First synthesis of racemic methylophiopogonanone B and its inhibitory activity of hypoxia-inducible factor-1α

Article abstract of DOI:10.3987/COM-08-11517

Methylophiopogonanone B, a constituent of Ophiopogonis tuber, found to be a potent inhibitor of hypoxia induced factor-1 α (HIF-1 α) activity, was synthesized from 2,4,6-trihydroxy-3,5-dimethylacetophenone in 57% yield. The synthetic methylophiopogonanone

Full text of DOI:10.3987/COM-08-11517

HETEROCYCLES, Vol. 78, No. 1, 2009
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HETEROCYCLES, Vol. 78, No. 1, 2009, pp. 207 - 212. © The Japan Institute of Heterocyclic Chemistry
Received, 7th August, 2008, Accepted, 2nd September, 2008, Published online, 4th September, 2008.
DOI: 10.3987/COM-08-11517
FIRST SYNTHESIS OF RACEMIC METHYLOPHIOPOGONANONE B
AND ITS INHIBITORY ACTIVITY OF HYPOXIA-INDUCIBLE
FACTOR-1α
Mikio Fujii,1,2 Kiyoshi Egawa,1 Yasuaki Hirai,1 Masato Kondo,1,3 Hiroyuki
Akita,2 Kiyoshi Nose,1 Kazuo Toriizuka,1 and Yoshiteru Ida1,4*
1School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai,
2
Shinagawa-ku, Tokyo 142-8555, Japan. Faculty of Pharmaceutical Sciences,
3
Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. School of
Nursing and Rehabilitation Sciences, Showa University, 1865 Tokaichiba-cho,
4
Midori-ku, Yokohama 226-8555, Japan. Yokohama College of Pharmacy, 601
Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan.
E-mail: yoshi.ida@nifty.com
Abstract – Methylophiopogonanone B, a constituent of Ophiopogonis tuber,
found to be a potent inhibitor of hypoxia induced factor-1α (HIF-1α) activity, was
synthesized from 2,4,6-trihydroxy-3,5-dimethylacetophenone in 57% yield. The
synthetic methylophiopogonanone B inhibited the reporter activity at 3 to 10
μg/mL.
Methylophiopogonanone B (1) (Figure 1) is a homoisoflavanone, found in Ophiopogonis tuber
(Ophiopogon japonicus KER-GAWLER var. genuinus Maxim. or Ophiopogon japonicus KER-GAWLER cv.
NANUS, Liliaceae) that was used as a crude drug.1-5
Me
1'
O
4
OMe
7'
HO
Me
5'
OH
O
Figure 1
1
. Structure of methylophiopogonanone B ( )
In a previous study, it was found that 1 inhibited hypoxia-inducible facter-1α (HIF-1α) activity in vitro.6
Hif-1α is a key transcription factor which regulates expression of several genes involved in the

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HETEROCYCLES, Vol. 78, No. 1, 2009
hypoxia-responses and angiogenesis during carcinogenesis, and 1 can be a potentially angiogenic
inhibitor. As 1 was a minor constituent in Ophiopogonis tubers (0.0002%), we could not continue in vivo
experiment. For further biological and pharmaceutical studies, the suppliment of 1 by the organic
synthesis was demanded. In this paper, we report the synthesis of rac-1 and its HIF-1α inhibitory activity.
2,4-Dimethoxy-3,5-dimethyl-6-hydroxyacetophenone (3) was obtained from 2,4,6-trihydroxyaceto-
phenone (2).7 Compound 3 was condensed with 4-methoxybenzaldehyde to afford chalcone 4 in 84%
yield. Then, hydrogenation of 4 gave dihydrochalcone 5 in 94% yield. Construction of C-ring of the
homoisoflanvone by the use of sodium and formaldehyde,3 or Kirkiacharian’s method8 was not achieved.
Instead, Jaspal’s method using formaldehyde and diethylamine was applied, and homoisoflavanone rac-6
was obtained.9
Me
Me
HO
OH
Me
MeO
Me
OH
Me
MeO
Me
OH
OMe
a
ref. 3
OH
O
OMe O
OMe O
4
2
3
Me
Me
7'
MeO
Me
O
OMe
MeO
Me
OH
OMe
c
b
5'
OMe O
OMe O
5
rac-6
Scheme 1. Conditions: a) 4-methoxybenzaldehyde, potassium hydroxide, MeOH - water, 84%. b) H2,
5% Pd/C, 94%. c) formaldehyde, diethylamine, 94%
The regioselective cleavages of methyl ether at 5’ and 7’ position of rac-6 are recommended for the
synthesis of 1. We tested several acidic conditions to obtain rac-1, and iodotrimethylsilane (TMSI) was
found to be effective to the selective cleavage. On treatment of rac-6 with 2.0 equivalents of TMSI at rt
for 8 h, demethylation was occurred selectively at 7’ position to afford rac-7 in 84% yield. While,
treatment of rac-6 with 4.0 equivalent of TMSI at 50 ˚C for 7 days gave rac-1 in 80% yield accompanied
with rac-7 (18%) and rac-810 (1.6%). This selectivity of TMSI against two methoxy groups can be
considered that the acidity of hydroxy group at 7’ of rac-1 is higher than that of normal phenol since the
C-7’ hydroxyl group is affected by the conjugation of C-4 carbonyl group and the steric effects by two
o-methyl groups.

