PET imaging of nobiletin based on a practical total synthesis

Article abstract of DOI:10.1039/c0cc04936k

A practical synthesis of nobiletin, a polymethoxylated citrus flavone, was accomplished by utilizing our novel flavone synthesis. Synthetic nobiletin was labelled by selective demethylation and rapid incorporation of ¹¹C atom. Positron emission tomography images successfully visualized the brain distribution, which may provide therapeutic benefits in the treatment of Alzheimer's disease. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0cc04936k

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 2868–2870
www.rsc.org/chemcomm
COMMUNICATION
PET imaging of nobiletin based on a practical total synthesisw
Tomohiro Asakawa,a Aiki Hiza,a Miho Nakayama,a Makoto Inai,a Dai Oyama,a
a
Hiroyuki Koide,a Kosuke Shimizu,a Toshiyuki Wakimoto,z Norihiro Harada,b
Hideo Tsukada,b Naoto Okua and Toshiyuki Kan*a
Received 12th November 2010, Accepted 20th December 2010
DOI: 10.1039/c0cc04936k
A practical synthesis of nobiletin, a polymethoxylated citrus
flavone, was accomplished by utilizing our novel flavone synthesis.
Synthetic nobiletin was labelled by selective demethylation and
rapid incorporation of 11C atom. Positron emission tomography
images successfully visualized the brain distribution, which may
provide therapeutic benefits in the treatment of Alzheimer’s
disease.
1: labelled compound 1 must be intact during PET imaging,
and fast synthetic procedures are necessary for the preparation
of labeled-1. We recently developed an efficient synthetic route
to the flavone skeleton through a b-diketone intermediate,7
which would fulfil these criteria. Here, we report the practical
total synthesis of 1, conversion to a 11C-PET probe, and PET
imaging analysis.
The heart of our synthetic plan is illustrated in Scheme 1.
Because construction of the flavone ring of 1 is performed by
condensation of 3 and 48 and subsequent cyclization from the
b-diketone intermediate 2, preparation of the polymethoxy
acetophenone derivatives 3 would be significant work.
As shown in Scheme 2, the preparation of methoxy aceto-
phenone commenced from 1,3,4,5-tetramethoxybenzene (5)
according to the reported procedure.7 5 was subjected to the
Friedel–Crafts acylation with acetyl chloride, and aluminium
trichloride. The concomitant demethylation and subsequent
O-methylation of the resultant phenol gave tetramethoxy-
acetophenone 6. Conversion to the phenol from the acyl group
was performed by the selenium dioxide-catalyzed Baeyer–
Villiger oxidation reaction. After hydrolysis of the acetate, a
labile phenol was methylated by treatment with methyl iodide
in the presence of potassium carbonate under an argon
atmosphere to give 7. Subsequent Friedel–Crafts reaction of
8 also accompanied the demethylation reaction to give the
acetophenone 3.9 Although the desired A-ring unit was
obtained, we investigated a more practical route using the
Few pharmacodynamic studies of synthetic labelled forms of
dietary flavonoids have been reported. The lack of comprehensive
data from such studies has hindered the precise modelling of
in vivo dynamics, even though several flavonoids have shown
positive health effects both in vitro and in vivo.1 Nobiletin (1),
isolated as a polymethylated flavone from citrus fruits,2 has
received special attention for its remarkable biological activity
enhancing PKA/ERK/CREB signalling in cell culture systems,
including cultured rat hippocampal neurons.3,4
This promising activity may provide therapeutic benefits for
the treatment of Alzheimer’s Disease (AD), and the restrictions
imposed by the blood–brain barrier may be circumvented.
However proof that 1 distributes throughout regions of the brain
is still lacking.5 Positron emission tomography (PET) imaging
studies would be appropriate for such analysis, although
they have not been applied to pharmacodynamic studies of
flavonoids. Thus, a PET probe of 1 would be significant
for analyzing the distribution and bioavailability of dietary
flavonoids.
Securing a supply of 1 sufficient for such studies has been
difficult because isolation of pure 1 from natural sources
requires labor-intensive and time-consuming processes.
Therefore, an efficient and flexible synthesis of 1 would
facilitate further development of this drug candidate.6 Two
critical criteria must be considered regarding the dynamics of
a School of Pharmaceutical Sciences, University of Shizuoka and
Global COE Program, 52-1 Yada, Suruga-ku, Shizuoka 422-8526,
Japan. E-mail: kant@u-shizuoka-ken.ac.jp; Fax: +81 54-264-5745;
Tel: +81 54-264-5746
b Central Research Laboratory, Hamamatsu Photonics K. K.,
5000 Hirakuchi, Hamakita-ku, Hamamatsu-shi, Shizuoka 434-8601,
Japan
w Electronic supplementary information (ESI) available: Experimental
Procedures and NMR spectral data. See DOI: 10.1039/c0cc04936k
z Present address: Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Scheme 1 Structure and synthetic strategy of nobiletin (1).
c
2868 Chem. Commun., 2011, 47, 2868–2870
This journal is The Royal Society of Chemistry 2011

