1)
In a 1 L flask, benzophenonehydrazone (10.0 g, 51.0 mmol),
yellow mercuric oxide (26.9 g, 124 mmol), anhydrous sodium sulfate
(11.5 g, 81.0 mmol) and a saturated ethanol solution of KOH
(3.80 mL) were suspended in ethyl ether (154 mL) and stirred for
75 min, according to the method published by Miller [27]. The
solution was filtered and the solvent was evaporated under vacuum at
room temperature. The dark red oil obtained was dissolved in
petroleum ether 40–60 °C and filtered once again. The solvent was
evaporated at room temperature and the residue was frozen. When
heated to room temperature, the diphenyldiazomethane (DDM, 1)
afforded dark red crystals (9.86 g, 50.8 mmol) in a yield of 99.6%.
In this work we envisaged the synthesis and the study of micelles-
forming triaza chelates of Ga(III) potentially interesting for diagnosis
in nuclear medicine. The synthetic route of this series of NOTA-based
chelators, with one of the acetate pendant arms bearing a α-alkyl
substituent with a variable number of carbon atoms (from four to
fourteen — NOTAC6, NOTAC8, NOTAC10, NOTAC16, see Scheme 1)
makes use of an orthogonal protection strategy, allowing their
coupling to targeting biomolecules.
2.2.2. 2-bromohexanoate tert-butyl ester (2a)
A solution of tert-butyltrichloroacetimidate (TBTA) (5.00 g,
22.9 mmol) in 11.0 mL of cyclohexane was added dropwise during
10 min to a solution of 2-bromohexanoic acid (0.815 mL, 5.72 mmol)
in 10.0 mL of dichloromethane (DCM). During the addition, a white
precipitate was formed, which was dissolved with the addition of
1.25 mL of N,N-dimethylacetamide. A catalyst, boron trifluoride ethyl
etherate (BF3.OEt2) (0.700 mL), was added to the reaction and a new
white precipitate was formed. The reaction was stirred during three
days, according to the method published by Nicolle et al. [20]. The
precipitate was removed by filtration and the solution was concen-
trated. The oil obtained was purified by chromatography (cyclohex-
ane/ethyl acetate 7:3) originating a volatile brown oil 2a (1.16 g
4.62 mmol) in a yield of 80.5%. 1H NMR (300 MHz, CDCl3, TMS,
δ(ppm)): 0.91 (3H, t, J=7.5 Hz, (CH2)2CH3), 1.29–1.40 (4H, m,
(CH2)2CH3), 1.48 (9H, s, C(CH3)3), 1.86–2.10 (2H, m, CH2 ABX), 4.10
(1H, t, J = 7.5 Hz, CH ABX).
A multinuclear NMR study (1H, 13C, 27Al and 71Ga) gave an insight
on the structure and dynamic behavior of the metal chelates in
solution. Al(III) chelates were used as models of the analogous Ga(III)
chelates, due to the more suitable magnetic resonance properties of
aluminum (100% abundance and fairly good receptivity) [4]. The
variation of the half-width of the 27Al NMR signal with concentration
allowed the assessment of the critical micellar concentration (cmc) of
the amphiphilic Al(III) chelates, consistent with the quadrupolar
nature of the Al(III) ion. The cmc results were confirmed using a
fluorescent method based on the use of ANS (8-anilino-1-napthalene
sulfonic acid) [25].
A very important indicator for in vivo applications is the
measurement of the rate of exchange of Ga(III) in blood serum
under physiological conditions [26]. For this purpose, the stability of
the 67Ga(III) labeled chelates in blood serum was investigated.
Biodistribution and γ-scintigraphic studies of the 67Ga(III) labeled
chelates were performed in Wistar rats.
2.2.3. 2-bromohexanoate benzhydryl ester (2b)
A solution of 2-bromohexanoic acid (3.70 mL, 26.0 mmol) in 160 mL
of acetone was added to a solution of compound 1 (4.50 g, 23.2 mmol)
in 160 mL of acetone. The reaction was stirred during 13 h in an ice bath
and 10 h at room temperature. The solution was then concentrated and
the yellow oil obtained was purified by chromatography (cyclohexane/
ethyl acetate 4:1) affording the expected ester 2b with a yield of 93.1%
(7.82 g, 21.6 mmol). 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.88 (3H,
t, J=6.6 Hz, (CH2)2CH3), 1.22–1.48 (4H, m, (CH2)2CH3), 1.97–2.18 (2H,
m, CH2 ABX), 4.33 (1H, t, J=7.2 Hz, CH ABX), 6.92 (1H, s, CH(Ph)2),
7.32–7.40 (10H, m, CH(Ph)2).
2. Experimental
2.1. Materials and methods
Analytical grade reagents were purchased from Sigma-Aldrich, Fluka,
Acros Organics, Macrocyclics and Chematech. [67Ga](citrate) was pur-
chased from CIS-BIO (Gif-sur — Yvette, France). The reactions were
monitored by thin layer chromatography (TLC) on aluminum plates coated
with silica gel 60 F254 (Macherey-Nagel).
Chromatography separations were performed on silica gel Whatman
230–240 Mesh. The NMR spectra were recorded on a Varian Unity Plus
300 spectrometer or on a Bruker Avance III 400 spectrometer. The 1H
NMR spectra were assigned using the two-dimensional COSY technique.
The 1H chemical shifts are reported in ppm, relative to tetramethylsilane
(TMS) or sodium 2,2-dimethylsilapentane-5-sulfonate (DSS) and the
following abbreviations are used: s=singlet; sb =broad singlet;
d=doublet; dd=double doublet; t=triplet; tb =broad triplet;
m=multiplet; mb=broad multiplet. 27Al NMR spectra were recorded
at 104.261 MHz (on the Bruker Avance III 400 spectrometer) using the
signal of [Al(H2O)6]3+ at 0 ppm as reference. 71Ga NMR spectra were
recorded at 122.026 MHz (on the same spectrometer) using the signal
2.2.4. 2-bromooctanoate benzhydryl ester (2c)
The title compound was prepared according to the method
described for the preparation of 2b, starting from 2-bromooctanoic
acid and using cyclohexane/ethyl acetate (9.5:0.5) as eluent in the
chromatography. The compound 2c (5.10 g, 13.1 mmol) was obtained
with a yield of 99.8%. 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.89 (3H,
t, J=6.6 Hz, (CH2)4CH3), 1.24–1.30 (8H, m, (CH2)4CH3), 1.96–2.18 (2H,
m, CH2 ABX), 4.34 (1H, t, J=7.2 Hz, CH ABX), 6.92 (1H, s, CH(Ph)2),
7.34–7.39 (10H, m, CH(Ph)2). 13C NMR (75.43 MHz, CDCl3, TMS,
δ(ppm)): 13.97 (1C, (CH2)4CH3), 22.40–31.41 (4C, (CH2)4CH3), 34.88
(1C, CH2 ABX), 46.12 (1C, CH ABX), 78.18 (1C, CH(Ph)2), 127.02–139.45
(12C, CH(Ph)2), 168.69 (1C, CHCO).
2.2.5. 2-bromodecanoate benzhydryl ester (2d)
The title compound was prepared according to the method
described for the preparation of 2b, starting from 2-bromodecanoic

A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
1053
Scheme 1. A — i) Solution of TBTA in cyclohexane added to the bromoacid dissolved in DCM; ii) DMA; iii) BF3.OEt2. B — Solution of DDM in acetone added to the bromoacid dissolved
in acetone. C — Addition of ester solution in DCM to the 1,4,7-triazacyclononane solution in DCM; D — K2CO3 and tert-butyl bromoacetate in MeCN; E — K2CO3, NO2AtBu and ester in
MeCN; F — TFA/DCM (1:1).
acid and using cyclohexane/ethyl acetate (4:1) as eluent in the
chromatography. The compound 2d (6.78 g, 16.2 mmol) was obtained
with a yield of 98.8%. 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.95 (3H,
t, J=6.3 Hz, (CH2)6CH3), 1.20–1.42 (12H, m, (CH2)6CH3), 2.00–2.22 (2H,
m, CH2 ABX), 4.38 (1H, t, J=7.8 Hz, CH ABX), 6.97 (1H, s, CH(Ph)2),
7.33–7.45 (10H, m, CH(Ph)2).

