NODAPA-OH and NODAPA-(NCS)n: Synthesis, 68Ga-radiolabelling and in vitro characterisation of novel versatile bifunctional chelators for molecular imaging

Article abstract of DOI:10.1016/j.bmcl.2008.09.054

This report concerns synthesis, ⁶⁸Ga-radiolabelling and stability data of 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-isothio-cyanatophenyl-acetic acid (NODAPA-NCS), 1,4,7-triazacyclononane-1-acetic acid-4,7-di-p-isothiocyanatophenyl-acetic acid (NODAPA-(NCS)₂) and 1,4,7-triazacyclononane-1,4-diacetic acid-7-p-hydroxyphenyl-acetic acid (NODAPA-OH), versatile bifunctional chelators with potential for molecular imaging. Protein binding and exemplified conjugation are also reported.

Full text of DOI:10.1016/j.bmcl.2008.09.054

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5364–5367
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
NODAPA-OH and NODAPA-(NCS)n: Synthesis, 68Ga-radiolabelling and in vitro
characterisation of novel versatile bifunctional chelators for molecular imaging
*
Patrick J. Riss , Carsten Kroll, Verena Nagel, Frank Rösch
Institute of Nuclear Chemistry, University of Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
This report concerns synthesis, 68Ga-radiolabelling and stability data of 1,4,7-triazacyclononane-1,4-
diacetic acid-7-p-isothio-cyanatophenyl-acetic acid (NODAPA-NCS), 1,4,7-triazacyclononane-1-acetic
acid-4,7-di-p-isothiocyanatophenyl-acetic acid (NODAPA-(NCS)2) and 1,4,7-triazacyclononane-1,4-diace-
tic acid-7-p-hydroxyphenyl-acetic acid (NODAPA-OH), versatile bifunctional chelators with potential for
molecular imaging. Protein binding and exemplified conjugation are also reported.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 31 July 2008
Revised 15 September 2008
Accepted 15 September 2008
Available online 18 September 2008
Keywords:
Macrocycles
Bifunctional chelators
Gallium-68
Molecular imaging
PET
Non-invasive molecular imaging of biochemical mechanisms
and pathologies in vivo is an emerging interdisciplinary field of re-
search. Among the available techniques for a sensitive, quantitative
visualisation of the biochemical and physiological function of bio-
logical tissue approached by suitable imaging probes, positron
emission tomography (PET) provides great potential in terms of
quantification, sensitivity, temporal, and lateral resolution. The
commercially available 68Ge/68Ga radionuclide generator system
and its recent improvement concerning on-line processing and
labelling,1 may provide a beneficial complement to nuclear imag-
ing with established, cyclotron produced PET nuclides like 11C
and 18F. 68Ga provides a high positron abundance of 89% and an
intermediate positron maximum energy. With its half-life
(1.13 h) lying perfectly in between the half-lives of the most fre-
quently used 11C (0.33 h) and 18F (1.82 h), it provides excellent de-
cay characteristics as a PET-radiolabel. In addition, its most
promising advantage is its availability via a radionuclide generator
system. The mother nuclide 68Ge has a half-life of 270.8 d, guaran-
teeing prolonged use of generator set-ups in action (ca. 1 year).
Consequently, 68Ga provides an economical alternative to the
cyclotron produced radionuclides. On the other hand, radiolabel-
ling of molecular probes with 68Ga requires a completely different
synthesis route. The main group metal rapidly forms chelate-com-
plexes with hard donor functions of four to six coordinating chela-
tors. Adequate radioligand precursors meeting the coordination
chemistry of gallium(III) with versatile conjugation possibilities
are of high interest. To reinforce the flow of novel tracer candidates
to biological evaluation, a convenient, time efficient route to vari-
ous chelator conjugated potential targeting vectors would be
desirable.
The macrocyclic chelators DOTA and NOTA (Fig. 1) are estab-
lished as frequently considered routes for the introduction of a
68Ga-tag. Compared to open chain acyclic analogues, both provide
complexes of superior kinetic and thermodynamic stability since
gallium is irreversibly complexed at room temperature. DOTA re-
mains the most frequently used chelator because of its better com-
mercial availability and less challenging synthesis. Its six-
coordinate nine-ring analogue NOTA forms slightly distorted octa-
hedral complexes with gallium which display higher stabilities and
faster incorporation of Ga(III) at lower temperatures.2 Thus, we
were interested in a time-saving and cost-effective access to a ver-
satile NOTA-based gallium chelator allowing convenient conjuga-
tion to various targeting molecules.
