Nobiletin metabolites: Synthesis and inhibitory activity against matrix metalloproteinase-9 production

Article abstract of DOI:10.1016/j.bmcl.2011.05.121

A divergent synthesis of nobiletin metabolites was developed through highly oxygenated acetophenone derivative. We used commercially available methyl 3,4,5-trimethoxybenzoate as a starting material for concise preparation of the key intermediate, 2′-hydro

Full text of DOI:10.1016/j.bmcl.2011.05.121

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4540–4544
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Nobiletin metabolites: Synthesis and inhibitory activity against matrix
metalloproteinase-9 production
⇑
⇑
Tetsuta Oshitari , Yuji Okuyama, Yoshiki Miyata, Hiroshi Kosano, Hideyo Takahashi, Hideaki Natsugari
School of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Midori-ku, Sgamihara, Kanagawa 252-5195, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A divergent synthesis of nobiletin metabolites was developed through highly oxygenated acetophenone
derivative. We used commercially available methyl 3,4,5-trimethoxybenzoate as a starting material for
concise preparation of the key intermediate, 20-hydroxy-30,40,50,60-tetramethoxyacetophenone (I). These
metabolites showed strong inhibitory activity against matrix metalloproteinase-9 production in human
lens epithelial cells.
Received 31 March 2011
Revised 27 May 2011
Accepted 31 May 2011
Available online 12 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Nobiletin
Polymethoxyflavone
Ulmann reaction
Baker–Venkataraman rearrangement
Matrix metalloproteinase-9
Nobiletin (30,40,5,6,7,8-hexamethoxyflavone) (1), isolated from
citrus fruits, has a broad spectrum of health-promoting properties
including anticancer,1 antimetastatic,2 antiinflammatory,3 antidia-
betic,4 and neurotrophic activities.5 In particular, increasing atten-
tion has been paid to its antitumor metastatic activity due to the
inhibition of gene expression and production of some matrix
metalloproteinases (MMP-1, -3, and -9).6 Recently, nobiletin metab-
olites have been found to possess potent antiinflammatory,7 antitu-
mor,8 and neurotrophic activities,9 which in some cases exceed
those of the parent compound.
Both approaches rely on 20-hydroxyacetophenone I as a key com-
pound. However, efficient synthesis of such a highly oxygenated
acetophenone derivative has not been reported. With this context
in mind, we commenced with the development of an alternative ac-
cess to key compound I.
We chose commercially available permethyl gallate (5) as a
starting material (Scheme 2). Our efforts concentrated on meth-
oxylation of the 2,6-positions of 5 using the Ulmann-type reaction.
Upon treatment with 2 equiv of NBS, ester 5 was brominated in
quantitative yield. The resulting 6 was subjected to the CuBr-med-
iated Ulmann-type reaction with a large excess amount of sodium
methoxide,22 which gave rise to 2,6-dihydroxybenzoate (7) instead
of the anticipated permethoxylated ester (8).23
Based on the result, we proposed a reaction mechanism includ-
ing catalytic cycles of Cu salt: (i) oxidative addition; and (ii) reduc-
tive elimination (Scheme 3). In this cycle, we recognized that
intermediate A, in which Cu(I) salt coordinates with the carboxyl
group and methoxy group, could be prepared for demethylation
to afford 7. Thus, we assumed that this demethylation process is
peculiar to the benzoate derivative.
In the course of metabolic studies of 1, 30-demethylnobiletin
(2),10 40-demethylnobiletin (3),11 and 30,40-didemethylnobiletin
(4)12 have been isolated from rodent urine and identified (Fig. 1).
Because of the limited availability of these metabolites in nature,
detailed investigations of the structure–activity relationship re-
main to be performed. We therefore initiated a divergent synthesis
of nobiletin metabolites 2–4 to confirm their structures unambig-
uously. In the present study, we developed a versatile method for
the synthesis of nobiletin metabolites and evaluated their inhibi-
tory activity against MMP-9 production.