1054
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
2.2.6. 2-bromohexadecanoate tert-butyl ester (2e)
2.2.12. 1,4,7-triazacyclononane-N-hexanoate tert-butyl ester-N′-N″-
diacetate tert-butyl ester (4a)
The title compoundwasprepared according to themethod described
for the preparation of 2a, starting from 2-bromohexadecanoic acid. The
purification was performed by recrystallization with methanol affording
the ester 2e (3.11 g, 7.94 mmol) with a yield of 73.5%. 1H NMR
(300 MHz, CDCl3, TMS, δ(ppm)): 0.89 (3H, t, J=5.1 Hz, (CH2)12CH3),
1.26 (24H, s, (CH2)12CH3), 1.49 (9H, s, C(CH3)3), 1.87–2.10 (2H, m, CH2
ABX), 4.11 (1H, t, J=9.9 Hz, CH ABX).
Potassium carbonate (0.279 g, 2.02 mmol) and 2-bromoacetate tert-
butyl ester (0.142 mL, 0.964 mmol) were added to a solution of 3a
(0.144 g, 0.481 mmol) in 21.0 mL of acetonitrile (MeCN). After 24 h, the
remaining potassium carbonate was filtered off and the solution was
concentrated. The yellow oil obtained was purified by chromatography
(DCM/EtOH 7:3). The compound 4a (0.147 g, 0.278 mmol) was obtained
with a yield of 57.8%. 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.89 (3H, t,
J=7 Hz, (CH2)2CH3), 1.24–1.39 (4H, m, (CH2)2CH3), 1.45 (27H, s, C(CH3)3),
1.50–1.70 (2H, m, CH2 ABX), 2.58–3.40 (17H, m, en, CH2CO, CH ABX). m/z
(ESI+) calculated for C28H54N3O6 (M+H)+ 528.40. Found 528.42.
2.2.7. 2-bromohexadecanoate benzhydryl ester (2f)
The title compoundwasprepared according to themethod described
for the preparation of 2b, starting from 2-bromohexadecanoic acid and
using cyclohexane/ethyl acetate (4:1) as eluent in the chromatography.
The compound 2f (6.04 g, 12.0 mmol) was obtained with a yield of
93.8%. 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.93 (3H, t, J=6.3 Hz,
(CH2)12CH3), 1.20–1.42 (24H, m, (CH2)12CH3), 1.98–2.20 (2H, m, CH2
ABX), 4.35 (1H, t, J=7.2 Hz, CH ABX), 6.94 (1H, s, CH(Ph)2), 7.32–7.42
(10H, m, CH(Ph)2). 13C NMR (75.43 MHz, CDCl3, TMS, δ(ppm)): 14.11
(1C, (CH2)12CH3), 22.67–31.90 (12C, (CH2)12CH3), 34.89 (1C, CH2 ABX),
46.12 (1C, CHABX), 78.19(1C, CH(Ph)2), 127.03–139.46 (12C, CH(Ph)2),
168.68 (1C, CHCO).
2.2.13. 1,4,7-triazacyclononane-N-hexanoate benzhydryl ester-N′-N″-
diacetate tert-butyl ester (4b)
The title compound was prepared according to the method described
for the preparation of 4a, starting from 3b. The compound 4b (0.137 g,
0.215 mmol) was obtained with a yield of 82.4%. 1H NMR (300 MHz, CDCl3,
TMS, δ(ppm)): 0.84 (3H, t, J=6.9 Hz, (CH2)2CH3), 1.20–1.40 (4H, m,
(CH2)2CH3), 1.44 (18H, s, C(CH3)3), 1.45–1.84 (2H, m, CH2 ABX), 2.60–3.60
(16H, m, en and CH2CO), 4.80 (1H, CH ABX), 6.88 (1H, s, CH(Ph)2), 7.27–
7.33 (10H, m, CH(Ph)2). 13C NMR (100.613 MHz, CDCl3, TMS, δ(ppm)):
13.81 (1C, (CH2)2CH3), 22.49 (1C, CH2CH2CH3), 27.87–28.06 (6C, C(CH3)3),
28.75 (1C, CH2CH2CH3), 31.15 (1C, CH2 ABX), 50.62–68.12 (8C, en and
CH2CO), 59.21 (1C, CH ABX), 77.00 (1C, CH(Ph)2), 81.62–85.28 ( 2C, C
(CH3)3), 126.52–139.88 (12C, CH(Ph)2), 164.03 (1C, CHCO), 170.93–172.06
(2C, CH2CO). m/z (ESI+) calculated for C37H56N3O6 (M+H)+ 638.41691.
Found 638.41671.
2.2.8. 1,4,7-triazacyclononane-N-hexanoate tert-butyl ester (3a)
A solution of 2a (0.747 g, 2.97 mmol) in 17.0 mL of DCM was added
dropwise to a solution of 1,4,7-triazacyclononane (0.500 g, 3.87 mmol)
in 26.0 mL of DCM. The reaction was stirred during 24 h. The white
precipitate was filtered off and the solution was concentrated. The
yellow residue obtained was purified by chromatography (DCM/EtOH
10:0 to 7:3 and DCM/EtOH/NH3 7:3:0.5) giving rise to compound 3a
(0.191 g, 0.638 mmol) with a yield of 21.5%. 1H NMR (300 MHz, CDCl3,
TMS, δ(ppm)): 0.95 (3H, t, J=6.6 Hz, (CH2)2CH3), 1.30–1.45 (4H, m,
(CH2)2CH3), 1.47 (9H, s, C(CH3)3), 1.70–1.85 (2H, m, CH2 ABX), 2.60–
3.65 (12H, m, en), 4.62 and 4.94 (1H, dd, J=9.6 Hz and J=10.2 Hz, CH
ABX). m/z (ESI+) calculated for C16H34N3O2 (M+H)+ 300.27. Found
300.25.
2.2.14. 1,4,7-triazacyclononane-N-octanoate benzhydryl ester-N′-N″-
diacetate tert-butyl ester (4c)
The title compound was prepared according to the method described
for the preparation of 4a, starting from 3c. The compound 4c (0.093 g,
0.140 mmol) was obtained with a yield of 48.6%. 1H NMR (300 MHz, CDCl3,
TMS, δ(ppm)): 0.86 (3H, t, J=6.3 Hz, (CH2)4CH3), 1.22–1.29 (8H, m,
(CH2)4CH3), 1.45 (18H, s, C(CH3)3), 1.55–1.80 (2H, m, CH2 ABX), 2.65–3.60
(16H, m, en and CH2CO), 3,99 and 4.30 (1H, dd, J=17.4 and 17.1 Hz, CH
ABX), 6.89 (1H, s, CH(Ph)2), 7.27–7.34 (10H, m, CH(Ph)2). 13C NMR
(100.613 MHz, CDCl3, TMS, δ(ppm)): 13.99 (1C, CH2CH2CH2CH2CH3),
22.48 (1C, CH2CH2CH2CH2CH3), 26.59 and 29.11 (2C, CH2CH2CH2CH2CH3),
27.97 (6C, C(CH3)3), 30.46 (1C, CH2 ABX), 31.63 (1C, CH2CH2CH2CH2CH3),
53.79–66.68 (8C, en and CH2CO), 62.91 (1C, CH ABX), 76.94 (1C, CH(Ph)2),
81.20 ( 2C, C(CH3)3), 127.09–140.06 (12C, CH(Ph)2), 170.76–172.66 (3C,
CH2CO and CHCO). m/z (ESI+) calculated for C39H60N3O6 (M+H)+
666.448. Found 666.500.
2.2.9. 1,4,7-triazacyclononane-N-hexanoate benzhydryl ester (3b)
The title compound was prepared according to the method
described for the preparation of 3a, starting from 2b. The yellow
residue obtained was purified by chromatography (DCM/EtOH 9:1).
