Use of 2-[18F]fluoroethylazide for the Staudinger ligation - Preparation and characterisation of GABAA receptor binding 4-quinolones

Article abstract of DOI:10.1016/j.bmcl.2010.05.106

The labelling reagent 2-[¹⁸F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was employed to obtain the GABA_A receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3- carboxylic acid (2-[¹⁸F]flu

Full text of DOI:10.1016/j.bmcl.2010.05.106

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4649–4652
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Use of 2-[18F]fluoroethylazide for the Staudinger ligation – Preparation
and characterisation of GABAA receptor binding 4-quinolones
Alessandra Gaeta a, John Woodcraft a, Stuart Plant a, Julian Goggi a, Paul Jones a, Mark Battle a, William Trigg a,
Sajinder K. Luthra a,b, Matthias Glaser b,
*
a GE Healthcare, Medical Diagnostics, White Lion Road, Amersham HP7 9LL, UK
b GE Healthcare, Medical Diagnostics, Hammersmith Hospital, London W12 0NN, UK
a r t i c l e i n f o
a b s t r a c t
The labelling reagent 2-[18F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was
employed to obtain the GABAA receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid
(2-[18F]fluoroethyl) amide. The radiotracer was prepared with a non-decay corrected radiochemical yield
Article history:
Received 15 April 2010
Revised 26 May 2010
Accepted 29 May 2010
Available online 8 June 2010
of 7%, a radiochemical purity >95% and a specific radioactivity of 0.9 GBq/lmol. The compound showed
low brain penetration in normal rats. A series of fluoroalkyl 4-quinolone analogues with nanomolar
to sub-nanomolar affinity for the GABAA receptor has been prepared as well.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Staudinger ligation
Click chemistry
GABA receptor
PET
Fluorine-18
The recently reported 2-[18F]fluoroethylazide (15) has demon-
strated its potential as a small labelling reagent for the copper(I)
catalysed 1,3-dipolar cycloaddition with alkyne bearing sub-
strates.1–5 An application of 15 beyond ‘click chemistry’ would be
the Staudinger ligation. This conjugation reaction offers a useful
alternative approach to construct or label peptides.6–8 The value
of this chemistry has been already recognised for fluorine-18 label-
ling.9 An elegant variant of the traceless Staudinger ligation, using
an azide and auxiliary 2-(diphenylphosphino)ethanethiol, has been
published recently by Soellner et al.10
have been reported in patients suffering from temporal lobe epi-
lepsy.15 The allosteric sites of the GABAA ion channel are the target
of various drugs such as barbiturates and benzodiazepines (BZ).16
The BZ allosteric site has been studied using positron emission
tomography (PET). PET is a modern non-invasive imaging modality
of superb sensitivity and it can be seen as the method of choice to
study brain function in vivo. At the present, the PET gold standards
for imaging of the GABAA-BZ site are 11C- and 18F-labelled versions
of Flumazenil (FMZ).17–19 FMZ is a GABAA neutral allosteric modula-
tor that binds to the BZ site with low nanomolar affinity. The radio-
synthesis of 18F analogues is either a low yielding nucleophilic
aromatic fluorine substitution18 or produces an alternative tracer
of poor metabolic stability.19 However in a clinical study, [18F]-
FMZ has recently shown similar binding and kinetic behaviour com-
pared to [11C]-FMZ.20
Currently, the only efficient method to access [18F]fluoroalkyl
amides is a two-step labelling process using [18F]fluoroalkyl amines
as building blocks. This could be due to the tendency of prospective
one-step sulphonate precursors to form 1,3-oxazoles under alkaline
conditions.11,12 Unfortunately, the present radiosynthesis of
[
18F]fluoroalkyl amines is based on a low yielding method involving
4-Quinolone derivatives with high affinity for the BZ site of GA-
BAA have been reported by Lager et al.21 Based on this work, we pre-
pared a series of fluoroalkyl substituted 4-quinolones as potential
new tracers for the GABAA receptor.
the use of protecting groups.13 We have exemplified the traceless
Staudinger ligation to access a new (2-[18F]fluoroethyl) amide as po-
tential radiotracer for the GABAA receptor.
The
c
-aminobutyric acid (GABA) neurotransmitter is involved in
The synthesis of the 4-quinolone fluoroethyl amide 5 was achieved
using the route described in Scheme 1. 4-Benzyl aniline 1 was reacted
with diethyl ethoxymethylenemalonate to afford the desired intermedi-
ate 2 in quantitative yield. Compound 2 was heated with diphenylether
under reflux conditions, to give 3 in good yield. The ester was hydrolysed
to the corresponding acid 4in quantitative yield. The desired amide 5was
synthesised using O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate (HATU) as coupling reagent. The same route
a number of neurological conditions such as epilepsy, traumatic
brain injury, anxiety and insomnia. For instance, in a model of Status
Epilepticus a rapid internalisation of the subtype GABAA receptors,
ligand gated ion channels, has been observed.14 Similar changes
* Corresponding author.
E-mail address: matthias.glaser@ge.com (M. Glaser).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.106