Syntheses of nobiletin and its derivatives reported thus far can be
classified into two categories as depicted in Scheme 1, that is, (A)
intramolecular Michael cyclization of 20-hydroxychalcone followed
by oxidative dehydrogenation;9,13–16 and (B) C-ring construction via
dehydration of 1-(2-hydroxyaryl)-3-arylpropane-1,3-dione.17–21
OR2
4'
CH3O
CH3O
: R1 = R2 = CH3 (nobiletin)
: R1 = H, R2 = CH3
B
1
2
3
O
C
OR1
3'
A
: R1 = CH3, R2 = H
CH3O
4: R1 = R2 = H
CH3O
O
⇑
Corresponding authors. Tel./fax: +81 426 85 3728.
E-mail addresses: ostr@pharm.teikyo-u.ac.jp (T. Oshitari), natsu@pharm.
Figure 1. Structures of nobiletin and its metabolites.
teikyo-u.ac.jp (H. Natsugari).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.121

T. Oshitari et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4540–4544
4541
OR2
CH3O
B
OR2
OR1
CH3O
A
B
CH3O
O
C
(A)
CH3O
OH
O
OR1
A
CH3O
CH3O
CH3O
O
CH3O
OR2
OR1
2'-hydroxychalcone
B
(B)
X
O
CH3O
A
CH3O
CH3O
OH
OR2
OR1
CH3O
CH3O
OH
CH3
B
A
CH3O
CH3O
O
O
CH3O
O
1,3-diketone
key intermediate I
Scheme 1. Classification of polymethoxyflavone synthesis.
CH3O
CH3O
CH3O
CH3O
CH3O
OH
OCH3
CH3O
Br
O
b)
a)
OCH3
OCH3
CH3O
CH3O
CH3O
O
OH
O
Br
5
6
7
Scheme 2. Synthesis of 2,6-dihydroxybenzoate. Reagents and conditions: (a) NBS (2.4 equiv), DMF, 50 °C, quant.; (b) CuBr (0.1 equiv), NaOMe (20 equiv), methanol, reflux,
90%.
CH3O
CH3O
Br
CH3O
CH3O
NaOCH3
CH3O
O
CuIIIBr2
O
CH3O
(i)
Br
OCH3
CH3O
6
Br
Br
OCH3
CuBr
OCH3
CH3Br
Na
CH3O
CuIIIBr
CH3O
CH3
O
CH3O
Br
O
CH3O
CH3O
(ii)
CH3O
CH3O
ONa
O
CuBr
Br
OCH3
O
O
CH3O
Br
OCH3
CH3
Intermediate A
2 nd Cycle
CH3O
CH3O
CH3O
NaO
OH
CH3O
CH3O
ONa
work up
O
O
CH3O
HO
OCH3
OCH3
7
Scheme 3. Proposed reaction mechanism of Ulmann-type reaction.
Unfortunately,
selective
monoetherification
of
2,6-
hydrolysis of ester 8 provided carboxylic acid 9 in 96% yield in
three steps.24 To provide methyl ketone functionality, 9 was then
treated with 2 equiv of methyllithium.25 However, unexpected
dihydroxybenzoate 7 failed. We therefore examined subsequent
permethylation of crude 7 to obtain 8 (Scheme 4). Alkaline

4542
T. Oshitari et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4540–4544
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
OCH3
OH
O
OCH3
OCH3
Br
O
b)
a)
OCH3
CH3O
CH3O
CH3O
O
Br
9
6
8
CH3O
CH3O
CH3O
CH3O
d)
CH3O
CH3O
OCH3
OH
CH3
c)
CH3
CH3O
O
CH3O
O
I
10
Scheme 4. Synthesis of 20-hydroxyacetophenone I. Reagents and conditions: (a) (i) CuBr (0.1 equiv), NaOMe (20 equiv), methanol, reflux; (ii) K2CO3 (5 equiv), CH3I (7 equiv),
DMF; (b) NaOH (3 equiv), ethanol/H2O, 70 °C, 96% in three steps; (c) (i) (COCl)2 (1.3 equiv), DMF (cat), CH2Cl2; (ii) Fe(acac)3 (0.05 equiv), MeMgBr (1.1 equiv), THF, 0 °C to rt,
74% in two steps; (d) BCl3 (1.1 equiv), CH2Cl2, ꢀ78 °C, 1 h, ꢀ15 °C, 2 h, 96%.
