A convenient synthesis of 13N-labelled azo compounds: A new route for the preparation of amyloid imaging PET probes

Article abstract of DOI:10.1016/j.ejmech.2010.08.053

In the present paper, a fast and automated method for the synthesis of ¹³N-labelled azo compounds is reported for the first time. The labelling strategy is based on trapping [¹³N]NO₂ ^- in an anion exchange resin. The reaction with primary aromatic amines in acidic media led to the formation of the corresponding diazonium salts, which were further reacted with aromatic amines and alcohols to yield the corresponding ¹³N-labelled azo derivatives with good radiochemical conversion (40.0-58.3%). Good radiochemical yields (20.4-47.2%, decay corrected) and radiochemical purities (>99.9%) were obtained after purification by HPLC. Such methodology can be easily applied to the preparation of a wide range of ¹³N-labelled azo derivatives by adequate selection of the non-radioactive precursors.

Full text of DOI:10.1016/j.ejmech.2010.08.053

European Journal of Medicinal Chemistry 45 (2010) 5318e5323
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
13
A convenient synthesis of N-labelled azo compounds: A new route for the
preparation of amyloid imaging PET probes
,
Vanessa Gómez-Vallejo a, José I. Borrell b, Jordi Llop a
*
a Radiochemistry Department, CIC biomaGUNE, Parque Tecnológico de San Sebastián, pꢀ Miramón 182, 20009 San Sebastián, Spain
b 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 paper, a fast and automated method for the synthesis of 13N-labelled azo compounds is
reported for the first time. The labelling strategy is based on trapping [13N]NO2ꢁ in an anion exchange
resin. The reaction with primary aromatic amines in acidic media led to the formation of the corre-
sponding diazonium salts, which were further reacted with aromatic amines and alcohols to yield the
corresponding 13N-labelled azo derivatives with good radiochemical conversion (40.0e58.3%). Good
radiochemical yields (20.4e47.2%, decay corrected) and radiochemical purities (>99.9%) were obtained
after purification by HPLC. Such methodology can be easily applied to the preparation of a wide range of
13N-labelled azo derivatives by adequate selection of the non-radioactive precursors.
Article history:
Received 9 June 2010
Received in revised form
19 August 2010
Accepted 25 August 2010
Available online 15 September 2010
Keywords:
Positron
Emission
Tomography
Amyloid
Azo
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
During the last years, the emergence of in vivo molecular
imaging techniques like positron emission tomography (PET) has
Alzheimer’s disease (AD) is a neurodegenerative disease of the
brain associated with irreversible cognitive decline, memory
impairment, and behavioural changes [1]. Neuropathologic features
like the presence of senile plaques (SPs) and neurofibrillary tangles
(NFTs), which contain amyloid peptides and highly phosphorylated
tau proteins, are revealed in post-mortem brains of AD patients [2].
Although the precise mechanism of neuronal death in AD
patients is still unknown, it is currently accepted that SPs and NFTs
play a central role in its development. SPs are predominantly
led many groups to develop imaging probes for the in vivo detection
of amyloid deposits, because comprehensive longitudinal studies
using A
b imaging agents likely would help in the elucidation of the
Ab
deposition process in normal subjects prior to the onset of the
clinical symptoms.
Some of these imaging probes are [11C]-2-40(methylaminophenyl)
-6-hydroxybenzothiazole ([11C]PIB) [6],
40hydroxystilbene ([11C]SB-13) [7],
[
11C]-4-N-methylamino-
[
18F]-4-(N-methylamino)-
40-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-stilbene ([18F]BAY94-9172)
composed of insoluble amyloid-beta (A
b) peptides, mostly 40 or 42
[8], [
11C]-2-(2-(2-dimethylaminothiazol-5-yl)ethenyl)-6-(2-(fluoro)
amino acids in length, with A
component [3].
b42 being the most prevalent
ethoxy)benzoxazole ([11C]BF-227) [9], and [18F]-2-(1-(2-(N-(2-fluo-
roethyl)-N-methylamino)naphthalene-6-yl)ethylidene)malononitri-
le ([18F]-FDDNP) [10]. All these probes are labelled either with
carbon-11 or fluorine-18, which are by far the most widely used
positron emitters for the preparation of PET tracers.
