Synthesis and radiopharmacological characterization of [11C]AL-438 as a nonsteroidal ligand for imaging brain glucocorticoid receptors

Article abstract of DOI:10.1016/j.bmcl.2007.04.094

The radiosynthesis and the radiopharmacological characterization of [¹¹C]AL-438 as a nonsteroidal ligand for the glucocorticoid receptor (GR) is described. Radiolabeling of the corresponding desmethyl precursor 10 with [¹¹C]MeI gave [¹¹C]AL-438 in decay-corrected radiochemical yields of 30 ± 4% (based upon [¹¹C]CO₂) within 35 min at a specific radioactivity of 10-15 GBq/μmol at the end-of-synthesis. The radiopharmacological evaluation of [¹¹C]AL-438 involved biodistribution and small animal PET imaging in rats, and autoradiography studies using rat brain sections. Biodistribution studies were performed in male Wistar rats and demonstrated high radioactivity uptake in pituitary and brain. However, the inability of high dose corticosterone to block binding would suggest that the radioactivity accumulation in the brain was not receptor-mediated.

Full text of DOI:10.1016/j.bmcl.2007.04.094

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4035–4039
Synthesis and radiopharmacological characterization of
[11C]AL-438 as a nonsteroidal ligand for imaging
brain glucocorticoid receptors
Frank Wuest,a,* Torsten Kniess,a Ralf Bergmann,a Brian Henryb and Jens Pietzscha
aInstitut fu¨r Radiopharmazie, Forschungszentrum Dresden-Rossendorf e.V., POB 51 01 19, D-01314 Dresden, Germany
bOrganon Laboratories Limited, Newhouse, Scotland, UK
Received 3 November 2006; revised 24 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—The radiosynthesis and the radiopharmacological characterization of [11C]AL-438 as a nonsteroidal ligand for the gluco-
corticoid receptor (GR) is described. Radiolabeling of the corresponding desmethyl precursor 10 with [11C]MeI gave [11C]AL-438 in
decay-corrected radiochemical yields of 30 4% (based upon [11C]CO2) within 35 min at a specific radioactivity of 10–15 GBq/lmol
at the end-of-synthesis. The radiopharmacological evaluation of [11C]AL-438 involved biodistribution and small animal PET imag-
ing in rats, and autoradiography studies using rat brain sections. Biodistribution studies were performed in male Wistar rats and
demonstrated high radioactivity uptake in pituitary and brain. However, the inability of high dose corticosterone to block binding
would suggest that the radioactivity accumulation in the brain was not receptor-mediated.
Ó 2007 Elsevier Ltd. All rights reserved.
Glucocorticoids which are appropriately labeled with
the short-lived positron emitters 11C (t1/2 = 20.4 min)
and 18F (t1/2 = 109.8 min) would allow the noninvasive
in vivo imaging of brain glucocorticoid receptors
(GRs) by means of positron emission tomography
(PET). PET imaging of brain GRs would provide
important information on the neurobiological basis of
GR-mediated abnormalities of hypothalamo–pituitary–
adrenocortical (HPA) axis function and regulation,
which has been suggested to play a crucial role in the
pathogenesis of depression.1–5 The accumulating evi-
dence of the important role of brain GRs in several neu-
ropsychiatric disorders, such as severe depression and
anxiety, has stimulated research on PET radiotracers
for studying brain GRs in vivo over the last 15 years.
Research was mainly focused on steroidal glucocorti-
coids, and numerous attempts have been made to
synthesize GR-binding steroids labeled with the short-
lived positron emitter 18F6–13 and, to a lesser extent, with
11C.14 Despite the excellent in vitro GR binding of some
of the synthesized compounds, none of the investigated
compounds are suitable for imaging brain GRs either
due to their rapid in vivo defluorination and/or insuffi-
cient blood–brain-barrier penetration. Moreover, the
steroidal GR ligands used (e.g. RU486) often show
binding to other steroid hormone receptors, mainly to
the progesterone receptor (PR). The lack of selectivity
of some of the steroidal compounds has further limited
their use as selective GR tracers.
In contrast, recently identified nonsteroidal compounds
with high affinity and selectivity toward the GR provide
an interesting alternative for the design and synthesis of
PET radiotracers for GRs. Nonsteroidal GR-binding li-
gands comprise a broad structural variety. Prominent
examples of nonsteroidal GR ligands are structurally
based on dibenzyl anilines, N-arylpyrazolo-based li-
gands or benzopyrano-quinolines.15
A selection of structures for nonsteroidal GR ligands
and the steroidal GR ligand RU486 is given in Figure 1.
Among the reported benzopyrano-quinolines, AL-438
was identified as one of the first compounds showing
both, high affinity and selectivity for the GR.16 More-
over, the methoxy group at position 10 of AL-438 makes
this compound an attractive candidate for isotopic
labeling with 11C through a methylation reaction of
Keywords: Positron emission tomography; Glucocorticoid receptor;
Radiolabelling.
*
Corresponding author. Tel.: +49 351 260 2760; fax: +49 351 260
2915; e-mail: f.wuest@fz-rossendorf.de
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.094

