Three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone derivatives enantiomeric to agarotetrol from agarwood

Article abstract of DOI:10.1007/s11418-018-1201-2

Agarwood (jinkoh in Japanese) is a resinous wood from Aquilaria species of the family Thymelaeaceae and has been used as incense and in traditional medicines. Characteristic chromone derivatives such as agarotetrol have been isolated from agarwood. In previous study, we isolated two new 2-(2-phenylethyl)chromones together with six known compounds from MeOH extract of agarwood. Further chemical investigation of the MeOH extract led to isolation of eighteen 2-(2-phenylethyl)chromones, including three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones with stereochemistry enantiomeric to agarotetrol-type, viz. (5R,6S,7S,8R)-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (2), (5R,6S,7S,8R)-2-[2-(4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (6), and (5R,6S,7S,8R)-2-[2-(4′-hydroxy-3′- methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (13). The absolute configurations of the new compounds were determined by exciton chirality method. All isolated compounds were tested for their phosphodiesterase (PDE)?3A inhibitory activity by fluorescence polarization method. Compounds 8, 12–15, 21–24 showed moderate PDE?3A inhibitory activity.

Full text of DOI:10.1007/s11418-018-1201-2

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1201-2
ORIGINAL PAPER
Three new 5,6,7,8‑tetrahydroxy‑5,6,7,8‑tetrahydrochromone
derivatives enantiomeric to agarotetrol from agarwood
Takuji Sugiyama1 · Yuji Narukawa1 · Shunsuke Shibata1 · Ryo Masui2 · Fumiyuki Kiuchi1
Received: 1 December 2017 / Accepted: 3 March 2018
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Agarwood (jinkoh in Japanese) is a resinous wood from Aquilaria species of the family Thymelaeaceae and has been used
as incense and in traditional medicines. Characteristic chromone derivatives such as agarotetrol have been isolated from
agarwood. In previous study, we isolated two new 2-(2-phenylethyl)chromones together with six known compounds from
MeOH extract of agarwood. Further chemical investigation of the MeOH extract led to isolation of eighteen 2-(2-pheny-
lethyl)chromones, including three new 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones with stereochemistry enantio-
meric to agarotetrol-type, viz. (5R,6S,7S,8R)-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tet-
rahydrochromone (2), (5R,6S,7S,8R)-2-[2-(4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (6),
and (5R,6S,7S,8R)-2-[2-(4′-hydroxy-3′- methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone (13). The
absolute confgurations of the new compounds were determined by exciton chirality method. All isolated compounds were
tested for their phosphodiesterase (PDE) 3A inhibitory activity by fuorescence polarization method. Compounds 8, 12–15,
21–24 showed moderate PDE 3A inhibitory activity.
Keywords Agarwood · 2-(2-Phenylethyl)chromone · 5,6,7,8-Tetrahydroxy-5,6,7,8-tetrahydrochromone · Enantiomer ·
Phosphodiesterase inhibitor
Introduction
(3) [1], aquilarone A (4) [1], AH2b (5) [2], isoagarotetrol (7)
[3], aquilarone B (8) [1], 6,7-dihydroxy-2-(2-phenylethyl)-
5,6,7,8-tetrahydrochromone (9) [4], 8-chloro-2-(2-
phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone
(10) [4], aquilarone F (12) [1], AH2a (14) [2], aquilarone C
(15) [1], aquilarone G (22) [1], 6-hydroxy-7-methoxy-2-[2-
(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone (23) [5],
aquilarone I (24) [1], findersiachromone (25) [6], AH4
(26) [7], AH3 (27) [7], 6-methoxy-2-[2-(3′-hydroxy-4′-
methoxyphenyl)ethyl]-chromone (28) [8], and AH6 (29) [7]
(Fig. 1). The new compounds (2, 6, 13) have stereochemistry
enantiomeric to agarotetrol (1).
Agarwood (jinkoh in Japanese) is a resinous wood of Aqui-
laria species of the family Thymelaeaceae and has been
used as incense and in traditional medicines. Character-
istic sesquiterpenes and chromone derivatives have been
isolated from agarwood [1–8]. In our previous study, two
new 2-(2-phenylethyl)chromones (19, 21) were isolated
from MeOH extract of agarwood, together with six known
compounds (1, 11, 16–18, 20), among which 20 showed
PDE 3A inhibitory activity with 50 % inhibitory concen-
tration (IC50) of 4.83 µM [9]. Further fractionation of the
extract led to isolation of three new 2-(2-phenylethyl)-
5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromones (2, 6, 13)
together with eighteen known compounds, i.e., aquilarone D
Results and discussion
Compound 2 was obtained as white amorphous pow-
der. High-resolution (HR) electrospray ionization (ESI)
mass spectrometry (MS) gave an [M + Na]+ ion peak
at m/z 387.1086 (calcd. for C18H20NaO8, 387.1056).
The nuclear magnetic resonance (NMR) spectroscopic
data of 2 (Tables 1, 2) were in good agreement with
* Fumiyuki Kiuchi
kiuchi-fm@pha.keio.ac.jp
1
Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen,
Minato-ku, Tokyo 105-8512, Japan
2
Tokyo Research Center, Kyushin Pharmaceutical Co. Ltd.,
1-22-10 Wada, Suginami-ku, Tokyo 166-0012, Japan
Vol.:(0123456789)
1 3

Journal of Natural Medicines
Fig. 1 Structure of compounds 1–29
Table 1 1H NMR data of compounds 2, 6, 13 (δ in ppm, J in Hz)
No.