Currently, the only definitive confirmation of AD is by post-
mortem histopathologic examination of A deposits in the brain.
The A neurotoxicity is dependent on aggregation of A into b-
b
b
b
sheet fibrils [4], and this structure has been used for years to
develop special markers to identify the presence of such amyloid
deposits. Among all markers, Congo Red (Fig. 1) is by far the most
widely used and has been applied for decades to the identification
of amyloid deposits in post-mortem tissue [5].
Recently, we have developed strategies for the generation (with
good radiochemical yields) of the radioactive precursor [13N]NO2ꢁ
starting from cyclotronproduced [13N]NO3ꢁ [11], and its incorporation
into bioactive molecules like N-[13N]nitrosamines [11] and S-[13N]
nitrosothiols [12,13]. Despite the short half-life of nitrogen-13, these
13N-labelled compounds have been prepared with good radioche-
mical yields in short times. Moreover, in one of our previous works
[13], an automatic remote controlled synthesis box for the prepara-
tion of 13N-labelled compoundshas been designed and implemented.
* Corresponding author. Tel.: þ34 943005333; fax: þ34 943005301.
E-mail address: jllop@cicbiomagune.es (J. Llop).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.053

V. Gómez-Vallejo et al. / European Journal of Medicinal Chemistry 45 (2010) 5318e5323
5319
Fig. 1. Chemical structure of Congo Red.
In the current paper, we present a simple and fast procedure for
2. Results and discussion
the manufacture of 13N-labelled azo compounds by following
a three-steps synthesis approach (Fig. 2). In the first step, cyclotron
generated [13N]NO3ꢁ is reduced to [13N]NOꢁ2 and trapped in an anion
exchange resin. The second step involves the reaction of the resin
trapped [13N]NOꢁ2 with primary aromatic amines in acidic condi-
tions to yield the corresponding diazonium salt, which is then
pushed to a reactor pre-charged with a solution of an aromatic
amine or phenol to yield the 13N-labelled azo compound (third
step). Purification by means of HPLC leads to chemically and
radiochemically pure radiotracers with good radiochemical
conversion and acceptable radiochemical yield values in short
production times.
The here reported new synthetic strategy could be used for the
preparation of 13N-labelled analogs of Congo Red with putative
application as PET imaging probes for the in vivo detection of
amyloid deposits. Although nitrogen-13 is not a commonly used
radioisotope for the preparation of radiotracers (mainly due to its
short half-life which restricts its use) the reported strategy not only
satisfies a radiochemical challenge with academic interest, but also
allows the incorporation of different functional groups to the
radiotracer by simply selecting the adequate non-radioactive
precursors. Thus, a wide range of azo derivatives can be quickly and
efficiently prepared (for further in vivo and/or ex vivo evaluation) by
following one single strategy.
It is well known that azo compounds can be synthesized by first
reacting an aromatic amine hydrochloride with nitrous acid (HNO2)
to produce an aryldiazonium ion. This ion can couple with
a nucleophile in basic solution to produce the azo derivative [14]. In
normal conditions, both reactions are carried out at low tempera-
ture (0 ꢀC) using relatively long reaction times (5e15 min) and the
final product is isolated by precipitation. Due to the short half-life of
nitrogen-13 (9.97 min), and the low concentration at which 13N can
be obtained in cyclotrons, this general procedure had to be modi-
fied to prepare 13N-labelled azo compounds.
