Radiosynthesis of novel 18F-labelled derivatives of indiplon as potential GABAA receptor imaging tracers for PET

Article abstract of DOI:10.1002/jlcr.1473

The involvement of gamma amino butyric acid (GABA) receptors in a variety of neurological and psychiatric diseases has promoted the development and use of radiolabelled benzodiazepines (BZ) for brain imaging by PET. However, these radioligands are unable

Full text of DOI:10.1002/jlcr.1473

Research Article
Received 5 July 2007,
Revised 26 September 2007,
Accepted 9 October 2007
Published online 5 February 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1473
18
Radiosynthesis of novel F-labelled
derivatives of indiplon as potential GABAA
receptor imaging tracers for PET
Ã
Steffen Fischer,a Achim Hiller,a Matthias Scheunemann,a
Winnie Deuther-Conrad,a Alexander Hoepping,b Michael Diekers,b
c
Florian Wegner, Peter Brust, and Jorg Steinbach
a
a,d
¨
The involvement of gamma amino butyric acid (GABA) receptors in a variety of neurological and psychiatric diseases has
promoted the development and use of radiolabelled benzodiazepines (BZ) for brain imaging by PET. However, these
radioligands are unable to distinguish between the various subtypes of GABAA receptors. Novel non-BZ such as the
pyrazolo-pyrimidine indiplon proved to be selective for the a1-subunit of the GABAA receptor. Here, we describe the
syntheses of four novel 18F-labelled indiplon derivatives. Radiosyntheses were performed via n.c.a. 18F-nucleophilic
substitution starting from the tosyl, bromo, and 4-nitrobenzoyl precursors to obtain fluorine substituted N-alkylamide side
chain derivatives of indiplon, followed by multistep purification using semi-preparative high-performance liquid
chromatography and solid phase extraction. Tosyl and bromo precursors were converted into 18F-labelled indiplon
derivatives with good and reproducible radiochemical yield (RCY) (35–70%, decay corrected), high radiochemical purity
(
X
98.5%), and high specific activity ( > 150 GBq/lmol). By contrast, a low RCY (5–10%) and specific activity (10–15 GBq/
lmol) were achieved for the 4-nitrobenzoyl precursor.
Keywords: GABAA receptor; 18F-labelled indiplon derivatives; PET
(N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-a] pyrimidin-
7-yl]phenyl]acetamide),13 which is currently approved for the
treatment of insomnia. Indiplon also acts as an allosteric agonist
at the BZ binding site, but demonstrates pharmacological
selectivity via high affinity to a1-containing GABAA receptors.14 It
has been shown by radioligand binding studies on rat cerebellar
and cerebral cortex membranes that [3H]indiplon has a higher
affinity compared with other related non-BZ such as zolpidem or
zaleplon.15 A first attempt of developing [11C]zolpidem as PET
radiotracer failed,16 most probably due to low affinity in vivo and
rapid, unfavourable metabolism.
To test the suitability of indiplon as a lead structure for
a1-subtype selective PET radiotracers, we have started with the
development of fluorinated derivatives.17,18
In this article, we describe the radiosynthesis of four novel
18F-labelled indiplon derivatives.
Introduction
Gamma amino butyric acid (GABA) is the major inhibitory
neurotransmitter in the human brain and exerts its actions via
ionotropic GABAA and GABAC receptors or metabotropic
G-protein-coupled GABAB receptors.1 Imbalances in the GABA-
ergic system are involved in numerous neurodegenerative and
psychiatric diseases such as epilepsy, anxiety, or insomnia.
Therefore, drugs that target GABAA receptors, are used as
therapeutics.1,2 Benzodiazepines (BZ), which act as positive
allosteric modulators at GABAA receptors, were introduced in
the late 1950s and are still used as the treatment of choice for
insomnia.1,3 The specific BZ binding site consists of a variable
a-subunit and the g2-subunit of the pentameric GABAA receptor.
Therefore, ‘classical’ BZ (e.g. flunitrazepam, diazepam) do not
discriminate between the various a-subunits.1,3 Nevertheless,
they were used as lead structures for the development of
radioligands for brain imaging of neuropsychiatric diseases with
PET or SPECT.4,5 Hence, the clinically used PET and SPECT tracers
aInstitute of Interdisciplinary Isotope Research, Leipzig, Germany
bABX Advanced Biochemical Compounds GmbH, Radeberg, Germany
cDepartment of Neurology, University of Leipzig, Leipzig, Germany
dInstitute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, Germany
[11C]flumazenil,6 [18F]fluoroethylflumazenil,7,8
[
123I]iomazenil,9
and other related compounds such as [11C]suriclone, [11C]ioma-
zenil, [18F]fluoroflumazenil, [11C]flunitrazepam, [11C]fludiazepam,
[11C]Ro 154513, and [18F]oxoquazepam (as previously re-
viewed10–12) also lack selectivity, which is a drawback for the
investigation of subtype-related brain functions and diseases.
Today, novel non-BZ with higher subtype selectivity are
available,3 among them the pyrazolo-pyrimidine indiplon
*Correspondence to: Steffen Fischer, Institut fu¨r Interdisziplina¨re Isotopen-
forschung, PermoserstraXe 15, D-04318, Leipzig, Germany.