The compound 3b (0.107 g, 0.261 mmol) was obtained with a yield
of 32.2%. 1H NMR (300 MHz, CDCl3, TMS, δ(ppm)): 0.83 (3H, t,
J=6.3 Hz, (CH2)2CH3), 1.20–1.37 (4H, m, (CH2)2CH3), 1.46–1.86
(2H, m, CH2 ABX), 2.69–3.42 (12H, m, en), 4.43 and 4.76 (1H, dd,
J=9.9 and 9.6 Hz, CH ABX), 6.86 (1H, s, CH(Ph)2), 7.20–7.30 (10H,
m, CH(Ph)2).
2.2.15. 1,4,7-triazacyclononane-N-decanoate benzhydryl ester-N′-N″-
diacetate tert-butyl ester (4d)
Compound 2d (0.467 g, 1.12 mmol) was added to a solution of
NO2AtBu (1,4,7-triazacyclononane-N,N′-diacetic acid tert-butyl ester)
(0.401 g, 1.12 mmol) and K2CO3 (0.310 g, 2.25 mmol) in 30.0 mL of
MeCN. The suspension was stirred during 4 h and was filtered to remove
the solid. The yellow oil obtained after concentration under vacuum was
purified by column chromatography (DCM/EtOH 7:3). Compound 4d
(0.753 g, 1.08 mmol) was obtained with a yield of 96.4%. 1H NMR
(300 MHz, CDCl3, TMS, δ(ppm)): 0.88 (3H, t, J=6.0 Hz, (CH2)6CH3), 1.15–
1.35 (12H, m, (CH2)6CH3), 1.45 (18H, s, C(CH3)3), 1.52–1.83 (2H, m, CH2
ABX), 2.60–4.00 (16H, m, en and CH2CO), 4.32 (1H, t, CH ABX), 6.90 (1H, s,
CH(Ph)2), 7.27–7.36 (10H, m, CH(Ph)2).
2.2.10. 1,4,7-triazacyclononane-N-octanoate benzhydryl ester (3c)
The title compound was prepared according to the method
described for the preparation of 3a, starting from 2c and was
obtained with 35.2% yield (0.126 g, 0.288 mmol). 1H NMR (300 MHz,
CDCl3, TMS, δ(ppm)): 0.86 (3H, t, J=5.7 Hz, (CH2)4CH3), 1.21–1.40
(8H, m, (CH2)4CH3), 1.50–1.90 (2H, m, CH2 ABX), 2.72–3.26 (12H, m,
en), 4.47 and 4.77 (1H, dd, J=9.6 and 9.9 Hz, CH ABX), 6.90 (1H, s,
CH(Ph)2), 7.27–7.36 (10H, m, CH(Ph)2).
2.2.11. 1,4,7-triazacyclononane-N-hexadecanoate tert-butyl ester (3e)
The title compound was prepared according to the method
described for the preparation of 3a, starting from 2d. Compound 3e
(0.132 mg, 0.300 mmol) was obtained with a yield of 17.2%. 1H NMR
(300 MHz, CDCl3, TMS, δ(ppm)): 0.87 (3H, t, J=6.9 Hz, (CH2)12CH3),
1.25 (24H, s, (CH2)12CH3), 1.45 (9H, s, C(CH3)3), 1.68–1.81 (2H, m,
CH2 ABX), 2.76–3.56 (12H, m, en), 4.59 and 4.92 (1H, dd, J=9.9 Hz,
CH ABX).
2.2.16. 1,4,7-triazacyclononane-N-hexadecanoate tert-butyl ester-N′-
N″-diacetate tert-butyl ester (4e)
The title compound was prepared according to the method described
for the preparation of 4a, starting from 3e. Compound 4e (0.025 g,
0.037 mmol) was obtained with a yield of 12.3%. 1H NMR (300 MHz, CDCl3,
TMS, δ(ppm)): 0.87 (3H, t, J=6.9 Hz, (CH2)12CH3), 1.24 (24H, s,

A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
1055
(CH2)12CH3), 1.45 (27H, s, C(CH3)3), 1.63–1.82 (2H, m, CH2 ABX), 2.64–
4.90 (17H, m, en, CH ABX and CH2CO).
(CH2)4CH3), 1.70–2.00 (2H, m, CH2 ABX), 3.28 (12H, s, en), 3.79 (1H, t,
J=7.8 Hz, CH ABX), 3.90 (4H, s, CH2CO). 13C NMR (75.43 MHz, D2O, DSS,
δ(ppm)): 13.54 (1C, (CH2)4CH3), 22.11 (1C, CH2CH2CH2CH2CH3), 26.04
and 28.46 (2C, CH2CH2CH2CH2CH3), 28.34 (1C, CH2 ABX), 30.96 (1C,
CH2CH2CH2CH2CH3), 47.35–50.40 (6C, en), 56.30 (2C, CH2CO), 66.62 (1C,
CH ABX), 173.04 (2C, CH2CO), 175.32 (1C, CHCO). m/z (ESI+) calculated for
C18H33N3O6 (M+H)+ 388.24. Found 388.50.
2.2.17. 1,4,7-triazacyclononane-N-hexadecanoate benzhydryl ester-N′-
N″-diacetate tert-butyl ester (4f)
The title compound was prepared according to the method described
for the preparation of 4d, starting from 2f. Compound 4f (0.113 g,
0.145 mmol) was obtained with a yield of 74.0%. 1H NMR (300 MHz, CDCl3,
TMS, δ(ppm)): 0.88 (3H, t, J=6.3 Hz, (CH2)12CH3), 1.26 (24H, s,
(CH2)12CH3), 1.45 (18H, s, C(CH3)3), 1.50–1.83 (2H, m, CH2 ABX), 2.60–
4.40 (17H, m, en, CH ABX and CH2CO), 6.89 (1H, s, CH(Ph)2), 7.27–7.36
(10H, m, CH(Ph)2).
2.2.20. 1,4,7-triazacyclononane-N-decanoic-N′-N″-diacetic acid
(NOTAC10)
The title compound was prepared according to the method described
for the preparation of NOTAC6, starting from 4d. NOTAC10 (0.569 g,
0.751 mmol) was obtained with a yield of 88.4%. 1H NMR (300 MHz, D2O,
DSS, δ(ppm)): 0.86 (3H, tb, (CH2)6CH3), 1.15–1.50 (8H, mb, (CH2)6CH3),
1.60–1.96 (2H, mb, CH2 ABX), 2.95–3.25 (12H, sb, en), 3.57 (1H, sb, CH
ABX), 3.88 (4H, sb, CH2CO). 13C NMR (75.43 MHz, D2O, DSS, δ(ppm)):
13.87 (1C, (CH2)6CH3), 22.62–31.98 (6C, (CH2)6CH3), 28.60 (1C, CH2 ABX),
46.64–50.93 (6C, en), 55.62 (2C, CH2CO), 64.92 (1C, CH ABX), 171.95–
172.28 (2C, CH2CO), 174.93 (1C, CHCO). m/z (ESI+) calculated for
C20H38N3O6 (M+H)+ 416.28. Found 416.50.
2.2.18. 1,4,7-triazacyclononane-N-hexanoic-N′-N″-diacetic acid
(NOTAC6)
5.00 mL of trifluoracetic acid (TFA) was added to a solution of 4a
(0.092 g, 0.174 mmol) in 5.00 mL of DCM and the reaction was stirred
during 17 h. After that, the solvent and the remaining TFA were
evaporated and the brown oil was washed three times with n-hexane
to assure that all the TFA was removed. NOTAC6 (0.090 mg,
0.128 mmol) was obtained with a yield of 73.6%. (The same method
could be applied to the pro-chelators orthogonally protected). 1H
NMR (300 MHz, D2O, DSS, pH=1.31, δ(ppm)): 0.87 (3H, t, J=7.0 Hz,
(CH2)2CH3), 1.28–1.44 (4H, m, (CH2)2CH3), 1.75–1.98 (2H, m, CH2
ABX), 3.28 (12H, s, en), 3.78 and 3.81 (1H, dd, J=6.3 and 6.3 Hz, CH
ABX), 3.92 (4H, s, CH2CO). 13C NMR (75.43 MHz, D2O, DSS, pH=1.31,
δ(ppm)): 13.19 (1C, (CH2)2CH3), 22.08 (1C, CH2CH2CH3), 28.08 (1C,
CH2 ABX), 28.26 (1C, CH2CH2CH3), 47.13–50.28 (6C, en), 55.99 (2C,
CH2CO), 66.23 (1C, CH ABX), 172.99 (2C, CH2CO), 175.38 (1C, CHCO).
m/z (ESI+) calculated for C16H30N3O6 (M+H)+ 360.21346. Found
360.21241.