View Article Online
Scheme 4 Synthesis of nobiletin (1).
Scheme 2 Synthesis of acetophenone 3 from 5.
Scheme 5 Conversion to the 11C-labeled nobiletin ([11C]-1).
possesses six methoxy groups,13 regioselective demethylation
could be useful for 11C substitution. Enough amount of
synthetic 1 allowed for an optimization of reaction conditions
to convert the corresponding radioactive compound. Upon
treatment under Node’s conditions,14 selective demethylation
at the 5-position of 1 proceeded smoothly to provide 5-desmethyl
nobiletin (13), as shown in Scheme 5. Presumably, this
reaction proceeded via coordination of the aluminium to the
ketone group. Radiosynthesis of the 11C-labeled compound
was subsequently carried out using an automated synthesis
apparatus. Treatment of 13 with [11C]-methyl iodide and
Scheme 3 Improved synthesis of pentamethoxybenzene 8.
inexpensive starting material 9 and avoiding the labile inter-
mediate 7.
tetrabutylammonium hydroxide furnished a methylation
reaction that, within 1 min, afforded the 11C-labeled nobiletin
([11C]-1). Purification of [11C]-1 was performed by HPLC, as
shown in ESIw.1
As shown in Scheme 3, the improved synthesis of penta-
methoxybenzene (8) started from 1,2,3-trimethoxybenzene
(9).10 Stepwise incorporation of the hydroxyl group was
achieved by sequential formylation and Baeyer–Villiger
oxidation reactions.11 Twice similar formylation reactions12
yielded 9 and 11, respectively, but different reaction conditions
were suitable for each. Fortunately, the phenol 12 was more
stable than 7, even though it featured the regioisomer
of a tetramethoxy phenol. The practical synthesis of 8 was
accomplished from 9 in 79% yield without the need for
chromatographic purification.
With the desired 11C-labeled nobiletin ([11C]-1) in hand, we
investigated the biodistribution of [11C]-1 in a rat model,
focusing on accumulation in the brain by imaging with a
Clairvivo PET system (Shimadzu Corporation). The solution
of [11C]-1 was intravenously injected into an 8-week-old SD
male rat via the tail vein, and PET scans were collected during
the following 60 min.
As shown in Fig. 1, accumulation of the positron emitter in
the brain was observed 5 min after injection although the
accumulation gradually decreased thereafter. Accumulation in
each organ demonstrated the transient accumulation of [11C]-1
in the brain (Fig. 2) and higher levels of accumulation in the
liver (Fig. 3). These observations suggested that nobiletin may
confer potential beneficial effects, not only for brain function,
but also for the treatment of AD because some flavonoids
have been shown to prevent fibril formation.15 Recently,
accumulation of 40-demethylnobiletin was detected by
UV-HPLC analysis of brain tissue.5 We thereby designed the
[11C]-nobiletin probe by replacing the methyl group of the
5-O-Me moiety to avoid false detection by endogenous
metabolism of 1. Our findings revealed that the 1 or its
metabolite was distributed in the brain.
According to our flavone synthesis, the benzotriazole
derivative 3 is a suitable acyl donor as well as readily obtained
from 3,4-dimethoxybenzoyl chloride. Upon treatment of 3 and
4 with LiHMDS at 0 1C, the desired C-acylation reaction
proceeded smoothly to give the desired b-diketone 2.
Subsequent cyclization and dehydration were performed by
heating in methanol with trifluoroacetic acid to afford 1, as
shown in Scheme 4. In summary, the total synthesis of 1
proceeded in 11 steps, starting from 9, in 35% total yield.
Next, we turned our attention toward incorporation of 11C
atoms into the synthesized 1 to prepare a PET probe. Although
PET imaging studies provide a powerful methodology for
investigating the bioavailability and pharmacodynamics
of biologically active compounds, the short half-life of the
radioisotope has prevented general application. Because 1
In conclusion, we have accomplished the efficient total
synthesis of nobiletin. Because our synthetic route did not
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2868–2870 2869