OR2
CH3O
O
OR2
OR1
CH3O
CH3O
OR1
CH3O
OH
CH3
a)
CH3O
CH3O
O
O
+
Cl
CH3O
CH3
O
CH3O
O
11a–c
I
12a–c
OR2
OR1
CH3O
CH3O
CH3O
OH
O
OR2
OR1
CH3O
CH3O
O
b)
c)
CH3O
CH3O
O
CH3O
O
14a–c
13a–c
d)
R
R
R
1 = H, R2 = CH3
1 = CH3, R2 = H
1 = H, R2 = H
11–14:
2:
3:
4:
1
: R = Bn, R2 = CH3
a
b
1
2
: R = CH3, R = Bn
c: R1 = Bn, R2 = Bn
Scheme 5. Synthesis of nobiletin metabolites 2–4. Reagents and conditions: (a) Et3N (1.1–1.6 equiv), CH2Cl2; 12a 74%; 12b 87%; 12c 92%; (b) t-BuOK (1.1 equiv), THF, reflux,
1–1.5 h; 13a 88%; 13b 61%; 13c 60%; (c) p-TsOHꢁH2O (0.25 equiv), benzene, reflux (Dean–Stark), 5–10 h; 14a 85%; 14b 84%; 14c 80%; (d) H2 (1 atm), 20% Pd(OH)2/C (cat), ethyl
acetate/ethanol (1:1), 0.5–1 h; 2 84%; 3 88%; 4 93%.
decarboxylation occurred, and 10 was not obtained. Therefore,
we next examined the two-step conversion protocol. Treatment
of 8 with oxalyl chloride led to the corresponding acid chloride,
which was coupled with methylmagnesium bromide in the pres-
ence of tris(acetylacetonato)iron(III)26 to give methyl ketone (10)
in 74% yield in two steps. Demethylation with boron trichloride
proceeded in a highly regioselective manner to afford the key
intermediate I27 in 96% yield (68% overall yield from permethyl
gallate [5]).
With the requisite methyl ketone I in hand, we followed ap-
proach B in Scheme 1: the introduction of the B-ring moiety
and the subsequent closure of the C ring. As shown in Scheme
5, acid chlorides 11a–c were unambiguously prepared from isova-
nillin, vanillin, and 3,4-dihydroxybenzoic acid, respectively. Acyl-
ation of I proceeded, and the resulting aryl esters 12a–c were
subjected to a modified protocol of Baker–Venkataraman rear-
rangement,28 which gave rise to 1,3-diketones 13a–c in good
yields, respectively. Acid-catalyzed dehydration constructed the
flavone skeleton 14a–c smoothly. Finally, catalytic hydrogenolysis
of the benzyl ether with Pearlman’s catalyst afforded nobiletin
metabolites 2–4,29 for which the spectroscopic data were identi-
cal to those reported previously.9,12a,13,15
We next investigated the effects of synthesized metabolites
2–4 on the inhibition of the production of proMMP-9 in the
PMA- or TNF-a-stimulated human lens epithelial cell line
SRA01/0430 (Table 1). It was revealed that compound 3 inhibits
the expression of proMMP-9 much more potently than 1, whereas
compounds 2 and 4 possess activity comparable to that of 1.
Although we have limited information on the structure–activity
relationship, the position of the hydroxyl group on the B ring of
nobiletin may be closely linked with their inhibitory activity
against proMMP-9 production.