2.2.21. 1,4,7-triazacyclononane-N-hexadecanoic-N′-N″-diacetic acid
(NOTAC16)
The title compound was prepared according to the method described
for the preparation of NOTAC6, starting from 4e or 4 f. NOTAC16 (0.021 g,
0.029 mmol) was obtained with a yield of 78.4%. 1H NMR (300 MHz,
CD3OD, TMS, δ(ppm)): 0.92 (3H, t, J=6.6 Hz, (CH2)12CH3), 1.31 (24H, s,
(CH2)12CH3), 1.70–2.20 (2H, m, CH2 ABX), 2.80–3.37 (12H, m, en), 3.62
(1H, t, J=7.2 Hz, CH ABX), 3.70–4.00 (4H, sb, CH2CO). m/z (ESI+)
calculated for C26H50N3O6 (M+H)+ 500.36996. Found 500.36711.
2.2.22. [Al(NOTAC6)]
2.2.19. 1,4,7-triazacyclononane-N-octanoic-N′-N″-diacetic acid
(NOTAC8)
The title compound was prepared according to the method described
for the preparation of NOTAC6, starting from 4c. NOTAC8 (0.034 g,
0.088 mmol) was obtained with a yield of 79.3%. 1H NMR (300 MHz, D2O,
DSS, δ(ppm)): 0.82 (3H, t, J=6.6 Hz, (CH2)4CH3), 1.24–1.44 (8H, m,
Aluminum nitrate and NOTAC6 dissolved in water were added in
equimolar amounts (Scheme 2). The pH was adjusted from 1.7 to 3.0
with 1 M KOH solution. The solution was stirred for 1 h at 75 °C. After
it reached room temperature, the pH was fixed to 4.0. The water was
evaporated and a 20.0 mM solution of the chelate in D2O was
prepared to perform the multinuclear NMR characterization. 1H NMR
Scheme 2. A — Al(NO3)3.9H2O in H2O; B — Ga(NO3)3.xH2O in H2O.

1056
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
(300 MHz, D2O, DSS, δ(ppm)): 0.92 (3H, t, J=7.2 Hz, (CH2)2CH3), 1.32–
1.50 (2H, m, CH2CH2CH3, 1.53–1.63 (2H, m, CH2CH2CH3), 1.75–1.90 and
2.04–2.16 (2H, m, CH2 ABX), 2.84–3.71 (13H, m, en and CH ABX), 3.82
(4H, d, J=10.8 Hz, CH2CO). 13C NMR (100.613 MHz, D2O, SiMe4,
pH=5.20, δ): 13.06 (1C, (CH2)2CH3), 22.04 (1C, CH2CH2CH3), 25.31
(1C, CH2 ABX), 29.09 (1C, CH2CH2CH3), 44.39–54.06 (6C, en), 62.92 (2C,
CH2CO), 68.61 (1C, CH ABX), 175.47 (2C, CH2CO), 176.94 (1C, CHCO).
27Al NMR (104.261 MHz, H2O/D2O (75/25%), [Al(H2O)6]3+, δ(ppm)):
47.58 ([Al(NOTAC6)]) (Δω1/2=51.31 Hz).
D2O (3:1) was prepared to perform the NMR characterization. 27Al
NMR (104.261 MHz, CD3OD/D2O (3:1), [Al(H2O)6]3+, δ(ppm)): 47.31
([Al(NOTAC10)]) (Δω1/2 =145.59 Hz).
2.2.27. [Al(NOTAC16)]
2.2.27.1. Method 1. Aluminum nitrate and NOTAC16 dissolved in water
were added in equimolar amounts. The pH was adjusted from 2.0 to
3.0 with 1 M KOH solution. The solution was stirred for 1 h at 75 °C.
After it reached room temperature, the pH was fixed to 4.0. The water
was evaporated and a solution of the solid in D2O was prepared to
perform the NMR characterization. 27Al NMR (104.261 MHz, D2O, [Al
(H2O)6]3+, δ(ppm)): no signal was found.
2.2.23. [Ga(NOTAC6)]
Gallium nitrate and NOTAC6 dissolved in water were added in
equimolar amounts. The pH was adjusted from 1.8 to 5.7 with 1 M
KOH solution. The solution was stirred for 1 h at 75 °C. The water was
evaporated and a 20.0 mM solution of the chelate in D2O was
prepared to perform the multinuclear NMR characterization. 1H NMR
(300 MHz, D2O, DSS, δ(ppm)): 0.93 (3H, t, J=7.2 Hz, (CH2)2CH3),
1.26–1.51 (2H, m, CH2CH2CH3), 1.53–1.64 (2H, m, CH2CH2CH3), 1.70–
1.90 and 2.06–2.18 (2H, m, CH2 ABX), 2.86–3.68 (13H, m, en and CH
ABX), 3.83 (4H, d, J=10.5 Hz, CH2CO). 13C NMR (75.43 MHz, D2O,
DSS, pH=5.20, δ): 13.21 (1C, (CH2)2CH3), 22.20 (1C, CH2CH2CH3),
25.91 (1C, CH2 ABX), 29.31 (1C, CH2CH2CH3), 44.05–53.84 (6C, en),
61.98 (2C, CH2CO), 68.67 (1C, CH ABX), 175.16 (2C, CH2CO), 176.85
(1C, CHCO). 71Ga NMR (122.026 MHz, D2O, [Ga(H2O)6]3+, δ): 165.48
([Ga(NOTAC6)]) (Δω1/2 =528.45 Hz). m/z (ESI+) calculated for
C16H26GaN3O6K (M+K)+ 464.07. Found 464.42.
2.2.27.2. Method 2. Aluminum nitrate dissolved in water and NOTAC16
dissolved in EtOH were added in equimolar amounts. The solution
was stirred for 1 h at 75 °C. After it reached room temperature, the pH
was fixed to 2.5 with 1 M KOH solution. The white precipitate formed
with the addition of base was filtered off and the solution was
evaporated. A solution of the solid in CD3OD was prepared to perform
the NMR characterization. 27Al NMR (104.261 MHz, CD3OD, [Al
(H2O)6]3+, δ(ppm)): 47.87 (weak signal).
2.2.28. [Ga(NOTAC16)]
Gallium nitrate dissolved in MeOH and NOTAC16 dissolved in
EtOH were added in equimolar amounts. The pH was adjusted from
0.8 to 2.8 with 1 M KOH solution. The solution was stirred for 1 h at
75 °C. The solvent was evaporated and a solution of the solid in CD3OD
was prepared to perform the NMR characterization. 71Ga NMR
(122.026 MHz, CD3OD, [Ga(H2O)6]3+, δ(ppm)): no signal was found.
2.2.24. [Al(NOTAC8)]
Aluminum nitrate and NOTAC8 dissolved in water were added in
equimolar amounts. The pH was adjusted from 1.9 to 2.7 with 1 M
KOH solution. The solution was stirred for 1 h at 75 °C. After it reached
room temperature, the pH was fixed to 4.1. The water was evaporated
and a 4.28 mM solution of the chelate in D2O was prepared to perform
the multinuclear NMR characterization. 1H NMR (300 MHz, D2O, DSS,
δ(ppm)): 0.88 (3H, t, J=6.9 Hz, (CH2)4CH3), 1.22–1.67 (8H, m,
(CH2)4CH3), 1.72–1.88 and 2.02–2.18 (2H, m, CH2 ABX), 2.82–3.62
2.3. Determination of the critical micellar concentration
The chelates were prepared by adding an appropriate amount of
metal nitrate to a weighted quantity of ligand, dissolving in water at
pH 4 and heating at 75 °C during one hour. The water was evaporated
and the solid was re-dissolved in phosphate buffer pH 7.4 or D2O. In
order to know the exact number of ligand equivalents existing in a
weighted amount of ligand, an excess of a standard Al(III) solution
was added to a weighted quantity of chelator, leaving the complex-
ation to occur during one hour at 75 °C. To this solution was added an
excess of standard EDTA solution and this was back-titrated with a
standard Ca(II) solution using eriochrome black T as indicator [28].
