Flavan-4-ol glycosides from the rhizomes of Abacopteris penangiana

Article abstract of DOI:10.1021/np050191p

Four new flavan-4-ol glycosides, abacopterins A-D (1-4), were isolated from a methanol extract of the rhizomes of Abacopteris penangiana, together with two known compounds, triphyllin A (5) and 6,8-dimethyl-7-hydroxy-4′- methoxyanthocyanidin-5-O-β-D-glucopyranoside (6). Their structures were elucidated by extensive spectroscopic analysis and chemical methods. The cytotoxic activity of 1-5 against HepG2 human hepatoma cells was investigated.

Full text of DOI:10.1021/np050191p

J. Nat. Prod. 2006, 69, 265-268
265
Flavan-4-ol Glycosides from the Rhizomes of Abacopteris penangiana
Zhongxiang Zhao,† Jinlan Ruan,*,† Jing Jin,‡ Jian Zou,§ Daonian Zhou,† Wei Fang,† and Fanbo Zeng†
College of Pharmacy, Tongji Medical Center, Huazhong UniVersity of Science and Technology, Wuhan 430030, People’s Republic of China,
Laboratory of Traditional Chinese Medicine and Marine Drugs, School of Pharmaceutical Sciences, Sun Yat-Sen UniVersity,
Guangzhou 510275, People’s Republic of China, and Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
ReceiVed May 31, 2005
Four new flavan-4-ol glycosides, abacopterins A-D (1-4), were isolated from a methanol extract of the rhizomes of
Abacopteris penangiana, together with two known compounds, triphyllin A (5) and 6,8-dimethyl-7-hydroxy-4′-
methoxyanthocyanidin-5-O-â-D-glucopyranoside (6). Their structures were elucidated by extensive spectroscopic analysis
and chemical methods. The cytotoxic activity of 1-5 against HepG2 human hepatoma cells was investigated.
The genus Abacopteris (Thelypteridaceae) comprises approxi-
mately 35 species that are found in tropical and subtropical zones.1
Abacopteris penangiana (Hook.) Ching is a fern widely distributed
in the south of mainland China. It has been used as a folk medicine
to treat upper respiratory tract infections and dysentery.2,3 Previous
phytochemical investigations on thelypteridaceous ferns showed the
presence of some unusual flavan-4-ol glycosides.4-6 No chemical
studies have been performed on A. penangiana. As a part of an
investigation on Abacopteris species, we examined the rhizomes
of A. penangiana. This study has resulted in four new flavan-4-ol
glycosides (1-4), together with a known flavan-4-ol glycoside (5)
and a known anthocyanidin glycoside (6). Herein, we report the
isolation and structural elucidation of 1-4, as well as the cytotoxic
activity of 1-5 against HepG2 human hepatoma cells.
The air-dried rhizomes of A. penangiana (5.0 kg) were extracted
with MeOH. The extract was concentrated under vacuum to leave
a residue, which was suspended in H2O and extracted with CHCl3,
EtOAc, and n-BuOH, sequentially. After being chromatographed
on silica gel, Sephadex LH-20, and RP C18 columns, the EtOAc
fraction afforded 1-3 and 6, and the n-BuOH fraction afforded
4 and 5. The structures of the new compounds were elucidated
on the basis of spectroscopic data (1H and 13C NMR, HSQC,
1
HMBC, H-1H COSY, and ROESY) interpretation and chemical
evidence.
