Concise synthesis of polymethoxyflavone sudachitin and its derivatives, and biological evaluations

Article abstract of DOI:10.1016/j.tetlet.2018.03.064

We accomplished a divergent synthesis of sudachitin (2), a polymethoxyflavone isolated from citrus fruits, and six derivatives from acetophenone 9, which was an intermediate in our previous synthesis of nobiletin (1). Compound 2 enhanced glucose-induced i

Full text of DOI:10.1016/j.tetlet.2018.03.064

Accepted Manuscript
Concise synthesis of polymethoxyflavone sudachitin and its derivatives, and
biological evaluations
Hiroto Sagara, Masaki Kanakogi, Yuki Tara, Hitoshi Ouchi, Junko Kimura,
Yukiko Kaneko, Makoto Inai, Tomohiro Asakawa, Tomohisa Ishikawa,
Toshiyuki Kan
PII:
S0040-4039(18)30391-5
https://doi.org/10.1016/j.tetlet.2018.03.064
TETL 49833
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
21 February 2018
15 March 2018
22 March 2018
Please cite this article as: Sagara, H., Kanakogi, M., Tara, Y., Ouchi, H., Kimura, J., Kaneko, Y., Inai, M., Asakawa,
T., Ishikawa, T., Kan, T., Concise synthesis of polymethoxyflavone sudachitin and its derivatives, and biological
evaluations, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.03.064
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Tetrahedron Letters
Concise synthesis of polymethoxyflavone sudachitin and its derivatives, and
biological evaluations
Hiroto Sagaraa , Masaki Kanakogia , Yuki Taraa , Hitoshi Ouchia , Junko Kimuraa , Yukiko Kanekoa ,
Makoto Inaia , Tomohiro Asakawab , Tomohisa Ishikawaa and Toshiyuki Kana, 
a
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
Tokai University Institute of Innovative Science and Technology, 4-1-1, Kitakaname, Hiratsuka-city, Kanagawa 259-1292, Japan
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We accomplished a divergent synthesis of sudachitin (2), a polymethoxyflavone isolated from
citrus fruits, and six derivatives from acetophenone 9, which was an intermediate in our previous
synthesis of nobiletin (1). Compound 2 enhanced glucose-induced insulin secretion in INS-1D
cells, but was less potent than 1. Compared with 1, compound 2 and two derivatives were more
potent inhibitors of cAMP-specific phosphodiesterase.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Sudachitin
Nobiletin
Flavone synthesis
Regioselective demethylation
Benzotriazole
Polymethylated flavones from citrus fruits1 have a variety of
interesting physiological actions, including anti-inflammatory,
anti-cancer, anti-apoptosis, anti-dementia and neuroprotective
activities. Therefore, practical and flexible synthetic methods are
needed to obtain sufficient amounts of derivatives for testing.
During the course of our investigation of flavone synthesis,2 we
accomplished a practical and economical synthesis of nobiletin
(1) on a hundred-gram scale.3, 4 Here, we turned our attention to
OMe
RO
OH
O
9 (R = Me)
10 (R = H)
11 (R = Bn)
LiCl, DMF, 120 °C, 59% (brsm 70%)
BnBr, K2CO3, DMF, 79%
MeO
MeO
OMe
OR'
OMe
OMe
N
N
BnO
MeO
OH
O
OBn
LiHMDS
THF
N
+
11
O
–78 °C to 0 °C
sudachitin (2), which is
a
5,7,4’-tridesmethyl nobiletin
MeO
O
12 (R' = Bn)
13 (R' = Me)
87%
derivative.5-7 Although synthesis and biological evaluation of
desmethyl derivatives on the A-ring of nobiletin have been
reported by Oshitari and Natsugari’s group, 8 there has been no
detailed comparison of the biological activities of 1 and 2.
Furthermore, in order to elucidate the biosynthetic pathway of 2
from 1, we planned to synthesize the possible biogenesis
intermediates 3–8 using our established methodology.2
14
OMe
OAc
OMe
1) Ac2O, DMAP
AcO
O
O
NH3
TsOH
pyridine
2
3
MeOH
69%
toluene
80 °C
93%
2) AlCl3, EtSH
CH2Cl2
MeO
HO
67% (2 steps)
15
Scheme 1. Synthesis of sudachitin (2).
As shown in Scheme 1, synthesis of sudachitin (2) was
commenced with acetophenone 9, which we have previously
synthesized on a large scale.3 Upon treatment of 9 with LiCl,
regioselective demethylation reaction occurred at the 4-methoxy
group to provide 10.9 Further treatment with benzyl bromide and
K2CO3 resulted in regioselective incorporation of the benzyl
group at the 4-hydroxy group to afford 11. In the presence of
LiHMDS, condensation reaction of acetophenone 11 and
Figure 1. Structure of nobiletin (1), sudachitin (2) and their demethyl
derivatives 3–8.
———
 Corresponding author. Fax: +81-54-264-5745; e-mail: kant@u-shizuoka-ken.ac.jp