E-mail: fischer@iif-leipzig.de
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S. Fischer et al.
The 4-nitro- and 4-fluorophenyl compounds 5a and 5b (with
R1 5 CH3, cf. Table 1) were obtained in a similar way. The
detailed synthesis strategy is described elsewhere.18
Results and discussion
Based on the chemical structure of indiplon (Figure 1), novel
derivatives with fluorine substituted N-alkylamide side chain
1b–5b were synthesized (Table 1). Prior to considering specific
18F-labelling procedures for the synthesis of radiotracers for PET
applications, biological binding studies with compounds 1b–5b
were made to select appropriate candidates.18 Their affinities
(IC50 between 2.8 and 18 nM) are comparable to indiplon
(IC50 5 3.3 nM).
The preparation of the precursors 1a–5a focused on
compounds suitable for one-step labelling to achieve high
radiochemical yields (RCY) and to afford a simple separation of
[18F]1b–5b.
Synthesis of 4a, 4b
A
second approach focused on a fluorinated derivative
by modifying the acetyl group of indiplon. Such a strategy
would allow a one-step radiolabelling process starting from
the corresponding bromo precursor. The synthesis of the
fluorinated reference compound 4b and the bromo derivative
4a is shown in Figure 3. Indiplon (VI) was prepared following
a synthetic route described in the literature.19 Both synthetic
building blocks I and V, available via two convergent syntheses
starting from 3-aminoacetophenone and 2-acetyl-thiophene,
respectively, were assembled to obtain compound VI (Figure 3).
The acetyl group of indiplon (VI) was removed under
acidic conditions to give deacetyl-indiplon (VII). The
latter compound served as the starting material for the synthesis
of the fluoroacetamide 4b and the bromoacetamide 4a
by applying a standard acylation under Schotten– Baumann
conditions.
Non-radioactive syntheses
Synthesis of 2a–3a, 5a and 1b–3b, 5b
In a first attempt we synthesized demethyl-indiplon (Figure 1)
and used this molecule for further derivatizations. The latter was
hampered by the low reactivity and the poor yields for the
alkylation of the amide group. Thus, the introduction of the
fluoroalkyl and hydroxyalkyl groups had to be performed at an
earlier stage of the synthesis. Hence, several enaminones II were
synthesized. Condensation of II with aminopyrazole I yielded
the fluoroalkyl and hydroxyalkyl derivatives III of indiplon
under acidic conditions. Finally, the hydroxyalkyl derivatives IIIa
were converted to the corresponding tosylate precursors IV
(Figure 2).18
Radiosyntheses
For n.c.a. nucleophilic substitution, 18F was transformed into
the K[18F]F-K222-carbonate or the K[18F]F-18-crown-6-carbonate
complex using
a A 2.3:1 molar
standard procedure.20,21
ratio of K222 (or 18-crown-6) to potassium carbonate proved
to be optimal. The nucleophilic insertion of 18F is shown in
Figure 4.
Figure 1. Indiplon N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]phenyl]acetamide, (left) and demethyl-indiplon, N-[3-[3-(2-thienylcarbonyl)-
pyrazolo[1,5-a]pyrimidin-7-yl]phenyl]acetamide (right).
Table 1. Fluorine derivatives of indiplon and precursors for 18F-labelling
Fluorinated
compound
Precursor
R1
R2
R1, R2
–CH3
–CH3
–CH3
–CH3
–CH3
–CH2Br
Indiplon
1a
–(CH2)2OTos
–(CH2)3OTos
–(CH2)4OTos
–CH3
R1: –(CH2)2F
R1: –(CH2)3F
R1: –(CH2)4F
R2: –CH2F
1b
2b
3b
4b
2a
3a
4a
–CH3
5a
R2:
5b
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S. Fischer et al.
Figure 2. Strategy for the synthesis of the compounds 1b–3b and the corresponding tosylate precursors 1a–3a for 18F-labelling.
Figure 3. General scheme of synthesis of the compound 4b (X 5 F) and the precursor 4a (X 5 Br) for 18F-labelling.
Syntheses of [18F]2b–3b via tosyl precursors 2a–3a
formamide (DMF) (approx. 1401C, 10 min) with reproducible
labelling efficiencies of 55–65 and 56–75%, respectively.
Because an O-tosyl function is a good leaving group for the Compared with the propyl derivatives 2a and 2b, precursor
nucleophilic aliphatic exchange with 18F,22 the precursors 3a and radiotracer [18F]3b have a higher thermal stability;
1a–3a could readily be converted into the labelled products therefore, allowing the application of higher temperatures at
[
18F]1b–3b. However, the precursors themselves have only a reduced reaction time and precursor amounts. Labelling yields
limited stability. Depending on the length of the alkyl chain, of [18F]2b and [18F]3b were lower in dimethyl sulphoxide
precursor molecules gradually decomposed over months, even (DMSO) than in DMF (Figure 5a). Almost no reaction was
if stored in a deep freezer. This is probably due to the observed in acetonitrile (MeCN) and acetone. Thin-layer
elimination of toluenesulphonic acid as indicated by analytical chromatography (TLC) analyses of the crude labelling product
data (1H-NMR, ESI-MS). Compound 1a was quite unstable and displayed a series of by-products and a highly intensive blue
decomposed rapidly within a few days. Therefore, a reasonable fluorescence for both reference substances 2b, 3b and the
and reproducible radiosynthesis for [18F]1b could not be precursors 2a, 3a.
established. For [18F]2b (Figure 5a, 5b) and [18F]3b, the
The separation of the remaining [18F]fluoride from the
optimum preparation conditions were achieved in dimethyl reaction mixtures by using an anion exchanger Acell Plus
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S. Fischer et al.