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F. Wuest et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4035–4039
methanolysis affording methyl acetal 8 in 58% yield for
both steps.
S
OH
Treatment of methyl acetal 8 with allytrimethylsilane in
the presence of the Lewis acid BF3ÆEt2O afforded allylat-
ed product 9 in excellent 95% yield. Removal of the
TBS-ether protecting group in 9 succeeded through
treatment with TBAF in 68% yield to give compound
10 as labeling precursor. Subsequent treatment of desm-
ethyl precursor 10 with Cs2CO3 as the base in the pres-
ence of an excess of MeI afforded AL-438 in 73% yield.
The total yields of desmethyl precursor 10 and reference
compound AL-438 were 24% and 18%, respectively,
based upon compound 6.
N
N
N
O
O
S
N
H
F
Dibenzyl aniline-based
GR ligands
N-arylpyrazolo-based
GR ligands
N
O
R
OH
MeO
N
H
The radiosynthesis of [11C]AL-438 is shown in Figure 3.
O
Benzopyrano-quinolines
RU486
R = allyl (AL-438)
The radiolabeling was accomplished by a methylation
reaction of desmethyl precursor 10 with [11C]methyl io-
dide. The radiosynthesis of [11C]AL-438 was performed
in a remotely controlled synthesis apparatus (Nuclear
Figure 1. Structures of nonsteroidal GR ligands15 and RU486.
the corresponding desmethyl precursor with [11C]methyl
iodide.
Interface, Munster). [11C]Methyl iodide was prepared
¨
starting from [11C]CO2 according to Crouzel and co-
workers.17 [11C]Methyl iodide was transferred in a
stream of nitrogen into the reaction vessel containing
the sodium salt of desmethyl precursor 10 (1 mg) in
DMF (400 ll) and 5 N NaOH (30 ll) at À20 °C. The
formation of the sodium salt of 10 could easily be mon-
itored by the yellow color of the DMF solution. After
completion of the [11C]MeI transfer, the reaction vessel
was sealed and heated at 100 °C for 5 min. The reaction
mixture was diluted with eluent (1 ml) and the mixture
was transferred from the reaction vessel onto a semi-
preparative C-18 column (83:17 acetonitrile/water
containing 0.1% ammonium formate, 4 ml/min). The
fraction eluting at 9–11 min was collected, diluted with
water (10 ml), and passed through a C-18 Sep-Pak Light
cartridge. The cartridge was washed with water (5 ml)
and the product was eluted with ethanol (0.5 ml).
Addition of 0.9% saline (9.5 ml) gave 5% EtOH solution
in saline suitable for the animal experiments.
In this paper we describe the radiosynthesis and radio-
pharmacological evaluation of [11C]AL-438 as a first
example of a nonsteroidal PET radiotracer for imaging
brain glucocorticoid receptors.
The synthesis of labeling precursor 10 and AL-438 as
reference compound is depicted in Figure 2.
A suitable starting material for synthesis of compounds
10 and AL-438 is tetracyclic lactone 6, which was pre-
pared according to literature procedure.16 The phenolic
OH-group at position 10 was protected as silyl ether
using TBDMSCl and KOtBu as the base in DMF in
65% yield. The TBS-protected tetracyclic compound 7
was further functionalized at position 5. For this pur-
pose lactone 7 was subjected to a controlled reduction
using DIBAL in toluene followed by acid-catalyzed
In a typical experiment, 30 GBq of [11C]CO2 could be
converted into 3.0 GBq of [11C]AL-438 (30 4% de-
cay-corrected radiochemical yield) after HPLC purifica-
tion at a specific radioactivity of 10–15 GBq/lmol at the
end-of-synthesis within 35 min. The radiochemical pur-
ity exceeded 95%.
O
O
O
O
a
OH
TBSO
N
N
H
H
6
7
O
O
b,c
d
The biodistribution results18 of [11C]AL-438 in normal
and corticosterone pretreated male Wistar rats are de-
picted in Table 1.
TBSO
N
H
8
O
O
e
O
O
TBSO
[
11C]MeI
RO
N
N
H
H
NaOH, DMF
4 min, 100˚C
9
10
R=H
HO
O
H311C
f
N
N
AL-438 R=Me
H
H
Figure 2. Reagents: (a) TBDMSCl, KOt-Bu, DMF, 65%; (b) DIBALH,
toluene; (c) MeOH, TsOHÆH2O, 58% for two steps; (d) allyltrimeth-
ylsilane, BF3ÆEt2O, 95%; (e)TBAF, 68%; (f) Cs2CO3, MeI, 73%.
[11C]AL-438
10
Figure 3. Radiosynthesis of compound [11C]AL-438.

