Synthesis of an 11C-labeled antiprion gn8 derivative and evaluation of its brain uptake by positron emission tomography

Full text of DOI:10.1002/cmdc.201300167

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DOI: 10.1002/cmdc.201300167
11
Synthesis of an C-Labeled Antiprion GN8 Derivative and
Evaluation of Its Brain Uptake by Positron Emission
Tomography
Tsutomu Kimura,[a] Takeo Sako,[b] Siqin,[b] Junji Hosokawa-Muto,[a] Yi Long Cui,[b]
Yasuhiro Wada,[b] Yosky Kataoka,[b] Hisashi Doi,[b] Suehiro Sakaguchi,[c] Masaaki Suzuki,[b]
Yasuyoshi Watanabe,[b] and Kazuo Kuwata*[a]
N,N’-(Methylenedi-4,1-phenylene)bis[2-(1-pyrrolidinyl)
acet-
ing a therapeutic agent for prion diseases. A wide range of
compounds have been identified as having antiprion activity
in TSE-infected cells.[3,4] However, current therapeutic agents
directed at prion diseases remain unsatisfactory[5,6] due to
a lack of blood–brain barrier (BBB) permeability. Although an
intraventricular injection of BBB-impermeable antiprion com-
pounds is an alternative route for drug administration, it is in-
vasive and harmful to the patients, and poses a risk for iatro-
genic prion infections. Therefore, it is essential to develop an-
tiprion agents that can cross the BBB.
amide] (GN8) is a promising candidate for the treatment of
prion diseases. The purpose of this study was to synthesize
a GN8 derivative labeled with a positron emitting radionuclide
and to clarify the blood–brain barrier (BBB) permeability of the
resultant derivative by positron emission tomography (PET). As
a key synthetic intermediate, a GN8 derivative bearing a tribu-
tylstannyl group was prepared from commercially available
materials in four steps. Palladium(0)-mediated rapid C-methyla-
tion of the aryltributylstannane using [11C]methyl iodide yield-
ed a [11C]methyl-substituted GN8 derivative ([11C]-1) with suffi-
cient radioactivity (0.5–2.0 GBq) and specific radioactivity in the
region of 60–126 GBqmmolÀ1. [11C]-1 was injected into the tail
vein of rats, and its biodistribution was determined by PET; the
results unambiguously demonstrated the brain penetration of
[11C]-1 in rat brain.
During the course of our antiprion drug discovery and de-
velopment studies,[7,8] GN8 was identified as a novel antiprion
compound (Figure 1a).[9–15] Subcutaneous administration of
GN8 offered a survival benefit to TSE-infected mice.[9] Further-
more, nonclinical safety assessment of GN8 in rats and dogs re-
vealed that GN8 could be used safely at the concentration nec-
Prion diseases, also referred to as transmissible spongiform
encephalopathies (TSEs), are a family of fatal neurodegenera-
tive disorders that affect both humans and animals.[1,2] Human
prion diseases include Creutzfeldt–Jakob disease (CJD), Gerst-
mann–Strꢀussler–Scheinker (GSS) syndrome, fatal familial in-
somnia, and kuru. The representatives of animal prion diseases
are scrapie in sheep and goats, bovine spongiform encephal-
opathy (BSE) in cattle, and chronic wasting disease in deer and
elk. These disorders are characterized by vacuolar degeneration
of the central nervous system (CNS). The causative agent of
the diseases is thought to be an infectious isoform of the
prion protein, designated PrPSc, and an accumulation of PrPSc
in the CNS gives rise to prion diseases.
Since the epidemic of BSE and the appearance of a new var-
iant of CJD, which seems to be caused by PrPSc-contaminated
beef consumption, much effort has been devoted to develop-
[a] Dr. T. Kimura, Dr. J. Hosokawa-Muto, Prof. K. Kuwata
Center for Emerging Infectious Diseases
United Graduate School of Drug Discovery & Medical Information Sciences
Gifu University, 1-1 Yanagido, Gifu 501-1194 (Japan)
E-mail: kuwata@gifu-u.ac.jp
[b] Dr. T. Sako, Dr. Siqin, Dr. Y. L. Cui, Dr. Y. Wada, Dr. Y. Kataoka, Dr. H. Doi,
Prof. M. Suzuki, Prof. Y. Watanabe
RIKEN Center for Molecular Imaging Science
6-7-3 Minatojima, Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan)
[c] Prof. S. Sakaguchi
Figure 1. a) Chemical structures of GN8, and the unlabeled and 11C-labeled
methylated derivative of GN8 (1). b) Western blotting of proteinase K-resist-
ant prion protein in GT+FK cells after treatment of the cells with GN8 and
1 at 10 mm.