Experimental conditions for the preparation of 13N-labelled azo
compounds were first optimized for the synthesis of compound 1
(see Fig. 2). In these optimization runs, no purification was carried
out, and the reaction crude (after performing steps 1e3, Fig. 2) was
directly collected into a vial, the radioactivity was measured in
a dose calibrator and the solution was analysed by HPLC. Our
approach consisted of generating the aryldiazonium salt by react-
ing, into the anion exchange SPE cartridge, a solution of aniline in
hydrochloric acid (1 M aqueous solution, added with a remote
controlled syringe driver, 400
NOꢁ2 . The diazonium salt was not isolated, but pushed into a reac-
tion vial pre-charged with an adequate volume (>400 L) of basic
(NaOH, 1 M aqueous) solution of phenol to perform step 3 (Fig. 2)
m
L) with the previously trapped [13N]
m
Fig. 2. General scheme for the synthesis of 13N-labelled azo compounds. The diazonium salt is generated by reaction of the adequate primary aromatic amine with [13N]NOꢁ2 under
acidic conditions. Further reaction with aromatic amines and/or alcohols yields the 13N-labelled azo compounds.

5320
V. Gómez-Vallejo et al. / European Journal of Medicinal Chemistry 45 (2010) 5318e5323
Table 1
Optimized experimental conditions for the production of 1e5.
CA (M)a
SolventA
Rt2 (s)c
CB (M)d
SolventB
Rt3 (s)f
Reaction
b
e
Compound
temperature (ꢀC)g,h
1
2
3
4
5
0.25
0.25
0.25
0.25
0.25
1.0 M aqueous HCl
1.0 M aqueous HCl
1.0 M aqueous HCl
0.1 M aqueous HCl
0.1 M aqueous HCl
60
60
60
30
30
0.45
0.60
5.00
0.45
1.25
1.0 M aqueous NaOH
30
120
120
60
25
90
90
90
90
2.5 M aqueous AMF/Formic acid (pH ¼ 4.0)
1.0 M aqueous AcOH/NaAcO (pH ¼ 4.0)
1.0 M aqueous NaOH
2.5 M aqueous AMF/formic acid (pH ¼ 4.0)
120
a
Concentration of precursor A.
Solvent used for precursor A solution.
Reaction time for step 2.
Concentration of precursor B.
Solvent used for precursor B solution.
Reaction time for step 3.
b
c
d
e
f
g
h
Reaction temperature for step 3.
Step 2 always performed at room temperature.
under moderate basic conditions. Different reaction times for steps
2 and 3, reaction temperatures (step 3) and NaOH solution volumes
were assayed. Surprisingly, short reaction times (60 and 30 s for
steps 2 and 3, respectively, see Table 1) gave good radiochemical
conversion values at room temperature (58.3 ꢂ 5.5%, Table 2) using
respectively, gave optimal results using a precursor concentration
of 0.60 M (Table 1). Higher reaction temperatures were not assayed
to avoid overpressure in the reaction vial. Radiochemical conver-
sion values of 40.9 ꢂ 11.0% were obtained under optimal conditions
(Table 2). After purification, 3.3 mCi were obtained with a total
synthesis time of 13 min (decay corrected radiochemical yield:
31.4 ꢂ7.5%, radiochemical purity: 100%; see Table 2).
550 mL of NaOH solution. Higher NaOH solution volumes increased
the formation of radioactive by-products (as determined by HPLC)
while NaOH volumes below 500 L drastically decreased the
m
The same experimental conditions (entry 2, Table 1) were first
applied for the preparation of 3; lower radiochemical conversion
values (<20%) were obtained, and thus the concentration of
precursor B was rised up to 5.0 M. Although good radiochemical
conversion values were obtained (comparable to those obtained for
compound 2) a chemical impurity (not identified) with almost
identical retention time to compound 3 was found. When acetic
acid/sodium acetate (pH ¼ 4) was used as precursor solvent, the
chemical impurity disappeared without affecting the radiochem-
ical conversion; thus, these experimental conditions were used to
run the syntheses with purification (Table 1). Under these condi-
tions, radiochemical conversion and radiochemical yield (decay
corrected) were, respectively, 40.0 ꢂ 8.4% and 23.9 ꢂ 6.3% (Table 2).
Final activity was 2.5 mCi with a total synthesis time of 15 min.