F. Wuest et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4035–4039
4037
Table 1. Radioactivity expressed as percent injected dose per gram tissue in different organs after single intravenous injection of 20 MBq of [11C]AL-
438 in 0.5 ml saline with 2% ethanol
%ID/g tissue (time p.i.)
Control
Blocked
5 min
60 min
5 min
60 min
Blood
Rest of brain
Cortex
0.52 0.10
1.52 0.41
1.67 0.32
1.39 0.37
1.30 0.34
2.49 0.69
7.63 1.38
2.79 0.55
0.97 0.16
1.72 0.32
1.19 0.42
5.22 2.50
0.94 0.29
1.70 0.38
1.44 0.35
1.57 0.47
1.68 0.32
2.65 0.38
0.90 0.20
0.47 0.14
0.60 0.19
0.56 0.10
0.60 0.18
0.53 0.06
0.61 0.09
1.77 1.04
4.40 1.07
1.99 0.48
1.06 0.26
1.19 0.29
3.11 1.30
8.54 1.43
0.59 0.08
0.75 0.15
1.11 0.21
1.48 0.32
3.35 0.92
2.54 0.54
0.88 0.15
0.52 0.15
0.52 0.07
1.46 0.21
1.55 0.23
1.46 0.26
1.35 0.29
2.45 0.93
8.68 1.19
2.81 0.38
1.21 0.18
2.06 0.11
1.51 0.32
5.00 1.09
0.90 0.11
1.91 0.43
1.44 0.11
1.46 0.38
1.65 0.0.35
3.04 0.54
0.85 0.07
0.43 0.11
0.74 0.30
0.61 0.14
0.74 0.11
0.64 0.11
0.68 0.11
1.76 0.82
3.80 0.34
1.43 0.19
0.91 0.28
1.13 0.08
2.43 0.37
7.44 1.57
0.52 0.06
0.67 0.06
1.03 0.20
1.60 0.43
2.87 0.63
3.84 1.46
0.70 0.12
0.50 0.08
Cerebellum
Hippocampus
Pituitary
Adrenals
Pancreas
Spleen
Kidneys
Fat
Brown fat
Muscle
Heart
Lungs
Thymus
Harderian glands
Liver
Femur
Testis
n = 4 for each time point; p.i., post injection.
The radioactivity concentration in the blood remains
stable over time reaching the maximum 5 min post injec-
tion (p.i.). The highest initial radioactivity uptake was
observed in the adrenals (7.63 1.38% ID/g at 5 min)
which decreased to 4.40 1.07% ID/g at 60 min. Com-
pared to other tissues and organs relatively high radio-
activity concentration was further found in the
pancreas, liver, and brown fat. The pituitary as target
organ known to express high levels of GRs showed high
radioactivity uptake of 2.49 0.69% ID/g at 5 min
which was slightly reduced to 1.77 1.04 ID/g after
60 min. In all organs, excluding the adrenals, changes
of the activity concentration within the first hour after
injection were relatively low. The compound readily
penetrates the blood–brain barrier as shown by the rel-
atively high radioactivity concentration in selected brain
regions, like cortex, hippocampus, and cerebellum.
However, radioactivity was steadily washed out from
the brain over time reaching comparable radioactivity
levels in the cortex (0.60 0.18% ID/g), hippocampus
(0.61 0.09% ID/g) and cerebellum (0.53 0.06% ID/g)
at 60 min p.i.
Consequently, the endogenous adrenal steroid capacity
of the animals was high during the experiments which
might have prevented sufficient blocking of radiotracer
uptake. It is known that under normal conditions the
GRs are occupied through endogenous corticosterone
by 28%.19 The effect of plasma corticosterone level in
normal rats (423 23 ng/mg) and in adrenalectomized
rats (23 3 ng/mg) was discussed recently.14 The almost
30% occupancy of the brain GR binding sites by endog-
enous corticosterone in addition to the rather low spe-
cific activity of AL-438 (5 GBq/lmol at the time of
injection) probably caused a substantial saturation of
the binding sites which may have prevented a measur-
able blocking effect.
Our data on the competition binding of AL-438 and the
potent antiprogestin RU486 for GR and PR (Table 2)
are in agreement with the proposed GR binding affinity
and selectivity properties of AL-438. The binding affin-
ity of AL-438 is lower but still in a comparable range
to that of the high affinity GR/PR ligand RU486. How-
ever, AL-438 demonstrates lower binding to the PR
compared to RU486 resulting in an improved GR selec-
tivity profile toward the PR. The calculated logP values
(6.13 for AL-438 and 4.84 for RU486, respectively) re-
flect a high lipophilicity for both compounds, being
especially high for the nonsteroidal compound AL-438.
The high logP value for compound AL-438 agrees with
The blocking experiments with corticosterone were per-
formed to study glucocorticoid binding site-mediated
uptake. However, no change of radioactivity concentra-
tion was observed for the GR-rich target organs (adre-
nals, thymus), the pituitary and selected brain regions
(cortex and hippocampus). Thus, it can be concluded
that the observed radioactivity uptake is not receptor-
mediated and the biodistribution reflects predominantly
nonspecific distribution of compound [11C]AL-438. This
is in keeping with the calculated high lipophilicity of this
compound (logP 6.13).
the biodistribution profile of
compound.
a highly lipophilic
Figure 4 shows an autoradiogram of the in vivo distribu-
tion of [11C]AL-438 in the median horizontal plane of
the rat brain at 5 min after injection.20 High radioactiv-
ity concentration was detected in the cortex and tha-
lamic region. The observed regional radioactivity
accumulation is in agreement with the results reported
This finding may be explained by the fact that in this
study normal nonadrenalectomized rats were used.