Compound 1 was obtained as white needles. Its molecular
formula was assigned as C26H30O10 on the basis of its 13C DEPT
1
and HREIMS (m/z 502.1854 [M]+) data. The H NMR spectrum
of 1 (Table 1) showed signals characteristic for three methyls as
singlets at δ 2.01, 2.05, and 2.14, a methoxy as a singlet at δ 3.80,
an anomeric proton at δ 4.38 (d, J ) 7.2 Hz), and four aromatic
protons as two doublets at δ 6.96 (2H, d, J ) 8.8 Hz) and 7.44
(2H, d, J ) 8.8 Hz), suggesting a para-substituted phenyl group
(Table 1). The 13C NMR spectrum of 1 (Table 1) analyzed with
the aid of the DEPT and HSQC spectra revealed there are one para-
substituted and one fully substituted phenyl group and two aromatic
methyl groups in the aglycon. A substructure, -CH-CH2-CH-,
was also confirmed in the aglycon through a 1H-1H COSY
experiment, from the correlations of H-2 (δ 4.93) with H2-3 (δ
2.11, 2.55), and H-4 (δ 5.00) with H2-3 (δ 2.11, 2.55). Analysis of
these structural characteristics indicated 1 is a flavan-4-ol-type
glycoside. Alkaline hydrolysis of 1 with 1% KOH gave a deacety-
lated glycoside, 3, which was identified by TLC analysis. On
hydrolysis with acid, 1 provided a degraded aglycon, 1a, which is
a known anthocyanidin,5 and D-glucose. The â-configuration of the
1
glycosidic bond was deduced from the H and 13C NMR data of
the sugar moiety. The sequence of the sugar and binding sites to
the aglycon were determined by 2D NMR experiments. As observed
in the HMBC NMR spectrum (Figure 1), correlations of H-1′′ of
the sugar unit with C-5 (δ 151.9) and H-4 with C-2′′ (δ 86.0) were
used to establish that the sugar has two binding sites to the aglycon,
with C-1′′ attached to C-5 and C-2′′ attached to C-4. Thus, C-5,
C-10, C-4, C-1′′, and C-2′′ formed a heptacyclic ring, inclusive of
two oxygen atoms. The acetyl group was attached to C-6′′ of the
sugar, and its binding site was also confirmed from the HMBC
spectrum according to the correlations of the carbonyl carbon (δ
171.0) with H-6′′ (δ 4.27, 4.40).
* To whom correspondence should be addressed. Tel/Fax: 86-27-
83692892. E-mail: jinlan8152@163.com.
† Huazhong University of Science and Technology.
‡ Sun Yat-Sen University.
§ Shanghai Institute of Materia Medica.
10.1021/np050191p CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/11/2006

266 Journal of Natural Products, 2006, Vol. 69, No. 2
Notes
1
Table 1. 13C and H NMR Data (δ values) of Compounds 1-3a
1
2
3
position
δC
δH
δC
δH
δC
δH
4.92 brd (10.0)
2.47 dd (10.0, 8.0)
2.03 m
2
3
77.0 d
38.2 t
4.93 brd (11.0)
2.55 dd (13.7, 7.8)
2.11 m
77.0 d
38.3 t
4.93 brd (12.0)
2.43 dd (13.5, 7.8)
2.03 m
76.0 d
37.3 t
4
5
6
7
8
9
73.1 d
151.9 s
110.3 s
154.1 s
108.5 s
152.4 s
113.0 s
9.0 q
5.00 brd (7.8)
72.9 d
151.7 s
110.4 s
154.2 s
108.6 s
152.4 s
112.8 s
9.0 q
4.97 brd (7.8)
72.1 d
151.1 s
110.0 s
153.4 s
107.9 s
151.5 s
112.4 s
9.5 q
4.97 brd (8.0)
10
Me-6
Me-8
1′
2′,6′
3′,5′
4′
OMe
1′′
2′′
3′′
4′′
5′′
6′′
2.01 s
2.14 s
2.01 s
2.13 s
1.94 s
2.07 s
8.5 q
8.5 q
8.9 q
133.6 s
127.9 d
114.2 d
159.9 s
55.2 q
102.2 d
86.0 d
74.8 d
70.3 d
75.3 d
63.8 t
133.5 s
127.9 d
114.2 d
160.0 s
55.2 q
102.1 d
83.9 d
75.4 d
68.4 d
78.2 d
61.5 t
132.7 s
127.5 d
113.8 d
159.0 s
55.1 q
101.7 d
85.6 d
73.8 d
69.6 d
77.9 d
60.6 t
7.44 d (8.8)
6.96 d (8.8)
7.42 d (8.8)
6.96 d (8.8)
7.41 d (8.8)
6.97 d (8.8)
3.80 s
4.38 d (7.2)
3.43 m
3.49 m
3.49 m
3.63 m
4.27 dd (5.7, 11.9)
4.40 dd (2.2, 11.9)
3.80 s
4.47 d (7.4)
3.49 m
5.02 m
3.68 m
3.54 m
3.77 dd (5.6, 11.9)
3.90 dd (2.2, 11.9)
3.77 s
4.30 d (6.6)
3.29 m
3.27 m
2.36 m
3.27 m
3.54 dd (3.8, 11.5)
3.73 br d (11.5)
CO
Me
171.0 s
20.4 q
170.3 s
20.6 q
2.05 s
2.05 s
a Compounds 1 and 2 were measured in acetone-d6 and compound 3 in DMSO-d6. Coupling constants (J in Hz) are in parentheses. Multiplicities
were determined by DEPT experiments.