HO2C
HO2C
HO2C
CO2H
CO2H
CO2H
N
N
N
N
N
N
N
CO2H
Figure
1. (4,7,10-tris-Carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic
* Corresponding author. Tel.: +49 6131 3925701.
E-mail address: riss@uni-mainz.de (P.J. Riss).
acid (DOTA) and (4,7-bis-carboxymethyl-[1,4,7]-triazanon-1-yl)-acetic acid
(NOTA).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.054

P. J. Riss et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5364–5367
5365
Specific bifunctional derivatives of NOTA, published so far, are
surveyed in Figure 2. NODAGA (1) and NODASA (2) are limited to
coupling peptides through an amide bond.3,4 The value of NODAGA
has been demonstrated convincingly. The isothio-cyanatophenyl
derivative 3 has been introduced by Brechbiel et al. but no further
use has been reported yet.5 The C-substituted analogue 4 is com-
mercially available. The synthesis of C-substituted NOTA derivative
5 was reported by Parker et al. in 7.7% yield.6 Brechbiel et al. also
reported very low cyclisation yields of C-substituted nine-mem-
bered rings due to transannular condensation.5 Consequently, we
were interested in a cost-effective concept for an easy access to a
bifunctional derivative of NOTA, starting from bulk chemicals.
Therefore a straightforward and economic synthesis of a bifunc-
tional chelator was developed. Orthogonally reactive phenol as
well as isothiocyanate functions were included for conjugation.
The intended bifunctional chelators are illustrated in Scheme 1.
The gallium core forms stable five-ring chelates with the Nitrogen
and the adjacent carboxylate donors in NOTA and its analogues.
Introduction of a conjugation functionality in a pendant arm
branch into NOTA–analogue chelators following nucleophilic N-
13) with sodium hydroxide. After exposure to activated carbon un-
der reflux followed by extraction with predistilled 1-butanol, 12
was obtained as colourless crystals displaying sufficient purity
for all further reactions. Statistical alkylation (b) with 9 or 10
was carried out in the presence of potassium carbonate over three
days in dichloromethane, using a threefold excess of TACN (12).
Employing a lower excess of 12, (d) the yield of the dialkylated
product 15 increased. Thereby, a convenient access to multivalent
[
68Ga]chelates is provided. The trialkylated product was not ob-
served. Introduction of the tert-butyl protected acetic acid donor
functions (c) was performed in acetonitrile with stoichiometric
amounts of 2-bromo-tert-butyl acetate and potassium carbonate
as base to afford 14, 16 or 18 in a yield of up to 90%.4 Although
all NODAPA derivatives presented herein were successfully syn-
thesised via statistical alkylation, the latter remains somewhat
unfavourable for the synthesis of mono-functionalised derivatives
14. Furthermore, an unreasonable excess of 12 has to be employed.
Therefore, an alternative route was examined. The key step in-
volved selective detosylation of two ring-nitrogens to facilitate sto-
ichiometrical alkylation. First, 1,4,7-tritosyl-TACN (11) was reacted
with HBr in glacial acetic acid containing an eightfold excess of
phenol (Scheme 2, e). The reaction proceeded straightforward to
afford 88% of 1-tosyl-1,4,7-triazacyclononane (17), as determined
by ESI-MS and proton-NMR. Subsequent exposure of 17 to tert-bu-
tyl bromoacetate in acetonitrile followed by SET-reduction (f) with
sodium naphtalenide in dimethoxyethane or lithium in propyl-
amine/ethylendiamine gave secondary amine 19. The latter was
converted into the protected chelators 14a and 14b by reaction
with bromides 9 and 10, respectively, in 27% overall yield. Catalytic
hydrogenation of 16 was conducted under basic conditions (g) to
give the desired aniline 20 in good yield (90%) as shown in scheme
3.8
Subsequent conversion to an isothiocyanate 21 was carried out
using thiophosgene in 85% yield (Scheme 3h).15c The tert-butyl
esters were deprotected in trifluoroacetic acid and the trifluoro-
acetic acid salts were removed using an ion exchange resin, to
afford 6 and 8 in up to 23% overall yield.15a,c Compound 7 was
obtained in similar yield after deacetylation in 10% KOH in meth-
anol, prior to deprotection in TFA.15b The desalted precursors
were directly used for radiolabelling without further purification.