Figure 4. Radiosyntheses of 18F-labelled indiplon derivatives 1b–5b.
Figure 6. Semi-preparative HPLC separation of crude 18F-labelling product [18F]2b.
The final product elutes at ꢁ63 min; inset: [18F]2b is well separated from impurities
(25-fold magnification of the y-axis). To obtain [18F]2b free from any impurities, a
high retention time has to be accepted (see text).
Figure 5a. Labelling efficiency of [18F]2b over time in dependence on solvent and
precursor concentration 2a (approx. 1401C). & 1 mg/0.5 ml DMSO, ꢀ 1 mg/0.5 ml
DMF, m 2.4 mg/0.5 ml DMF, n 5 5–7 for each point.
[18F]3b on solid phase extraction (SPE), for example, an RP-18
cartridge, was incomplete due to their hydrophilic properties
and was associated with the enrichment of by-products. Hence,
the mixture was directly purified via semi-preparative radio-
high-performance liquid chromatography (radio-HPLC) using an
RP column with MeCN/water mixtures including ammonium
acetate as eluent. By means of isocratic radio-HPLC, a very good
separation of [18F]2b and 3b was obtained (Figure 6). However,
further improvement of this method is needed to reduce the
retention time. Figure 7 shows that high radiochemical and
chemical purity of [18F]2b (after solvent change (EtOH) with SPE)
was achieved as analysed by HPLC.
It has been demonstrated that the syntheses can be
transferred into a commercially available automated synthesis
module (see Experimental).
Synthesis of [18F]4b via bromo precursor 4a
Because both compounds, [18F]4b and its bromo precursor 4a
(Figure 4), are alkali sensitive, the phase transfer catalyst (PTC)
Kryptofix K222 was replaced by 18-crown-6.23 A change of the
carbonate was not necessary. The nucleophilic 18F substitution
succeeded in MeCN at 801C with good labelling yields (50–60%).
Higher temperatures and application of other solvents led to
Figure 5b. Labelling efficiency of [18F]2b over time in dependence on reaction
temperature (1 mg precursor 2a in DMF). & 801C, ꢀ 1101C, m 1401C, n 5 3–5.
QMA, or PS-HCO3, or Sep-Pak Alumina N Plus cartridges was
accompanied by a considerable loss (about 12–28%) of the final
products. On the other hand, the adsorption of [18F]2b and
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S. Fischer et al.
Figure 7. Radiochemical and chemical purity of [18F]2b, suitable for animal
experiments: (a) radio-chromatogram and (b) UV (228 nm) (isocratic HPLC, 35%
MeCN120 mM ammonium acetate).
both decreased labelling efficiency and decomposition of
[18F]4b due to the limited chemical stability of the precursor
molecules. Purification of the crude reaction mixture on Sep-Pak
Alumina N and C18 Plus cartridges permitted the removal of
radioactive impurities without significant loss of labelled radio-
tracer. Nonetheless, non-radioactive by-products were simulta-
neously enriched. Therefore, processing of [18F]4b was
performed with an isocratic semi-preparative HPLC (Figure 8)
as described above.
Synthesis of [18F]5b via nitro precursor 5a
Figure 8. Semi-preparative HPLC separation of [18F]4b. Radio-chromatogram (a) of
crude 18F-labelling mixture without SPE purification, final product elutes at
ꢁ65 min, and after SPE purification with Sep-Pak Alumina N/C18 Plus cartridges:
radio-chromatogram (b), corresponding UV chromatogram (c).
For the preparation of the 18F-labelled benzoyl derivative 5b,
the easily accessible nitro precursor 5a (Figure 4) was used.
The nucleophilic aromatic substitution of the nitro group by 18
F
is difficult to achieve by conventional thermal heating.24,25
Here, the microwave-assisted synthesis in DMF under
defined reaction conditions (140–150 W, 1551C, 12 min) afforded
[18F]5b in low yields of up to 14%. Numerous radioactive
products were found under such harsh conditions making a
time-consuming purification of the reaction mixture necessary
(Figure 9). A decrease in the labelling yield with increasing
reaction time was observed. HPLC analyses show an increasing
content of more lipophilic components with low retention time.
One of them is consistent with [18F]fluoride (proved by
[18F]fluoride spike) and therefore, a strong evidence for the
defluorination process of [18F]5b. The application of solvents
with better dielectric properties (e.g. DMSO, ethylene glycol) and
an increase in reaction temperature did not result in higher
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S. Fischer et al.
(SYKAM Chromatographie, Germany), UV detector (WellChrom
K-2001; KNAUER, Germany), NaI(Tl)-counter, and data acquisition
by Nina Chromatografix system (Nuclear Interface, Germany)
using Kromasil 100-5 C18 (20 Â 4 mm i.d.) and Kromasil
100-5 C18 (250 Â 8 mm) columns with a particle size of
5 mm; solvent: various mixtures of MeCN/water with 20
(40) mmol ammonium acetate and a flow gradient from 0.75
to 1.75 mL/min.