Institute for Enzyme Research, Tokushima University
3-18-15 Kuramoto-cyo, Tokushima, 770-8503 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300167.
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essary to exert antiprion activity.[15] PET is a powerful technique
to assess the clinical dose, regimen selection, as well as brain
penetration of the emitter-labeled compounds in prion disease
patients. PET analysis of a GN8 derivative labeled with a posi-
tron-emitting radionuclide would give us important clues for
antiprion drug development.[16] Herein, we report the synthesis
of a 11C-labeled GN8 derivative ([11C]-1) by palladium-catalyzed
rapid methylation of aryltributylstannane, and its subsequent
assessment for BBB permeability and biodistribution in rat by
PET.
dine, 2-iodoaniline hydrochloride, and paraformaldehyde.[14,25]
Bromoacetylation of diamine 2 and subsequent substitution
reaction of the resulting bis(2-bromoacetamide) with pyrroli-
dine yielded compound 3.[14] Unfortunately, several attempts to
convert 3 to 4 by palladium-catalyzed tributylstannylation with
bis(tributylstannane) were unsuccessful, presumably because
of the substituent effect of the neighboring acylamino group.
Alternatively, we examined the synthesis of 4 via tributylstan-
nylation at an earlier stage. Aryl iodide 2 underwent a palladi-
um-catalyzed coupling reaction with bis(tributylstannane) to
afford aryltributylstannane 5 at a yield of 67%. Chloroacetyla-
tion of 5 gave bis(2-chloroacetamide) 6, and the reaction of 6
with pyrrolidine in the presence of K2CO3 provided the desired
aryltributylstannane 4.
With the key precursor in hand, rapid methylation of aryltri-
butylstannane 4 with [11C]methyl iodide was carried out in the
presence of palladium catalyst (Scheme 1b).[19–23] After several
experiments, we found that N-methylpyrrolidone (NMP) was
an effective solvent for the rapid methylation of aryltributyl-
stannane 4. The optimized conditions for the reaction of aryl-
tributylstannane 4 with [11C]methyl iodide were NMP for five
minutes to give the 11C-labeled GN8 derivative [11C]-1 with suf-
ficient radioactivity (0.5–2.0 GBq) for PET and a specific radioac-
tivity of 60–126 GBqmmolÀ1. The chemical purity analyzed at
254 nm was 82–92%, and the radiochemical purity was deter-
mined to be greater than 95%. The decay-corrected radio-
chemical yield, which was calculated from the radioactivity of
[11C]methyl iodide trapped in the palladium catalyst-containing
reaction mixture, was approximately 20%. The total synthesis
time, including HPLC purification and radiopharmaceutical for-
mulation for intravenous administration, was 43 min.