proton and the 13C NMR data of the sugar moiety. The 1H and 13
C
NMR signals of the sugar moiety were assigned by a combination
of HSQC, HMBC, and 1H-1H COSY experiments. The attachment
of the acetyl group at C-3′′ of the sugar was evidenced from the
correlation of the carbonyl carbon (δ 170.3) with H-3′′ (δ 5.02) in
the HMBC spectrum. The configurations at C-2 and C-4 were
determined as 2S and 4S by comparing the CD spectrum of 2 with
that of 1, in which the negative Cotton effects at 222 (∆ꢀ -6.17)
and 278 nm (∆ꢀ -1.30) were very similar to 226 (∆ꢀ -6.83) and
279 nm (∆ꢀ -1.69) of 1. From these results, 2 was characterized
as (2S,4S)-6,8-dimethyl-7-hydroxy-4′-methoxy-4,2′′-oxidoflavan-
5-O-â-D-3-O-acetyl-glucopyranoside.
Figure 1. Selected HMBC and ROESY correlations for compounds
1 and 4.
Comparison of the above NMR spectroscopic data of 1 with those
of eruberin A, which is a flavan-4-ol glycoside isolated from
Glaphyropteridopsis erubescens (Wall.) Ching (Thelypteridaceae),5
showed that 1 has a very similar structure, with an additional acetyl
group. Since the optical rotation of 1 (-15°) is different from that
of eruberin A (+88°),5 it was proposed that the configurations at
C-2 and C-4 of 1 are different from the C-2 (R) and C-4 (S)
configurations of eruberin A. The coupling constants of H-2 (brd,
J ) 11.0 Hz) and H-4 (brd, J ) 7.8 Hz) suggested that the
stereochemical relationship between H-2 and H-4 was cis.7 To
further determine the configurations at C-2 and C-4, a ROESY
experiment was performed, where cross-peaks at H-4/H-2′′ and
H-4/H-2 were observed (Figure 1). On the basis of the above
spectroscopic evidence, the configurations at C-2 and C-4 were
established as 2S and 4S. Thus, the structure of 1 was elucidated
as (2S,4S)-6,8-dimethyl-7-hydroxy-4′-methoxy-4,2′′-oxidoflavan-
5-O-â-D-6-O-acetyl-glucopyranoside.
Compound 3 was isolated as white needles and was assigned
the molecular formula C24H28O9, which was determined from its
HREIMS (m/z 460.1725 [M]+) and confirmed by its 13C NMR and
DEPT data. The IR, EIMS, and NMR spectroscopic data indicated
that 3 is the deacetylated derivative of 1 or 2, which was confirmed
1
by the 2D NMR (HSQC, H-1H COSY, and HMBC) spectral
analysis of 3. The configurations at C-2 and C-4 were determined
as 2S and 4S by comparing the optical rotation, CD spectrum, and
coupling constants of key protons of 3 with those of 1. Thus
compound 3 was elucidated as (2S,4S)-6,8-dimethyl-7-hydroxy-4′-
methoxy-4,2′′-oxidoflavan-5-O-â-D-glucopyranoside.
