Efficient system for the preparation of [13N]labeled nitrosamines

Article abstract of DOI:10.1016/j.bmcl.2009.02.066

In the present Letter, a fast and reproducible method for the synthesis of N-[¹³N]nitrosamines is reported. The labeling strategy is based on trapping [¹³N]NO₂^- in an anion exchange resin. The reaction with secondary amines in the presence of Ph₃P and Br₂ led to the formation of the desired nitrosamines in short reaction times (2 min) with excellent radiochemical conversion (>45%). Final radiotracers were obtained after purification in good radiochemical yields (>30%, decay corrected). Radiochemical purity was above 99% in all cases.

Full text of DOI:10.1016/j.bmcl.2009.02.066

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1913–1915
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient system for the preparation of [13N]labeled nitrosamines
Vanessa Gómez-Vallejo a, Koichi Kato b, Masayuki Hanyu b, Katsuyuki Minegishi b,
José I. Borrell c, Jordi Llop a,
*
a Radiochemistry Department, CIC biomaGUNE, Parque Tecnológico de San Sebastián, p° Miramón 182, Edificio Empresarial C, 20009 San Sebastián, Guipúzcoa, Spain
b Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
c Grup d’Enginyeria Molecular, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
In the present Letter, a fast and reproducible method for the synthesis of N-[13N]nitrosamines is reported.
Article history:
The labeling strategy is based on trapping [13N]NO2 in an anion exchange resin. The reaction with sec-
ꢀ
Received 23 January 2009
Revised 13 February 2009
Accepted 15 February 2009
Available online 21 February 2009
ondary amines in the presence of Ph3P and Br2 led to the formation of the desired nitrosamines in short
reaction times (2 min) with excellent radiochemical conversion (>45%). Final radiotracers were obtained
after purification in good radiochemical yields (>30%, decay corrected). Radiochemical purity was above
99% in all cases.
Keywords:
Nitrosamines
Ó 2009 Elsevier Ltd. All rights reserved.
Nitrogen-13
Positron emission tomography
Positron emission tomography (PET) is a non-invasive in vivo
imaging technique which produces a three-dimensional image of
functional processes in a living organism. Commonly used radio-
nuclides for PET imaging include carbon-11 (11C), fluorine-18
with tumor induction in different sites like oral cavity, esophagus,
stomach, urinary bladder and brain.3 In spite of the large number
of studies in the preclinical area, a better understanding of the
in vivo mechanism by which nitrosamines exert their carcinogenic
effect is highly desirable. Especially, the carcinogenic potential of
nitrosamines has driven the studies about the influence of tobacco
and food additives. In this sense, 13N labeling (either combined
with 11C labeling or not) would represent a powerful tool to per-
form in vivo pharmacokinetic studies in animals, including bio-dis-
tribution and metabolism.
(
18F), oxygen-15 (15O), nitrogen-13 (13N) or bromine-76 (76Br),
which can be easily produced in good yields in cyclotrons for fur-
ther incorporation into organic molecules to be used as radiotra-
cers.1 Due to radioactive decay, the preparation of the
radiotracers requires the development of fast and efficient syn-
thetic strategies to be carried out in automatic-remote controlled
synthesis modules.
N-Nitrosation of amines is a well explored reaction in organic
synthesis. In the last decades, different nitrosating agents including
nitrous acid (HNO2), nitrosyl chloride (NOCl), dinitrogen trioxide
(N2O3), dinitrogen tetroxide (N2O4), nitrosonium tetrafluoroborate
and alkyl nitrites have been applied widely for the synthesis of
nitrosamines. More recently, heterogeneous reagent systems have
been demonstrated to be an excellent alternative for the synthesis
of nitrosamines following simple experimental procedures under
mild conditions.4 However, the [13N]nitrosation of amines is
strongly restricted due to the limited availability of cyclotron-gen-
Among all PET radionuclides, 11C (half-life of 20.4 min and max-
imum positron energy of 960.5 keV) and 18F (half-life of 109.8 min
and maximum positron energy of 633.5 keV) have been by far the
most widely used, due to their versatile chemistry, relatively long
half-life and high production yields in commercially available
cyclotrons. In spite of the historical importance of 11C and 18F,
the use of other radioisotopes (e.g., 13N, with a half-life of
9.97 min and maximum positron energy of 1.19 MeV) might be
very advantageous for the development of alternative synthetic
strategies to label molecules in different positions, thus giving fur-
ther understanding of specific biological and/or physiological pro-
cesses. A clear example is found in nitrosamines. Since Magee and
Barnes showed the carcinogenic potential of N-nitrosodimethyl-
amine (NDMA) in rats in 1956,2 a high number of nitrosamines
have shown potent carcinogenic effects in experimental studies
erated radioactive precursors ([13N]NH4 and [13N]NO3ꢀ) and the
þ
low amount (sub-micro molar scale) in which they can be
produced.
In a recent work, we presented a fast and simple strategy for the
generation of the radioactive precursor [13N]NO2 and further
ꢀ
reaction with glutathione to yield S-[13N]nitrosoglutathione.5 In
continuation of our work, we have used the same radioactive pre-
cursor to efficiently synthesize different N-[13N]nitrosamines fol-
* Corresponding author. Tel.: +34 943 00 53 33.
E-mail address: jllop@cicbiomagune.es (J. Llop).
ꢀ
lowing the Ph3P/Br2/NO2
strategy recently described by
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.066