2 (DMSO-d6, 400 MHz)
2 (CD3OD, 600 MHz)
6 (CD3OD, 600 MHz)
13 (CD3OD, 600 MHz)
3
5
6.08 (1H, s)
4.47 (1H, m)
6.11 (1H, s)
4.74 (1H, d, 3.8)
6.10 (1H, s)
4.74 (1H, d, 4.0)
6.09 (1H, s)
4.73 (1H, d, 3.9)
5-OH
6
6-OH
7
7-OH
8
8-OH
2′
3′
5′
6′
7′
5.19 (1H, d, 4.4)
3.72* (1H)
4.96 (1H, d, 4.4)
3.83 (1H, m)
5.10 (1H, d, 4.8)
4.31 (1H, dd, 7.6, 6.0)
5.81 (1H, d, 6.0)
6.67 (1H, d, 2.4)
4.02 (1H, dd, 4.1, 2.5)
4.04 (1H, dd, 7.4, 2.2)
4.57 (1H, d, 7.5)
4.01 (1H, dd, 4.0, 2.3)
4.04 (1H, dd, 7.4, 2.3)
4.56 (1H, d, 7.4)
4.00 (1H, dd, 3,9, 2.3)
4.03 (1H, dd, 7.3, 2.3)
4.57 (1H, d, 7.3)
6.68 (1H, d, 2.2)
7.12 (1H, d, 8.5)
6.82 (1H, d, 8.5)
6.82 (1H, d, 8.5)
7.12 (1H, d, 8.5)
2.96 (2H, m)
6.75 (1H, d, 1.7)
6.81 (1H, d, 8.4)
6.61 (1H, dd, 8.4, 2.4)
2.80 (2H, m)
6.81 (1H, d, 8.3)
6.64 (1H, d, 8.3, 2.2)
2.90 (2H, m)
6.68 (1H, d, 7.9)
6.64 (dd, 7.9, 1.7)
2.95 (2H, m)
8′
2.80 (2H, m)
8.85 (1H, s)
2.89 (2H, m)
2.89 (2H, m)
2.89 (2H, m)
3′-OH
3′-OCH3
4′-OCH3
3.79 (3H, s)
3.72* (3H, s)
3.80 (3H, s)
3.73 (3H, s)
* overlapped
those of aquilarone E [1]. However, the optical rotation
([α]2D0 = –16.0). These data indicate that 2 was an enantiomer
of aquilarone E. The absolute confguration of aquilarone E
([α]2D2 = +17.3) had the opposite sign to that of aquilarone E
1 3

Journal of Natural Medicines
Table 2 13C NMR data of compounds 2, 6, 13 (150 MHz, CD3OD, δ
confrmed by the downfeld shift of C-6 and C-7 carbon
chemical shifts (4.4 and 7.4 ppm, respectively) similar to
those observed for the monoacetonide of 1 (1a) [10]. Acety-
lation of 2a aforded diacetate of the acetonide (2b), which
was hydrolyzed with trifuoroacetic acid (TFA) to yield diac-
etate derivative (2c). Finally, p-methoxybenzoylation of 2c
with p-methoxybenzoyl chloride in presence of 4-dimeth-
ylaminopyridine (DMAP) gave the target 6,7-di-p-meth-
in ppm)
Carbon no.
2
6
13
2
3
4
5
171.4
114.2
182.0
66.8
171.5
114.2
182.0
66.8
171.4
114.3
182.0
66.7
6
7
74.0
72.5
74.0
72.5
74.0
72.5
1
oxybenzoate derivative (2d). The H NMR spectroscopic
data and circular dichroism (CD) spectrum (Fig. 2) of 1d,
prepared by the same procedure from 1, were consistent with
8
9
10
1′
2′
3′
4′
5′
70.2
70.2
70.1
165.4
121.8
134.1
116.5
147.7
147.7
113.0
120.6
33.2
165.4
121.8
133.2
130.4
115.0
159.8
115.0
130.4
32.9
165.4
121.8
132.6
116.2
148.9
146.1
113.1
121.8
33.4
1
previous report [10], and the H NMR signals of the four
methine protons of 2d at δH 6.10 (1H, d, J = 4.0 Hz, H-5),
5.80 (1H, dd, J = 4.0, 2.5 Hz, H-6), 5.75 (1H, dd, J = 8.0,
2.5 Hz, H-7), and 6.32 (1H, d, J = 8.0 Hz, H-8) were in
good agreement with those of 1d. These data indicate that
the C5–C10 ring of 2d had the same half-chair conforma-
tion as that of 1d as shown in Fig. 2. The CD spectrum of
2d showed positive Cotton efect, opposite to that observed
for 1d (Fig. 2), indicating that the absolute confguration
of 2d was opposite to that of 1d. Thus, the structure of 2
was determined to be (5R,6S,7S,8R)-2-[2-(3′-hydroxy-
4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-
tetrahydrochromone (2). As the absolute confguration of
2 is opposite to that of agarotetrol (1), the optical purity
of the dibenzoates 1d and 2d was evaluated by high-per-
formance liquid chromatography (HPLC) analysis with a
chiral column and circular dichroism detector. Both com-
pounds contained a small amount of the enantiomer, with
(5R,6S,7S,8R):(5S,6R,7R,8S) ratio of 10.5:89.5 for 1d and
(5R,6S,7S,8R):(5S,6R,7R,8S) ratio of 94:6 for 2d.
6′
7
8′
36.5
36.6
36.7
56.4
3′-OCH3
4′-OCH3
56.5
55.7
[1] has been determined by comparison of its optical rota-
tion value with that of agarotetrol (1) [3, 10], whose absolute
confguration was determined by exciton chirality method
[11]. To examine the absolute confguration of 2, its 6,7-di-
p-methoxybenzoate derivative, together with that of 1 (1d)
[3, 10], were prepared by a procedure similar to that of Chen
et al. [1]. The derivatization method is shown in Scheme 1.