HETEROCYCLES, Vol. 78, No. 1, 2009
209
1
13
The H and C NMR of synthetic rac-1 was identical with the authentic sample of natural 1, and this is
the first synthesis of rac-1.
Next, we examined HIF-1α inhibitory activity of synthetic rac-1 by the reporter assay.6,11 A4-4 cells
were cultured in the presence of various concentrations of synthetic rac-1 for 16 h under hypoxic
condition, and then luciferase activity (filled bar) and cell viability (open squares) were measured. As can
be seen in Figure 2, synthetic rac-1 was effective at 3 - 10 μg/mL in inhibiting the reporter activity
without affecting cellular survival significantly.
Me
MeO
Me
O
OMe
OH
O
rac-7 (18%)
Me
Me
MeO
Me
O
OMe
HO
Me
O
OMe
TMSI
OMe O
OH
O
rac-1 (80%)
rac-6
Me
HO
Me
O
OH
OH
O
rac-8 (1.6%)
Scheme 2
Hif-1 activity
Cell viability
120
100
80
60
40
20
0
cnt
0.3
1
3
10
1 μ
Concentration of ( g / mL)
Figure 2
1
. Effect of synthetic on reporter activity induced by hypoxia.
We succeeded in synthesizing rac-methylophiopogonanone B (rac-1) from 3 in 57% overall yield, and
the synthetic rac-1 showed HIF-1α inhibitory activity in the same concentration range as natural 1.6

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HETEROCYCLES, Vol. 78, No. 1, 2009
In addition, it was reported that 1 promoted Rho activation and tublin depolymerization.12 Our synthetic
route is important to investigate the anticancer activity of 1 and its derivatives. Study of structure and the
activity relationship of 1 are on progress.
EXPERIMENTAL
General
The NMR spectra were recorded in CDCl3 on JEOL-AL-400 spectrometer. Mass spectra were measured
by JEOL JMS-AM II 50. Elemental analysis was achieved by EA-1108 Elemental Analyzer (Fisons Inc.).
Compound 6 was synthesized as following the reported procedure.3
5’,7’-Dimethoxy-6’,8’-dimethyl-3’-(4-methoxybenzyl)chroman-4’-one (rac-6)
To the solution of 5 (1.00 g, 2.91 mmol) in EtOH (75 mL) were added paraformaldehyde (0.30 g, 0.01
mol) and diethylamine (3.00 mL, 2.11 g, 28.8 mmol) and the reaction mixture was refluxed for 24 h. The
mixture was evaporated under reduced pressure, the residue was dissolved in Et2O and washed with water.
The ethereal solution was dried over MgSO4 and evaporated under reduced pressure to afford
5,7-dimethoxy-6,8-dimethyl-3-(4-methoxybenzyl)chroman-4-one (6) (0.97 g, 2.72 mmol, 94%) as a
colorless oil.１H NMR (400 MHz,CDCl3) δ: 2.08 (3H, s), 2.13 (3H, s), 2.63 (1H, dd, J = 10.8, 14 Hz),
2.71-2.77 (1H, m), 3.17 (1H, dd, J = 4.0, 14 Hz), 3.71 (3H, s), 3.77 (3H, s), 3.78 (3H, s), 4.10 (1H, dd, J
= 8.0, 11.2 Hz), 4.30 (1H, dd, J = 4.4, 11.2 Hz), 6.83 (2H, d, J = 8.4 Hz), 7.13 (2H, d, J = 8.4 Hz). 13C
NMR (100 MHz, CDCl3) δ: 8.9, 9.1, 32.1, 48.8, 55.6, 60.4, 61.4, 69.1, 111.7, 114.3, 114.3, 115.5, 118.8,
130.4, 130.4, 130.8, 158.2, 158.6, 160.0, 163.4, 192.6. EI-HR-MS calcd for C21H24O5: 356.1624. Found:
356.1628.
Selective cleavage of methoxy group of 6 by TMSI for the synthesis of rac-1
To the solution of rac-6 (234 mg, 0.659 mmol) in CHCl3 (1.0 mL) under N2 atmosphere was added TMSI
(0.80 mL, 2.6 mmol), and the resultant mixture was stirred at 50 ˚C. The course of the reaction was
monitored by TLC and the reaction proceeded slowly and stopped after 7 d. The resulting mixture was
added to aq. NaCl (20 mL) and extracted by Et2O (20 mL) three times. The ethereal solution was dried
over MgSO4 and evaporated under reduced pressure. The residue was purified by column
chromatography (silica gel (10 g), hexane - AcOEt (20:1, v/v)) to afford rac-7 (43.3 mg, 0.126 mmol,
18%), rac-1 (174 mg, 0.530 mmol, 80%) and 5,7-dihydroxy-6,8-dimethyl-3-(4-hydroxybenzyl)-
chroman-4-one (rac-8) (3.3 mg, 0.110 mmol, 1.6%).
１
rac-7: H NMR (400 MHz, CDCl3) δ: 2.03 (3H, s), 2.08 (3H, s), 2.69 (1H, dd, J = 10, 13.6 Hz),
2.78-2.83 (1H, m), 3.16 (1H, dd, J = 4.0, 13.6 Hz), 3.71 (3H, s), 3.78 (3H, s), 4.10 (1H, dd, J = 7.6, 11.4

