Biotransformation of isoimperatorin and imperatorin by Glomerella cingulata and β-secretase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2009.10.004

Biotransformation studies conducted on the furanocoumarins isoimperatorin (1) and imperatorin (3) have revealed that 1 was metabolized by Glomerella cingulata to give the corresponding reduced acid, 6,7-furano-5-prenyloxy hydrocoumaric acid (2), and 3 was transformed by G. cingulata to give the dealkylated metabolite, xanthotoxol (4) in high yields (83% and 81%), respectively. The structures of the new compound 2 have been established on the basis of spectral data. The metabolites 2 and 4 were tested for the β-secretase (BACE1) inhibitory activity in vitro, and metabolite 2 slightly inhibited the β-secretase activity with an IC₅₀ value of 185.6 ± 6.8 μM. The metabolite 4 was less potent activity than compounds 1-3. In addition, methyl ester (2Me), methyl ether (2a) and methyl ester and ether (2aMe) of 2 were synthesized, and investigated for the ability to inhibit β-secretase. Compound 2aMe exhibited the best β-secretase inhibitory activity at the IC₅₀ value 16.2 ± 1.2 μM and found to be the 2aMe showed competitive mode of inhibition against β-secretase with K_i value 11.3 ± 2.8 μM.

Full text of DOI:10.1016/j.bmc.2009.10.004

Bioorganic & Medicinal Chemistry 18 (2010) 455–459
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biotransformation of isoimperatorin and imperatorin by Glomerella cingulata
and b-secretase inhibitory activity
*
Shinsuke Marumoto, Mitsuo Miyazawa
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotransformation studies conducted on the furanocoumarins isoimperatorin (1) and imperatorin (3)
have revealed that 1 was metabolized by Glomerella cingulata to give the corresponding reduced acid,
6,7-furano-5-prenyloxy hydrocoumaric acid (2), and 3 was transformed by G. cingulata to give the deal-
kylated metabolite, xanthotoxol (4) in high yields (83% and 81%), respectively. The structures of the new
compound 2 have been established on the basis of spectral data. The metabolites 2 and 4 were tested for
the b-secretase (BACE1) inhibitory activity in vitro, and metabolite 2 slightly inhibited the b-secretase
activity with an IC50 value of 185.6 6.8 lM. The metabolite 4 was less potent activity than compounds
1–3. In addition, methyl ester (2Me), methyl ether (2a) and methyl ester and ether (2aMe) of 2 were syn-
thesized, and investigated for the ability to inhibit b-secretase. Compound 2aMe exhibited the best b-
Received 7 August 2009
Revised 1 October 2009
Accepted 2 October 2009
Available online 8 October 2009
Keywords:
Biotransformation
Glomerella cingulata
Furanocoumarin
secretase inhibitory activity at the IC50 value 16.2 1.2
lM and found to be the 2aMe showed competi-
Isoimperatorin
tive mode of inhibition against b-secretase with Ki value 11.3 2.8
lM.
Imperatorin
b-Secretase (BACE1) inhibitory activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and heterocyclic compounds have been designed and evaluated
as BACE inhibitors, none of them have been successfully developed
as anti-AD agents to date.5 There are only very few reports on nat-
ural products-based BACE inhibitors.4,6
Furanocoumarins are abundant in citrus fruits, umbelliferous
vegetables, and certain herbal medicines.1 Interest in these com-
pounds has long been limited to psoralen, the mainstay of photo-
dynamic therapy, but over the past few years there has been a
growing interest in its prenylated derivatives spurred by the po-
tent activity on drug metabolism of dietary compounds such as
bergamottin and its dimeric analogs. While bergamottin is mainly
contained in citrus plants, its isomers isoimperatorin (1) and impe-
ratorin (3) are more widespread, occurring not only in lemon and
lime oils, but also in the medicinal plant Angelica dahurica and its
popular culinary herbs such as parsnip, parsley, and fennel.2 De-
spite its occurrence in edible plants, isoimperatorin (1) and impe-
ratorin (3) shows potent pharmacological activity and has been
studied for its anti-inflammatory and antitumoral activities.3 Re-
cently, we reported the b-secretase (BACE1) inhibitory activity of
isoimperatorin (1) and imperatorin (3).4
Alzheimer’s disease (AD) has become an increasingly severe
medical and social problem, due to the rapid growth of aging peo-
ple populations in industrialized countries, and even in some
developing countries. The b-secretase (BACE1) has been recognized
as a valuable target for the treatment of AD. The BACE1 inhibitors
have promised to be a class of disease disrupting rather than symp-
tom relieving agents.5 Although many peptides, peptidomimetics
Biotransformation is an alternative tool in the structural modi-
fication of complex natural products due to its great capabilities to
catalyze novel reactions and its regio- and stereo-selectivity.