2
Tetrahedron
acylbenzotriazole 12,10 which was readily obtained from 4-
Compound
2
is known to inhibit cAMP-specific
hydroxy-3-methoxybenzoic acid and benzotriazole,11 proceeded
smoothly to give 14. Without purification, acidic treatment of 14
phosphodiesterase (PDE). The intracellular second messenger
cAMP directly modulates protein kinase A (PKA) and exchange
protein directly activated by cAMP (Epac), which regulate
various cellular signal transduction pathways. cAMP is
catabolized by PDE, resulting in down-regulation of cAMP/PKA
and cAMP/Epac signaling. Hence, we used an established assay
to evaluate the inhibitory effects of 2 and its derivatives 3–8 on
PDE activity, with nobiletin (1) as a positive control.15, 16 We
found that 2 and its derivatives inhibited PDE activity, with the
rank order of 2 = 3 > 5 > 1 > 8 >> 4 > 6 > 7 at the concentration
of 10 μM, as shown in Figure 3.
resulted in simultaneous cyclization and dehydration in one pot
with concomitant cleavage of the benzyl ether to afford 3. After
acetylation of the phenol groups of 3, removal of the methyl
group was carried out by means of Node’s protocol.12 Treatment
with EtSH in the presence of AlCl3 resulted in regioselective
removal of the methyl group to afford 15. In this reaction,
chelation of the Lewis acid by the carbonyl group played a key
role in the regioselectivity and reactivity.9 Finally, ammonolysis
of the acetate provided 2. All spectral data (1H NMR, 13C NMR
and HRMS) were in full agreement with reported values.
Figure 3. Inhibitory effects of polymethylated flavones on PDE activity.16
With 2 in hand, we next turned our attention to the synthesis
of the other desmethyl nobiletin derivatives as shown in Scheme
2. Firstly, 4’-desmethyl derivatives 4 and 5 were synthesized by
condensation of 9 and 12 in the same manner as described for the
synthesis of 2 and 3. Selective deprotection of the benzyl ether of
16 by hydrogenation provided 4 and simultaneous removal of the
benzyl and 5-methyl groups was performed by treatment with
EtSH and AlCl3 to give 5. Compound 6 was readily obtained
from 1 by applying our methodology. Condensation of 11 and 13
provided 7, which was readily converted to 8 by similar
treatment to that employed for 2.
In conclusion, we have developed efficient and flexible
methodology for the synthesis of methylated flavone derivatives.
Free hydroxyl group(s) in these compounds should be suitable