In summary, we succeeded in developing a new synthetic meth-
od for nobiletin metabolites 2–4, which involves easier access to
the highly oxygenated A-ring moiety. The metabolites thus synthe-
sized showed potent inhibitory activity against proMMP-9 produc-
tion. Since MMP-9 expression plays an important role in various
pathological states including cataract, rheumatoid arthritis,7 tumor
metastasis,8 etc., these metabolites could be potent lead com-
pounds for chemotherapeutic agents for the treatment of those

T. Oshitari et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4540–4544
4543
Table 1
20. Tsukayama, M.; Kawamura, Y.; Ishizuka, T.; Hayashi, S.; Torii, F. Heterocycles
2003, 60, 2775.
IC50 values (l
M) of nobiletin (1) and compounds 2–4 for proMMP-9 production31
21. 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.
22. (a) Aalten, H. L.; van Koten, G.; Grove, D. M.; Kuilman, T.; Piekstra, O. G.;
Hulshof, L. A.; Sheldon, R. A. Tetrahedron 1989, 45, 5565; (b) Majetich, G.; Li, Y.;
Zou, G. Heterocycles 2007, 73, 217.
Entry
Compd
PMA-treated cells
TNF-a-treated cells
1
2
3
4
1
2
3
4
20.9 6.5
16.9 2.0
3.7 0.3
17.0 1.6
16.4 6.6
5.5 0.8
12.3 1.2
10.8 1.9
23. For a similar example, see: Saito, S.; Gao, H.; Kawabata, J. Helv. Chim. Acta 2006,
89, 821.
24. For the catalyst, CuCl, CuCl2, and CuI were found to work as well as CuBr to give
the same 2,6-dihydroxybenzoate. On the other hand, no reaction occurred in
the absence of the Cu catalyst. A mixture of 6 (1.00 g, 2.60 mmol), CuBr (37 mg,
0.26 mmol), and 28 wt % sodium methoxide solution in methanol (11 ml,
52 mmol) was placed into a 100-ml round-bottomed flask fitted with a reflux
condenser. The reaction mixture was heated with vigorous stirring at reflux
(bath temperature: 110 °C) for 3.5 h. Upon cooling to room temperature, the
reaction mixture was carefully poured into 2 M HCl (40 mL). After the
methanol evaporated, the aqueous layer was extracted with ethyl acetate
(3 ꢂ 20 mL). Combined organic layers were washed with brine (2 ꢂ 50 mL),
dried (MgSO4), and concentrated in vacuo. The residue (700 mg) was dissolved
in DMF (8 mL), to which were successively added K2CO3 (1.08 g, 7.81 mmol)
and iodomethane (0.80 mL, 13 mmol). The reaction mixture was stirred at
room temperature for 12 h before the addition of water (50 mL) and extracted
with a mixture of toluene (20 mL) and ethyl acetate (20 mL). The extracts were
washed with brine (2 ꢂ 50 mL), dried (MgSO4), and concentrated in vacuo to
afford crude 8 (830 mg), a small amount of which was purified on a silica gel
column (hexane/EtOAc = 10/1–8/1) to give colorless solids: mp 48–49 °C; IR
diseases. Further synthesis and evaluation of nobiletin analogues
are currently underway in our laboratory.
Acknowledgments
We are grateful to Dr. Nobuhiro Ibaraki (Jichi Medical Univer-
sity, Tochigi, Japan) for a generous gift of the human lens epithelial
cell line SRA01/04. We thank Ms. Junko Shimode and Ms. Miki
Takahashi for spectroscopic measurements.