4650
A. Gaeta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4649–4652
O
O
NH2
O
O
O
O
EtO
OEt
i
ii
iii
OH
HN
OEt
N
H
N
H
3
4
iv
1
O
O
2
NH
F
N
H
5
Scheme 1. Synthesis of quinolone 5. Reagents and conditions: (i) ethoxymethylene malonate, 3 h, 120 °C; (ii) diphenylether, 1 h, reflux; (iii) NaOH (2 N), ethanol, 2 h, reflux;
(iv) 2-fluoroethylamine, HATU, DIPEA, DMF, 12 h, rt.
was applied for the synthesis of analogues 6–8 (Table 1), using 4-bromo
aniline and 4-ethyl aniline, respectively.
diate and then successfully converted to compound 10 in good
overall yield (28%) using the conditions described above.
Compound 5 was identified in vitro as a potent ligand for the
GABAA receptor with a Ki of 0.70 0.66 nM (Table 1). Further var-
iation at the 6-position of the quinolone system did not signifi-
cantly alter the affinity of the test compounds. The same applied
for extending the 2-fluoroethyl chain to a 3-fluoropropyl function.
Quinolone 5 was selected for in vivo evaluation.
In order to obtain quinolone 5 via Staudinger ligation, the re-
quired precursor 10 was generated from commercially available
thioester 11. This was achieved in two high-yielding steps follow-
ing a previously reported approach (Scheme 2).10,22,23 Initially, 13
was employed with the quinolone acid 4 using literature condi-
tions22 to form the desired quinolone thioester. Unfortunately, this
method failed to give the desired product, with no reaction ob-
served. This was most likely due to the low reactivity of the quin-
olone acid 4 under the standard coupling conditions. However,
treatment of quinolone acid 4 with 1 equiv of oxalyl chloride gave
quantitative conversion to the acyl chloride 9. The latter was char-
acterised by NMR to confirm the formation of the desired interme-
Initial attempts to radiolabel compound 5 by one-step labelling
of the ethyl tosylate precursor using fluorine-18 failed. We hypoth-
esised that this was caused by the acidic nature of the tautomeric
quinolone proton, effectively quenching the 18F-fluoride during the
labelling reaction.
An alternative approach, based on the use of 2-[18F]fluoroethy-
lazide 15 for the Staudinger ligation, was explored for the labelling
of this class of compounds. The optimised synthetic route to suit-
able Staudinger precursors is exemplified in Scheme 3. Reagent
15 was prepared from tosylate 14 according to a published meth-
od1 and subsequently reacted with phosphine 10. The desired
product 18F-5 was formed in 75% radiochemical incorporation as
observed by analytical HPLC. The tracer was subsequently isolated
using preparative HPLC with a non-decay-corrected radiochemical
yield of 7% (based on starting [18F]fluoride) after 105 min (see Sup-
plementary information, Fig. 1).24 The radiochemical purity of the
formulated product (30–50 MBq) was >95% with a specific radioac-
tivity of 0.9 GBq/lmol. The PBS formulated compound was found
to be stable at room temperature for at least 2 h (see Supplemen-
tary information, Figs. 5 and 6). This chemistry has therefore pro-
vided a convenient method to introduce a 2-[18F]fluoroethylamide
functionality into a molecule of interest.
Table 1
Structures of fluoroalkyl 4-quinolones 5–8 and measured Ki data using
inhibition of [3H]Ro151788 (FMZ) binding to GABAA receptors on rat
cerebellar membrane preparations.
The biodistribution of 18F-labelled 5 was evaluated in naïve
Sprague–Dawley rats (Table 2). The results show that only a mod-
est fraction of the compound crossed the blood–brain barrier, with
peak uptake no greater than 0.12% of the injected dose (0.11% ID/g).
In the periphery, there was high initial uptake to the muscle with
clearance over the 60-min study period. Excretion was primarily
via the hepatobiliary rather than urinary system, as shown by
increasing levels in the small intestines with time. There was no in-
crease in the radioactivity in the bone that would indicate de-fluo-
rination. Ex vivo autoradiographic analysis of prefrontal cortex,
striatum and thalamus slices indicated significant differential
binding of 18F-5 consistent with the distribution of GABAA recep-
tors (Fig. 1, and Supplementary information, Fig. 8). The specific
tracer binding in these areas was high as found by blocking with
cold Flumazenil (ꢀ75% in high expressing regions and ꢀ60% in
low expressing regions). Thus, 18F-5 demonstrated affinity for the
BZ sites (see Supplementary information, Table 1).
Compound
Ki (nM)
O
O
NH
5
0.70 0.66
N
H
F
O
O
NH
6
0.13 0.09
N
H
F
O
O
Br
NH
7
8
1.30 0.87
3.60 2.24
N
H
F
O
O
The c log D value of 5 at pH 7.0 was found to be 2.1 1.0. There-
fore, the tracer would be expected to penetrate the blood–brain
barrier.25 Further biological evaluation will be required to investi-
gate the metabolic profile of 18F-5 and whether the compound is a
PgP substrate.
NH
N
H
F