Microorganisms are well known as efficient and selective catalysts.
In previous papers, we reported the biotransformation of some
complex natural products and obtained a series of new products.7
Therefore, it is envisioned that biotransformation of isoimperatorin
(1) and imperatorin (3) may provide some analogues which could
be utilized for screening for new activities or better activity/side ef-
fects profile. By studying the b-secretase inhibitory activities of ob-
tained products, we can explore the structure–activity
relationships of such compounds.
2. Results and discussion
2.1. The biotransformation of isoimperatorin (1) with
Glomerella cingulata
G. cingulata is widely distributed in the world, infecting various
plants and causing anthracnose. We investigated the microbial
transformation of terpenoids and flavonoids using G. cingulata.8
Then we tried the microbial transformation of furanocoumarins 1
and 3 using G. cingulata.
* Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6727 2024.
E-mail address: miyazawa@apch.kindai.ac.jp (M. Miyazawa).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.004

456
S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 18 (2010) 455–459
HRFABMS of compound
2
showed [M+H]+ peaks at m/z
291.1209 (calcd for m/z 291.1231), which established a molecular
formula of C16H19O5. The presence of a broad absorption band at
3335 cmꢀ1 and a strong absorption band at 1704 cmꢀ1 in the IR
spectrum suggested the conversion of the original isoimperatorin
lactone into phenol and carboxylic acid functionalities. The 13C
NMR spectrum showed 16 resonances distributed as two primary,
three secondary, four tertiary and seven quaternary carbons. The
conversion of original two olefinic doublets (C-3, dC 112.5 and C-
4, dC 139.5) into two aliphatic secondary carbons (C-3, dC 34.6
and C-4, dC 20.1) indicated the reduction of the C-3,4 double bond.
Except for the downfield shift of C-2 and upfield shift of C-4a, the
13C resonances remained the same as those of the starting material
(Table 1). Furthermore, the 1H NMR spectrum showed the conver-
sion of two doublets (d 6.27, d, J = 9.8 Hz, H-3 and d 8.16, d,
J = 9.8 Hz, H-4) into two multiplets at d 2.97–3.00 (H-3) and d
2.52–2.55 (H-4). Thus, chemical structure of metabolite 2 was
established as 6,7-furano-5-prenyloxy hydrocoumaric acid, a new
compound. The protons and carbons assignments were unambigu-
ously made from the H–H COSY, HMQC and HMBC spectra.
Figure 1. Biotransformation of isoimperatorin (1) and imperatorin (3), and
chemical structures of all the compounds.
2.2. The biotransformation of imperatorin (3) with G. cingulata
To clarify the time course of the microbial transformation of iso-
imperatorin (1) by G. cingulata, a small amount of 1 was incubated
for 5 days, and one metabolite was detected by thin layer chroma-
tography (TLC) and high performance liquid chromatography
(HPLC). The time course of metabolite was measured by HPLC. Fig-
ure 2 shows the time course of biotransformation of 1 and the pH
gradient. The pH value in the biotransformation changed from 4.7
to 6.2. In this system, 1 was transformed to 2 at the conversion rate
of 83% for 5 days. To isolate the metabolite, a large-scale incubation
of 1 using G. cingulata was carried out for 5 days. After the biotrans-
formation, the culture was extracted as described in Materials and
methods section and methylated metabolite 2 (2Me) was isolated
from the EtOAc extract. Metabolite 2 was obtained by the hydrolysis
of 2Me. The structures of these compounds were determined by
spectral data.