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F. Wuest et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4035–4039
Table 2. Competition binding of AL-438 or RU486 at GR and PR.
Binding data were generated using fluorescence polarization assay kits
available from Invitrogen (PolarScreen Glucocorticoid Receptor
Competitor Assay Kit, Green and PolarScreen Progesterone Receptor
Competitor Assay Kit, Green)
Compound
GR
PR
logP
AL-438
RU486
8.1 0.21
8.6 0.01
6.9 0.17
7.8 0.16
6.13
4.84
Data are expressed as pEC50 with 95% confidence interval of the mean
of N = 3 determinations. RU 486 was purchased from Sigma. Calcu-
lated logP values are based on ACDLab predictions.
Figure 5. Sagittal, coronal, and transaxial images of a male Wistar rat
brain after intraveneous injection of 30 MBq of [11C]AL-438.
GRs could be conveniently synthesized in sufficient
radiochemical yield via 11C-methylation of the
corresponding desmethyl precursor 10. Compound
[11C]AL-438 represents the first example of a nonsteroi-
dal glucocorticoid ligand labeled with a short-lived
positron emitter. The radiopharmacological investigation
of [11C]AL-438 showed promising brain uptake and accu-
mulation in brain region known to express high levels of
GRs. However, the failure to demonstrate effective block-
ade by corticosterone pre-treatment suggests that the up-
take of [11C]AL-438 into target tissues may not be GR-
mediated. For further experiments, optimization of the
benzopyrano-quinolines-based lead structure will be nec-
essary to reduce the lipophilicity. For this purpose several
recently described novel ligands bearing a sulfonamide
moiety22 should be envisaged for further 11C and 18F
radiolabeling experiments to provide compounds with
high affinity and selectivity for the GR while showing a
significantly reduced lipophilicity.
Figure 4. Digital autoradiograph of a rat brain section at 5 min after
single iv administration of [11C]AL-438.
3
by Ahima et al. using H-labeled corticosteroids. In this
study the authors report high densities of GRs in all re-
gions of the cerebellar cortex.21
Small animal PET imaging with [11C]AL-438 was per-
formed using a microPETÒ P4 primate model scanner
(CTI Concorde Microsystems Inc., Knoxville, TN).
The raw data were sorted into 3D sinograms followed
by Fourier re-binning and two-dimensional ordered sub-
sets expectation maximization (OSEM) image recon-
struction. No correction for recovery and partial
volume effects was applied. General anesthesia of the
animal was induced and maintained by inhalation of
halothane (3%) and N2O (65%) in O2. The animal was
positioned with the brain in the center of the 22 cm
transaxial and 8 cm axial field of view (FOV) of the
scanner. The rat was injected with 30 MBq of [11C]AL-
438 via the tail vein and sacrificed at 2 h p.i. by intrave-
nous application of KCl. The rat was scanned for
50 min, and the raw data were sorted into 30 frames
with photon attenuation correction. Two-dimensional
projections of sagittal, transversal, and coronal small
animal PET images are displayed in Figure 5.
Acknowledgments
Financial support from the Deutsche Forschungsgeme-
inschaft is gratefully acknowledged. The authors are
grateful to Mareike Barth, Regine Herrlich, Stephan
Preusche, and Tilow Krauß for their expert technical
assistance. The authors also thank Moira Elmore and
Michael Speake, Organon Laboratories Limited, for
the in vitro GR and PR binding data.
References and notes
The brain is clearly visible in the microPET images,
which is consistent with the obtained regional brain dis-
tribution data. The initial high radioactivity uptake in
the brain declined over time. Summation of frames be-
tween 30 and 60 min shows local accumulation of radio-
activity consistent with the regional radioactivity
distribution found in the ex vivo autoradiography.
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