Compounds 4 and 5 required the formation, in step 2, of the
diazonium salt of 4-aminobenzenesulfonic acid. This precursor was
not soluble in 1 M HCl and thus lower acid concentrations were used
(0.1 M HCl solution, Table 1). Increasing amounts of precursor B were
assayed until no significant improvement in radiochemical conver-
sion was observed. Reaction times of 30 and 60 s for steps 2 and 3,
respectively, gave optimal results for compound 4 at 90 ꢀC, while for
compound 5 a longer reaction time had to be applied to perform step
3 (120 s, Table 1). Radiochemical conversions of 40.3 ꢂ1.5% and
54.0 ꢂ 9.0% (see Table 2) were obtained for compounds 4 and 5,
respectively, giving radiochemical yields (after purification, decay
radiochemical conversion. In these cases, the presence of a major
wide radioactive peak at short retention time (RT ¼ 1.7 min) sug-
gested the presence of unreacted 13N-labelled diazonium salt.
Under optimal conditions (Table 1), 4.9 mCi were obtained after
purification (decay corrected radiochemical yield: 47.2 ꢂ 2.0%) with
a total synthesis time of 13 min (Table 2). Radiochemical purity was
100% as determined by radio-HPLC.
In view of these results, the same experimental conditions were
applied to the preparation of compound 2 (see Fig. 2). However, in
this case, a major radioactive peak at RT ¼ 12 min suggested the
formation of a triazene specie (Fig. 3) probably due to strong basic
conditions in step 3. To prevent this undesired reaction, sodium
acetate (pH ¼ 9) was used for step 3, but low radiochemical
conversion values were obtained (<10%) irrespectively of the
precursor B concentration (0.45e5.0 M). The presence of a wide
major radioactive peak at short RT (1.7 min) suggested again the
presence of unreacted 13N-labelled diazonium salt, and the pres-
ence of the undesired radioactive peak at RT ¼ 12 min was still
detected. In a third set of experiments, 2.5 M aqueous AMF/formic
acid buffer (pH ¼ 4.0) was used. Low radiochemical conversion was
observed, but the peak at RT ¼ 12 min completely disappeared.
Thus, heating was applied to the reaction vial (step 3), longer times
were used for step 3, and higher concentrations of precursor B were
also assayed. Reaction time and temperature of 120 s and 90 ꢀC,
Table 2
Radiochemical conversion, radiochemical yield (corrected and uncorrected), radiochemical purity, specific radioactivity, final activity and synthesis time for the preparation of
13N-labelled azo compounds 1e5.
c,f
c,d,g
Compound
RCC (%) a,b,c
RCY (%)b,c,d
RCP (%)e
SR (GBq/
m
mol)
Final activity (mCi)
RCY (%)
Total synthesis timeh (min)
1
2
3
4
5
58.3 ꢂ 5.5
40.9 ꢂ 11.0
40.0 ꢂ 8.4
40.3 ꢂ 1.5
54.0 ꢂ 9.0
47.2 ꢂ 2.0
31.4 ꢂ 7.5
23.9 ꢂ 6.3
20.4 ꢂ 2.5
24.4 ꢂ 5.3
100
100
100
100
100
4.6 ꢂ 1.1
8.3 ꢂ 2.1
6.3 ꢂ 1.8
8.8 ꢂ 2.4
5.6 ꢂ 0.9
4.9
3.3
2.5
2.1
2.6
19.1 ꢂ 0.8
12.7 ꢂ 3.0
8.4 ꢂ 2.2
7.7 ꢂ 0.9
8.0 ꢂ 1.7
13
13
15
14
16
a
Calculated as the ratio between the amount of radiotracer before purification and the amount of [13N]NO2ꢁ obtained after the reduction step.
b
c
Decay corrected values.
AVG ꢂ STDV.
d
e
f
Calculated as the ratio between the amount of radiotracer after purification and the amount of 13N generated in the cyclotron.
Calculated from chromatographic profiles after purification.
End of synthesis.
End of bombardment.
g
h
Including purification; RCC: Radiochemical conversion; RCY: Radiochemical yield; RCP: Radiochemical Purity; SR: Specific radioactivity.