1914
V. Gómez-Vallejo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1913–1915
Nowrouzi and co-workers.6 Although very high yields were de-
scribed in this work, in the particular case of using non carrier
added-cyclotron produced [13N]NO2ꢀ, the anion is obtained in very
low concentration aqueous solution, and thus application of the
methodology is not trivial.
Table 1
Entry
R1R2NH
R1R2NNO
RCCa,c,d
RCYb,c,d
1
2
3
4
1
2
3
4
5
6
7
8
53.4 1.3 (51.9–54.4)
50.2 9.8 (38.8–56.2)
45.6 7.4 (38.1–52.8)e
45.9 5.4 (42.0–49.7)
37.8 3.1 (34.7–40.9)
40.7 8.0 (31.6–46.1)
34.0 7.3 (27.3–41.9)e
36.4 5.0 (32.9–40.0)
Our first attempts to synthesize N-[13N]nitrosamines followed
the general procedure in which nitrous acid, (generated from ni-
trite solution and mineral acid in water) is reacted with secondary
amines. In this case, using piperidine (1) as secondary amine, under
a
RCC: Radiochemical conversion (%), calculated as the ratio between the amount
of radioactivity present as N-[13N]nitrosamine and the amount of radioactivity
trapped in the SPE cartridge.
non radioactive conditions and for a wide range of temperatures
b
RCY: Radiochemical yield (%), calculated as the ratio between the amount of
ꢀ
(0 °C 6 T 6 100 °C) and acid concentrations (1 6 pH 6 4.5, NO2
/
radioactivity present as N-[13N]nitrosamine (after purification) and the amount of
amine ratio = 1.2/1), the reaction of formation of N-nitrosopiperi-
dine (5) was very slow and chemical conversion (NO2 into 5)
radioactivity generated in the cyclotron.
ꢀ
c
Decay corrected values.
AVG STDV (range).
Amount of Ph3P/Br2/amine was 50/50/40 lmol.
d
was very poor (under 5% in all cases) after 10 min of reaction.
The reaction was also carried out at lower NO2ꢀ/amine ratios (to
simulate radioactive conditions) and chemical conversion was
strongly decreased. These results were confirmed, also using 1 as
secondary amine, under radioactive conditions. No radioactive
peak corresponding to [13N]5 was detected in any case.
e
In Table 1, the radiochemical conversion of [13N]NO2 into
N-[13N]nitrosamine is shown for amines 1–4. In all cases, radio-
chemical conversion (decay corrected) was above 45%. Impor-
tantly, irrespectively of the structure of the secondary amine,
only the presence of one major chemical impurity (retention
time = 10.5 min) was detected in all cases in the final solution be-
fore purification. As previously reported in the literature,11 Ph3P
quantitatively reacts with Br2 to immediately yield bromotriphe-
nylphosphonium bromide. The slow reaction of this reagent with
the secondary amine yields the precursor for the nitrosation reac-
tion (triphenyl(amin-1-yl)phosphonium bromide, see Scheme 2 for
structure). The excess of bromotriphenylphosphonium bromide
constituted the undesired impurity observed in the HPLC at the
end of the synthesis, and could be removed from the bulk solution
by eluting the reaction mixture through a C-18 SPE cartridge, rins-
ing with purified water and eluting the desired radiotracer with
3 mL of ethanol/water solution (25/75 in the case of [13N]7, 20/
80 in the case of [13N]5 and 15/85 in the other cases). This purifi-
ꢀ
With the aim of improving our results, the resin-supported
methodology for the synthesis of N-[13N]nitrosamines previously
reported by Vavrek and Mulholland7 was used;8 under these con-
ditions, the formation of [13N]5 could not be detected after
10 min of reaction. Due to this fact, the combined approach
ꢀ
(resin-supported NO2 + Ph3P/Br2/amine strategy) was assayed
(Scheme 1).
In a typical experiment, 13N (25 mCi, 925 GBq) was produced in
a cyclotron via the 16O(p, 13N nuclear reaction. The target (con-
a)
taining 3.2 mL of purified water) was irradiated with 18 MeV pro-
tons at a beam current of 10
l
A for 2 min. The resulting solution,
containing [13N]NO2 (16.5 3.8%), [13N]NO3 (74.1 3.5%) and
ꢀ
ꢀ
þ
[
13N]NH4 (9.4 0.4%) was passed through a glass column filled
with cadmium/sand 3:1 mixture (w/w, column length = 16.5 cm)
ꢀ
to obtain
[
a
virtually
[
13N]NO3
free solution (95.5 1.1%
13N]NO2ꢀ, 0.7 0.45% [13N]NO3 and 3.8 1.4% [13N]NH4þ).
9
ꢀ
cation step was also successful in removing unreacted [13N]NO2
ꢀ
The solution was directed to an anion exchange solid phase
13N]NO3
.
Final reconstitution with 5 mL of injectable
m filter left the
ꢀ
and
[
extraction (SPE) cartridge (Sep-PakÒ Accell Plus QMA, Waters) to
physiologic solution and filtering through a 0.22
radiotracer ready for putative in vivo studies in animals.
l
trap [13N]NO2 quantitatively. The cartridge was dried with nitro-
ꢀ
gen, washed with tetrahydrofuran (2 mL) and dried again. A freshly
Average radiochemical yields (calculated as the ratio between
the amount of radiotracer and the amount of radioactivity gener-
ated in the cyclotron, decay corrected) for the preparation of
N-[13N]nitrosamines were in the range 34.0–40.7% (Table 1). Total
synthesis time (including purification) was less than 10 min and
radiochemical purity was above 99% in all cases.
As can be seen in Table 1, the lowest radiochemical conversion
and yield were obtained for [13N]7, despite a higher concentration
of Ph3P/Br2/amine was used. We believe that this is due to steric
hindrance exerted by diisopropylamine after formation of tri-
phenyl(diisopropylamin-1-yl)phosphonium bromide.
prepared
solution
(0.8 mL)
containing
Ph3P/Br2/amine
(25:25:20
lmol) in dry dichloromethane was circulated through
the cartridge at a flow rate of 0.4 mL/min. The eluted solution
was recovered in a vial, dried under continuous nitrogen flow
and the residue was reconstituted with water (2 mL).
The resulting solution was analyzed by HPLC using a YMC
J’sphere ODS-H80 column (4
l
m particle size, 15 ꢁ 0.46 mm) as
stationary phase and water/acetonitrile/methanol (28:18.5:3.5)
as mobile phase.10 Simultaneous UV (k = 254 nm) and isotopic
detection were used. The presence of N-[13N]nitrosamine was con-
firmed by co-elution with reference compound solution (retention
times = 3.43, 2.56, 5.62, and 3.23 min for 5, 6, 7, and 8,
respectively).
In summary, we present here a simple, fast and easy to auto-
mate procedure for the preparation of [13N]labeled nitrosamines.
ꢀ
By using a combined approach (resin-supported 13NO2 + Ph3P/
ꢀ
Br2/amine), 13NO2 (aqueous solution) can be reacted with second-
ary amines to synthesize nitrosamines with excellent conversion
and good radiochemical yields. Despite [13N]NO2 is obtained as
ꢀ
aqueous solution from the cyclotron, the amine is solved in dichlo-
romethane; thus, the method is applicable to the preparation of
Scheme 1. Synthesis of N-[13N]nitrosamines by following the combined approach
(resin-supported NO2 + Ph3P/Br2/amine).
Scheme 2. Reaction of formation of the precursor triphenyl(amin-1-yl)phospho-
nium bromide.
ꢀ