for incorporation of various probes moieties, which would be
useful for chemical-biology studies.2b Detailed evaluation of the
biological activities and biosynthetic route to 2 from 1 are in
progress.
Acknowledgments
H2, Pd/C
1) LiHMDS, THF
–30 °C to 0 °C
91%
OBn
OMe
4
OMe
We thank Dr. Kazuo Okamoto, CEO of Ushio ChemiX
Corporation for kindly providing acetophenone 9 (synthetic
intermediate for nobiletin). This work was financially supported
by MEXT/JSPS KAKENHI Grant Numbers JP17H03973 and
JP17K15424, Grants-in-Aid for Scientific Research on Priority
Areas JP16H01160 from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan, and the
Platform Project for Supporting in Drug Discovery and Life
Science Research (Platform for Drug Discovery, Informatics and
Structural Life Science) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT).
EtOAc
93%
MeO
MeO
O
+
12
9
2) TsOH, toluene
80 °C, 92%
AlCl3, EtSH
5
MeO
O
CH2Cl2
47%
16
AlCl3, EtSH, CH2Cl2
6
1
0 °C to rt, 76%
1) LiHMDS, THF
–78 °C to 0 °C
70%
1) Ac2O, DMAP, pyridine
2) AlCl3, EtSH, CH2Cl2
0 °C to rt, 80% (2 steps)
7
8
+
11
13
2) TsOH, toluene
80 °C, 63%
3) K2CO3, MeOH, 99%
References and notes
Scheme 2. Synthesis of demethyl nobiletin derivatives 4–8.
1. (a) Tseng, KF. J. Chem. Soc. 1938, 1003. (b) Lai, CS.; Li, S.;
Chai, CY.; Lo, CY.; Dushenkov, S.; Ho, CT.; Pan, MH.; Wang,
YJ. Carcinog. 2008, 29, 2415–2424. (c) Li, S.; Wang, H.; Guo, L.;
Zhao, H.; Ho, CT. J. Funct. Foods 2014, 6, 2–10.
2. Our flavone synthesis: (a) Furuta, T.; Nakayama, M.; Suzuki, H.;
Tajimi, H.; Inai, M.; Nukaya, H.; Wakimoto, T.; Kan, T. Org.
Lett. 2009, 11, 2233–2236. (b) Hiza, A.; Tsukaguchi, Y.; Ogawa,
T.; Inai, M.; Asakawa, T.; Hamashima, Y.; Kan, T. Heterocycles
2014, 88, 1371–1396.
3. Our nobiletin synthesis: Asakawa, T.; Hiza, A.; Nakayama, M.;
Inai, M.; Oyama, D.; Koide, H.; Shimizu, K.; Wakimoto, T.;
Harada, N.; Tsukada, H.; Oku, N.; Kan, T. Chem. Commun. 2011,
47, 2868–2870.
Next, we compared the biological activity of 1 and 2 to
enhance insulin secretion from rat β-cell line INS-1D. Compound
2 significantly enhanced glucose-induced insulin secretion at 100
μM, but not at 10 μM. Since we previously showed that 1
significantly promotes glucose-induced insulin secretion at 10
μM,13 2 seems to be slightly less potent than 1, as shown in
Figure 2.14
Figure 2. Effect of sudachitin (SDC) 2 on glucose-induced insulin secretion
in INS-1D cells. The effect of nobiletin (NOB) 1 (100 μM) is also shown for
comparison.