V. Gómez-Vallejo et al. / European Journal of Medicinal Chemistry 45 (2010) 5318e5323
5321
Fig. 3. Reaction of benzenediazonium salt with aniline. The formation of the triazene structure is favoured under basic conditions.
corrected) of 20.4 ꢂ 2.5% and 24.4 ꢂ 5.3% (final activity values of 2.1
and 2.6 mCi in total synthesis times of 14 and 16 min, respectively).
In all cases (compounds 1e5), the stability of the resulting pure
radiotracers was checked at 25 ꢀC. Radiochemical purity values
>99.9% were obtained 60 min after the end of the synthesis.
Specific radioactivity values were checked by HPLC. Values
Ethanol, acetonitrile, and methanol were HPLC grade and purchased
from Scharlab. Solid phase anion exchange cartridges (Sep-PakÒ
Accell Plus QMA, Waters) were conditioned before use with saturated
aqueous KHCO3 solution (10 mL). Water (ultrapure, Type I water, ISO
3696) was obtained from a Millipore purification system.
between 4.6 and 8.8 GBq/mmol (corrected to the end of the
4.2. General method for the production of 13N-labelled azo
compounds
synthesis) were obtained (Table 2). These values are in concordance
with previous results obtained by our group in the preparation of
13N-labelled S-nitrosothiols [13].
All 13N-labelled azo compounds were synthesized by following
the same synthetic approach and using an automatic remote
controlled synthesis box (Fig. 4). The valve-opening sequence is
shown in Table 3.
Although reformulation was not performed on the final radio-
tracer solutions, no undesired peaks were detected in the UV
chromatograms in any case. Such reformulation step should be
implemented in order to eliminate the solvents introduced in the
HPLC purification process before in vivo and/or ex vivo application
of the tracers. On the other hand, although relatively low activity
and specific radioactivity values were obtained for the here
reported 13N-labelled radiotracers, preliminary assays performed
with a new target (results not shown) confirm that both the
amount of activity and the specific radioactivity at the end of the
bombardment can be at least twelve- and two-fold increased,
respectively. These results suggest that both the dose and the
specific radioactivity of the radiotracers should be easily increased
up to useful values for in vivo and/or ex vivo applications.
In a general experiment, nitrogen-13 (30 mCi, 1110 GBq) was
produced in an IBA Cyclone 18/9 cyclotron via the 16O(p, 13N
a)
nuclear reaction. The target (containing 1.75 mL of water) was
irradiated with 18 MeV protons at a beam current of 20 A for
1.5 min (integrated current of 0.5 Ah).
m
m
The irradiated solution (containing a mixture of [13N]NO3ꢁ, [13N]
NOꢁ2 and minute amounts of [13N]NHþ4 ) was transferred to the
synthesis box, collected in a vial (step 1, Table 3), passed through
a glass column filled with cadmium to quantitatively reduce [13N]
NOꢁ3 into [13N]NOꢁ2 (step 2, Table 3) and further eluted through the
anion exchange cartridge to selectively retain [13N]NO2ꢁ (step 3,
Table 3). The cadmium column was rinsed with 2 mL of water (steps
4 and 5). After drying the cartridge with inert gas flow (step 6),
a solution containing the adequate primary aromatic amine (aniline
for 1e3 and 4-aminobenzenesulfonic acid for 4 and 5) was loaded
into the cartridge in the adequate media (step 7) and reaction for
the formation of the diazonium salt was allowed to occur (step 8).
The reaction mixture was pushed into a vial (step 9) pre-charged
with a solution of the corresponding aromatic amine (aniline for 2,
N,N-dimethylaniline for 3 and 5) or phenol (1 and 4), and reaction
was allowed to occur (step 10). The reaction crude was pushed to
the HPLC loop (step 11) and injected into the HPLC column (step
12). The desired fraction was collected (step 13) when the radio-
active peak corresponding to the desired radiotracer was detected.