In order to analyse whether the chain branch, containing the
coupling moiety in one pendant arm, affects the kinetic and
thermodynamic characteristics of [68Ga]NOTA-complex forma-
tion, labelling of NODAPA-NCS (6), NODAPA-OH (7), NODAP-
A(NCS)2 (8), and NODAPA-NO2 (14b) with generator produced
and purified gallium-68 was carried out in aqueuous solution at
pH 2.8.
alkylation, requires a secondary leaving group in
a-position to
the carboxylate function. In addition, the planar phenylen subunit
provides a non-flexible initial spacer, complementary to 2, that can
be combined with a variety of additional spacer functions via the
included conjugation functionality. Both linkers, 2-bromo-(4-acet-
oxy-phenyl)-tert-butylacetate (9) and 2-bromo-2-(p-nitrophenyl)-
tert-butylacetate (10) were easily synthesised from the corre-
sponding acetic acid derivatives via tert-butyl protection under
Steglich conditions (i) in up to 80% yield, followed by Wohl–Ziegler
bromination (ii) in 90% yield (Scheme 2).7–10,14 Cyclisation of 1,4,7-
tritosyl-1,4,7-triazacyclononane (11) was achieved according to
the well-known procedure reported by Richman and Atkins.11 Full
detosylation (a) was performed in concentrated sulphuric acid
(110 °C, 2 h; Scheme 2).12 The polyhydrogensulfate of 1,4,7-triaza-
cyclononane (TACN, 12) was precipitated in ethanol and diethyl-
ether,13 dissolved in a small amount of water and basified (pH
R'
R
CO2H
N
CO2H
R
1
2
3
4
5
CH2 CO2H
H
N
(CH2)2 CO2H
H
CH2-p-Ph NCS
H
N
H
H
p-Ph NCS
(CH2)4 NH2
R'
HO2C
Figure 2. NOTA-based bifunctional chelators.
Quality control was performed using an Agilent Zorbax C 8 col-
umn using 50 mM phosphate buffer and MeOH as eluent at 0.5 ml/
min.
O
N
OH
O
N
OH
Yields were very high (85 5% after 3 min) and comparable to
those achieved for NOTA (Fig. 3).
O
O
N
N
The stability of both novel 68Ga chelates was determined in a
DTPA-challenge study at 25 °C and 37 °C employing 1 mM,
10 mM and 100 mM solutions of DTPA in water, indicating >94%
complex stability, in a similar range as the congener NOTA
(Fig. 4).
OH
OH
OH
N
N
NCS
OH
OH
OH
O
O
O
OH O
6
7
Plasma protein binding and transchelation to serum proteins
in vitro was examined under physiological conditions in rat plas-
ma. Four MegaBequerels [68Ga]NODAPA-OH were incubated in
N
N
N
SCN
NCS
300
lL of rat plasma from male adult Wistar rats, obtained via cen-
OH
trifugation of full blood. Samples of 50
lL were withdrawn after 1,
O
30, 60, 90, and 180 min and analysed by radio-TLC (silica gel 60, 5%
aq. NaCl–EtOH, 3:1).
8
In correlation to the DTPA-challenge, less than 2% of non-
Scheme 1. Novel NOTA-derived bifunctional chelators NODAPA-NCS (6), NODAPA-
OH (7) and NODAPA-(NCS)2 (8).
[
68Ga]NODAPA-OH radioactivity was observed after 3 h.