In brief, treatment of 2 with 2,2-dimethoxypropane in pres-
ence of pyridinium p-toluenesulfonate (PPTS) afforded
6,7-acetonide derivative 2a. The position of acetonide was
Compound 6 was obtained as pale-yellow amorphous
powder. HR-ESI–MS gave an [M + Na]+ ion peak at m/z
371.1141 (calcd. for C18H20NaO7, 371.1107). The NMR of
6 (Tables 1, 2) showed very similar signals to the chromone
Scheme 1 Synthesis of 6,7-dimethoxybenzoate derivatives of compounds 2, 6
1 3

Journal of Natural Medicines
Fig. 2 Conformation and CD spectra of compounds 1d, 2d, 6d
moiety of 2, with signals of four methine protons at δH 4.74
(1H, d, J = 4.0 Hz), 4.01 (1H, dd, J = 4.0, 2.3 Hz), 4.04
(1H, dd, J = 7.4, 2.3 Hz), and 4.56 (1H, d, J = 7.4 Hz),
which showed correlation signals to δC 66.8, 74.0, 72.5,
and 70.2, respectively, in the C–H correlation spectroscopy
(COSY) spectrum. However, 6 showed para-substituted
phenyl signals at δH 7.12 (2H, d, J = 8.5 Hz) and 6.82
(2H, d, J = 8.5 Hz) instead of the signals for 3′-hydroxy-
4′-methoxyphenyl group in 2. These data indicate that 6
was a deacetyl derivative of AH1A [2]. However, the sign
of the optical rotation ([α]2D2 = +13.5) was opposite to that
of AH1A ([α]1D4 = –14.3). These data indicate that 6 was the
enantiomer of deacetyl derivative of AH1A. To confrm the
absolute confguration of 6, its 6,7-di-p-methoxybenzoate
derivative (6d) was prepared using the same procedure as
2d (Fig. 2). The 1H NMR spectroscopic data of 6d showed
similar signals for four methine protons as those of 1d,
indicating that 6d has the same conformation of C5–C10
ring as that of 1d (Fig. 2). The CD spectrum of 6d indi-
cated positive chirality of 6,7-dibenzoate groups, the same
as for 2d (Fig. 2). Thus, the structure of 6 was determined
to be (5R,6S,7S,8R)-2-[2-(4′-methoxyphenyl)ethyl]-5,6,7,8-
tetrahydroxy-5,6,7,8-tetrahydrochromone (6). Chiral HPLC
analysis revealed that 6d was also a mixture of enantiomers
with (5R,6S,7S,8R):(5S,6R,7R,8S) ratio of 76:24.
NMR spectroscopic data of 13 (Tables 1, 2) were similar to
those of 2, showing signals for four oxymethine protons at
δH 4.73 (1H, d, J = 4.0 Hz), 4.00 (1H, dd, J = 4.0, 2.2 Hz),
4.03 (1H, dd, J = 7.4, 2.2 Hz), and 4.57 (1H, d, J = 7.4 Hz),
which showed correlation signals to δC 66.7, 74.0, 72.5, and
70.1, respectively, in the heteronuclear multiple-quantum
coherence (HMQC) spectrum. These data indicate that 13
had the same (5R*,6S*,7S*,8R*)-5,6,7,8- tetrahydroxy-
1
5,6,7,8-tetrahydrochromone structure as 2. The H NMR
spectrum also showed a methoxy signal at δH 3.79 (3H, s).
However, the 1H NMR pattern of the aromatic protons at δH
6.75 (1H, d, J = 1.9 Hz), 6.68 (1H, d, J = 8.0 Hz), and 6.64
(1H, dd, J = 8.0, 1.8 Hz) difered from that of 2, indicating
that the methoxy group was present at a diferent position.
The locations of methoxy and hydroxy groups on the ben-
zene ring were determined from the heteronuclear multi-
ple-bond connectivity (HMBC) spectrum and the nuclear
Overhauser efect (NOE) diference experiment, as shown
in Fig. 3. The absolute confguration of 13 was assumed to
be the same as that of 2, because 13 showed the same sign
of optical rotation as 2. Consequently, the structure of 13
was determined to be (5R,6S,7S,8R)-2-[2-(4′-hydroxyl-3′-
methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahy-
drochromone (13). As compounds 2d and 6d were a mix-
ture of enantiomers and 2, 6, and 13 showed similar optical
rotation values ([α]2D2 = +17.3, +13.5, +7.27, respectively),
13 may also be a mixture of enantiomers with excess of
(5R,6S,7S,8R) isomer.
Compound 13 was obtained as colorless oil. ESI–MS
gave an [M + Na]+ ion peak at m/z = 387, and HR-fast
atom bombardment (FAB)-MS gave an [M + H]+ ion peak
at m/z = 365.1252 (calcd. for C18H21O8, 365.1236), showing
All isolated compounds were tested for their PDE 3A
inhibitory activity. Compounds 8, 12–15, 21–24 showed
that 13 had the same molecular formula as 2. The 1H and 13
C
1 3

Journal of Natural Medicines
using Chiralcel OD-RH 4.6 mm I.D. × 150 mm (Daicel,
Osaka, Japan). Preparative HPLC was performed with LC-
10AD, SPD-10A, FCV-10AL, and DGU-12A equipment
(Shimadzu) using ODS columns of CAPCELL PAK C18
MG-II, 10 mm I.D. × 150 mm (Shiseido, Tokyo, Japan) and
COSMOSIL 5C18-MG-II, 10 mm I.D. × 250 mm (Nacalai,
Kyoto, Japan). Column chromatography was performed with
silica gel (40–50, 40–100, and 100–210 µm, Kanto Chemi-
cal, Tokyo, Japan), Diaion HP-20 (Mitsubishi chemical,
Tokyo, Japan), and MCI-Gel CHP20P (Mitsubishi chemi-
cal). Silica gel 60 F-254 and Silica gel 60 RP-18 F-254S
(Merck, Darmstadt, Germany) were used for thin-layer chro-
matography (TLC), and spots were detected under UV light
(254 and/or 365 nm).
Fig. 3 Selected HMBC and NOE correlations of compound 13
moderate PDE 3A inhibitory activity (Table 3). Interest-
ingly, the only structural diference between compounds 8
(IC50: 41.8 µM) and 15 (IC50: 33.2 µM) lies in the presence
or absence of methoxy group at C-4′ position. These results
verify our previous structure–activity relationship hypoth-
esis that methoxy group at C-4′ position plays an important
role to increase PDE 3A inhibitory activity [9].
Plant material
Agarwood was purchased from Uchida Wakanyaku Ltd. (lot
D2R3219, from Indonesia).