Radioluminescence thin-layer chromatograms were recorded
using a BAS-1800 II system Bioimaging Analyzer (Fuji Film, Japan)
and images were evaluated with AIDA 2.31 software (raytest).
Analytical radio-HPLC was performed on a Merck-Hitachi
LaChrome device (Merck, Darmstadt) with a binary pump, UV
detector, and a GINA Radio-Chromato-Graphik-System (raytest);
column: Multisorb RP 18-5 (250 Â 4 mm), particle size 5 mm;
solvent: various mixtures of MeCN and water with 20 mM
ammonium acetate and a flow rate of 1.0 mL/min. Chromato-
graphic data (UV absorption at wavelengths 228, 254, 220 nm,
radioactivity in cps) were recorded using a D-7000 HPLC System
Manager version 3.1 (Merck, Hitachi).
Figure 9. Semi-preparative HPLC separation of crude [18F]5b. The chromatogram
displays numerous major radioactive by-products.
45.5–47.5 min.
[
18F]5b appears at
yields. One of the major decomposition products, deacetyl-
indiplon, resulting from cleavage of the nitro-benzoyl moiety,
could be identified by electrospray ionization (ESI-MS) and was
1
confirmed by H-NMR.
Stability
The stability of [18F]2b–5b was tested in 0.9% NaCl, phosphate-
buffered saline, and inactivated plasma at 371C for up to 5 h.
According to TLC and analytical HPLC, [18F]2b–5b proved to be
very stable (impurities o1.2%). No decomposition or defluor-
ination was observed.
Chemical syntheses
N-(2-Fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-phenyl}-acetamide (1b)
The synthesis of 1b was carried out as described in detail for 2b.
Yield: 44% of a yellow solid. m.p. 5 179–1801C. TLC: Rf (EtOAc/
CH2Cl2/ MeOH 10:10:1) 5 0.39. H-NMR (CDCl3, 500 MHz) d 2.02
1
Experimental
(s, 3H, CH3), 4.05 (td, 1H, J3F 5 26 Hz, J 5 4.8 Hz, N-CH2), 4.69 (td,
2H, J2F 5 47 Hz, J 5 4.7 Hz, CH2F), 7.16 (d, 1H, J 5 4.4 Hz, HAr),
7.21 (m, 1H, HAr), 7.52 (m, 1H, HAr), 7.66–7.73 (m, 2H, HAr), 8.03
(m, 2H, HAr), 8.10 (m, 1H, HAr), 8.72 (s, 1H, HAr), 8.84 (d, 1H,
J 5 4.4 Hz, HAr). 19F-NMR (CDCl3, 470 MHz) d –222.24 (1F). Anal.
(C21H17FN4O2S): Calculated C, 61.75; H, 4.20; N, 13.72; S, 7.85.
Found C, 61.49; H, 4.06; N, 13.50; S, 8.12.
General
Solvents were purchased from Merck and Fisher Scientific.
Chemicals were obtained from Merck, Fisher Scientific, Johnson
Matthey, and Sigma-Aldrich. All chemical reagents were of
highest commercially available quality and applied without
further purification.
TLC was performed on silica gel precoated plates (Polygrams SIL
G/UV254), and spots were visualized under UV light (254, 366 nm).
1H- and 19F-NMR spectra were recorded with a Varian Gemini
300BB, Bruker DRX-400 or Bruker AV500 Ultra Shielded spectro-
N-{3-[3-(Thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]-
phenyl}-N-[4-(toluene-4-sulphonyloxy)-propyl]-acetamide (2a)
meter. Chemical shifts are reported as d values. Coupling N-(3-Hydroxy-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo
constants are denoted in Hertz.
[1,5-a]-pyrimidin-7-yl]-phenyl}-acetamide18 (1 g, 2.4 mmol) and
Elemental analyses were performed with a vario EL analyser pyridine (0.57 mL, 7.1 mmol) were dissolved in 100 mL abs.
(Elementar Analysensysteme, Germany).
CH2Cl2 under argon atmosphere, and the mixture was
Low-resolution mass spectra were recorded on a Mariner cooled in an ice bath. Toluene sulphonic acid anhydride (1.0 g,
Biospectrometry Workstation (Applied Biosystems) using ESI. 3.1 mmol) was added, and stirring was continued for 30 min.
Aqueous [18F]fluoride was generated on PETtraces The ice bath was removed and stirring was continued
16.5 MeV cyclotron (GE Healthcare-Siemens, Germany) and used for 45 min. The mixture was washed with 100 mL 1 N hydro-
directly or after recovery on an anionic exchange resin.26
chloric acid solution, half-saturated NaHCO3 solution, and brine.
18F-labelling was performed in 5 mL silicon-coated glass vials The organic phase was dried over Na2SO4 and the solvent
sealed by a rubber septum under controlled Ar atmosphere.
was evaporated (bath temperature o301C). The product was
Microwave-assisted radiosyntheses were done in a closed purified by flash chromatography (silica gel, EtOAc).
standard 10 mL vial using research microwave reactor After evaporation of the solvent, the product was washed with
equipment Discovers (CEM, Germany).
diethyl ether and pentane, and dried in high vacuum. Yield:
a
a
First experiments towards an automated manufacturing were 0.88 g (65%) of a yellow, temperature-sensitive solid (handle
performed using a slightly modified TraceLab FxNs synthesis o251C, storage at À201C). m.p. 5 123–1271C (decomp.). TLC: Rf
module (GE Healthcare-Nuclear Interface, Germany).