For purification, an isocratic pump (Knauer) was used. A Medi-
3. Conclusions
From this investigation it can be concluded that the radiosyn-
thesis of 13N-labelled azo compounds can be achieved by (i) reaction
of the labelled precursor [13N]NOꢁ2 with aromatic primary amines to
generate the corresponding diazonium salts and (ii) further reaction
of the diazonium salts with aromatic amines or phenols. Reasonable
radiochemical yields (20.4e47.2%, decay corrected) and excellent
radiochemical purity values (>99.9%) are obtained after HPLC
purification step. The versatility of the here reported method should
permit the preparation of a wide range of 13N-labelled azo deriva-
tives, with potential application as b-Amyloid markers.
terranean Sea18 column (5 mm, 250 mm, 10 mm) was used as
4. Experimental section
stationary phase. Mobile phases (6 mL/min) were: Compound 1,
AMF 0.1 M aqueous solution (pH ¼ 3.9)/Ethanol/Acetonitrile (30/15/
55); compound 2, AMF 0.1 M aqueous solution (pH ¼ 3.9)/Ethanol/
Acetonitrile (35/15/50); compound 3, AMF 0.1 M aqueous solution
(pH ¼ 3.9)/Ethanol/Acetonitrile (25/15/60); compound 4, AMF
0.1 M aqueous solution (pH ¼ 3.9)/Acetonitrile (75/25); compound
5, AMF 0.1 M aqueous solution (pH ¼ 3.9)/Acetonitrile (65/35).
4.1. General
Aniline (ACS reagent), 4-aminobenzenesulfonic acid (ACS
reagent), N,N-dimethyl-N-phenylamine (puriss. p. a., ꢃ99.5%), Phenol
(ReagentPlusÒ, ꢃ99.5%), 4-phenylazophenol (98%), p-phenylazoani-
line, 4-(dimethylamino)azobenzene (butter yellow, analytical
standard,ꢃ98.0%), 4-[4-(dimethylamino)phenylazo]benzenesulfonic
acid sodium salt (methyl orange, Ph. Eur. Grade), 4-(4-hydroxy-
phenylazo)-benzenesulfonic acid sodium salt, ammonium formate
(reagent grade, 97%), formic acid (ACS reagent, >96%), acetic acid
(purum, >99.0%) and sodium acetate (ACS reagent, ꢃ99.0%) were
purchased from Sigma-Aldrich and used without further purification.
4.3. Identification of the radiotracers
Identification of the radiotracers after purification was carried
outbyHPLC and co-elutionwith reference standard. AnAgilent 1200
series HPLC equipped with
a quaternary pump, a variable

5322
V. Gómez-Vallejo et al. / European Journal of Medicinal Chemistry 45 (2010) 5318e5323
Fig. 4. Schematic drawing of the automated synthesis module implemented for the synthesis of 13N-labelled azo compounds. V1eV13 are 2 and 3-way valves. All 2-way valves are
normally closed. Normally Open paths in 3-way valves are shown in black. Radiation detectors were positioned in the SPE cartridge (D1), injection loop (D2) and detection loop (D3).
wavelength detector (
Raytest) was used. A Zorbax Eclipse XDB C18 column (4.6 ꢄ 150 mm,
m) was used as stationary phase. Mobile phases (1 mL/min)
l
¼ 254 nm) and a radiometric detector (Gabi,
aqueous solution (pH ¼ 3.9)/Methanol/Acetonitrile (40/15/45),
RT ¼ 6.0 min; compound 3, AMF 0.1 M aqueous solution (pH ¼ 3.9)/
Methanol/Acetonitrile (20/15/65), RT ¼ 5.7 min; compound 4,
AMF 0.1 M aqueous solution (pH ¼ 3.9)/Acetonitrile (83/17),
RT ¼ 6.0 min.; compound 5, AMF 0.1 M aqueous solution (pH ¼ 3.9)/
Acetonitrile (70/30), RT ¼ 5.0 min.