Extraction and isolation
Powdered agarwood (80.0 g) was extracted twice with
MeOH (2.0 L) under refux for 2 h. The combined fltrates
were concentrated under reduced pressure to give brown
MeOH extract (20.2 g). MeOH extract (18.2 g) was sub-
jected to Diaion HP-20 column chromatography (CC)
(4.4φ × 24 cm) and successively eluted with H2O, MeOH,
acetone, and CHCl3. The MeOH fraction (14.0 g) was
applied to MCI-Gel CHP20P CC (4.0φ × 17 cm) and eluted
with a gradient of H2O/MeOH (1:0, 1:4, 2:3, 3:2, 4:1, 0:1)
and acetone to yield seven fractions (M-1 to M-7). Frac-
tion M-2 (128 mg) was then separated by semipreparative
HPLC (MeCN/H2O, 1:9–0:1) to give six fractions (M-2-1
to M-2-6). Fraction M-2-1 (4.9 mg) was purifed by semi-
preparative HPLC (MeCN/H2O, 8:92) to give 12 (3.4 mg).
Fraction M-2-3 (3.6 mg) was purifed by semipreparative
HPLC (MeCN/H2O, 8:92) to give 13 (2.3 mg). Fraction M-3
(1.1 g) was separated by semipreparative HPLC (MeCN/
H2O, 1:9–3:7) to give eight fractions (M-3-1 to M-3-
8). Compounds 2 (47.9 mg), 4 (23.6 mg), 5 (24.4 mg), 6
(45.0 mg), 7 (29.2 mg), and 8 (34.9 mg) were obtained from
Experimental
General experimental procedures
Optical rotations were measured using a JASCO P-1020
(Tokyo, Japan). Circular dichroism spectra were recorded
using a JASCO J-1100 spectrometer. Ultraviolet (UV)
spectra were obtained on a UV–visible spectrophotometer
UV-1600 (Shimadzu, Kyoto, Japan), and infrared (IR) spec-
tra were obtained with FT-IR SPX 60 (JASCO). ESI–MS
was carried out using a JMS-T100LP AccuTOF LC-plus
(JEOL, Tokyo, Japan). HR-FAB-MS was measured with a
JEOL JMS-7000. JEOL ECP-600 FT-NMR, VARIAN Unity
Inova 500-MR (Palo Alto, CA, USA), and VARIAN 400-
MR were used for measurement of NMR spectra with tetra-
methylsilane (TMS) as internal standard. Chiral HPLC was
performed with Pu-2089plus, As-2055plus, Co-2065plus,
and circular dichroism detector CD-2095plus (JASCO)
Table 3 PDE 3A inhibitory activity of compounds 1–29
Compound
IC50 (μM)
Compound
IC50 (μM)
Compound
IC50 (μM)
Compound
IC50 (μM)
Compound
IC50 (μM)
Milrinone
0.92
6
>100
>100
41.8
>100
>100
>100
12
13
14
15
16
17
90.5
40.1
54.0
33.2
26.6
32.8
18
19
20
21
22
23
31.2
24.9
4.83
32.9
11.9
18.9
24
25
26
27
28
29
19.9
1
2
3
4
5
>100
>100
>100
>100
>100
7
>100
>100
>100
>100
>100
8
9
10
11
1 3

Journal of Natural Medicines
M-3-1, M-3-3, M-3-4, M-3-6, M-3-7, and M-3-8, respec-
tively. Fraction M-3-2 (10.2 mg) was purifed by silica gel
CC (1.0φ × 14.5 cm) with CHCl3/MeOH (9:1) to yield 3
(4.2 mg). Crystallization of fraction M-3-5 from 2-propanol
gave 1 (87.4 mg). Fraction M-4 was subjected to silica
gel CC (2.5φ × 22 cm) with a gradient of CHCl3/MeOH
(19:1–1:4) to give ten fractions (M-4-1 to M-4-10). Frac-
tion M-4-5 was separated by semipreparative HPLC (MeCN/
H2O, 1:4–3:7) to give three fractions (M-4-5-1 to M-4-5-
3). Fraction M-4-6 was separated by semipreparative HPLC
(MeCN/H2O, 1:4–3:7) to give seven fractions (M-4-6-1 to
M-4-6-7). Fraction M-4-6-4 was subjected to silica gel CC
(1.0φ × 13 cm) with a gradient of hexane/EtOAc (2:3–1:3)
to give 9 (3.7 mg). Fractions M-4-5-3 and M-4-6-7 were
combined and purifed by semipreparative HPLC (MeCN/
H2O, 3:7) to give 10 (18.1 mg). Fraction M-4-8 was sepa-
rated by semipreparative HPLC (MeCN/H2O, 15:85) to give
three fractions (M-4-8-1 to M-4-8-3). Fraction M-4-8-3
gave compound 15 (12.4 mg). Fraction M-4-8-2 was puri-
fed by semipreparative HPLC (MeCN/H2O, 14:86) to give
14 (11.0 mg). Fraction M-5 (2.0 g) was subjected to silica
gel CC (3.0φ × 19 cm) with a gradient of CHCl3/MeOH
(49:1–0:1) to give eleven fractions (M-5-1 to M-5-11).
Fraction M-5-2 (241 mg) was separated by semipreparative
HPLC (MeCN/H2O, 3:7–5:5) to give nine fractions (M-5-
2-1 to M-5-2-9). Fraction M-5-2-3 (19.0 mg) was subjected
to silica gel CC (1.0φ × 15 cm) with a gradient of hexane/
EtOAc (1:1–0:1) to give 16 (5.4 mg). Fraction M-5-2-6
(23.5 mg) was subjected to silica gel CC (1.0φ × 17 cm)
with hexane/EtOAc (1:1) to obtain 11 (7.1 mg). Fraction
M-5-4 (66.3 mg) was purifed by semipreparative HPLC
(MeCN/H2O, 35:65) to give 17 (5.9 mg) and 21 (4.2 mg).