A. Gaeta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4649–4652
4651
O
O
O
O
i
S
P
OH
N
H
N
H
4
10
ii
iii
O
O
Cl
N
H
9
S
O
iv
v
SH
PPh
S
O
Ph
+
P
BH
3
2
PPh
2
-
Ph
11
12
13
Scheme 2. Synthesis of quinolone 10. Reagents and conditions: (i) HOBt, DCC, DMF, 13, 3 h, rt; (ii) oxalyl chloride, DCM, cat DMF, 1 h, rt; (iii) 13, DCM, 12 h, rt; (iv) DABCO,
toluene, 3 h, 40 °C; (v) NaOH, methanol, 2 h, rt.
18F
Ph
—
N
+
i
ii
P
O
O
N3
N3
Ph
TsO
18F
-N2
S
14
15
N
H
H2O
- P(O)Ph2CH2SH
O
O
NH
18F
N
H
18F-5
Scheme 3. Radiosynthesis of 18F-5. Reagents and conditions: (i) [18F]KF/Kryptofix/K2CO3, MeCN, 20 min, 80 °C, distillation; (ii) 10, MeCN, DMF, 15 min, 130 °C.
Table 2
Biodistribution data of 18F-5 in naïve Sprague–Dawley rats (n = 3)
Radioactivity levels SD (% injected dose)
0.5 min pi
2 min pi
10 min pi
30 min pi
60 min pi
Blood
Brain
Heart
Bone
Liver
Lung
Spleen
Muscle
Kidneys
Stomach
Small intestine
Large intestine
Bladder and Urine
Fat
8.7 1.8
0.1 0.0
2.0 0.4
3.0 0.3
14.6 8.2
2.9 0.3
0.2 0.1
56.1 14.1
4.0 1.7
0.8 0.3
4.7 2.8
0.9 0.6
0.2 0.3
0.6 0.9
4.2 0.6
0.1 0.0
1.1 0.2
3.0 0.4
28.5 4.1
1.5 0.7
0.3 0.0
47.3 10.3
3.6 0.5
1.2 0.5
7.0 0.5
1.3 0.1
0.0 0.0
1.8 0.9
2.8 0.2
0.1 0.0
0.3 0.2
1.7 0.2
33.7 1.7
0.6 0.0
0.2 0.1
27.2 1.6
2.1 0.1
1.0 0.3
24.5 3.1
1.7 0.4
0.8 0.2
1.8 0.5
1.5 0.1
0.1 0.0
0.2 0.0
1.5 0.2
18.7 2.7
0.4 0.0
0.1 0.0
16.5 1.3
1.5 0.2
0.5 0.2
50.3 4.1
1.7 0.1
4.2 0.8
1.7 0.2
0.7 0.1
0.0 0.0
0.1 0.0
1.4 0.2
4.8 0.9
0.1 0.0
0.0 0.0
4.6 0.8
0.5 0.1
0.2 0.0
74.5 0.5
1.9 0.4
7.3 1.4
0.3 0.6