5
m
were: Compound 1, AMF 0.1 M aqueous solution (pH ¼ 3.9)/Meth-
anol/Acetonitrile (40/15/45), RT ¼ 6.2 min; compound 2, AMF 0.1 M
Identification of the 13N-labelled structures was also carried out
by UPLC-MS (ESI-TOF) analysis. UPLC/MS analyses were performed,
after complete decay, using an Acquity UPLC separation module
coupled to a LCT TOF Premier XE mass spectrometer (Waters,
Table 3
Valve-opening sequence and synthetic process step by step. Coding for 2-way
valves: O, open; C, closed; coding for 3-way valves: L, left; R, right; U, up; D, down.
STEPa V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13
Manchester, UK). An Acquity BEH C18 column (1.7
m
m, 50 ꢄ 2.1 mm)
1
2
3
4
5
6
C
C
C
C
O
O
C
C
C
C
C
C
C
C
C
O
O
C
C
O
C
C
C
C
O
C
O
C
O
O
C
O
O
C
C
C
C
O
C
O
O
C
C
C
C
C
C
C
C
C
C
C
C
O
C
C
O
C
C
C
C
C
C
C
C
C
L
R
L
L
L
L
R
R
L
L
L
L
L
L
L
L
L
L
L
L
L
R
R
L
R
R
L
L
U
U
U
U
U
U
U
U
U
U
U
U
D
C
C
C
C
C
C
C
C
O
C
O
C
C
C
C
C
C
C
C
C
C
O
C
C
C
C
C
C
C
C
C
C
C
C
C
C
O
C
C
was used as stationary phase. The elution buffers were A (water and
0.1% formic acid) and B (methanol and 0.1% formic acid). The column
was eluted with a linear gradient consisted of 95% A to 1% over
2.3 min,1% over 2.3e4.0 min, returned to 95 for 0.1 min and kept for
O
O
O
O
O
C
C
C
C
O
C
R
R
R
R
R
R
L
L
L
L
L
a further 50 s. Total runwas 5 min, injectionvolumewas 5
mL and the
7b
8c
9d
10e
11f
12g
13h
flow rate 600
m
L/min. Detection was performed in: ESꢁ V mode
(compounds 1 and 4) and ESþ W mode (compounds 2, 3 and 5) in the
range of 50e1000, with a scan time of 1 s and a delay time of 0.1 s.
Compounds 2 and 3 were detected as protonated molecules (m/
z ¼ 198.09 and 226.14 and retention times ¼ 2.14 and 2.58 min,
respectively); compound 5 was detected as diprotonated MeNaþ
(m/z ¼ 306.9, retention time ¼ 1.11 min); Compounds 1 and 4 were
detected as deprotonated molecules (m/z ¼ 197.31 and 277.09,
retention times ¼ 2.28 and 0.89 min, respectively).
O
a
The 5 mL vial must be loaded with purified water (2 mL), the syringe driver
must be loaded with the precursor A solution and the 5 mL vial (with heating
system) must be filled with the precursor B solution before starting the process.
b
Syringe driver is started.
Syringe driver is stopped.
Peristaltic pump is started.
Peristaltic pump is stopped.
Rheodyne valve is in load position.
Rheodyne valve is switched to inject position, HPLC pump is started.
When signal is detected in radioactive detector.
c
d
Acknowledgments
e
f
g
The authors would like to thank Javier Calvo for fruitful
discussion and support in UPLC-MS (ESI-TOF) analyses and Iosu
h

V. Gómez-Vallejo et al. / European Journal of Medicinal Chemistry 45 (2010) 5318e5323
5323
[7] M. Ono, A. Wilson, J. Nobrega, D. Westaway, P. Verhoeff, Z.P. Zhuang,
M.P. Kung, H.F. Kung, 11C-labeled stilbene derivatives as A
-aggregate-specific
Oliden for actively participating in the implementation and fine
tuning of the automatic synthesis box. The authors would also like
to thank the Departamento de Industria, Comercio y Turismo of the
Vasc Government for financial support.
b
PET imaging agents for Alzheimer’s disease, Nucl. Med. Biol. 30 (2003)
565e571.