As a positron emitting radionuclide-labeled compound, we
designed [11C]-1 in which two methyl groups are linked to the
diphenylmethane unit of GN8 at the 2,2’-positions via carbon–
carbon bonds (Figure 1a). To confirm the antiprion activity of
the dimethyl-modified GN8 derivative, cold GN8 derivative
1 was prepared by bromoacetylation of 4,4’-diamino-3,3’-dime-
thyldiphenylmethane and subsequent substitution of the
bromo groups with pyrrolidine (see Supporting Information).[14]
The antiprion activity of 1 was tested in GT+FK cells, which
are mouse neuronal cells (GT1–7) persistently infected with
a
mouse-adapted GSS agent (Fukuoka-1 strain) (Fig-
ure 1b).[7,17,18] GN8 derivative 1 exhibited a similar order of ac-
tivity to that of GN8 with an IC50 value of 2.35Æ0.12 mm. Thus,
the modification of GN8 by the introduction of two methyl
groups did not substantially affect its antiprion activity.[9]
11C is a short-lived positron emitting radionuclide (t1/2
=
20.4 min). A 11C-labeled methyl group can be incorporated into
an aromatic framework via a carbon–carbon bond in a short
synthesis time using palladium(0)-mediated rapid cross-cou-
pling of aryltributylstannane with [11C]methyl iodide.[19–23] Aryl-
tributylstannane 4 is a key synthetic intermediate for the syn-
thesis of [11C]-1 by rapid C-[11C]methylation (Scheme 1a). Initial-
ly, synthesis of 4 was examined via palladium-catalyzed tribu-
tylstannylation of aryl iodide 3.[24] Unsymmetrically substituted
4,4’-diaminodiphenylmethane 2 was prepared from ortho-tolui-
To investigate the BBB permeability and the biodistribution
of [11C]-1, consecutive PET scans of the brain and whole body
of Sprague–Dawley rats (n=4) were conducted. A 90 minute
emission scan of the brain revealed that, after intravenous ad-
Scheme 1. Synthesis of aryltributylstannane 4 and radiosynthesis of 11C-labeled GN8 derivative [11C]-1 by palladium-catalyzed rapid methylation with [11C]-
CH3I. Reagents and conditions: a) CH3OH, reflux, 18 h, 22%; b) 1. BrCH2C(O)Br, pyridine, DMAP, CH2Cl2, 258C, 3 h; 2. pyrrolidine, K2CO3, THF, 608C, 12 h, 69%
(two steps); c) (Bu3Sn)2, Pd(PPh3)4 (10 mol%), toluene, reflux, 48 h, 10%; d) (Bu3Sn)2, Pd(PPh3)4 (5 mol%), toluene, 1008C, 24 h, 67%; e) ClCH2C(O)Cl, Et3N,
CH2Cl2, 08C, 10 min, 83%; f) pyrrolidine, K2CO3, THF, 608C, 12 h, 92%; g) Pd2(dba)3, (o-tolyl)3P, CuCl, K2CO3, NMP, 808C, 5 min.
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ministration of [11C]-1, radioactivity was observed in the pituita-
ry gland, pineal body, cerebral cortex, and choroid plexus (Fig-
ure 2a). Around the head and neck, accumulation of [11C]-
1 was observed in the thyroid gland, submandibular gland,
and Harderian gland (Figure 2a). Whole-body PET images indi-
cated that [11C]-1 uptake was scanned in the lung, liver, kidney,
intestine, and spleen (Figure 2b). After the PET scan, the
Table 1. Biodistribution data for [11C]-1.
Tissue
ID [%][a]
Lung
Kidney
Spleen
Liver
Brain
Heart
Muscle
Fat
4.98Æ0.08
2.17Æ0.11
2.11Æ0.11
1.21Æ0.06
0.36Æ0.01
0.23Æ0.03
0.093Æ0.005
0.04Æ0.02
0.03Æ0.002
Blood
[a] Data are the percent injected dose per gram of tissue Æ standard
error of the mean (SEM) of n=4 independent experiments.
Figure 3. a) Photographic images of coronal sections of rat brain, and b) the
corresponding auto-radiographic images at 60 min postinjection of [11C]-1.
Abbreviations: pineal body (P.B.) and pituitary gland (P.G.).
Figure 2. Positron emission tomography (PET) images of the brain and
whole body. a) Maximum a posteriori probability (MAP) algorithm recon-
structed static image taken 5–90 min after [11C]-1 injection (sagittal). b) Maxi-
mum intensity projection (MIP) image of 90–120 min after [11C]-1 injection.
Standardized uptake values (SUV) are a semi-quantitative measure, derived
from determination of tissue activity obtained from a single static image.
Transition-metal-catalyzed cross-coupling reactions are useful
methods for constructing carbon–carbon bonds with excellent
functional group tolerance.[26] We adopted palladium(0)-medi-
ated rapid C-[11C]-methylation using arylstannane
4 and
organs of the animal were dissected and the radioactivity of
each tissue was measured with a gamma counter.
[11C]methyl iodide as a 11C-labeling method,[19–23] since the con-
ventional palladium-catalyzed cross-coupling reaction generally
takes several hours. In addition, in practical terms, the 11C-label-
ing must typically be carried out with small quantities of
[11C]methyl iodide (less than micromolar amounts) and excess
amounts of stannyl precursor (greater than micromolar
amounts). Thus, the palladium(0)-mediated rapid C-
[11C]methylation is an efficient method for 11C-labeling that can
meet the chemically difficult demands of radiolabeling condi-
tions.[27,28]
The tissue distribution data were in good agreement with
the PET images, and the radioactivity of the brain was found
to be higher than that of the blood or muscle (Table 1). These
results demonstrated that [11C]-1 administered intravenously
crosses the BBB and is retained in the brain.