V. Gómez-Vallejo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1913–1915
1915
Bamoniri, A. Synlett 2002, 1621; (d) Zolfigol, M. A.; Habibi, D.; Mirjalili, B. F.;
Bamoniri, A. Tetrahedron Lett. 2003, 44, 3345; (e) Hou, J. Y.; Wang, Y. L.; Wang, J.
Y. J. Chem. Res. 2003, 2003(10), 626.
other nitrosamines with more complex structure, even if they are
not water-soluble.
5. Llop, J.; Gómez-Vallejo, V.; Bosque, M.; Quincoces, G.; Peñuelas, I. Appl. Radiat.
Isot. 2009, 671, 95.
6. Iranpoor, N.; Firouzabadi, H.; Nowrouzi, N. Tetrahedron Lett. 2008, 49(27),
4242.
Acknowledgments
The authors would like to thank Dr. Fujiko Konno, the staff of
cyclotron section and personnel of the department of molecular
probes of National Institute of Radiological Sciences for helping
throughout experimental work. We also thank the Departamento
de Industria, Comercio y Turismo of the Vasc Government for finan-
cial support.
7. Vavrek, M. T.; Mulholland, G. K. J. Labelled Compd. Radiopharm. 1995, 37, 118.
8. In the previous work by Vavrek and Mullholand many details about the
procedure are omitted and reaction conversions are not specified for the
particular cases. In the present work, for the synthesis of N-
[
13N]nitrosopiperidine the solution coming from the cyclotron, after
reduction step, was directed to an anion exchange solid phase extraction
cartridge (Accell plus QMA, Waters). A freshly prepared solution (2.0 mL)
containing piperidine (20–200 lmol) and hydrochloric acid (1.0 6 final pH 6 4)
in water was circulated through the cartridge at a flow rate of 0.4 mL/min. The
eluted solution was analyzed by HPLC under the same conditions as described
in the main text.
References and notes
9. Stationary phase: HP Asahipak ODP-50 column (5
lm particle size, 125 ꢁ 4 mm,
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water/acetonitrile (86/14, 1L), adjusted to pH = 8.6 with 1 M NaOH solution;
flow rate 1 mL/min; simultaneous UV (k = 254 nm) and isotopic detection.
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