For SPE, Sep-Paks cartridges Plus (Waters, USA), and CH2, CH3), 2.43 (s, 3H, CH3), 3.83 (m, 2H, CH2), 4.11 (t, 2H,
(EtOAc) 5 0.40. 1H-NMR (CDCl3, 500 MHz) d 1.92–2.05 (m, 5H,
alternatively Chromafixs cartridges (MACHEREY-NAGEL, Ger- J 5 6.3 Hz, CH2), 7.22 (m, 2H, HAr), 7.32 (d, 1H, J 5 8.1 Hz, HAr),
many), were used.
The crude labelling product was separated on
7.42 (m, 1H, HAr), 7.66–7.76 (m, 4H, HAr), 7.96–8.06 (m, 2H, HAr),
8.12 (m, 1H, HAr), 8.73 (s, 1H, HAr), 8.86 (d, 1H, J 5 4.4 Hz, HAr).
a
semi-preparative radio-HPLC consisting of pump S1021 Anal. (C29H26N4O5S2): Calculated C, 60.61; H, 4.56; N, 9.75; S,
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S. Fischer et al.
11.16. Found C, 60.28; H, 4.57; N, 9.76; S, 11.28. LRMS (ESI pos.) was further purified by flash chromatography (EtOAc) to
m/z 575.16 [M1H]1.
afford 4a (112 mg, 82%) as pale yellow powder.
m.p. 5 160–1651C. TLC: Rf (EtOAc) 5 0.39. 1H-NMR (CDCl3,
(N-(3-Fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]- 400 MHz) d 3.39 (s, 3H, NCH3), 3.80 (s, 2H, –N(CO)CH2Br), 7.18
pyrimidin-7-yl]-phenyl}-acetamide) (2b)
(d, J 5 3.9 Hz, 1HAr), 7.20 (d, J 5 3.9 Hz, 1HAr), 7.56 (d, J 5 7.8 Hz,
1HAr), 7.68–7.73 (m, 2HAr), 8.00–8.10 (m, 2HAr), 8.09 (dd, J 5 3.9,
1.6 Hz, 1HAr), 8.71 (s, 1HAr), 8.83 (d, J 5 4.7 Hz, 1HAr). Anal.
(C20H15BrN4O2S): Calculated C, 52.76; H, 3.32; N, 12.30; S, 7.04.
Found C, 52.86; H, 3.20; N, 12.35; S, 7.29. LRMS (ESI pos.) m/z
455.00 [M1H]1.
N-[3-(3-Dimethylamino-acryloyl)-phenyl]-N-(3-fluoro-propyl)-
acetamide (800 mg, 2.74 mmol) and (5-amino-1H-pyrazol-4-yl)-
thiophen-2-yl-methanone (530 mg, 2.74 mmol) were suspended
in 20 mL acetic acid, and the suspension was heated to 651C for
1.5 h. After cooling to room temperature, the mixture was added
to ice–water and the pH value was adjusted to 8 with NaHCO3.
The mixture was extracted three times with CH2Cl2. The
combined organic phases were dried with Na2SO4, and
2-Fluoro-N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-phenyl}-acetamide (4b, representative procedure)
the solvent was evaporated. Purification of the product Indiplon (VI) and deacetyl-indiplon (VII) were synthesized
was achieved by flash chromatography (silica gel, EtOAc) to utilizing the methods reported.18,19
yield 535 mg (46%) of
a
yellow solid. m.p. 5 194–1951C.
Fluoroacetylchloride (10 mg, 7.2 mL, 0.103 mmol) in CH2Cl2
TLC: Rf (EtOAc) 5 0.45. 1H-NMR (CDCl3, 500 MHz) d 1.95–2.09 (1.5 mL) was added in the course of 20 min to a stirred mixture
(m, 5H, CH2, CH3), 3.92 (t, 2H, J 5 7.3 Hz, N-CH2), 4.53 (dt, of VII (30 mg, 0.09 mmol) in CH2Cl2 (3.5 mL) and an aqueous
2H, J2F 5 47 Hz, J 5 5.8 Hz, CH2F), 7.17 (d, 1H, J 5 4.4 Hz, HAr), solution of NaHCO3 (0.35 M, 3 mL) at 0–21C. The mixture was
7.21 (m, 1H, HAr), 7.47 (m, 1H, HAr), 7.67–7.73 (m, 2H, HAr), 8.00 warmed to ambient temperature, and after being stirred for 3 h
(m, 2H, HAr), 8.10 (m, 1H, HAr), 8.72 (s, 1H, HAr), 8.84 (d, 1H, at 201C, the organic layer was separated and the aqueous layer
J 5 4.4 Hz, HAr). 19F-NMR (CDCl3, 470 MHz) d À219,99 (1F). Anal. was extracted with CH2Cl2 (2 Â 3 mL). The combined organic
(C22H19FN4O2S): Calculated C, 62.54; H, 4.53; N, 13.26; S, 7.59. phases were dried (Na2SO4), filtered, and evaporated to leave a
Found C, 62.42; H, 4.57; N, 13.15; S, 8.09. LRMS (ESI pos.) m/z yellowish film (39 mg). This was triturated with MeOH (1 mL) to
423.12 [M1H]1.
yield 4b (26 mg, 73%) as pale yellow powder, m.p. 5 171–1731C.