(12H, m, en), 3.67 (1H, t, J = 6.3 Hz, CH ABX), 3.82 (4H, s, CH2CO). 13
C
NMR (100.613 MHz, D2O, DSS, δ(ppm)): 13.27 (1C, (CH2)4CH3), 21.79
and 30.72 (2C, CH2CH2CH2CH2CH3), 25.50 (1C, CH2 ABX), 26.80 and
28.24 (2C, CH2CH2CH2CH2CH3), 44.56–54.18 (6C, en), 63.10 (2C,
CH2CO), 68.82 (1C, CH ABX), 175.34 (2C, CH2CO), 176.63 (1C, CHCO).
27Al NMR (104.261 MHz, D2O, [Al(H2O)6]3+, δ(ppm)): 47.47 ([Al
(NOTAC8)]) (Δω1/2 =54.87 Hz).
2.2.25. [Ga(NOTAC8)]
2.3.1. 27Al NMR method
Gallium nitrate and NOTAC8 dissolved in water were added in
equimolar amounts. The pH was adjusted from 2.1 to 3.0 with 1 M
KOH solution. The solution was stirred for 1 h at 75 °C. After it reached
room temperature, the pH was fixed to 4.8. The water was evaporated
and a 4.20 mM solution of the chelate in D2O was prepared to perform
the multinuclear NMR characterization. 1H NMR (300 MHz, D2O, DSS,
δ(ppm)): 0.88 (3H, t, J=6.9 Hz, (CH2)4CH3), 1.31–1.53 (8H, m,
(CH2)4CH3), 1.75–1.90 and 2.05–2.20 (2H, m, CH2 ABX), 2.90–3.55
The determination of the cmc was performed by monitoring the
variation of the half-width of the 27Al NMR signal with the variation of
the chelate concentration [4]. Stock solutions of [Al(NOTAC6)] and [Al
(NOTAC8)] in D2O, 13.0 mM and 4.28 mM respectively, were
prepared at room temperature at pH 4.0. These concentrations were
close to the limit of solubility of the chelates. The stock solutions were
then gradually diluted in D2O and 27Al NMR spectra were recorded for
each concentration of chelate.
(12H, m, en), 3.65 (1H, t, J=5.7 Hz, CH ABX), 3.83 (4H, m, CH2CO). 13
C
NMR (100.613 MHz, D2O, DSS, δ(ppm)): 13.27 (1C, (CH2)4CH3), 21.79
(1C, CH2CH2CH2CH2CH3), 26.03 (1C, CH2 ABX), 26.85 and 28.23 (2C,
CH2CH2CH2CH2CH3), 30.71 (1C, CH2CH2CH2CH2CH3), 44.16–53.71
(6C, en), 61.95 (2C, CH2CO), 68.83 (1C, CH ABX), 174.79 (2C,
CH2CO), 176.39 (1C, CHCO). 71Ga NMR (122.026 MHz, D2O, [Ga
(H2O)6]3+, δ(ppm)): 165.75 ([Ga(NOTAC8)]) (Δω1/2 =621.80 Hz).
2.3.2. Fluorescence method
The estimation of the cmc was also performed by fluorescence
using ANS as fluorescence probe [29]. Stock solutions of 16.63 mM of
[Ga(NOTAC6)] and 3.36 mM of [Ga(NOTAC8)] were prepared in 0.1 M
and 0.2 M phosphate buffer pH 7.4, respectively. These concentrations
were close to the limit of solubility of the chelates. Solutions with
different chelate concentrations, prepared by dilution of the stock
solutions, containing 1×10−5 M ANS were used in this study. The
fluorescence was measured at 480 nm upon excitation at 350 nm at
room temperature. The fluorescence measurements were recorded on
a Bio-Tek® SynergyTM HT spectrofluorimeter using the software
2.2.26. [Al(NOTAC10)]
Aluminum nitrate and NOTAC10 dissolved in water were added in
equimolar amounts. The pH was adjusted to 3.0 with 1 M KOH
solution, occurring precipitation. The solution was stirred for 1 h at
75 °C. The water was evaporated and a solution of the solid in CD3OD/
KC4TM
.

A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
1057
2.4. Radiochemistry
3. Results and discussion
[
67Ga] chelates for in vivo and in vitro experiments were prepared
3.1. Synthesis
by adding 1 mCi of [67Ga](citrate) to a solution of 1 mg of the chelator
in HEPES (0.150 mL, 0.1 M, pH 5) and heated at 80 °C for ca 1 h. The
radiochemical purity of the [67Ga]L solutions were determined by TLC,
and the percentage of bound metal averaged 96%.
We followed two different synthetic approaches for the prepara-
tion of the chelators. In the first method both alkylating agents were
protected in the form of tert-butyl ester, which allowed obtaining pro-
chelators 4a and 4e (C and D in Scheme 1). Their reaction with TFA
afforded the chelators NOTAC6 and NOTAC16. The other method
involved the mono-alkylation of the triazacyclononane moiety using
the alkylating agents protected in the form of benzhydryl ester (C in
Scheme 1) originating intermediates 3b and 3c. Their reaction with 2-
bromoacetate tert-butyl ester afforded the pro-chelators 4b and 4c,
which are orthogonally protected. Treating 4b and 4c with TFA
afforded the totally unprotected chelators NOTAC6 and NOTAC8. As
the preparation of the mono-alkylated intermediates involves low
yield synthesis we alternatively started with NO2AtB (a triazacyclo-
nonane moiety with two acetate pendant arms protected with tert-
butyl groups) shortening the synthetic pathway and affording the
pro-chelators 4d and 4f in good yield (E in Scheme 1). Their treatment
with TFA afforded the chelators NOTAC10 and NOTAC16.
2.4.1. Determination of logP and stability in blood serum
The octanol/water partition coefficient (logP) of [67Ga](NOTAC6)
and [67Ga](NOTAC8) was determined using the shake-flask method.
The partition coefficient was determined by adding 25 μL of the
chelate solution to a tube containing 1 mL of saline solution and 1 mL
of 1-octanol. The resulting mixture was shaken at room temperature
for 1 h and then centrifuged at 3000 rpm during 3 min. After the
centrifugation, 100 μL of each phase was collected and the activity was
measured. The partition coefficient was calculated as a ratio of the
counts in the octanol fraction to the counts in the water fraction being
this the result of the average of 5 determinations (S.D.b0.01).
For the blood serum stability studies, 5 μCi of the standard solution
of [67Ga](NOTAC8) were added to 5 mL of fresh human serum,
previously equilibrated in 5% CO2 (95% air) environment at 37 °C. The
mixture was stored in the same environment conditions, and aliquots
of 100 mL (in triplicate) were taken at appropriate periods of time
(0 min, 30 min, 1 h and 3 h). The aliquots were treated with 200 μL of
ethanol, cooled (4 °C), and centrifuged during 15 min at 4000 rpm, at
4 °C, in order to precipitate the serum proteins. A 100 μL aliquot of
supernatant was collected for activity counting in a γ well-counter.
The sediment was washed twice with 1 mL of EtOH and its activity
was counted. The activity of the supernatant was compared to that of
the sediment in order to determine the percentage of the chelate
associated to the proteins. The activity of the supernatant at 3 h was
evaluated by TLC in order to check whether the chelate remained
intact.