To clarify the time course of the microbial transformation of
imperatorin (3) by G. cingulata, a small amount of 3 was incubated
for 12 days. One metabolite was detected by TLC and HPLC. The
time course of metabolites was measured by HPLC. Figure 3 shows
the time course of biotransformation of 3 and the pH gradient. The
pH value in the biotransformation changed from 5.1 to 6.9. In this
system, 3 was transformed to 4 at the conversion rate of 81% for
12 days. To isolate this metabolite, a large-scale incubation of 3
by G. cingulata for 12 days. After the biotransformation, the culture
was extracted as described in Experimental section and metabolite
4 was isolated from the EtOAc extract. The structure of 4 was
shown to be identical with xanthotoxol (4) which is the dealkylat-
ed product of 3 by comparison of its spectroscopic data.9
The biotransformation pathway is illustrated in Figure 1.
Through the previous works,8 we guessed that prenyloxy side-
chain group in 1 could be oxidized or hydrolyzed by G. cingulata.
Although the expected products were not obtained, G. cingulata
used in this work did possess ability to reduction of
a,b-unsatu-
rated lactone on isoimperatorin (1). Fugal reduction of 1 probably
proceeds by opening of the lactone ring followed by isomerization
and reduction or by hydrogenation of the 3,4-double bond fol-
Table 1
The 13C and 1H NMR spectroscopic data of compounds 1 and 2a
Atom
1
2
dC
dH
dC
dH
2
3
161.3 (C)b
175.5 (C)
112.5 (CH) 6.27 1H, d, (9.8)c 34.6 (CH2)
2.97–3.00 2H, m
2.52–2.55 2H, m
4
4a
5
6
7
8
8a
9
10
11
12
13
14
15
139.5 (CH) 8.16 1H, d, (9.8)
107.5 (C)
148.9 (C)
114.2 (C)
158.5 (C)
20.1 (CH2)
115.6 (C)
151.5 (C)
112.4 (C)
156.4 (C)
93.9 (CH)
155.0 (C)
94.2 (CH)
152.6 (C)
7.15 1H, s
6.90 1H, d (1.1)
144.8 (CH) 7.59 1H, d, (2.3)
105.0 (CH) 6.95 1H, d, (2.3)
143.2 (CH) 7.56 1H, d (2.3)
105.5 (CH) 6.73 1H, dd (2.3, 1.1)
4.76 2H, d (6.8)
121.6 (CH) 5.54–5.56 1H, m
138.0 (C)
69.7 (CH2)
4.92 2H, d, (6.9)
69.8 (CH2)
119.1 (CH) 5.52–5.54 1H, m
139.8 (C)
25.8 (CH3)
18.2 (CH3)
1.80 3H, s
1.70 3H, s
25.7 (CH3)
18.1 (CH3)
1.75 3H, d (0.7)
1.68 3H, br s
Figure 2. Time course in the biotransformation of 1 by G. cingulata: (d) isoimpe-
ratorin (1); (j) Metabolite 2; (N) pH. The obtained sample solution was analyzed
a
b
c
Spectra for 1 and 2 were recorded in CDCl3 and acetone-d6, respectively.
13C multiplicities were determined by DEPT 135°.
The J values are in Hertz in parentheses.
under the following conditions: RP-18 GP (4.6
lmf ꢁ 150 lm), solvent: water and
acetonitrile (50:50), detection: UV 254 nm, flow: 0.5 ml/min.

S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 18 (2010) 455–459
457
Figure 4. Inhibition of b-secretase activity by compounds 1–4. -d- Isoimperatorin
(1); -N- 6,7-furano-5-prenyloxy hydrocoumaric acid (2); -.- 6,7-furano-5-prenyl-
oxy hydrocoumaric acid methyl ester (2Me); -ꢀ- 6,7-furano-8a-methoxy-5-prenyl-
oxy hydrocoumaric acid methyl ester (2a); -s- 6,7-furano-8a-methoxy-
5-prenyloxy hydrocoumaric acid methyl ester (2aMe); -j- imperatorin (3);
-h- xanthotoxol (4).