[8] W. Zhang, S. Oya, M.P. Kung, C. Hou, D.L. Maier, H.F. Kung, F-18 Poly-
ethyleneglycol stilbenes as PET imaging agents targeting Abeta aggregates in
the brain, Nucl. Med. Biol. 32 (2005) 799e809.
[9] Y. Kudo, N. Okamura, S. Furumoto, M. Tashiro, K. Furukawa, R. Maruyama,
H. Itoh, R. Iwata, K. Yanai, H. Arai, -(2-[2-Dimethylaminothiazol-5-yl]ethenyl)-
6- (2-[fluoro]ethoxy)benzoxazole: a novel PET agent for in vivo detection of
dense amyloid plaques in Alzheimer’s disease patients, J. Nucl. Med. 48 (2007)
553e561.
[10] E.D. Agdeppa, V. Kepe, J. Liu, S. Flores-Torres, N. Satyamurthy, A. Petric,
G.M. Cole, G.W. Small, S.C. Huang, J.R. Barrio, Binding characteristics of radi-
ofluorinated 6-dialkylamino-2-naphthylethylidene derivatives as positron
emission tomography imaging probes for beta-amyloid plaques in
Alzheimer’s disease, J. Neurosci. 21 (2001) RC189.
References
[1] M. Ono, Development of positron-emission tomography/single-photon emis-
sion computed tomography imaging probes for in vivo detection of b-amyloid
plaques in Alzheimer’s brains, Chem. Pharm. Bull. 57 (2009) 1029e1039.
[2] W.E. Klunk, Biological markers of Alzheimer’s disease, Neurobiol. Aging 19
(1999) 145e147.
[3] T. Iwatsubo, A. Odaka, N. Suzuki, H. Mizusawa, N. Nukina, Y. Ihara, Visuali-
zation of A beta 42(43) and A beta 40 in senile plaques with end-specific A
beta monoclonals: evidence that an initially deposited species is A beta 42
(43), Neuron 13 (1994) 45e53.
[11] V. Gómez-Vallejo, K. Kato, M. Hanyu, K. Minegishi, J.I. Borrell, J. Llop, Efficient
system for the preparation of [13N]labeled nitrosamines, Bioorg. Med. Chem.
Lett. 19 (2009) 1913e1915.
[4] B. Seilheimer, B. Bohrmann, L. Bondolfi, F. Muller, D. Stuber, H. Dobeli, The
toxicity of the Alzheimer’s b-amyloid peptide correlates with a distinct fiber
[12] J. Llop, V. Gómez-Vallejo, M. Bosque, G. Quincoces, I. Peñuelas, Synthesis of
S-[13N]nitrosoglutathione (13N-GSNO) as a new potential PET imaging agent,
Appl. Radiat. Isot. 67 (2009) 95e99.
[13] V. Gómez-Vallejo, K. Kato, I. Oliden, J. Calvo, Z. Baz, J.I. Borrell, J. Llop, Fully
automated synthesis of 13N-labeled nitrosothiols, Tetrahedron Lett. 51 (2010)
2990e2993.
[14] A.O. Alnajjar, M.E. El-Zaria, Synthesis and characterization of novel azo-
morphine derivatives for possible use in abused drugs analysis, Eur. J. Med.
Chem. 43 (2008) 357e363.
morphology, J. Struct. Biol. 119 (1997) 59e71.
[5] J. Sajid, A. Elhaddaoui, S. Turrell, Investigation of the binding of Congo red to
amyloid in Alzheimer’s diseased tissue, J. Mol. Struct. 408e409 (1997)
181e184.
[6] W.E. Klunk, H. Engler, A. Nordberg, Y. Wang, G. Blomqvist, D.P. Holt,
M. Bergstrom, I. Savitcheva, G.F. Huang, S. Estrada, B. Ausen, M.L. Debnath,
J. Barletta, J.C. Proce, J. Sandell, B.J. Lopresti, A. Wall, P. Koivisto, G. Antoni,
G.A. Matthis, B. Langstrom, Imaging brain amyloid in Alzheimer’s disease with
Pittsburgh compound-B, Ann. Neurol. 55 (2004) 306e319.