3.2. Characterization of the Al(III) and Ga(III) chelates by multinuclear
NMR
Octahedral metal chelates of NOTA can have two possible
arrangements for the coordination of the acetate groups, while the
ethylenic bridges of the macrocyclic ring can also adopt two different
orientations [30]. According to this, the chelates of NOTA become
chiral. Depending on the torsion angle of the acetate groups, the
isomers can be clockwise (Δ) or anticlockwise (Λ). The orientation of
the ethylenic bridges originates the isomers δ and λ. Therefore, two
enantiomeric pairs are possible: type I [Δ(λλλ) and Λ(δδδ)] and type
II [Δ(δδδ) and Λ( λλλ)]. The solid state structures of Ga(NOTA) and Al
(NOTA) have been previously determined [6,31], showing coordina-
tion of the metal ion to the three ring nitrogen atoms, as well as to
three carboxylate oxygen atoms, yielding a pseudo-octahedral
arrangement corresponding to the type I enantiomeric pair. The
introduction of the aliphatic chain in the structures of the ligands
produces a chiral carbon (R and S configurations), leading to another
factor for enantiomerism. Accordingly, each of the chelates of
NOTAC6, NOTAC8, NOTAC10 and NOTAC16 may originate four
diastereoisomeric pairs of enantiomers in solution.
2.4.2. Biodistribution
2.4.2.1. Methods. A gamma camera-computer system (GE 400
GenieAcq, from General Electric, Milwaukee, WI, USA) was used for
acquisition and pre-processing the in vivo gamma imaging. Data
processing and display were performed on a personal computer using
homemade software developed for the IDL 6.3 (Interactive Data
Language, Research Systems, Boulder, CO, USA) computer tool.
The 1H NMR results obtained for the Al(III) and Ga(III) chelates of
NOTAC6 and NOTAC8 in aqueous solution (illustrated in Figs. 1, S3–S4
for NOTAC8) are in agreement with the structures determined for the
respective NOTA chelates by X-ray crystallography. These systems
show symmetrical and well resolved resonances corresponding to the
ring protons which create AA′MM′ multiplet patterns arising from the
(λλλ)↔(δδδ) ring conformational interconvertions. The β-CH2
protons on the alkyl side chain become unequivalent upon coordina-
tion. The very similar patterns in the 1H NMR spectra of the Al(III) and
Ga(III) chelates of NOTAC6 and NOTAC8 observed at two different
temperatures (25 °C and 75 °C, see Figs. 1, S3–S4) are consistent with
a slow exchange regime and with the high rigidity of NOTA-based
systems in solution, with two possible configurational arrangements
for the coordinated acetate groups and the puckering of the
ethylenediamine metal-chelate rings [30]. The 1H NMR spectra
recorded at chelate concentrations above and below the cmc show
no differences (Fig. S4), indicating that the dynamic behavior of the
chelates is not influenced by the micellization.
2.4.2.2. Biodistribution studies. Groups of four animals (Wistar rat
males weighting ca 200 g) were anaesthetized with Ketamine
(50.0 mg/ml)/chloropromazine (2.5%) (10:3) and injected in the tail
vein with ca 100 μCi of the tracer and sacrificed 30 min and 24 h later.
The major organs were removed, weighted and counted in a γ well-
counter.
2.4.2.3. In vivo gamma imaging. The animals were positioned in ventral
decubitus over the detector. Image acquisition was initiated imme-
diately before radiotracer injection in the tail vein. Sequences of 30
images (of 60 s each), were acquired to 64×64 matrices. In addition,
static data were acquired 24 h after the radiotracer injection
(180×180 matrices, time=10 min). Images were subsequently
processed using an IDL based program. In order to analyze the
transport of radiotracer over time, three regions of interest (ROI) were
drawn on the image files, corresponding to the thorax, liver and left
kidney. From these regions, time-activity curves were obtained and
normalized to the maximum activity in all the organs.
Despite the fact that it was not possible to obtain mono crystals
suitable for X-ray diffraction, some useful structural information
about the coordination site of the cations can be obtained from metal
NMR data. The 27Al and 71Ga NMR chemical shifts of the chelates in
aqueous solution (Table 1) are within the range usually found for

1058
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
Fig. 1. A — 1H NMR spectrum of [Al(NOTAC8)] (400 MHz, D2O, 75 °C, pH 4.10). B — 1H COSY spectrum of [Al(NOTAC8)].

A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
1059
Table 1
27Al and 71Ga NMR chemical shifts and signal half-widths of Al(III) and Ga(III) chelates
of NOTA derivatives in aqueous solution (25 °C) compared to those for NOTA.
27Al NMR
71Ga NMR
Ligand
δ (ppm)
Δω1/2 (Hz)
δ (ppm)
Δω1/2 (Hz)
NOTA
49 [38]
47.6a
47.5c
47.3e
47.9f
60 [38]
51.3
55.0
145.6
–
171 [36]
165.5b
165.8d
–
210 [36]
528.5
621.8
–
NOTAC6
NOTAC8
NOTAC10
NOTAC16
–
–
a
20.0 mM, pH 4.0.
20.0 mM, pH 5.7.
4.28 mM, pH 4.1.
4.20 mM, pH 4.8.
Concentration not defined, pH 3.0.
Methanol solution with concentration not defined, pH 2.5.
b
c
d
e
f
octahedral or pseudo-octahedral species with a C3 symmetry axis,
responsible for the high symmetry at the coordination metal centre
[4]. X-ray crystallography studies of Al(III) and Ga(III) chelates of
NOTA [3,31–34] and NOTA-based ligands [35–37] showed that these
chelates have pseudo-octahedral geometries.
The complexation process of the metal ions with NOTAC10 and
NOTAC16 was limited by the poor water solubility of the ligands, as
monitored by the 27Al NMR spectra. The [Al(NOTAC10)] complex was
formed in aqueous solution, originating a 27Al NMR signal at 47.31 ppm,
much weaker than the main resonance from [Al(H2O)6]3+ (0 ppm).
NOTAC16 had very poor solubility in water and it was not possible to
obtain the Al(III) and Ga(III) chelates in aqueous solution. However,
when the Al(III) chelate was prepared in ethanol, a weak 27Al NMR
signal at 47.87 ppm could be detected.
Fig. 2. Fluorescence intensity of the ANS fluorophore at 480 nm versus: A — [Ga
(NOTAC6)] concentration;
B — [Ga(NOTAC8)] concentration. The cmc values are
established from graphical break points.
The 27Al NMR signals of the Al(III) chelates bearing short side
chains (NOTAC6 and NOTAC8) show half-widths (Δω1/2) similar to
those found for the Al(III) chelate of NOTA, but they become
significantly broader for the chelates with longer side chains [38].
Such an increased broadening with side chain length increase is more
marked for the 71Ga resonance of the Ga(III) complexes [36] (Table 1).
These observations result from the quadrupolar relaxation of the
metal nuclei and the slowing down of the rotational dynamics of the
heavier complexes in solution.
tic acid) with dimensions superior to 100 nm below the cmc. The
formation of aggregates at concentrations below the cmc was also
verified by fluorescence correlation spectroscopy in such reduced
amount that the conventional techniques were not able to detect it
[39].
On the other hand, the cmc values of [Al(NOTAC6)] and [Al
(NOTAC8)] were assessed using 27Al NMR spectroscopy. Due to the
quadrupolar nature of the 27Al nuclide, whose nuclear relaxation is
dependent on the rotational correlation time of the chelate in solution
[4], the linear least-square fitting of the plot of the half-width of the
27Al NMR signal as a function of the chelate concentration (Fig. 3)
allowed obtaining the cmc values. The plot for [Al(NOTAC8)], with a
single break, reflected a simple micellization process with a cmc value
of 0.25 mM, consistent with the value found by fluorescence for the
analogous Ga(III) chelate. The corresponding plot for [Al(NOTAC6)]
showed two breaks, at 1.16 mM and 4.30 mM, reflecting a more
complex micellization behavior. While the second value (4.30 mM)
corresponds quite well to the cmc value detected for the Ga(III)
complex by fluorescence, the first value (1.16 mM) suggests the
formation of pre-micellar aggregates in solution, in agreement with
what has been found by fluorescence for the corresponding Ga(III)
complex.