HETEROCYCLES, Vol. 78, No. 1, 2009
211
Hz), 4.28 (1H, dd, J = 4.4, 11.4 Hz), 6.84 (2H, d, J = 8.8 Hz), 7.13 (1H, d, J = 8.8 Hz), 12.12 (1H, s). 13C
NMR (100 MHz, CDCl3) δ: 7.8, 8.2, 31.8, 47.0, 55.2, 60.2, 68.8, 104.4, 109.1, 111.0, 114.0, 114.0, 129.8,
130.0, 130.0, 157.8, 158.4, 159.5, 165.1, 199.3. Anal.Calcd for C20H22O5: C, 70.16; H, 6.48. Found: C,
13
１
70.00; H, 6.50 %. The H and C NMR spectra of rac-8 were superimposable with those reported
value.10
rac-1:１H NMR (400 MHz, CDCl3) δ: 2.01 (3H, s), 2.05 (3H, s), 2.68 (1H, dd, J = 10.8, 13.8 Hz),
2.75-2.80 (1H, m), 3.16 (1H, dd, J = 4.4, 13.8 Hz), 3.78 (3H, s), 4.10 (1H, dd, J = 7.2, 11.6 Hz), 4.26 (1H,
dd, J = 4.4, 11.6 Hz), 5.38 (1H, s), 6.84 (2H, d, J = 8.8 Hz), 7.13 (2H, d, J = 8.8 Hz), 12.36 (1H, s). 13C
NMR (100 MHz, CDCl3) δ: 6.8, 6.8, 7.3, 32.0, 46.8, 55.3, 68.9, 101.5, 102.3, 114.1, 120.5, 129.6, 130.1,
130.1, 156.5, 158.4, 159.6, 160.6, 198.5. EI-HR-MS calcd for C19H20O5: 328.1311. Found: 328.1315.
Measurement for HIF-1α inhibitory activiy of rac-1.
Cell Culture: A stable transformant of CHO cells (clone A4-4) was established by the transfection of
HIF-1-dependent luciferease (5XHRE/pGL3/VEGF/E1b) and neomycin-resistant genes as described
previously.13
Luciferase assay: Cells were plated into 96-well tissue culture plates (Falcon) at a density of 1 X 104
cells/well, and treated with synthetic methylophiopgonanone (1) 16 h later. They were incubated further
48 h under hypoxic conditions, and harvested for the determination of luciferase activity. The assay was
carried out using a kit provided by Promega Corp. (Madison, WI, USA) following the manufacturer’s
manual. The cytotoxicity was estimated by the MTT method as previously reported.13
ACKNOWLEDGEMENTS
We thank Mr. Seiji Utsumi, Ms. Nozomi Komoriya, Mr. Takuya Genkai, Mr. Shintaro Koike and Mr.
Kijyu Konno for their technical contribution to this research,also Ms. Kimiko Shiohara for measurement
of Mass spectra and Ms. Yuki Odanaka for measurement of elemental analysis.
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