TLC: Rf (EtOAc) 5 0.27. 1H-NMR (CDCl3, 300 MHz) d 3.39 (s, 3H,
N-{3-[3-(Thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]- NCH3), 4.76, 4.92 (2s [rotamers], 2H, N(CO)CH2F), 7.18 (d,
phenyl}-N-[4-(toluene-4-sulphonyloxy)-butyl]-acetamide (3a)
J 5 4.4 Hz, 1HAr), 7.21 (dd, J 5 4.9, 3.8 Hz, 1HAr), 7.49 (d,
J 5 7.1 Hz, 1HAr), 7.67–7.75 (m, 2HAr), 8.00 (d, J 5 8.2 Hz, 1HAr),
8.05 (dd, J 5 2.2, 1.6 Hz, 1HAr), 8.08 (dd, J 5 3.8, 1.1 Hz, 1HAr), 8.71
(s, 1HAr), 8.84 (d, J 5 4.4 Hz, 1HAr). 19F-NMR (CDCl3, 282 MHz) d
À47.46 (t, J 5 48.8 Hz, 1F). Anal. (C20H15FN4O2S): Calculated C,
60.90; H, 3.83; N, 14.20; S, 8.13. Found C, 60.65; H, 3.80; N, 14.10;
S, 8.36. LRMS (ESI pos.) m/z 395.08 [M1H]1.
The synthesis of 3a was performed in analogy with 2a. Yield
55%; yellow, temperature-sensitive solid (handle o251C, storage
À201C), m.p. 5 83–851C (decomp.). TLC: Rf (EtOAc) 5 0.28. 1H-
NMR (CDCl3, 500 MHz) d 1.61–1.76 (m, 4H, CH2), 1.95 (m, 3H,
CH3), 2.43 (s, 3H, CH3), 3.78 (t, 2H, J 5 6.9 Hz, CH2), 4.04 (t, 2H,
J 5 5.9 Hz, CH2), 7.22 (m, 2H, HAr), 7.31 (d, 1H, J 5 8.1 Hz, HAr),
7.42 (m, 1H, HAr), 7.67–7.76 (m, 4H, HAr), 7.95–8.04 (m, 2H, HAr),
8.12 (m, 1H, HAr), 8.74 (s, 1H, HAr), 8.86 (d, 1H, J 5 4.4 Hz, HAr).
Anal. (C30H28N4O5S2): Calculated C, 61.21; H, 4.79; N, 9.52; S,
10.89. Found C, 60.54; H, 4.82; N, 9.29; S, 10.84. LRMS (ESI pos.)
m/z 589.14 [M1H]1.
(4-Nitro-N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-phenyl}-benzamide) (5a)
A
mixture of (E)-N-(3-(3-(dimethylamino)acryloyl)phenyl)-N-
methyl-4-nitrobenzamide (915 mg, 2.59 mmol) and (5-amino-
1H-pyrazol-4-yl)-thiophen-2-yl-methanone (500 mg, 2.59 mmol)
was refluxed in 10 mL acetic acid for 4 h. After cooling, the
mixture was poured into ice–water, neutralized with NaHCO3
solution (pH 8), and extracted three times with CH2Cl2. The
combined organic phases were dried, evaporated, and purified
by column chromatography (silica gel; EtOAc/petroleum ether
4:1) to give 989 mg (89%) 5a as yellow solid, Rf (silica gel, EtOAc/
petroleum ether 4:1) 5 0.28. m.p. 5 160.5–162.51C. TLC: Rf
(EtOAc) 5 0.52. 1H-NMR (DMSO-d6, 500 MHz) d 3.48 (s, 3H,
NCH3), 7.30 (dd, 1HAr, J 5 3.9, 4.9 Hz), 7.42 (d, 1HAr, J 5 4.4 Hz),
7.54 (m, 1HAr), 7.63 (d, 1HAr, J 5 8.2 Hz), 7.93 (m, 1HAr), 8.01
(s, 1HAr), 8.05 (dd, 1HAr, J 5 1.0, 4.9 Hz), 8.14 (d, 1HAr, J 5 8.5 Hz),
8.19 (dd, 1HAr, J 5 1.0, 3.8 Hz), 8.74 (s, 1HAr), 8.87 (d, 1HAr,
J 5 4.4 Hz). Anal. (C25H17N5O4S): Calculated C, 62.10; H, 3.54; N,
14.48; S, 6.63. Found C, 62.11; H, 3.37; N, 14.40; S, 7.02. LRMS (ESI
pos.) m/z 484.2 [M1H]1.