Fraction M-5-5 (136.2 mg) was purifed by semiprepara-
tive HPLC (MeCN/H2O, 35:65) to give 18 (11.3 mg), 19
(3.7 mg), 20 (3.2 mg), and 21 (6.4 mg). Fraction M-5 (2.3 g)
was subjected to silica gel CC (3.0φ × 19 cm) with a gradi-
ent of CHCl3/MeOH (49:1–0:1) to give ten fractions (M-5-1
to M-5-10). Fraction M-5-3 (64.0 mg) was purifed by semi-
preparative HPLC (MeCN/H2O, 32:68) to give 22 (2.6 mg),
23 (4.7 mg), 17 (11.4 mg), and 24 (4.0 mg). Fraction M-6
(2.6 g) was subjected to silica gel CC (3.1φ × 18 cm) with
a gradient of CHCl3/MeOH (49:1–0:1) to give ten frac-
tions (M-6-1 to M-6-10). Fractions M-6-2 (341 mg) and
M-6-3 (147 mg) were combined, and a part of the mixture
(428 mg) was subjected to silica gel CC (3.1φ × 18 cm)
with a gradient of hexane/EtOAc (9:1–0:1) and MeOH to
give thirteen fractions (M-6-2-1 to M-6-2-13). Fraction
M-6-2-5 (31.6 mg) was purifed by semipreparative HPLC
(MeCN/H2O, 3:2) to give 25 (6.1 mg). Fraction M-6-2-6
(15.4 mg) was purifed by semipreparative HPLC (MeCN/
H2O, 1:1–1:0) to give 26 (8.1 mg). Fraction M-6-2-7
(34.4 mg) was purifed by semipreparative HPLC (MeCN/
H2O, 1:1) to give 26 (2.3 mg). Fraction M-6-2-8 (19.3 mg)
was purifed by semipreparative HPLC (MeCN/H2O, 2:3)
to give 27 (6.1 mg). Fraction M-6-2-9 (17.6 mg) was puri-
fed by semipreparative HPLC (MeCN/H2O, 2:3) to give
28 (3.4 mg). Fraction M-6-2-10 (84.3 mg) was separated
by semipreparative HPLC (MeCN/H2O, 2:3) to give 11
(11.3 mg), 28 (3.6 mg), and 29 (43.0 mg).
(5R,6S,7S,8R)‑2‑[2‑(3′‑Hydroxy‑4′‑methoxyphenyl)
ethyl]‑5,6,7,8‑tetrahydroxy‑ 5,6,7,8‑tetrahydrochromone
(2) White amorphous powder; [α]2D2 = +17.3 (c = 1.0,
MeOH); IR (ATR, cm−1) 3291, 1653, 1575, 1514, 1446; HR-
ESI–MS m/z: 387.1086 [M + Na]+ (calcd. for C18H20NaO8,
387.1056); 1H and 13C NMR (see Tables 1, 2).
(5R,6S,7S,8R)‑2‑[2‑(4′‑Methoxyphenyl)ethyl]‑5,6,7,8‑tet‑
rahydroxy‑5,6,7,8‑tetrahydrochromone (6) Pale-yellow
amorphous powder; [α]2D2 = +13.5 (c = 1.0, MeOH); IR
(ATR, cm−1) 3290, 1653, 1583, 1512, 1440, 1178; HR-
ESI–MS m/z: 371.1141 [M + Na]+ (calcd. for C18H20NaO7,
371.1107); 1H and 13C NMR (see Tables 1, 2).
(5R,6S,7S,8R)‑2‑[2‑(4′‑Hydroxy‑3′‑methoxyphenyl)
ethyl]5,6,7,8‑tetrahydroxy‑5,6,7,8‑tetrahydrochromone
(13) Colorless oil; [α]2D2 = +7.27 (c 0.22, MeOH); UV λmax
(MeOH) nm (log ε): 282 (3.10), 250 (3.71), 221 (3.75); HR-
FAB-MS m/z: 365.1252 [M + H]+ (calcd. for C18H21O8,
365.1236); 1H and 13C NMR (see Tables 1, 2).
Compound 2a A mixture of 2 (20.0 mg), PPTS (5.8 mg),
and 2,2-DMP (1.3 mL) in dry acetone (5 mL) was stirred
at room temperature overnight. The solvent was removed
under reduced pressure. The residue was then partitioned
between EtOAc and saturated NaHCO3. The organic layer
was dried over MgSO4, fltered, and concentrated. The resi-
due was purifed by silica gel CC (CHCl3/MeOH = 14:1) to
give 2a (6.4 mg) as colorless oil. HR-ESI–MS m/z: 427.1273
1
[M + Na]+ (calcd. for C21H24NaO8, 427.1369). H NMR
(CD3OD, 500 MHz) δ 1.15, 1.32 (each 3H, s, –CH3), 2.94
(4H, br s, H-7′, 8′), 3.79 (3H, s, –OCH3), 4.46 (1H, d,
J = 2.0 Hz, H-8), 4.68 (1H, dd, J = 6.5, 2.0 Hz, H-6), 4.72
(1H, dd, J = 6.5, 2.0 Hz, H-7), 5.00 (1H, d, J = 2.0 Hz,
H-5), 6.16 (1H, s, H-3), 6.60 (1H, dd, J = 8.0, 2.0 Hz, H-6′),
6.67 (1H, d, J = 2.0 Hz, H-2′), 6.78 (1H, d, J = 8.0 Hz,
H-5′). 13C NMR (CD3OD, 125 MHz) δ 24.2 (–CH3), 26.7
(–CH3), 33.8 (C-7′), 36.3 (C-8′), 56.4 (4′-OCH3), 63.3 (C-5),
69.5 (C-8), 78.8 (C-6), 79.9 (C-7), 109.7 [–(O)2–C–(CH3)2],
112.8 (C-5′), 115.1 (C-3), 116.4 (C-2′), 120.6 (C-6′), 123.7
(C-10), 133.9 (C-1′), 147.6 (C-4′), 147.7 (C-3′), 167.9 (C-9),
171.1 (C-2), 180.2 (C-4).