3.3. Determination of the critical micellar concentration
The amphiphilic behavior of the metal chelates of NOTAC6 and
NOTAC8 was demonstrated by determining their critical micellar
concentration. For this purpose, two distinct methods were used. In
one method, the fluorescence of ANS, which is sensitive to the
polarity of the environment, showing no fluorescence in water and
high fluorescence in non-polar environments, was used as a probe
for the Ga(III) chelates. Entrapping ANS in the inner part of the
micelle, which is non-polar, increases the intensity of its fluores-
cence [29]. The cmc value was estimated by linear least-square
fitting of the fluorescence emission at 480 nm versus the concen-
tration of the chelates (Fig. 2). The calculated cmc values were
5.20 mM for [Ga(NOTAC6)] and 0.36 mM for [Ga(NOTAC8)], show-
ing that the two chelates have quite different aggregation behaviors,
as an increase of alkyl chain length from four to six carbons
decreases the cmc by an order of magnitude, reflecting a strong
increase of the self-aggregation capacity of the chelates. The fact that
the fluorescence intensity of the [Ga(NOTAC6)] system increases
with the increase of concentration of chelate until the cmc value is
reached suggests the formation of pre-micellar aggregates, contrary
to what happens for [Ga(NOTAC8)]. The existence of pre-micellar
aggregation has been detected before [23], by using DLS to prove the
existence of aggregates of [Gd(EPTPAC16)]2− (EPTPAC16 =(hydro-
xymethylhexadecanoyl ester) ethylenepropylenetriaminepentaace-
The cmc values obtained for the studied chelates are within the
range of those found for amphiphilic Ni(II) chelates of triaza-based
ligands with comparable alkyl chain lengths [40].
3.4. Determination of logP and stability in blood serum
The measured octanol/water partition coefficients (logP) of
[
67Ga](NOTAC6) and [67Ga](NOTAC8), respectively logP=−2.25
and logP=−1.19, revealed that the chelates present low lipophi-
licity which, as expected, increases when the chain length of the α-
alkyl substituent at one of the pendant acetate arms increases from
C4 to C6.

1060
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
radiotracer, where various organs are enhanced. Time–activity curves
for the [67Ga](NOTAC6) tracer (Fig. 6) were obtained from scinti-
graphic dynamic acquisition experiments. The curves were smoothed
and normalized in relation to the maximum radioactivity obtained.
From these curves one can notice that the activity increased
sharply in the thorax, liver and kidneys immediately after the
injection, possibly corresponding essentially to blood activity. The
highest value for liver and thorax was obtained after one minute,
while it took about two minutes to reach maximum activity at the
kidneys. After reaching this maximum, the radioactivity in the
liver and thorax decayed relatively slowly while for the kidneys it
was almost constant during all the acquisition. At 30 min, the
activity at the kidneys remained at 99% of the maximum attained
while for liver and kidneys these values were 69% and 38%,
respectively. We obtained the same information from the images
presented in Fig. 5 where only the kidneys and bladder are clearly
visible. From these findings we can conclude that at least for
[
67Ga](NOTAC6), despite the contribution of the hepatobiliary
mechanism for the radiotracer elimination, the main excretion
mechanism is the kidney pathway. This is in accordance with the
logP obtained for [67Ga](NOTAC6).
From the biodistribution data (for the two compounds) and from
the imaging results for the NOTAC6 radiotracer, it is noticeable that
most of the radioactivity was cleared off from tissues and organs
within 24 h with virtually no deposition in the bones and liver/spleen,
demonstrating the high in vivo stability of [67Ga](NOTAC6) and [67Ga]
(NOTAC8).
From the specifications of the supplier of [67Ga](citrate) (CIS-BIO,
Gift-sur — Yvette, France), less than 4.5 ng of Ga(III) (1 mCi) of Ga(III)
should be present in the final solution of [67Ga](NOTAC8) or [67Ga]
(NOTAC6). Thus, in both cases, the concentrations were well below
the cmc determined for the complexes, and no micelles were
supposed to be present in solution.
Fig. 3. Half-width of the 27Al NMR signal of Al(III) chelates versus chelate
concentration: A — [Al(NOTAC6)]; B — [Al(NOTAC8)].
3.5. Stability in blood serum
Incubation studies of [67Ga](NOTAC8) in fresh human serum,
followed by precipitation of its protein content, showed that the
percentage of the activity in the protein pellet steadily increased
with the incubation time (13.8% at 30 min, 30.8% at 60 min),
reaching a value of 36.7% at 3 h. This increasing activity associated
to the blood proteins reflects the hydrophobic interactions
involving the α-alkyl substituent at one of the acetate pendant
arms and hydrophobic regions of the proteins. Nevertheless, it was
found by TLC analysis that the radioactivity present in this
fraction, after 3 h of incubation, represents the intact radiochelate,
reflecting the high stability of [67Ga](NOTAC8) with regard to
transchelation.
4. Conclusions
We developed a small library of four amphiphilic NOTA-based
chelators presenting a α-alkyl chain of variable size. The synthetic
strategy that was used in this work may also be compatible with the
preparation of bifunctional chelators which can be covalently coupled
to targeting biomolecules bearing an amine function (eg. peptides).
This is of paramount importance regarding receptor mediated medical
imaging and radiotherapeutic applications.
The metal chelates of NOTAC6 and NOTAC8 show distinct
amphiphilic behavior. The shorter-size-chain chelate shows some
level of pre-aggregation additionally to the formation of micelles.
When the α-alkyl chain has more than six carbon atoms, the chelators
show low solubility in water, thus reducing the possibility of using
their neutral chelates in biological applications.
3.6. Biodistribution and imaging studies
The biodistribution data for [67Ga](NOTAC8) and [67Ga](NOTAC6)
are expressed as the percentage of injected dose per gram of tissue (%
ID/g) in Fig. 4. Both chelates are excreted by kidneys but the
hepatobiliary pathway (stated as the sum of liver and intestines)
has also a strong contribution. This is particularly evident from the
scintigraphic studies. However, from our results, [67Ga](NOTAC8)
seems to be more hepatospecific (1.28% of the activity is present in
hepatobiliary transit at 30 min) than the corresponding C6 compound
(hepatobiliary contribution at the same time equals 0.26%). These
findings correlate with the higher lipophilicity of [67Ga](NOTAC8).
This chelate also shows a much higher lung uptake than [67Ga]
(NOTAC6) chelate at 30 min, which is not persistent at longer times,
as opposed to what has been found for the long chain tracer [153Sm]
(EPTPAC16)]2− [23].
The increase in the size of the α-alkyl chain, and the corresponding
increase in the lipophilicity of the chelates (logP values), are
responsible for a higher uptake of the Ga(III) chelates in the liver.
From the imaging and biodistribution studies we can conclude that
[
67Ga](NOTAC8) and [67Ga](NOTAC6) present high in vivo stability
and are excreted quite quickly, co-existing the kidney and hepato-
biliary pathway.
The fact that the radiolabeled chelates were injected at
concentrations below the determined cmc, precluded the forma-
tion of micelles in vivo with consequent lower hepatic uptake than
expected.
Abbreviations
Δω1/2
%ID/g
ANS
signal half-width
percentage of injected dose per gram of tissue
8-anilino-1-naphtalene sulfonic acid
Scintigraphic γ images of Wistar rats were obtained as a function
of time after injection of the [67Ga](NOTAC6) tracer. Fig. 5 compares
the images obtained 2 min, 4 min and 24 h after injection of the
BF3.OEt2 boron trifluoride ethyl etherate

A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
1061
Fig. 4. Biodistribution profile for Wistar rats (percentage of the injected dose/g of organ) of [67Ga](NOTAC6) (white bars) and [67Ga](NOTAC8) (gray bars) at 30 min (A) and 24 h (B)
after injection.
cmc
COSY
DDM
DLS
critical micellar concentration
correlation spectroscopy
diphenyldiazomethane
NOTA
1,4,7-triazacyclononane-N,N′,N″-triacetic acid
NOTAC6 1,4,7-triazacyclononane-N-hexanoic acid-N′,N″-diacetic
acid
dynamic light scattering
NOTAC8 1,4,7-triazacyclononane-N-octanoic acid-N′,N″-diacetic
DMA
DSS
en
N,N-dimethylacetamide
sodium 2,2-dimethylsilapentane-5-sulfonate
ethylenic bridges
acid
NOTAC10 1,4,7-triazacyclononane-N-decanoic acid-N′,N″-diacetic
acid
EPTPAC16 (hydroxymethylhexadecanoyl ester) ethylenepropylene-
triaminepentaacetic acid
NOTAC16 1,4,7-triazacyclononane-N-hexadecanoic acid-N′,N″-dia-
cetic acid
ESI+
HEPES
IDL
positive electrospray ionization
N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)
interactive data language
PET
ROI
TBTA
Tf
positron emission tomography
regions of interest
tert-butyltrichloroacetimidate
transferrin
L
ligand
MRI
magnetic resonance imaging
TFA
TLC
TMS
trifluoracetic acid
thin layer chromatography
tetramethylsilane
NO2AtB 1,4,7-triazacyclononane-N,N′-diacetic acid tert-butyl ester
NODASA 1,4,7-triazacyclononane-N-succinic acid-N′,N″-diacetic acid
Fig. 5. Scintigraphic images at 2 min, 4 min and 24 h of a Wistar rat injected with [67Ga](NOTAC6).