3. Materials and methods
Figure 3. Time course in the biotransformation of 3 by G. cingulata: (d) imperatorin
(3); (j) metabolite 4; (N) pH. The obtained sample solution was analyzed under the
3.1. General experimental procedures
following conditions: RP-18 GP (4.6
lmf ꢁ 150 lm), solvent: water and acetonitrile
Thin layer chromatography (TLC) was performed on precoated
plates (Si Gel 60 F254, 0.25 mm, Merck). The mobile phase was hex-
ane–EtOAc (3:7). Compounds were visualized by spraying plates
with 1% vanillin in 96% H2SO4 followed by brief heating. High per-
formance liquid chromatography (HPLC) analysis with a LC-CCPS
system (Tosoh, Tokyo) with a spectrometer UV-8020 (Tosoh, To-
(50:50), detection: UV 254 nm, flow: 0.5 ml/min.
lowed by, or concomitant with, opening of the lactone ring. How-
ever, intermediary metabolites in each pathway were not observed
during the time course experimental. On the other hand, biotrans-
formation of 3 by G. cingulata produced metabolite hydrolyzed at
prenyloxy side-chain. In previously report,10 imperatorin (3) was
metabolized by Aspergillus flavus to give five products, which main
compound was specifically oxidized at the C-14 and C-15 posi-
tions, in addition on the cleavage of the prenyloxy side-chain,
and xanthotoxol was produced as minor metabolite. Surprisingly,
biotransformation of imperatorin (3) by G. cingulata was selectivity
offered the one metabolite, xanthotoxol (4), in high yield. In con-
clusion, the incubation of isoimperatorin (1) and imperatorin (3)
with G. cingulata were produced the one metabolic product in high
yields (83% and 81%), respectively. In the bioconversion of isoimpe-
ratorin (1) and imperatorin (3) which are different the position of
prenyloxy side-chain combined to C-5 or C-8 at furanocoumarin
skeleton by G. cingulata, there was a difference in the metabolic
pathway.
kyo). Separation was done with a C18 5-
(Mightsil RP-18, 150 ꢁ 4.6 m, Kanto Chemical, Tokyo) equipped
with a C18 5- m guard column (Mightsil RP-18, 5–4.6 m, Kanto
lm analytical column
l
l
l
Chemical), mobile phase was 50% acetonitrile. EIMS, HREIMS, FAB-
MS and HRFABMS were obtained on a JEOL the Tandem Ms station
JMS-700 TKM. NMR spectra were recorded at 500 MHz for 1H and
125 MHz for 13C on a JEOL ECA-500 spectrometer. Tetramethylsil-
ane (TMS) was used as the internal standard reference (d, 0.00) for
1H NMR spectra measured in CDCl3. IR spectra were determined
with a JASCO FT/ IR-470 plus Fourier transform infrared spectrom-
eter. BACE1 (recombinant human BACE1) assay kit was purchased
from the PanVera Co., USA.
3.2. Chemicals
Isoimperatorin (1) and imperatorin (3) were isolated from A.
dahurica as the b-secretase inhibitors.4
All compounds inhibited b-secretase (BACE1) in a concentra-
tion-dependent manner (Fig. 4). The BACE1 inhibitory activities
of compounds 1–4 in addition methyl ester (2Me), methyl ether
(2a) and methyl ester and ether (2aMe) of 2 were determined
the IC50 values, respectively. Among them, compound 2aMe
showed the best b-secretase inhibitory activity at IC50 value
16.2 1.2
activity against b-secretase with IC50 values 244.2 8.2,
185.6 6.8, 91.8 7.5, 30.5 2.7 and 203.3 4.1 M, respectively.
lM. Compounds 1, 2, 3, 2Me and 2a showed inhibitory
l
Metabolite 4 was found to be inactive. The inhibitory activity of
metabolite 2, with corresponding reduced acid of 1, was slightly
stronger than that of substrate 1. The activities of compounds
2Me and 2aMe, with a carboxylic acid methyl ester, showed the in-
creased inhibitory effects, whereas in the latter, an additional
methyl ether group seems to have no significant influence. This
evidence was confirmed by comparing the structure and activities
of 2 and 2a. From the kinetics analysis (Fig. 5), compound 2aMe
was estimated as being a competitive-type inhibitor against b-
Figure 5. Dixon plots for inhibition of compound 2aMe on b-secretase for the
proteolysis of substrate. In the presence of different concentration of substrate:
750 nM (d), 500 nM (j) and 250 nM (N).
secretase with Ki value 11.3 2.8 lM.