Compound 2b A mixture of 2a (6.4 mg), pyridine (1 mL),
and acetic anhydride (1 mL) was stirred at room temperature
until 2a disappeared according to TLC. The mixture was
1 3

Journal of Natural Medicines
concentrated under reduced pressure to give 2b (9.3 mg)
as colorless oil that was used without purifcation for the
next step. HR-ESI–MS m/z: 553.1655 [M + Na]+ (calcd. for
C27H30NaO11, 553.1686). 1H NMR (CD3OD, 400 MHz) δ
1.23, 1.33 (each 3H, s, –CH3), 2.03, 2.11, 2.23 (each 3H, s, –
OCOCH3), 2.93 (4H, m, H-7′, 8′), 3.77 (3H, s, –OCH3), 4.62
(1H, dd, J = 6.4, 1.6 Hz, H-6), 4.69 (1H, dd, J = 6.4, 2.0 Hz,
H-7), 5.64 (1H, d, J = 2.0 Hz, H-8), 6.07 (1H, d, J = 1.6 Hz,
H-5), 6.21 (1H, s, H-3), 6.90 (1H, d, J = 2.4 Hz, H-2′), 6.96
(1H, d, J = 8.4 Hz, H-5′), 7.05 (1H, dd, J = 8.4, 2.4 Hz,
H-6′).13C NMR (CD3OD, 100 MHz) δ 20.5 (–OCOCH3),
20.6 (–OCOCH3), 20.8 (–OCOCH3), 24.2 (–CH3), 26.7
(–CH3), 32.7 (C-7′), 36.1 (C-8′), 56.4 (4′-OCH3), 65.2 (C-5),
69.6 (C-8), 75.9 (C-6), 77.1 (C-7), 110.2 [–(O)2–C–(CH3)2],
113.6 (C-5′), 115.3 (C-3), 121.6 (C-10′), 123.8 (C-2′), 127.8
(C-6′), 133.5 (C-1′), 141.1 (C-3′), 151.4 (C-4′), 164.2 (C-9),
170.7 (–OCOCH3), 171.0 (–OCOCH3), 171.3 (–OCOCH3),
171.5 (C-2), 179.2 (C-4).
J = 8.5 Hz, H-5′), 7.01 (2H, d, J = 9.1 Hz), 7.08 (1H, dd,
J = 8.5, 2.2 Hz, H-6′), 7.84 (2H, d, J = 9.1 Hz), 7.89 (2H,
d, J = 9.1 Hz).
Compounds 1d and 6a–d were prepared by same proce-
dure as compounds 2a–d.
Compound 1d Colorless oil, [α]2D4 = −76.9 (c = 0.82,
MeOH), UV λmax (MeOH) nm (log ε): 259 (4.15), CD λext
(MeOH) nm (Δε): 251 (1.71), 272 (–11.7), HR-ESI–MS
(positive) m/z: 693.1967 [M + Na]+ (calcd. for C37H34NaO12,
693.1948). 1H NMR (CD3OD, 600 MHz) δ 2.12, 2.18 (each
3H, s, –OCOCH3), 2.98 (4H, m, H-7′, 8′), 3.85, 3.86 (each
3H, s, –OCH3), 5.74 (1H, dd, J = 8.3, 2.5 Hz, H-7), 5.81
(1H, dd, J = 4.0, 2.5 Hz, H-6), 6.10 (1H, d, J = 4.0 Hz,
H-5), 6.21 (1H, s, H-3), 6.31 (1H, d, J = 8.3 Hz, H-8), 6.95
(2H, d, J = 9.1 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.20 (3H, m,
J = 7.5 Hz, H-2′, 4′, 6′), 7.27 (2H, t, J = 7.4 Hz, H-3′, 5′),
7.84 (2H, d, J = 9.1 Hz), 7.89 (2H, d, J = 9.0 Hz). 13C NMR
(CD3OD, 150 MHz) δ 20.5 (–OCOCH3), 20.6 (–OCOCH3),
33.6 (C-7′), 36.0 (C-8′), 56.1 (–OCH3), 56.1 (–OCH3), 65.3
(C-5), 67.9 (C-8), 69.9 (C-6), 70.9 (C-7), 114.9 (C-3), 115.0,
115.1, 120.0 (C-10), 122.1, 122.2, 127.6 (C-4′), 129.4 (C-2′,
6′), 129.7 (C-3′, 5′), 132.9, 133.0, 140.9 (C-1′), 161.5 (C-9),
165.7, 165.8, 166.0, 166.4, 171.0 (C-2), 171.3 (–OCOCH3),
171.5 (–OCOCH3), 179.2 (C-4).
Compound 2c A mixture of 2b (9.3 mg), dry CH2Cl2
(1 mL), and TFA (0.2 mL) was stirred at room tempera-
ture until 2b disappeared according to TLC. The reaction
mixture was concentrated under reduced pressure, and the
residue was purifed by silica gel CC (CHCl3/MeOH = 14:1)
to give 2c (6.9 mg) as colorless oil. HR-ESI–MS m/z:
513.1380 [M + Na]+ (calcd. for C24H26NaO11, 513.1373).
1H NMR (CDCl3, 500 MHz) δ 2.07, 2.22, 2.31 (each 3H, s,
OCOCH3), 2.77 (2H, m, H-8′), 2.86 (2H, m, H-7′), 3.82 (3H,
s, 4′-OCH3), 4.13 (1H, br d, J = 7.0 Hz, H-7), 4.22 (1H, dd,
J = 3.5, 2.5 Hz, H-6), 5.97 (1H, d, J = 3.5 Hz, H-5), 5.97
(1H, d, J = 7.0 Hz, H-8), 6.12 (1H, s, H-3), 6.86 (1H, d,
J = 2.0 Hz, H-2′), 6.90 (1H, d, J = 8.5 Hz, H-5′), 6.99 (1H,
dd, J = 8.5, 2.0 Hz, H-6′).
Compound 6a Colorless oil, [α]2D3 = −8.6 (c = 1.0,
MeOH), HR-ESI–MS m/z: 411.1471 [M + Na]+ (calcd. for
1
C21H24NaO7, 411.1420). H NMR (CD3OD, 500 MHz) δ
1.15, 1.32 (each 3H, s, –CH3), 2.90 (2H, m, H-8′), 2.95 (2H,
m, H-7′), 3.73 (3H, s, 4′-OCH3), 4.46 (1H, d, J = 2.0 Hz,
H-8), 4.68 (1H, dd, J = 7.0, 2.0 Hz, H-6), 4.72 (1H, dd,
J = 7.0, 2.0 Hz, H-7), 5.01 (1H, d, J = 2.0 Hz, H-5), 6.14
(1H, s, H-3), 6.80 (2H, d, J = 8.5 Hz, H-3′, 5′), 7.09 (2H,
d, J = 8.5 Hz, H-2′, 6′). 13C NMR (CD3OD, 125 MHz) δ
22.8 (–CH3), 25.3 (–CH3), 31.6 (C-7′), 35.1 (C-8′), 54.2
(4′–OCH3), 61.9 (C-5), 68.1 (C-8), 77.4 (C-6), 78.4 (C-7),
108.2 [–(O)2–C–(CH3)2], 113.6 (C-3′, 5′), 113.8 (C-3), 122.4
(C-10), 129.0 (C-2′, 6′), 131.5 (C-1′), 158.4 (C-4′), 166.5
(C-9), 169.5 (C-2), 178.7 (C-4).