(N-(4-)Fluoro-butyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-phenyl}-acetamide) (3b)
The synthesis of 3b was performed according to 2b. Yield 47%,
1
yellow solid, m.p. 5 151–1521C, TLC: Rf (EtOAc) 5 0.26. H-NMR
(CDCl3, 500 MHz) d 1.68–1.81 (m, 4H, CH2), 1.97 (s, 3H, CH3), 3.84
(t, 2H, J 5 7.1 Hz, N-CH2), 4.47 (dt, 2H, J2F 5 41.5 Hz, J 5 5.7 Hz,
CH2F), 7.18 (d, 1H, J 5 4.3 Hz, HAr), 7.23 (m, 1H, HAr), 7.45 (m, 1H,
HAr), 7.66–7.73 (m, 2H, HAr), 7.99–8.03 (m, 2H, HAr), 8.11 (m, 1H,
HAr), 8.73 (s, 1H, HAr), 8.87 (d, 1H, J 5 4.3 Hz, HAr). 19F-NMR (CDCl3,
470 MHz) d –218.57 (1F). Anal. (C23H21FN4O2S): Calculated C,
63.29; H, 4.85; N, 12.84; S, 7.35. Found C, 63.17; H, 4.87; N, 12.68;
S, 7.82. LRMS (ESI pos.) m/z 437.13 [M1H]1.
2-Bromo-N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-phenyl}-acetamide (4a)
(4-Fluoro-N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]-
pyrimidine-7-yl]-phenyl}-benzamide) (5b)
Bromoacetylbromide (66 mg, 28.5 ml, 0.325 mmol) was
reacted with VII (100 mg, 0.3 mmol) according to the procedure
described for 4b to give a yellowish solid (125 mg) after
aqueous work-up and trituration with MeOH. The solid
Compound 5b was prepared according to 5a. (E)-N-(3-(3-(di-
methylamino)acryloyl) phenyl)-N-methyl-4-flurobenzamide (843 mg,
J. Label Compd. Radiopharm 2008, 51 123–131
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S. Fischer et al.
2.59 mmol) and (5-amino-1H-pyrazol-4-yl)-thiophen-2-yl-metha- min, 10–17.5 min: 1.25 mL/min; 17.5–30 min: 1.5 mL/min,
none (500 mg, 2.59 mmol) were reacted to yield 918 mg (78%) of 30–60 min: 1.75 mL/min; tR(final product) 5 37.5–40.5 min.
a yellow solid, Rf (silica gel, EtOAc) 5 0.30. m.p. 5 170.7–171.81C. Combined separated fractions of [18F]4b were highly diluted
TLC: Rf (EtOAc) 5 0.57. 1H-NMR (CDCl3, 500 MHz) d 3.57 (s, 3H, with water, adsorbed on
a Sep-Pak C18 Plus cartridge,
NCH3), 6.92 (m, 3HAr), 7.20 (dd, J 5 4.9, 3.8 Hz, 1HAr), 7.30 and almost quantitatively desorbed with diethyl ether. Before
(m, 1HAr), 7.39 (m, 2HAr), 7.50 (t, J 5 7.9 Hz, 1HAr), 7.71 (dd, injection, diethyl ether was removed under reduced pressure.
J 5 4.9, 1.1 Hz, 1HAr), 7.80 (m, 2HAr), 8.07 (dd, J 5 3.7, 1.0 Hz, The purified final product was obtained by semi-preparative
1HAr), 8.65
( s, 1HAr), 8.77 (d, J 5 4.4 Hz, 1HAr). Anal. HPLC, SPE and formulation (overall synthesis time 100 min), and
(C25H17FN4O2S): Calculated C, 65.78; H, 3.75; N, 12.27; S, 7.02. analysed by HPLC and TLC: RCY of [18F]4b 38–43%, radio-
Found C, 65.68; H, 3.76; N, 12.01; S, 7.49. LRMS (ESI pos.) chemical purity of 499%, and a specific activity of 4150 GBq/
m/z 457.1 [M1H]1.
mmol (n 5 21).
Radiochemistry
Representative procedure for [18F]5b
RCY are decay-corrected data based on EOB. The radiochemical To the anhydrous K[18F]F-K222–carbonate complex, 4 mg
purity and specific activity are calculated to EOS.
precursor and 0.75–1 mL DMF were added and stirred for
complete dissolution (5 min, 751C). The reaction mixture was
transferred to a 10 mL standard vial sealed by a rubber septum.
The microwave-assisted radiosynthesis was carried out with an
optimized temperature program under continuous PC control-
ling system (heat-up phase: 1.5 min, 175 W; reaction: 13 min,
145–150 W, 150–1551C). The colour of the reaction solution
turned to yellow. After cooling, the solution was diluted with
3.0 mL 20% MeCN/water and applied to semi-preparative HPLC
(column: Kromasil, solvent: 48% MeCN and 20 mmol ammonium
acetate, flow gradient 0.5–1.5 mL/min). The collected final
fractions (tR 5 45.5–47.5 min) were diluted with 150 mL water
and transferred to a Sep-Pak C18 Plus cartridge. After trapping
and washing (about 10 mL water), [18F]5b was eluted with
1.25 mL EtOH. RCY of 5–10% (total preparation time ꢁ2 h),
Procedure for [18F]F transfer to organic phase
[18F]Fluoride (typically approx. 0.8–2.5 GBq) was trapped on an
anion exchanger (Acell Plus QMA or 30-PS-HCO3 cartridges),
eluted with aqueous K2CO3 solution, and transferred into the
reaction vial with a septum that contains K222 in MeCN. The
mixture was completely dried under reduced pressure in argon
atmosphere at 85–1051C, and [18F]F– was converted to the
reactive K[18F]F-K222–carbonate complex. For a standard pro-
tocol, 11.2 mg (29.7 mmol) K222 (or 7.86 mg 5 29.7 mmol of the
18-crown-6 complex) and 1.78 mg (12.9 mmol) K2CO3 were used.