5366
P. J. Riss et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5364–5367
H
H
Ts
Ts
H
Ts
N
N
N
N
N
N
a
e
R
N
H
N
H
N
Ts
O
HO
12
11
17
d
c
b
i
O
N
O
N
O
O
O
O
Ts
O
O
N
N
O
H
R
N
N
O
N
f
O
O
N
H
N
H
O N
2
R
NO
2
15
13
18
c
ii
R = OAc ; 14a
R = NO ; 14b
c
2
Br
O
O
N
O
N
O
O
N
O
R
b
O
H
O
O
N
N
O
N
O
O
O
N
N
N
O N
2
NO
R
2
O
O
O
R = OAc 9
R = NO 10
O
O
2
16
14
19
Scheme 2. Synthesis route to mono-functionalised and di-functionalised NOTA derivatives. Reagents and conditions: (a) H2SO4, 2 h, 110 °C; (b) tert-butyl 2-bromo-2-(40-
nitrophenyl) acetate, CH2Cl2, K2CO3, 3 d; (c) tert-butyl bromoacetate, MeCN, K2CO3; (d) 2 equiv tert-butyl 2-bromo-2-(40-nitrophenyl) acetate, CH2Cl2, K2CO3, 3 d; (e) PhOH,
HBr/AcOH, 1.5 d, 90 °C; (f) dimethoxyethane, Na, C10H8; i—t-BuOH, CH2Cl2, DCC, DMAP, 3 h; ii—CCl4 (distilled from P4O10), NBS, AIBN.
O
O
N
O
N
O
O
O
N
O
N
O
O
O
N
O
N
O
g
h
N
N
N
O2N
NO2
H2N
NH2
SCN
NCS
O
O
O
O
O
O
16
20
21
Scheme 3. Synthesis of di-functionalised NOTA derivatives. Reagents and conditions: (g) 2 mM KOH in MeOH, 10% Pd/C, H2, rt, 4 h; (h) Ca2CO3, Me2CO, H2O, thiophosgene.
100
99
98
97
96
95
94
93
100
80
60
40
20
0
(a) - 13.5 nM -45 ˚C
(b) - 13.5 nM - 45 ˚C
(c) - 5 nM - 75 ˚C
(d) - 5 nM - 75 ˚C
(a)
15
(b)
(c)
(d)
60
0
2
4
6
8
10
12
30
120
180
reaction time / min
incubation time / min
Figure 3. Time-dependency of 68Ga-labelling of (a) NOTA, (b) NODAPA-OH (7), (c)
NODAPA-NCS (6) and (d) NODAPA(NCS)2 (8) pH 2.8, 5–13.5 nmol ligand, 5 ml H2O,
Figure 4. Stability of [68Ga]NOTA (a), [68Ga]NODAPA-OH (b), [68Ga]NODAPA-NCS
(c), and [68Ga]NODAPA-(NCS)2: (d); 10 mM DTPA in DPBS (pH 7.4) at 37 °C (n = 3).
400 l
L [68Ga]GaCl3 in N2-solution.1
In conclusion, three novel NOTA-based bifunctional chelators
have been obtained via a simple and efficient synthesis route. 6,
7, and 8 provide excellent 68Ga labelling and stability parameters.
While offering –NCS and –OH functionalities, covalent coupling to
various potential targeting vectors is possible. As a proof-of-con-
cept, NODAPA-NCS was conjugated to L-lysine (22) and glucosa-
mine (23) (Scheme 4).15d The latter symbolise the first
bioconjugates with the promising novel NOTA-based bifunctional
chelators introduced in the present work.

P. J. Riss et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5364–5367
5367
7. Ziegler, K.; Schenck, G.; Krockow, E. W.; Siebert, A.; Wenz, A.; Weber, H. Liebigs
Ann. 1942, 551, 1.
CO tBu CO tBu
CO tBu CO tBu
2
2
2
2
8. Helps, I. M.; Parker, D.; Morphy, J. R.; Chapman, J. Tetrahedron 1989, 45,
219.
9. Neises, A.; Steglich, W. Angew. Chem. Int., Ed. Engl. 1978, 17, 52213.
10. Wohl, A. Chem. Ber. 1919, 52, 51.
11. Richman, J. E.; Atkins, T. J. J. Am. Chem. Soc. 1974, 96, 2268.
12. Raßhofer, W.; Vögtle, F. Liebigs Ann. Chem. 1977, 1340.
13. Diril, H.; Chang, H. R.; Nilges, M. J.; Zhang, X.; Potenza, J. A.; Schugar, H. J.; Isied,
S. S.; Hendrickson, D. N. J. Am. Chem. Soc. 1989, 111, 5102.
14. Horner, L.; Winkelmann, E. H. Angew. Chem. 1959, 7, 349.
15. (a) (14b) dH (300 MHz; CDCl3) 1.40 (9H, s, t-Bu), 1.42 (18H, s, t-Bu), 2.78–3.25
(16H, m, N–CH2, N–CH), 4.10 (1H, s, N–CH–Ar), 7.15 (2H, d, J = 8 Hz, Ar), 7.40 (2
H, d, J = 8 Hz, Ar); m/z (ESI) 606.32 ([M+H]+ C31H48N4O6S requires 606.33,
100%), 606.34 (96.1), 607.38 (58.0), 608.40 (12.9), 549.29.