Compound 2d To a mixture of 2c (6.9 mg) and DMAP
(8.9 mg) in pyridine (1 mL), p-methoxybenzoyl chloride
(0.5 mL) was added dropwise at 0 °C, and the mixture was
stirred at room temperature overnight. The resulting solu-
tion was diluted with EtOAc and saturated NH4Cl. The
organic layer was washed with saturated NaHCO3, dried
over MgSO4, fltered, and concentrated. The residue was
purifed by silica gel CC (CHCl3/MeOH = 39:1) and HPLC
(MeCN/H2O = 3:7–1:0) to give 2d (1.5 mg) as colorless
oil, [α]2D4 = +28.4 (c = 0.15, MeOH), UV λmax (MeOH) nm
(log ε): 257 (4.21), CD λext (MeOH) nm (Δε): 254 (–4.53),
271 (4.81), HR-ESI–MS m/z: 781.2182 [M + Na]+ (calcd.
for C40H38NaO15, 781.2108). 1H NMR (CD3OD, 600 MHz)
δ 2.13, 2.18, 2.21 (each 3H, s, –OCOCH3), 2.98 (4H, d,
J = 4.4 Hz, –CH2–CH2–), 3.77, 3.84, 3.87 (each 3H, s, –
OCH3), 5.75 (1H, dd, J = 8.0, 2.5 Hz, H-7), 5.80 (1H, dd,
J = 4.0, 2.5 Hz, H-6), 6.10 (1H, d, J = 4.0 Hz, H-5), 6.24
(1H, s, H-3), 6.32 (1H, d, J = 8.0 Hz, H-8), 6.93 (1H, d,
J = 2.2 Hz, H-2′), 6.95 (2H, d, J = 9.1 Hz), 6.98 (1H, d,
Compound 6b Colorless oil, [α]2D3 = +5.4 (c = 1.0,
MeOH), HR-ESI–MS m/z: 495.1675 [M + Na]+ (calcd. for
1
C25H28NaO9, 495.1631). H NMR (CD3OD, 500 MHz) δ
1.22, 1.33 (each 3H, s, –CH3), 2.03 (3H, s, 5-OCOCH3),
2.11 (3H, s, 8-OCOCH3), 2.91 (2H, m, H-8′), 2.94 (2H,
m, H-7′), 3.73 (3H, s, 4′-OCH3), 4.62 (1H, dd, J = 6.5,
1.5 Hz, H-6), 4.69 (1H, dd, J = 6.5, 2.0 Hz, H-7), 5.64 (1H,
d, J = 2.0 Hz, H-8), 6.07 (1H, d, J = 1.5 Hz, H-5), 6.17
(1H, s, H-3), 6.80 (2H, d, J = 8.5 Hz, H-3′, 5′), 7.08 (2H,
d, J = 8.5 Hz, H-2′, 6′). 13C NMR (CD3OD, 125 MHz) δ
20.6 (8-OCOCH3), 20.8 (5-OCOCH3), 24.2 (–CH3), 26.7
1 3

Journal of Natural Medicines
(–CH3), 32.9 (C-7′), 36.4 (C-8′), 55.6 (4′-OCH3), 65.2 (C-5),
69.6 (C-8), 75.8 (C-6), 77.1 (C-7), 110.1 [–(O)2–C–(CH3)2],
115.0 (C-3′, 5′), 115.3 (C-3), 121.6 (C-10), 130.4 (C-2′, 6′),
132.8 (C-1′), 159.8 (C-4′), 164.1 (C-9), 171.0 (8-OCOCH3),
171.4 (5-OCOCH3), 171.5 (C-2), 179.2 (C-4).
were processed using i-control software (Tecan). Percent-
age inhibition was calculated by taking the polarization of
without-enzyme group and control group as 0 and 100 % of
PDE activity, respectively. All compounds were screened at
100 µM, and active compounds were further tested at 10 µM.
Compounds 20 and 21 were tested at 3, 10, 30, and 100 µM.
Milrinone, a representative PDE 3 inhibitor, was used as
positive control, and the inhibitory activity was measured at
0.5, 5, and 50 µM. The assay was triplicated. Fifty percent
inhibitory concentration (IC50) values were calculated by a
line segment through 50 %, i.e., using the following equa-
tion: IC50 = 10[log(A/B) × (50 − C)/(D − C) + log(B)], where A = high
concentration through 50 %, B = low concentration through
50 %, C = inhibitory activity (%) at B, and D = inhibitory
activity (%) at A.
Compound 6c Colorless oil, [α]2D3 = +12.8 (c = 1.0,
MeOH), HR-ESI–MS m/z: 455.1377 [M + Na]+ (calcd. for
C22H24NaO9, 495.1318). 1H NMR (CDCl3, 400 MHz) δ 2.06
(3H, s, OCOCH3), 2.22 (3H, s, OCOCH3), 2.77 (2H, m,
H-8′), 2.86 (2H, t, J = 7.4 Hz, H-7′), 3.71 (3H, s, 4′-OCH3),
4.13 (1H, dd, J = 7.8, 2.4 Hz, H-7), 4.22 (1H, dd, J = 3.2,
2.4 Hz, H-6), 5.97 (1H, d, J = 3.2 Hz, H-5), 6.02 (1H, d,
J = 7.8 Hz, H-8), 6.13 (1H, s, H-3), 6.83 (2H, d, J = 8.6 Hz,
H-3′, 5′), 7.07 (2H, d, J = 8.6 Hz, H-2′, 6′). 13C NMR
(CDCl3, 100 MHz) δ 20.8 (OCOCH3), 20.9 (OCOCH3), 31.7
(C-7′), 35.5 (C-8′), 55.2 (4′-OCH3), 66.7 (C-5), 69.7 (C-8),
69.9 (C-7), 70.0 (C-6), 113.7 (C-3), 114.1 (C-3′, 5′), 118.6
(C-10), 129.1 (C-2′, 6′), 131.3 (C-1′), 158.3 (C-4′), 160.3
(C-9), 168.7 (C-2), 170.6 (OCOCH3), 170.6 (OCOCH3),
178.0 (C-4).