To evaluate the degradation of 1 in vivo, we monitored its
intravenous as well as intrabrain concentration in a time-de-
pendent manner using a gas chromatography–mass spectrom-
etry (GC/MS). The results showed that the elimination half-life
of 1 is much longer than 24 hours, indicating that radioactivity
is not originated from the degraded [11C]-1 (data not shown).
To confirm the distribution in brain, [11C]-1 was injected into
rats, which were then sacrificed 40 minutes after administra-
tion. Auto-radiographic images of coronal sections of rat brain
at 60 minutes post-injection are shown in Figure 3 along with
their photographs. The auto-radiographic images clearly dem-
onstrated that [11C]-1 is primarily localized in the cerebral
cortex, pineal body, pituitary gland, and choroid plexus.
Introduction of 11C-lableled methyl group through a carbon–
carbon bond is fascinating because it is resistant to in vivo me-
tabolism in marked contrast to carbon–heteroatom bonds.
As mentioned above, to date, a variety of compounds have
already been identified as antiprion compounds in TSE-infect-
ed cells.[3,4] However, most of these compounds were ineffec-
tive in vivo, and only a limited number of compounds includ-
ing amphotericin B and its derivative, pentosan polysulfate,
and porphyrin derivatives have been reported to be effective
in TSE-infected animals.[29–31] In clinical trials, pentosan polysul-
fate had to be administered intraventricularly since it did not
penetrate the BBB, and no apparent improvement of clinical
features was observed in the patients treated with this
agent.[32] Treatment with quinacrine also failed to provide
a therapeutic benefit, rather, it led to liver dysfunction.[33–35]
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Supporting Information: Instrument details, synthetic protocols
and characterization data including NMR spectra for compounds
1–6 and the radiosynthesis of [11C]-1 are given in the Supporting
Information along with the ex vivo assay procedure for evaluation
in GT+FK cells.
These agents were originally developed for the treatment of
interstitial cystitis and malaria, respectively, and would not be
suitable for the treatment of CNS disorders. Therefore, the
pharmacokinetic properties, especially the BBB permeability, of
the compounds should be carefully considered in drug devel-
opment for prion diseases.[36,37] Previously, we reported that
the subcutaneous administration of GN8 prolonged the life-
time of prion-infected mice, indicating that GN8 can enter the
brain across the BBB. In this study, we confirmed that [11C]-1 ad-
ministered intravenously also reaches the brain.
Acknowledgements
The authors thank Ms. Tomomi Saeki and Ms. Miku Yamada
(Center for Emerging Infectious Diseases, Gifu University) for out-
standing research assistance. The authors also thank Dr. Hiroko
Koyama (United Graduate School of Drug Discovery and Medical
Information Science, Gifu University) for helpful discussion. This
work was supported by the Program for the Promotion of Funda-
mental Studies in Health Sciences of the National Institute of Bio-
medical Innovation and the Molecular Imaging Research Pro-
gram of the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
In summary, an 11C-labeled GN8 derivative [11C]-1, in which
an 11C-labeled methyl group was connected to the aromatic
ring via a carbon–carbon bond, was successfully synthesized
by
palladium-catalyzed
rapid
methylation
of
aryl-
(tributyl)stannane with [11C]methyl iodide. PET analysis using
the 11C-labeled compound unequivocally demonstrated that
the GN8 derivative penetrated into the brain. These findings
will facilitate further refinement of GN8 as a therapeutic agent
for prion diseases. Further studies on the application of the
11C-labeled GN8 derivative as a molecular imaging probe for
detecting cellular prion protein[38–40] in vivo are currently ongo-
ing and will be reported in due course.
Keywords: antiprion agents · brain uptake · positron emission
tomography (PET) · prion diseases · radiolabeling
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Experimental Section
PET studies using rats: Male Sprague–Dawley rats (Japan SLC, Inc.,
Hamamatsu, Shizuoka, Japan) at 8–9 weeks old and weighing ap-
proximately 250 g each were used in the study. All PET scans were
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Received: April 18, 2013
Published online on May 24, 2013
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