References and notes
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(KBr) 2944, 1740 cmꢀ1 1H NMR (CDCl3) d: 3.96 (3H, s), 3.92 (3H, s), 3.87 (12H,
;
s); 13C NMR (CDCl3) d: 165.7, 149.1, 146.2 (2C), 142.9 (2C), 118.4, 61.8 (2C),
61.4, 61.2 (2C), 52.4; HRMS calcd. for [M+H]+ of C13H18O7: 287.1125; found:
287.1112. A mixture of crude 8 (825 mg), NaOH (0.312 g, 7.8 mmol), ethanol
(8 mL), and water (2 mL) was heated at 70 °C for 10 h and cooled to room
temperature. After the ethanol was evaporated, the residue was partitioned
between toluene (20 mL) and water (30 mL). After the organic layer was
discarded, the aqueous layer was acidified with 2 M HCl (20 mL) and extracted
with chloroform (2 ꢂ 20 mL). The combined organic layers were washed with
brine (30 mL), dried (MgSO4), and concentrated in vacuo to afford a crude off-
white solid (800 mg). Recrystallization (twice) from hot hexane/toluene (1:1)
gave 9 as colorless prisms (679 mg, 2.49 mmol, 96% yield from 6): mp 94–
96 °C; IR (KBr) 2990, 1706 cmꢀ1 1H NMR (CDCl3) d: 3.99 (3H, s), 3.94 (6H, s),
;
3.89 (6H, s); 13C NMR (CDCl3) d: 169.2, 149.9, 147.1 (2C), 143.1 (2C), 116.7, 62.2
(2C), 61.5, 61.3 (2C). HRMS calcd. for [M+H]+ of C12H16O7: 273.0969. Found:
273.0960.
25. Gilman, H.; van Ess, P. R. J. Am. Chem. Soc. 1933, 55, 1258.
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4805.
27. Intermediate I: obtained as a yellow oil. IR (neat) 2938, 1622, 1589 cmꢀ1 ꢀ1H
;
NMR (CDCl3) d: 13.17 (1H, s), 4.09 (3H, s), 3.96 (3H, s), 3.86 (3H, s), 3.81 (3H, s),
2.68 (3H, s); 13C NMR (CDCl3) d: 203.9, 154.3 (2C), 153.5, 151.2, 137.8, 136.6,
110.3, 61.2, 61.1, 61.0, 60.9, 32.2: HRMS calcd. for [M+H]+ of C12H16O6:
257.1020. Found: 257.1006.
5. (a) Matsuzaki, K.; Miyazaki, K.; Sakai, S.; Yawo, H.; Nakata, N.; Moriguchi, S.;
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58, 7903.
29. Compound 2: mp 170–171 °C; IR (KBr) 3309, 2937, 1638 cmꢀ1 1H NMR (CDCl3)
;
d: 7.51 (1H, d, J = 2.2 Hz), 7.47 (1H, dd, J = 2.2 Hz, 8.5 Hz), 6.96 (1H, d,
J = 8.5 Hz), 6.61 (1H, s), 6.17 (1H, br s), 4.10 (3H, s), 4.02 (3H, s), 3.97 (3H, s),
3.95 (6H, s); 13C NMR (CDCl3) d: 177.2, 161.0, 151.3, 149.3, 148.2, 147.6, 146.0,
143.9, 138.0, 124.6, 118.7, 114.8, 112.2, 110.7, 106.9, 62.2, 62.0, 61.8, 61.6,
56.1. HRMS calcd. for [M+H]+ of C20H20O8: 389.1231. Found: 389.1221.
Compound 3: mp 153–154 °C; IR (KBr) 3250, 2935, 1625 cmꢀ1 1H NMR
;
(CDCl3) d: 7.53 (1H, dd, J = 2.0 Hz, 8.3 Hz), 7.39 (1H, d, J = 2.0 Hz), 7.05 (1H, d,
J = 8.3 Hz), 6.61 (1H, s), 6.07 (1H, br s), 4.11 (3H, s), 4.03 (3H, s), 3.99 (3H, s),
3.96 (6H, s); 13C NMR (CDCl3) d: 177.2, 161.0, 151.3, 148.8, 148.3, 147.4, 146.8,
144.0, 138.0, 123.5, 120.2, 114.9, 114.7, 108.1, 106.7, 62.3, 62.0, 61.8, 61.7,
56.1; HRMS calcd. for [MꢀH]ꢀ of C20H20O8: 387.1085. Found: 387.1084.
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Compound 4: mp 209–211 °C; IR (KBr) 3379, 2941, 1631, 1605 cmꢀ1 1H NMR
;
10. (a) Murakami, A.; Kuwahara, S.; Takahashi, Y.; Ito, C.; Furukawa, H.; Ju-ichi, M.;
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291; For a review: (b) Li, S.; Tan, D.; Dushenkov, S.; Ho, C.-T. ACS Symp. Ser.