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S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 18 (2010) 455–459
3.3. Preculture of G. cingulata
3.6.2. 6,7-Furano-5-prenyloxy hydrocoumaric acid methyl ester
(2Me)
Pale yellow oil; IR (film) mmax 3367, 1735, 1622, 1599 cmꢀ1 1H
;
Spores of G. cingulata (the strain isolated from diseased grape
was a gift from Dr. M. Hyakumachi, Gifu University, Gifu, Japan),
which had been preserved on potato dextrose agar (PDA) at 4 °C,
were inoculated into 200 mL of sterilized culture medium (1.5%
saccharose, 1.5% glucose, 0.5% polypeptone, 0.05% MgSO4ꢂ7H2O,
0.05% KCl, 0.1% K2HPO4, and 0.001% FeSO4ꢂ7H2O in distilled H2O)
in a 500-mL shaking flask, and the flask was shaken (reciprocating
shaker, 100 rpm) at 27 °C for 3 days.
NMR (CDCl3, 500 MHz) d 7.78 (1H, br s, OH), 7.41 (1H, d,
J = 2.3 Hz, H-9), 6.82 (1H, d, J = 0.8 Hz, H-8), 6.77 (1H, dd, J = 2.3,
0.8 Hz, H-10), 5.50–5.54 (1H, m, H-12), 4.77 (2H, d, J = 6.9 Hz,
H-11), 3.66 (3H, s, COOCH3), 2.98–2.95 (2H, m, H-4), 2.75–2.73
(2H, m, H-3), 1.79 (3H, d, J = 0.85 Hz, H-14), 1.70 (3H, br s, H-
15); 13C NMR (CDCl3, 125 MHz) d 177.4 (C, C-2) 155.9 (C, C-7),
153.2 (C, C-8a), 150.5 (C, C-5), 142.4 (CH, C-9), 137.8 (C, C-13),
120.3 (CH, C-12), 114.3 (C, C-4a), 111.7 (C, C-6), 104.6 (CH, C-
10), 95.1 (CH, C-8), 68.9 (CH2, C-11), 52.2 (CH3, COOCH3), 34.1
(CH2, C-3), 25.9 (CH3, C-14), 18.6 (CH2, C-4), 18.1 (CH3, C-15);
EIMS m/z 304 [M]+(4), 236 (28), 204 (100), 176 (12), 162 (71),
64 (46), 41 (19); HREIMS m/z 304.1293 [M]+ (calcd for
C17H20O5, 304.1311).
3.4. Time course of biotransformation and quantification of
metabolite
Precultured G. cingulata (3 mL) was transferred into two 300-mL
Erlenmeyer flasks containing 100 mL of medium and was stirred
(ca. 100 rpm) for 3 days. After the growth of G. cingulata, 1
(10 mg, 37
l
mol) and 3 (10 mg, 37
l
mol) in 0.5 mL of dimethyl
3.6.3. 6,7-Furano-8a-methoxy-5-prenyloxy hydrocoumaric acid
(2a)
sulfoxide (DMSO) was added into the medium, respectively, and
cultivated for 5 (for 1) or 12 (for 3) more days. Every other day,
5 mL of the culture medium was extracted with EtOAc. This extract
was analyzed by TLC and HPLC. The mobile phase and detector
used were the same as above. The contents of these compounds
were calculated by means of the absolute calibration curves. The
time course of biotransformation is shown in Figures 2 and 3.
Compound 2aMe (20 mg, 0.06 mmol) was dissolved in MeOH
(0.5 mL), 5% NaOH (1 mL) added to the solution, and the solution
was refluxed for 30 min. The solution was acidified with 2 N HCl
and distributed between EtOAc and water, The EtOAc phase was
evaporated and purified by silica-gel open-column chromatogra-
phy (Silica Gel 60, 230–400 mesh, Merck) with a hexane–EtOAc
gradient (9:1–1:1) to yield compound 2a (15 mg, 74%) obtained
3.5. Preparative biotransformation of isoimperatorin (1)
as colorless powder; IR (KBr) mmax 1706, 1620, 1590 cmꢀ1 1H
;
NMR data (CDCl3, 500 MHz) d 7.45 (1H, d, J = 2.3 Hz, H-9), 6.79
(1H, dd, J = 2.3, 0.8 Hz, H-10), 6.78 (1H, br s, H-8), 5.54–5.57 (1H,
m, H-12), 4.70 (2H, d, J = 6.8 Hz, H-11), 3.84 (3H, s, OCH3), 3.08–
3.05 (2H, m, H-4), 2.60–2.56 (2H, m, H-3), 1.78 (3H, br s, H-14),
1.69 (3H, br s, H-15); 13C NMR (CDCl3, 125 MHz) d 177.8 (C, C-2)
156.6 (C, C-7), 155.6 (C, C-8a), 150.3 (C, C-5), 142.5 (CH, C-9),
138.1 (C, C-13), 120.3 (CH, C-12), 115.3 (C, C-4a), 112.4 (C, C-6),
104.6 (CH, C-10), 89.5 (CH, C-8), 69.5 (CH2, C-11), 55.8 (CH3,
OCH3), 33.7 (CH2, C-3), 25.7 (CH3, C-14), 19.2 (CH2, C-4), 18.1
(CH3, C-15); EIMS m/z 304 [M]+(5), 286 (86), 271 (47), 218 (60),
176 (100), 69 (24), 41 (18); HREIMS m/z 304.1295 (calcd for
C17H20O5, 304.1310).