Acknowledgements This work was supported in part by Keio Gijyuku
Academic Development Funds and MEXT-Supported Program for the
Strategic Research Foundation at Private Universities.
References
Compound 6d Colorless oil, [α]2D4 = +64.7 (c = 1.0,
MeOH), UV λmax (MeOH) nm (log ε): 258 (4.54), CD λext
(MeOH) nm (Δε): 254 (–6.90), 273 (6.63), HR-ESI–MS m/z:
723.2106 [M + Na]+ (calcd. for C38H36NaO13, 723.2054).
1H NMR (CD3OD, 600 MHz) δ 2.12, 2.18 (each 3H, s, –
OCOCH3), 2.98 (4H, m, –CH2–CH2–), 3.85, 3.86 (each
3H, s, -OCH3), 5.74 (1H, dd, J = 8.3, 2.5 Hz, H-7), 5.81
(1H, dd, J = 4.0, 2.5 Hz, H-6), 6.10 (1H, d, J = 4.0 Hz,
H-5), 6.21 (1H, s, H-3), 6.31 (1H, d, J = 8.3 Hz, H-8), 6.95
(2H, d, J = 9.1 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.20 (3H, m,
J = 7.5 Hz, H-2′, 6′, H-4′), 7.27 (2H, t, J = 7.4 Hz, H-3′, 5′),
7.84 (2H, d, J = 9.1 Hz), 7.89 (2H, d, J = 9.0 Hz). 13C NMR
(CD3OD, 150 MHz) δ 20.5 (–OCOCH3), 20.6 (–OCOCH3),
33.6 (C-7′), 36.0 (C-8′), 55.6 (–OCH3), 56.1 (–OCH3), 56.1
(–OCH3), 65.3 (C-5), 67.9 (C-8), 69.9 (C-6), 70.9 (C-7),
114.9 (C-3), 115.0, 115.1, 120.0, 122.1, 122.2, 127.6 (C-4′),
129.4 (C-2′, 6′), 129.7 (C-3′, 5′), 132.9, 133.0, 140.9 (C-1′),
161.5 (C-9), 165.7, 165.8, 166.0, 166.4, 171.0 (C-2), 171.3
(–OCOCH3), 171.5 (–OCOCH3), 179.2 (C-4).
1. Chen D, Xu ZR, Chai XY, Zeng KW, Jia YX, Bi D, Ma ZZ, Tu
PF (2012) Nine 2-(2-phenylethyl)chromone derivatives from the
resinous wood of Aquilaria sinensis and their inhibition of LPS-
induced NO production in RAW 264.7 cells. Eur J Org Chem
2012:5389–5397
2. Shimada Y, Konishi T, Kiyosawa S (1986) Studies on the agal-
wood (Jinkō) VI. Structures of three 2-(2-phenylethyl)-5,6,7,8-
tetrahydrochromone derivatives, AH1A, AH2a and AH2b. Chem
Pharm Bull 34:3033–3037
3. Shimada Y, Konishi T, Kiyosawa S, Nishi M, Miyahara K, Kawa-
saki T (1986) Studies on the agalwood (Jinkō) IV. Structures of
2-(2-phenylethyl)chromone derivatives, agarotetrol and isoagaro-
tetrol. Chem Pharm Bull 34:2766–2773
4. Yagura T, Ito M, Kiuchi F, Honda G, Shimada Y (2003) Four new
2-(2-phenylethyl) chromone derivatives from withered wood of
Aquilaria sinensis. Chem Pharm Bull 51:560–564
5. Yang L, Qiao LR, Xie D, Yuan YH, Chen NH, Dai JG, Guo SX
(2012) 2-(2-Phenylethyl)chromones from Chinese eaglewood.
Phytochemistry 76:92–97
6. Hashimoto K, Nakahara S, Inoue T, Sumida Y, Takahashi M, Mas-
ada Y (1985) A new chromone from agarwood and pyrolysis prod-
ucts of chromone derivatives. Chem Pharm Bull 33:5088–5091
7. Shimada Y, Tominaga T, Konishi T, Kiyosawa S (1982) Studies on
the Agarwood (Jinkō) I. Structures of 2-(2-phenylethyl)chromone
derivatives. Chem Pharm Bull 30:3791–3795
PDE 3A inhibitory activity
8. Li W, Cai CH, Dong WH, Guo ZK, Wang H, Mei WL, Dai HF
(2014) 2-(2-Phenylethyl)chromone derivatives from Chinese agar-
wood induced by artifcial holing. Fitoterapia 98:117–123
9. Sugiyama T, Narukawa Y, Shibata S, Masui R, Kiuchi F (2016)
New 2-(2-phenylethyl)chromone derivatives and inhibitors of
phosphodiesterase (PDE) 3A from agarwood. Nat Prod Commun
11:795–797
PDE 3A inhibitory activity was measured with PDE 3A
assay kit provided by BPS Bioscience (San Diego, USA).
In brief, reaction mixture containing PDE assay bufer,
200 nM FAM-cAMP, 400 pg human recombinant PDE 3A,
and a test compound was incubated at room temperature
for 60 min. Then, binding agent (1:100 dilution by binding
agent diluent) was added and incubated for 60 min. Fluores-
cence was measured by M-1000 (Tecan, Männedorf, Swiss)
at 475 nm (excitation) and 518 nm (emission). The data
10. Yoshii E, Koizumi T, Oribe T (1978) The structure of agarotet-
rol, a novel highly oxygenated chromone from agarwood (Jinko).
Tetrahedron Lett 41:3921–3924
11. Harada N, Nakanishi K (1972) The exciton chirality method and
its application to confgurational and conformational studies of
natural products. Acc Chem Res 5:257–263
1 3