4. See Wako Pure Chemicals Product List. The price of synthetic
nobiletin is JPY 55,000 per 500 mg, compared with JPY 20,000
per 10 mg for the natural product.
5. Isolation and structure determination of sudachitin (2); Horie, T.;
Masumura, M.; Okumura, FS. Bull. Chem. Soc. Jpn. 1961, 34,
1547–1548.
11. (a) Katritzky, AR.; Shobana, N.; Pernak, J.; Afridi, AS.; Fan, WQ.
Tetrahedron 1992, 48, 7817–7822. (b) Katritzky, AR.; Pastor, A.
J. Org. Chem. 2000, 65, 3679–3682.
12. Node, M.; Nishida, K.; Fuji, K.; Fujita, E. J. Org. Chem. 1980, 45,
4275–4277.
6. Synthesis of sudachitin; Farkas, L.; Nogradi, M.; Zubovics, Z.
Acta Chim. Acad. Sci. Hung. 1967, 52, 301–304.
13. Takii, M.; Kaneko, YK.; Akiyama, K.; Aoyagi, Y.; Tara, Y.;
Asakawa, T.; Inai, M.; Kan, T.; Nemoto, K.; Ishikawa, T. J.
Funct. Foods 2017, 30, 8–15.
14. INS-1D cells were incubated for 1 h in HEPES-buffered Krebs
solution containing 2.8 or 11.1 mM glucose in the absence or
presence of the indicated concentrations of sudachitin (2) and
nobiletin (1). Each value is the mean ± SEM of eight independent
experiments. **P < 0.01 vs. 11.1 mM glucose without any
addition.
15. Nagase, H.; Yamakuni, T.; Matsuzaki, K.; Maruyama, Y.;
Kasahara, J.; Hinohara, Y.; Kondo, S.; Mimaki, Y.; Sashida, Y.;
Tank, AW.; Fukunaga, K.; Ohizumi, Y. Biochemistry 2005, 44,
13683–13691.
16. The assays were performed using a cyclic nucleotide PDE assay
kit (Enzo Life Sciences) according to the manufacturer’s protocol.
The 3’,5’-cAMP substrate was cleaved by cAMP-specific PDE,
resulting in the release of 5’-AMP. The 5’-nucleotidase cleaved
the released 5’-AMP to nucleoside and phosphate. Phosphate was
determined with a colorimetric assay using BIOMOL® GREEN
reagent. The final concentration of DMSO was 1% (v/v) and the
DMSO control was subtracted from the values of test compounds.
The data were analyzed using a one-way ANOVA with Tukey’s
multiple-comparison test. C: control (absence of the test
compounds and DMSO), IB: 3-isobutyl-1-methylxanthine (IBMX:
PDE inhibitor, at a concentration of 40 μM), NOB (1): nobiletin
(1), SDC (2): sudachitin (2). Histogram indicated the mean ±
SEM. *P < 0.05, ***P < 0.001 vs. control.
7. Recent evaluation of biological activities of sudachitin, (a)
Tsutsumi, R.; Yoshida, T.; Nii, Y.; Okahisa, N.; Iwata, S.;
Tsukayama, M.; Hashimoto, R.; Taniguchi, Y.; Sakaue, H.;
Hosaka, T.; Shuto, E.; Sakai, T. Nutr. Metab. 2014, 11:32. (b)
Xing, TT.; Zhao, XJ.; Zhang, YD.; Li, YF. J. Agric. Food Chem.
2017, 65, 2615–2627.
8. Oshitari, T.; Okuyama, Y.; Miyata, Y.; Kosano, H.; Takahashi, H.;
Natsugari, H. Bioorg. Med. Chem. Lett. 2011, 21, 4540–4544.
9. Regioselective demethylation reaction of 9 and 3 proceeded
through the intermediates 17 and 18.
OMe
OMe
AcO
O
O
Ar
Li
O
OH
+ MeSEt
+ MeCl
MeO
MeO
O
OMe O
17
Al
18
10. Synthesis of 12 from 19 was performed as follows, similarly to the
preparation of 13.
OMe
OMe
1) SOCl2, MeOH
2) BnBr, K2CO3, DMF
OH
OBn
N
N
N
HO
3) NaOH, MeOH, reflux
4) EDCI, benzotriazole
CH2Cl2
O
O
19
12
66% (4 steps)

4
Tetrahedron
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Concise synthesis of polymethoxyflavone
sudachitin and its derivatives, and biological
evaluations
Leave this area blank for abstract info.
Hiroto Sagara, Masaki Kanakogi, Yuki Tara, Hitoshi Ouchi, Junko Kimura, Yukiko Kaneko, Makoto Inai,
Tomohiro Asakawa, Tomohisa Ishikawa, Toshiyuki Kan
OMe
OH
OMe
OMe
7 steps
MeO
MeO
OH
O
HO
O
O
17.4% yield
MeO
MeO
HO




The divergent total synthesis of sudachitin and
its derivatives were accomplished.
Sudachitin enhanced glucose-induced insulin
secretion in INS-1D cells.
Sudachitin and two derivatives were inhibited
a cAMP-specific phosphodiesterase.