Precultured G. cingulata (5 mL) was transferred into a 500 mL
Erlenmeyer flask containing 300 mL of medium. Cultivation was
carried out at 27 °C with stirring (ca. 100 rpm) for 3 days. After
the growth of G. cingulata, 50 mg of 1 in 1.0 mL of dimethyl sulfox-
ide (DMSO) was added into the medium and cultivated for an addi-
tional 5 days, together with two controls, which contained either
mycelia with medium or substrate dissolved in DMSO with med-
ium. No metabolic product was observed in two controls.
3.6. Isolation of metabolite in 1
After the fermentation, the culture medium and mycelia were
separated by filtration. The medium was saturated with NaCl,
and extracted with EtOAc. The mycelia were also extracted with
EtOAc. Each EtOAc extract was combined, the solvent was evapo-
rated, and a crude extract (320 mg) was obtained. The extract
was distributed between 5% NaHCO3 aq and EtOAc, and EtOAc
phase was evaporated to give the neutral fraction (112 mg). No
metabolic compounds were detected from neutral fraction by
TLC and HPLC. The alkali phase was acidified to pH 3 with 2 N
HCl and distributed between water and EtOAc. The EtOAc phase
was evaporated, and the acidic fraction (268 mg) was obtained.
The acidic fraction was dissolved in CH2Cl2 (1 mL), and CH2N2
(1 mL) was added to the fraction. The solution was evaporated,
and the methylation fraction was obtained. The methylation frac-
tion was subjected to silica-gel open-column chromatography (Sil-
ica Gel 60, 230–400 mesh, Merck) with a hexane–EtOAc gradient
(9:1–1:9) to yield compound 2Me (41 mg, 73%). Compound 2Me
(20 mg) was dissolved in MeOH (0.3 mL), 5% NaOH (1 mL) added
to the solution, and the solution was refluxed for 30 min. The solu-
tion was acidified to pH 3 with 2 N HCl and distributed between
EtOAc and water. The EtOAc phase was evaporated to give 2
(16 mg).
3.6.4. 6,7-Furano-8a-methoxy-5-prenyloxy hydrocoumaric acid
methyl ester (2aMe)
A solution of compound 2Me (50 mg, 0.16 mmol) in N,N-
dimethylformamide (DMF, 0.5 mL) was treated with methyl iodide
(0.3 mmol) in the presence of sodium hydride (0.3 mmol) and mix-
ture was stirred at room temperature for 12 h. The reaction mix-
ture was poured into ice-water and the whole was extracted
with CH2Cl2. The CH2Cl2 extract was successively washed brine,
then dried over Na2SO4 and the filtrate under reduced pressure fur-
nished a residue, which was purified by silica-gel open-column
chromatography (Silica Gel 60, 230–400 mesh, Merck) with a hex-
ane–Et2O gradient (1:0–7:3) to yield compound 2aMe (43 mg,
82%) was obtained as pale yellow powder; IR (KBr) mmax 1736,
1682, 1552 cmꢀ1 1H NMR data (CDCl3, 500 MHz) d 7.45 (1H, d,
;
J = 2.2 Hz, H-9), 6.78 (1H, dd, J = 2.2, 0.8 Hz, H-10), 6.77 (1H, br s,
H-8), 5.53–5.56 (1H, m, H-12), 4.68 (2H, d, J = 6.8 Hz, H-11), 3.83
(3H, s, OCH3), 3.68 (OOCH3), 3.06–3.03 (2H, m, H-4), 2.54–2.50
(2H, m, H-3), 1.78 (3H, br s, H-14), 1.68 (3H, br s, H-15); 13C
NMR (CDCl3, 125 MHz) d 174.1 (C, C-2) 156.6 (C, C-7), 155.5 (C,
C-8a), 150.3 (C, C-5), 142.4 (CH, C-9), 137.9 (C, C-13), 120.4 (CH,
C-12), 115.6 (C, C-4a), 112.5 (C, C-6), 104.6 (CH, C-10), 89.5 (CH,
C-8), 69.5 (CH2, C-11), 55.8 (CH3, OCH3), 51.3 (CH3, COOCH3),
34.0 (CH2, C-3), 25.7 (CH3, C-14), 19.4 (CH2, C-4), 18.0 (CH3, C-
15); EIMS m/z 318 [M]+(3), 286 (7), 250(63), 218 (82), 176 (100),
69 (21), 41 (16); HREIMS m/z 318.1482 (calcd for C18H22O5,
318.1467).