View Article Online
Fig. 1 PET imaging of [11C]-1 accumulation in the rat brain. [11C]-1 was intravenously injected via the tail vein of an 8-month-old male SD rat.
The distribution of [11C]-1 was imaged using a Clairvivo PET system (Shimadzu Corporation). The data were acquired for 60 min and integrated
every 5 min. The figure shows a CT image (A) and representative PET images obtained in the experiment (B). White arrows indicate the brain
region.
This work was supported by a grant from the Research and
Development Projects for Application in Promoting New
Policy of Agriculture, Forestry and Fisheries from the
Ministry of Agriculture, Forestry and Fisheries, and by a
Grant-in-Aid for Scientific Research on Priority from the
Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan. We also thank Professors Yasushi Ohizumi
and Shizuo Yamada (School of Pharmaceutical Sciences,
University of Shizuoka) for valuable discussions.
Notes and references
Fig. 2 Accumulation of [11C]-1 after intravenous injection into an SD
1 A. Scalbert, C. Manach, C. Morand, C. Remesy and L. Jimenez,
´ ´ ´
Crit. Rev. Food Sci. Nutr., 2005, 45, 287.
2 K. Tseng, J. Chem. Soc., 1938, 1003.
3 A. Murakami, Y. Nakamura, K. Torikai, T. Tanaka, T. Koshiba,
K. Koshimizu, S. Kuwahara, Y. Takahashi, K. Ogawa, M. Yano,
H. Tokuda, H. Nishino, Y. Mikami, Y. Sashida, S. Kitanaka and
H. Ohigashi, Cancer Res., 2000, 60, 5059.
rat (accumulation in the brain). The time–activity curve of 11C in the
brain was calculated from the mean voxel intensity in the region of
interest of the images.
4 H. K. Rooprai, A. Kandanearatchi, S. L. Maidment, M. Christidou,
G. Trillo-Pazos, D. T. Dexter, G. J. Rucklidge, W. Widmer and
G. J. Pilkington, Neuropathol. Appl. Neurobiol., 2001, 27, 29.
5 M. Al Rahim, A. Nakajima, D. Saigusa, N. Tetsu, Y. Maruyama,
M. Shibuya, H. Yamakoshi, Y. Tomioka, Y. Iwabuchi,
Y. Ohizumi and T. Yamakuni, Biochemistry, 2009, 48, 7713.
6 M. Tsukayama, Y. Kawamura, T. Ishizuka, S. Hayashi and
F. Torii, Heterocycles, 2003, 60, 2775.
7 T. Furuta, M. Nakayama, H. Suzuki, H. Tajimi, M. Inai,
H. Nukaya, T. Wakimoto and T. Kan, Org. Lett., 2009, 11, 2233.
8 (a) A. R. Katritzky, N. Shobana, J. Pernak, A. S. Afridi and
W.-Q. Fan, Tetrahedron, 1992, 48, 7817; (b) A. R. Katritzky and
A. Pastor, J. Org. Chem., 2000, 65, 3679; (c) T. Furuta,
Y. Hirooka, A. Abe, Y. Sugata, M. Ueda, K. Murakami,
T. Suzuki, K. Tanaka and T. Kan, Bioorg. Med. Chem. Lett.,
2007, 17, 3095.
9 Although excess AlCl3 should be required to complete the reaction,
excess reagent caused decomposition of the phenol 3. Thus, the
reaction was stopped before all starting material 8 had reacted.
Separation of 3 and 8 was readily performed by basic aqueous
work up.
10 Although both starting materials are commercially available, 9 was
rather inexpensive ($29.6 in 25 g) from Aldrich, as compared to 5
($78.4 in 5 g).
11 T. Fukuyama and L. Yang, Tetrahedron Lett., 1986, 27, 6299.
12 G. Jones and S. P. Stanforth, Org. React., 2000, 56, 355.
13 N. Koga, M. Matsuo, C. Ohta, K. Haraguchi, M. Matsuoka,
Y. Kato, T. Ishii, M. Yano and H. Ohta, Biol. Pharm. Bull., 2007,
30, 2317.
Fig. 3 Accumulation of [11C]-1 after intravenous injection into SD
rats (each organ). [11C]-1 was intravenously injected into SD rats (n = 4),
and the accumulation of 11C in each organ at 5 min after the injection
was measured using a g counter. Data are presented as the percent of
the injected dose per whole tissue and S.D.
require tedious purification steps, it enabled preparation of 1
in our laboratory on a scale in excess of 5 grams. Furthermore,
conversion to the PET probe was accomplished by rapid
incorporation of 11C atoms into the synthetic 1. To our
knowledge, this is the first report of direct PET analysis of
intact natural flavonoid in vivo distribution. Thus, these results
will contribute not only to the development of a new drug
candidate for AD, but also to the studies of bioavailability and
pharmacodynamic of natural flavonoids having positive
health effects. A more detailed PET analysis of the distribution
of 1 is currently being investigated in our laboratories, and the
results will be reported in due course.
14 M. Node, K. Nishida, K. Fuji and E. Fujita, J. Org. Chem., 1980,
45, 4275.
15 (a) H. Kim, B. S. Park, K. G. Lee, C. Y. Choi, S. S. Jang, Y. H. Kim
and S. E. Lee, J. Agric. Food Chem., 2005, 53, 8537; (b) T.
Akaishi, T. Morimoto, M. Shibao, S. Watanabe, K. Sakai-Kato,
N. Utsunomiya-Tate and K. Abe, Neurosci. Lett., 2008, 444, 280.
c
2870 Chem. Commun., 2011, 47, 2868–2870
This journal is The Royal Society of Chemistry 2011