Representative procedure for [18F]2b and [18F]3b
To the anhydrous K[18F]F-K222–carbonate complex, 2 mg radiochemical purity of 498.5%, and a specific activity of
precursor 2a in 0.5 mL DMF was added and the pale yellow 10–15 GBq/mmol were achieved (n 5 7).
solution was reacted at 1351C for 8 min. The labelling yield,
The complex crude labelling mixture was also investigated by
determined by radio TLC, was 55–65%. After dilution with 3.5 mL analytical HPLC. One of the isolated compounds could be
water, the crude product was directly applied to semi- identified as deacetyl-indiplon (ESI-MS: [M1H]1 5 335.10), a
preparative HPLC under isocratic conditions (column: Kromasil major non-radioactive decomposition product.
100-5 C18 7 mm (20 Â 4 mm i.d.) 1(250 Â 4.6 mm); eluent: 37%
Representative automated module synthesis of [18F]2b
MeCN120 mM ammonium acetate; flow gradient: 0.75–1.5 mL/
min. A solvents change is necessary for future animal experi-
The following solutions were prepared and filled into reagent
ments. Hence, combined separated fractions of [18F]2b were
vials: (a) 1.78 mg K2CO3 (12.9 mmol) in 0.4 mL water, (b) 11.2 mg
highly diluted with water, adsorbed on a Sep-Pak C18 Plus
K222 (29.7 mmol) in 0.75 mL anhydrous MeCN, (c) 3.0 mL MeCN,
cartridge, and almost quantitatively desorbed with ethanol. The
(d) 2 mg precursor 2a in 0.75 mL anhydrous DMF, (e) 3 mL 25%
final product was analysed by HPLC and TLC. RCY of 30–40%
MeCN/water, (f) 100 mL water (150 mL flask), and (g) 2 mL
(total synthesis time: 90–115 min), radiochemical purity of
absolute EtOH.
X99.0%, and a specific activity of X250 GBq/mmol were
The automated radiosynthesis was initiated and started on
achieved with good reproducibility (n 5 17). Chemical purity
program-controlled equipment and online recording of process
was sufficient for pharmaceutical requirements.
parameters.
The radiosynthesis of [18F]3b (N-(3-[18F]fluorobutyl)-N-{3-[3-
No-carrier-added [18F]fluoride was trapped on an anion
(thiophen-2-carbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]-phenyl}-
exchange resin (30-PS-HCO3 cartridge) and eluted into the
acetamide) was done according to [18F]2b (labelling yield:
reaction vessel as K[18F]F by adding 1.78 mg K2CO3 in 0.4 mL
56–75%, RCY: 32–43%, time: 90–115 min, radiochemical purity
water (solution (a)). After the addition of solution (b), water was
X98.5%, specific activity of X250 GBq/mmol, n 5 13).
removed by heating (80 and 951C), reduced pressure, and He
carrier gas flow. Three azeotropic distillations by repeated
Representative procedure for [18F]4b
addition of MeCN (1 mL each) were necessary to remove trace
The radiosynthesis of [18F]4b was performed using 18-C-6 as amounts of residual water. Solution (d) was transferred into the
PTC (7 mg, 26.5 mmol) under optimized conditions with a reaction vessel. Labelling reaction afforded [18F]2b with 25–42%
mixture of 2 mg of 4a in 0.8 mL MeCN at 801C for 20 min efficiency at 1351C in 10 min. The reaction mixture was dissolved
to achieve labelling efficiencies of 50–60%. The crude in 3.0 mL of 25% aqueous MeCN (solution (e)), injected onto
product was immediately applied to semi-preparative HPLC: semi-preparative HPLC (column: Kromasil, as described above),
Kromasil 100-5 C18 column (20 Â 4 mm i.d.)1Kromasil 100-5 C18 and eluted with 40% MeCN/water containing 20 mmol ammo-
pre-column (250 Â 8 mm), particle size 7 mm; eluent: 37% MeCN nium acetate at a flow rate of 1.5 mL/min. [18F]2b eluted at
120 mM ammonium acetate; flow gradient: 0–10 min: 0.75 mL/ 63 min and was collected in a flask containing 100 mL water. The
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J. Label Compd. Radiopharm 2008, 51 123–131

S. Fischer et al.
mixture was transferred onto a Sep-Pak C18 Plus cartridge and
[18F]2b was eluted with solution (g). After adding 15 mL sterile
saline, the solution was passed through a sterile filter. Radio-
chemical purity of 498.5%, specific activity 4150 GBq/mmol, an
overall RCY of about 15–23% (without decay correction), and
synthesis time of about 85 min (n 5 5) were achieved.
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compounds and primarily used for the selection of appropriate
candidates for 18F-labelling. 18F-labelling approaches were
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Acknowledgements
¨
This work was supported by a grant from the Sachsische
Aufbaubank. We wish to thank the cyclotron operator team of
the Department of Nuclear Medicine, University of Leipzig for
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