3.6.1. 6,7-Furano-5-prenyloxy hydrocoumaric acid (2)
White powder; IR (KBr) mmax 3335, 1704, 1622, 1599 cmꢀ1 1H
;
and 13C NMR shown as Table 1; HRFABMS (pos) m/z 291.1209
[M+H]+ (calcd for C16H19O5, 291.1231).

S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 18 (2010) 455–459
459
3.7. Preparative biotransformation of imperatorin (3)
590 nm was recorded. The inhibition ratio was obtained by the fol-
lowing equation:
Precultured G. cingulata (5 mL) was transferred into a 500 mL
Erlenmeyer flask containing 300 mL of medium. Cultivation was
carried out at 27 °C with stirring (ca. 100 rpm) for 3 days. After
the growth of G. cingulata, 50 mg of 3 in 1.0 mL of dimethyl sulf-
oxide (DMSO) was added into the medium and cultivated for an
additional 12 days, together with two controls, which contained
either mycelia with medium or substrate dissolved in DMSO
with medium. No metabolic product was observed in two
controls.
Inhibition ð%Þ ¼ ½1 ꢀ fðS ꢀ S0Þ=ðC ꢀ C0Þgꢃ ꢁ 100
where C was the fluorescence of the control (enzyme, buffer, and
substrate) after 60 min of incubation, C0 was the fluorescence of
control at zero time, S was the fluorescence of the tested samples
(enzyme, sample solution, and substrate) after incubation, and S0
was the fluorescence of the tested samples at zero time. To allow
for the quenching effect of the samples, the sample solution was
added to the reaction mixture C, and any reduction in fluorescence
by the sample was then investigated. All data are the mean of three
experiments.
3.8. Isolation of metabolite in 3
After the fermentation, the culture medium and mycelia were
separated by filtration. The medium was saturated with NaCl,
and extracted with EtOAc. The mycelia were also extracted with
EtOAc. Each EtOAc extract was combined, the solvent was evapo-
rated, and a crude extract (375 mg) was obtained. The extract
was subjected to silica-gel open-column chromatography (Silica
Gel 60, 230–400 mesh, Merck) with a hexane–Et2O gradient
(9:1–3:7) to yield metabolite 4 (24 mg, 64%).
Acknowledgment
This work was supported by Sumitomo foundation.
References and notes
1. Murray, R.; Mendez, J.; Brown, S.. The Natural Coumarins: Occurrence. In
Chemistry and Biochemistry; John Wiley & Sons: New York, 1982.
2. (a) Baek, N. I.; Ahn, E. M.; Kim, H. Y.; Perk, Y. D. Arch. Pharm. Res. 2000, 23, 467;
(b) Ceska, O.; Chaudhary, S.; Warrington, P.; Ashwood Smith, M. Phytochemistry
1987, 26, 165.
3. (a) Garcia-Argaez, A. N.; Ramirez Apan, T. O.; Parra Delgado, H.; Velazquez, G.;
Martinez-Vazquez, M. Planta Med. 2000, 66, 279; (b) Ban, H. S.; Lim, S. S.;
Suzuki, K.; Jung, S. H.; Lee, Y. S.; Shin, K. H.; Ohuchi, K. Planta Med. 2003, 69,
408; (c) Kawaii, S.; Tomono, Y.; Ogawa, K.; Sugiurra, M.; Yano, M.; Yoshizawa,
Y.; Ito, C.; Furukawa, H. Anticancer Res. 2001, 21, 1905.