N
N
N
N
j
N
N
NCS
NH
CO tBu
2
CO tBu
2
S
R
14
OH
HO
O
O
or
NHBOC
R=
HO
HO
OH
N
H
NH
22
23
(b) (14a) dH (300 MHz; CDCl3) 1.41 (9H, s, t-Bu), 1.42 (9H, s, t-Bu), 1.43 (9H, s, t-
Bu) 2.70–3.04 (12H, m, N–CH2), 3.25 (2H, br s, CH2–C(O)) 3.30 (2H, s, CH2–
C(O)) 4.41 (1H, dd, J = 6 Hz, N–CH–Ar), 6.74 (2H, d, J = 8 Hz, ArH), 7.20 (2H, d,
J = 8 Hz, ArH); m/z (ESI) 564.37 ([M+H]+ C30H49N3O7 requires 563.36, 100%),
565.37 (35%), 556.38 (8%).
Scheme 4. Conjugation of NODAPA-NCS to model substrates. Reagents: (j) Ca2CO3,
MeOH/H2O/NEt3, 7:1:1 (65–73%).
(c) (21) dH (300 MHz; CDCl3) 1.41 (s, 9H, t-Bu), 1.415 (s, 9H, t-Bu), 1.42 (s, 9H, t-
Bu), 3.10–2.60 (m, 12H), 3.2 (m, 4H, N–CH2–COOt-Bu), 4.41 (s, 1H, N–CH’–Ar),
4.41 (s, 1H, N–CH’–Ar), 4.42 (s, 1H, NCH–Ar); 7.57 (m, 4H, Ar); 8.16 (m, 4H, Ar)
m/z (ESI) 738.30 (100%), 739.33 (947%), 740.35 (58.8%), 741.37 (10.8%),
([M+H]+ C38H51N5O6S2 requires 737.33, 100.0%), 738.33 (41,1%), 739.32
(9.0%), 739.33 (8.2%), 740.33 (37%).
References and notes
1. Zhernosekov, K. P.; Filosofov, D. V.; Baum, R. P.; Aschoff, P.; Bihl, H.; Razbash, A.;
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2. Craig, A. S.; Parker, D.; Adams, H.; Bailey, N. A. J. Chem. Soc. Chem. Commun.
1989, 1793.
3. Eisenwiener, K.-P.; Prata, M. I. M.; Buschmann, I.; Zhang, H. W.; Santos, A. C.;
Wenger, S.; Reubi, J. C.; Mäcke, H. R. Bioconjugate Chem. 2002, 13, 530.
4. André, J. P.; Maecke, H. R.; Zehnder, M.; Macko, L.; Akyel, K. G. Chem. Commun.
1998, 1301.
(d) (22) dH (300 MHz; CDCl3): 1.25 (m, 2H, Lys-g-CH2), 1.34 (s, 9H, Ot-Bu), 1.40
(s, 18H, Ot-Bu) 1.42 (s, 9H, Ot-Bu), 1.7–1.5 (m, 4H, b-CH2, d-CH2), 3.0–2.65 (m,
12H, N–CH2–CH2), 3.25 (4H, s, N–CH2–COOt-Bu); 3.50 (t, J = 5 Hz, 2H,
4.05 (s, 1H, N–CH–Ar); 4.34 (t, 1H, -CH); 7.40–7.25 (m, 4H, Ar); m/z (ESI) 851.6
[M+H]+ C42H70N6O10 requires 850.49. (23) m/z (ESI): 784.43 [M+H]+
C37H61N5O11S requires 783.41.
e-CH2);
a
S
5. Brechbiel, M. W.; McMurry, T. J.; Gansow, O. A. Tetrahedron 1993, 34, 3691.
6. Cox, J.; Craig, A.; Helps, I. M.; Jankowski, K.; Parker, D.; Eaton, M.; Millican, A.;
Millar, K.; Beeley, N.; Boyce, B. J. Chem. Soc., Perkin Trans. 1 1990, 2567.