1062
A. de Sá et al. / Journal of Inorganic Biochemistry 104 (2010) 1051–1062
[8] M.I.M. Prata, A.C. Santos, C.F.G.C. Geraldes, J.J.P. de Lima, J. Inorg. Biochem. 79
(2000) 359–363.
[9] J.P. André, H.R. Maecke, M. Zehnder, L. Macko, K.G. Akyel, Chem. Commun. (1998)
1301–1302.
[10] J.P. André, H. Mäcke, A. Kaspar, B. Kunnecke, M. Zehnder, L. Macko, J. Inorg.
Biochem. 88 (2002) 1–6.
[11] J. Notni, K. Pohle, J.A. Peters, H. Gorls, C. Platas-Iglesias, Inorg. Chem. 48 (2009)
3257–3267.
[12] E.T. Clarke, A.E. Martell, Inorg. Chim. Acta 181 (1991) 273–280.
[13] H.R. Maecke, J.P. André, PET Chemistry The Driving Force in Molecular Imaging,
Springer, 2006.
[14] S.M. Moghimi, A.C. Hunter, J.C. Murray, Pharmacol. Rev. 53 (2001) 283–318.
[15] V.P. Torchilin, A.N. Lukyanov, Z. Gao, B. Papahadjopoulos-Sternberg, Proc. Natl.
Acad. Sci. U. S. A. 100 (2003) 6039–6044.
[16] J. Wang, D. Mongayt, V.P. Torchilin, J. Drug Target. 13 (2005) 73–80.
[17] V.P. Torchilin, Adv. Drug Deliv. Rev. 54 (2002) 235–252.
[18] K. Kostarelos, D. Emfietzoglou, J. Liposome Res. 9 (1999) 429–460.
[19] W.J. Mulder, G.J. Strijkers, G.A. van Tilborg, A.W. Griffioen, K. Nicolay, NMR
Biomed. 19 (2006) 142–164.
Fig. 6. Time/activity curves of [67Ga](NOTAC6) obtained from dynamic images of the
regions of interest of a Wistar rat. Kidneys (●); Liver (■); Thorax (▲).
[20] G.M. Nicolle, E. Toth, K.P. Eisenwiener, H.R. Macke, A.E. Merbach, J. Biol. Inorg.
Chem. 7 (2002) 757–769.
[21] S.M. Moghimi, A.E. Hawley, N.M. Christy, T. Gray, L. Illum, S.S. Davis, FEBS Lett. 344
(1994) 25–30.
[22] V.S. Trubetskoy, M.D. Frank-Kamenetsky, K.R. Whiteman, G.L. Wolf, V.P. Torchilin,
Acad. Radiol. 3 (1996) 232–238.
[23] S. Torres, M.I. Prata, A.C. Santos, J.P. André, J.A. Martins, L. Helm, E. Tóth, M.L.
Garcia-Martin, T.B. Rodrigues, P. Lopez-Larrubia, S. Cerdan, C.F.G.C. Geraldes, NMR
Biomed. 21 (2008) 322–336.
Acknowledgements
We thank the support from Fundação para a Ciência e a Tecnologia
(F.C.T.), Portugal, (project PTDC/QUI/70063/2006) and FEDER. The
Bruker Avance III 400 NMR spectrometer in Braga was acquired with
the support of the Programa Nacional de Reequipamento Científico of
the F.C.T., Portugal (contract REDE/1517/RMN/2005 — as part of
RNRMN-Rede Nacional de RMN). This work was carried out in the
framework of the COST D38 Action and EU-FP6 “Network of
Excellence” EMIL (grant no. LSHC-2004-503569) project.
[24] Z.R. Zhang, K.X. Liang, S. Bloch, M. Berezin, S. Achilefu, Bioconjug. Chem. 16 (2005)
1232–1239.
[25] K.S. Birdi, H.N. Singh, S.U. Dalsager, J. Phys. Chem. 83 (1979) 2733–2737.
[26] A. Riesen, T.A. Kaden, W. Ritter, H.R. Macke, J. Chem. Soc. Chem. Commun. (1989)
460–462.
[27] J.B. Miller, J. Org. Chem. 24 (1959) 560–561.
[28] J. Bassett, R.C. Denney, G.H. Jeffery, J. Mendham, Vogel's: Textbook of Quantitative
Inorganic Analysis, Longman, New York, 1978.
[29] E. De Vendittis, G. Palumbo, G. Parlato, V. Bocchini, Anal. Biochem. 115 (1981)
278–286.
[30] M.J.V. Merwe, J.C.A. Boeyens, R.D. Hancock, Inorg. Chem. 24 (1985) 1208–1213.
[31] U. Bossek, D. Hanke, K. Wieghardt, B. Nuber, Polyhedron 12 (1993) 1–5.
[32] A. Jyo, T. Kohno, Y. Terazono, S. Kawano, Anal. Sci. 6 (1990) 629–630.
[33] T.W. Duma, F. Marsicano, R.D. Hancock, J. Coord. Chem. 23 (1991) 221–232.
[34] K. Wieghardt, U. Bossek, P. Chaudhuri, W. Herrmann, B.C. Menke, J. Weiss, Inorg.
Chem. 21 (1982) 4308–4314.
[35] J.P. André, Synthesis, Characterization and Applications Relevant to Magnetic
Resonance Imaging and Radiopharmacy of New Complexes of Al(III), Ga(III) and
Gd(III), PhD Thesis, Basel, 1999.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jinorgbio.2010.06.002.
References
[36] E. Cole, R.C.B. Copley, J.A.K. Howard, D. Parker, G. Ferguson, J.F. Gallagher, B.
Kaitner, A. Harrison, L. Royle, Dalton Trans. (1994) 1619–1629.
[37] D.A. Moore, P.E. Fanwick, M.J. Welch, Inorg. Chem. 29 (1990) 672–676.
[38] M.P.M.M. Catarro, Estudos de Ligandos Poliazamacrocíclicos e de seus Complexos
Metálicos, Tese de Mestrado, Coimbra, 1995.
[1] M. Fani, J.P. André, H.R. Maecke, Contrast Media Mol. Imaging 3 (2008) 67–77.
[2] W.R. Harris, V.L. Pecoraro, Biochemistry 22 (1983) 292–299.
[3] C.J. Broan, J.P.L. Cox, A.S. Craig, R. Kataky, D. Parker, A. Harrison, A.M. Randall, G.
Ferguson, J. Chem. Soc., Perkin Trans. 2 (1991) 87–99.
[4] J.P. André, H.R. Mäcke, J. Inorg. Biochem. 97 (2003) 315–323.
[5] R. Ma, M.J. Welch, J. Reibenspies, A.E. Martell, Inorg. Chim. Acta 236 (1995) 75–82.
[6] A.S. Craig, D. Parker, H. Adams, N.A. Bailey, J. Chem. Soc. Chem. Commun. (1989)
1793–1794.
[39] H. Zettl, Y. Portnoy, M. Gottlieb, G. Krausch, J. Phys. Chem.
B 109 (2005)
13397–13401.
[40] P.C. Griffiths, I.A. Fallis, T. Chuenpratoom, R. Watanesk, Adv. Colloid Interface Sci.
122 (2006) 107–117.
[7] M.I.M. Prata, A.C. Santos, C.F.G.C. Geraldes, J.J.P. de Lima, Nucl. Med. Biol. 26 (1999)
707–710.