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A. Gaeta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4649–4652
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Figure 1. Autoradiography of striatal brain slice using [18F]-5: indicates highest
receptor expression in outer cortex and lower expression in the inner cortex. The
laminar distribution is consistent with the high alpha1 subunit distribution in
lamina III. Lowest expression observed in striatal areas (and thalamic) consistent
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24. Preparation of 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (2-
In conclusion, the reagent 2-[18F]fluoroethylazide is starting to
show versatility as a building block for PET radiochemistry beyond
the established field of ‘Click Labelling’. Here, the traceless Stauding-
er ligation proved to be compatible with the half-life of fluorine-18.
This reaction can thus be seen as useful tool to readily obtain 2-
[
18F]fluoroethylamides without the need for protective groups. The
investigated series of fluoroalkyl 4-quinolones demonstrated high
GABAA receptor affinity. However, the selected 18F-5 showed a brain
uptake in rats that was not suitable for PET imaging.
[
18F]fluoroethyl) amide
(
18F-5)
–
[
18F]fluoride was transferred to
a
3 mL
mol), MeCN (0.5 mL),
L, 0.1 M). The solution was dried at 100 °C
under a flow of N2 (0.3 L/min) for 20 min and cooled to room temperature. To
the dried L,
18F]fluoride was added 2-azidoethyl toluenesulfonate 14 (3
15.0
Wheaton vial containing Kryptofix K222 (5 mg, 13.4
and potassium carbonate (50
l
Supplementary data
l
[
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.106.
l
mol)1 in MeCN (0.2 mL), the Wheaton vial sealed and heated to 80 °C for
20 min. The temperature was increased to 130 °C and [18F]fluoroethylazide 15
was allowed to distil into a 1 mL Wheaton vial that was chilled in ice using a
stream of nitrogen (0.1 mL/min). To the Staudinger precursor 10 (2 mg,
References and notes
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l
mol) in a mixture of water/DMF (1:9 v/v, 100
(0.2 mL). The reaction mixture was heated at 130 °C for 15 min and allowed to
cool to room temperature. Water (100 L) was added and the resulting
colourless precipitate was removed by filtration (0.45 m, PALL ACRODISK
lL) was added 15 in MeCN
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l
CR13) to give the crude product. The reaction mixture was diluted into water
(3 mL) and was purified by preparative HPLC (Luna C18(2) 100 ꢁ 10 mm, A:
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