2008, 987, 216 (Dietary Supplements); Inhibitory activity of 4 against lens
aldose reductases was previously reported. See also: (c) Okuda, J.; Miwa, I.;
Inagaki, K.; Horie, T.; Nakayama, M. Chem. Pharm. Bull. 1984, 32, 767.
13. Li, S.; Pan, M.-H.; Lai, C.-S.; Lo, C.-Y.; Dushenkov, S.; Ho, C.-T. Bioorg. Med. Chem.
2007, 15, 3381.
(CDCl3) d: 7.74 (1H, d, J = 2.2 Hz), 7.51 (1H, dd, J = 2.2 Hz, 8.3 Hz), 7.03 (1H, d,
J = 8.3 Hz), 6.74 (1H, s), 4.12 (3H, s), 4.03 (3H, s), 3.99 (3H, s), 3.96 (3H, s); 13C
NMR (DMSO-d6): 175.7, 160.9, 150.8, 149.2, 147.5, 147.1, 145.7, 143.5, 137.7,
121.7, 118.4, 116.1, 114.3, 113.0, 105.4, 61.94, 61.90, 61.5, 61.4; HRMS calcd.
for [MꢀH]ꢀ of C19H18O8: 373.0929. Found: 373.0928.
30. Ibaraki, N.; Chen, S.-C.; Lin, L.-R.; Okamoto, H.; Pipas, J. M.; Reddy, V. N. Exp. Eye
Res. 1998, 67, 577.
31. Experimental procedure in Table 1: The human lens epithelial cell line SRA01/
04 was a kind gift from Dr. Nobuhiro Ibaraki (Jichi Medical University, Tochigi,
Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium
(Invitrogen, Carlsbad, CA) supplemented with 20% (v/v) heat-inactivated
(56 °C for 30 min) fetal bovine serum (Biowest, Nuaille, France) including
PSN antibiotic mixture (Invitrogen, penicillin/streptomycin/neomycin: 100
mL each) at 37 °C in humidified 5% CO2 atmosphere. After reaching
confluence, the cells were treated with the test sample in the presence of
PMA (Sigma–Aldrich, 10 nM) or TNF- (Sigma–Aldrich, 10 ng/mL) for 24 h. The
harvested culture media were stored at 4 °C until just before use. Aliquots
(20 L) of the harvested culture media were subjected to SDS–PAGE with 10%
acrylamide gel containing gelatin (0.6 mg/mL) (Difco Laboratories, Detroit, MI).
lg/
14. Oliverio, A.; Casinovi, C. Gazz. Chim. Ital. 1950, 80, 798.
15. Mizuno, M.; Matoba, Y.; Tanaka, T.; Tachibana, H.; Inuma, M.; Iwasa, M. J. Nat.
Prod. 1987, 50, 751.
16. Chen, W. M. Indian Heterocycl. Chem. 1997, 6, 221.
17. Horii, Z. Yakugaku Zasshi 1940, 60, 614.
a
a
l
18. Krishnamurti, M.; Seshadri, T. R.; Sharma, N. D. Indian J. Chem. 1970, 8, 575.
19. Wagner, H.; Maurer, G.; Hörhammer, L.; Farkas, L. Chem. Ber. 1971, 104, 3357.

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T. Oshitari et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4540–4544
The gel was washed with 50 mM Tris–HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2,
M ZnCl2, and 0.1% Triton X-100, and then incubated in 50 mM Tris–HCl (pH
7.5), 0.15 M NaCl, 10 mM CaCl2, and 1 M ZnCl2 at 37 °C. Thereafter, the gel
was stained with 0.1% Coomassie Brilliant Blue R-250, and gelatinolytic activity
was detected as unstained bands on a blue background. The IC50 values (means
of triplicate measurements) shown in Table 1 were estimated from the relative
amount of proMMP-9 (quantified by Image-J) at various concentrations (0.25,
1
l
l
1, 4, 16, or 64 lM) of the test samples.