3.8.1. Xanthoxol (4)
yellowish needles: IR (KBr) mmax 3324, 1705, 1594 cmꢀ1 1H
;
NMR data (CDCl3, 500 MHz) d 8.10 (1H, d, J = 9.8 Hz, H-4), 8.06
(1H, d, J = 2.3 Hz, H-9), 7.44 (1H, s, H-5), 7.03 (1H, d, J = 2.3 Hz,
H10), 3.35 (1H, br s, OH); 13C NMR (CDCl3, 125 MHz) d 160.2 (C,
C-2) 147.6 (CH2, C-9), 145.7 (CH2, C-4), 145.1 (C, C-7), 139.9 (C,
C-8a), 130.3 (C, C-8), 125.4 (C, C-6), 116.4 (C, C-4a), 114.0 (CH2,
C-3), 110.2 (CH2, C-5), 107.2 (CH2, C-30); EIMS m/z 202 [M]+(100),
174 (63), 149 (10), 146 (9), 89 (15); HREIMS m/z 202.0262 [M]+
(calcd for C11H6O4, 202.0325).
4. Marumoto, S., Miyazawa, M. Phytother. Res., in press.
5. (a) John, V.; Beck, J. P.; Bienkowski, M. J.; Sinha, S.; Heinrikson, R. L. J. Med.
Chem. 2003, 46, 4625; (b) Ghosh, A. K.; Hong, L.; Tang, J. Curr. Med. Chem. 2002,
9, 1135.
6. (a) Jeon, S. Y.; Bae, K. H.; Scog, Y. H.; Song, K. S. Bioorg. Med. Chem. Lett. 2003, 13,
3905; (b) Jeon, S. Y.; Kwon, S. H.; Seong, Y. H.; Bae, K.; Hur, J. M.; Lee, Y. Y.; Suh,
D. Y.; Song, K. S. Phytomedicine 2007, 14, 403; (c) Lee, H. J.; Seong, Y. H.; Bae, K.
H.; Kwon, S. H.; Kwak, H. M.; Nho, S. K.; Kim, K. A.; Hur, J. M.; Lee, K. B.; Kang, Y.
H.; Song, K. S. Arch. Pharm. Res. 2005, 28, 799; (d) Park, I. H.; Jeon, S. Y.; Lee, H. J.;
Kim, S. J.; Song, K. S. Planta Med. 2004, 70, 143.
3.9. b-Secretase (BACE1) enzyme assay
7. (a) Okuno, Y.; Miyazawa, M. J. Nat. Prod. 2004, 67, 1876; (b) Okuno, Y.;
Miyazawa, M. J. Chem. Technol. Biotechnol. 2006, 67, 29.
The assay was carried out according to the supplied manual
with modifications.4,10 Briefly, a mixture of 10
l
l of assay buffer
l of BACE1 (1.0 U/ml), 10
of the substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM
ammonium bicarbonate), and 10 l of sample dissolved in 30%
8. (a) Nankai, H.; Miyazawa, M.; Kameoka, H. J. Nat. Prod. 1997, 60, 287; (b)
Miyazawa, M.; Nankai, H.; Kameoka, H. Phytochemistry 1996, 43, 105; (c)
Miyazawa, M.; Takahashi, K.; Araki, H. Nat. Prod. Res. 2006, 20, 311.
9. (a) Harkar, S.; Razdan, T.; Waight, E. S. Phytochemistry 1984, 23, 419; (b)
Elgamal, A. H. M.; Elewa, H. N.; Elkhrisy, M. A. E.; Helmut, D. Phytochemistry
1979, 18, 139.
(50 mM sodium acetate, pH 4.5), 10
l
ll
l
DMSO was incubated for 60 min at room temperature in the dark.
The mixture was irradiated at 550 nm and the emission intensity at
10. Wen-Yuh, T.; Yu-Ling, H.; Ray-Ling, H.; Ren-Shin, C.; Chien-Chih, C. J. Nat. Prod.
2004, 67, 1014.