Compound 4 was isolated as white needles, and its molecular
formula was deduced as C30H38O15 on the basis of the ESITOFMS
1
(m/z 661.2103 [M + Na]+). On comparing the H and 13C NMR
spectroscopic data of 4 with those of triphyllin A (5), it was evident
that their aglycons were similar. The 1H NMR spectrum of 4
exhibited two anomeric proton signals at δ 4.33 (d, J ) 7.2 Hz)
and 4.44 (d, J ) 7.6 Hz). On acid hydrolysis of 4, D-glucose was
detected. In conjunction with the 1H and 13C NMR data of the sugar
moiety (Table 2), the absolute configuration of the sugar was thus
determined as â-D-glucose by GC. As observed in the HMBC
spectrum, the correlation of H-11a (δ 5.43, brd, J ) 13.0 Hz) with
C-6 (δ 119.8) suggested that C-11 (δ 64.1) was bonded to C-6,
and the correlations of H-1′′ (δ 4.44) with C-5 (δ 150.3) and H-1′′′
(δ 4.33) with C-7 (δ 153.9) suggested that two D-glucoses were
attached to C-5 and C-7, respectively. After complete assignment
Compound 2 was obtained as white needles. The molecular
formula (C26H30O10) was deduced from the 13C NMR data and the
ESITOFMS (m/z 503.1928 [M + H]+). Compounds 1 and 2 have
1
the same elemental formula, and their H and 13C NMR spectra
(Table 1) for the aglycon moiety are very similar, suggesting that
they have the same aglycon. The EIMS of 2 further confirmed this,
in which 2 had the same base peak m/z 299 (100) as 1. The
differences between their structures are in their sugar moieties.
Alkaline hydrolysis of 2 gave 3, and acid hydrolysis of 2 gave 1a
and D-glucose. The â-configuration of the glycosidic bond was
deduced from the coupling constant (J ) 7.4 Hz) of the anomeric

Notes
Journal of Natural Products, 2006, Vol. 69, No. 2 267
1
Table 2. 13C and H NMR Data (δ values) of Compound 4 in
Table 3. Cytotoxicity of Compounds 1-5 against HepG2
Human Hepatoblastoma Cells
DMSO-d6
position
δC
δH
1
2
3
4
5
5-fluorouracil
1.3
2
3
72.4 d
36.5 t
5.25 dd (1.6, 12.0)
2.06 m
IC50 (µg/mL)
3.5
4.1
4.0
3.1
5.7
1.86 brt (12.0)
4.95 brs
seven-membered ring and sugar ring, the stability of this newly
formed ring is poor. When compound 3 was treated with weak
acid, the ether linkage was broken, and it mainly converted to
compound 6.
Compounds 1-5 were tested for in vitro cytotoxicity against a
human hepatoblastoma cell line (HepG2) using the MTT method.9
The IC50 values of 1-5 are listed in Table 3. They all showed
weak cytotoxicity against HepG2 cells.
4
5
6
7
8
9
10
11
56.7 d
150.3 s
119.8 s
153.9 s
114.8 s
152.7 s
115.5 s
64.1 t
5.43 brd (13.0)
4.34 brd (13.0)
2.02 s
Me-8
1′
2′,6′
3′,5′
4′
OMe
O-glc-5
1′′
2′′
3′′
4′′
5′′
9.1 q
133.3 s
127.7 d
114.8 d
159.0 s
55.2 q
Experimental Section
7.38 d (8.8)
6.96 d (8.8)
General Experimental Procedures. Melting points were determined
on an X4 micro melting point apparatus and are uncorrected. Optical
rotations were measured on a Perkin-Elmer Model 341 polarimeter,
and CD spectra were recorded on a JASCO J-810 spectropolarimeter.
UV spectra were measured with a Shimadzu UV-260 UV-visible
spectrophotometer. IR spectra were determined on a Perkin-Elmer
Spectrum One FT-IR spectrometer, as KBr pellets. NMR spectra were
taken with TMS as internal standard on a Bruker AM-400 spectrom-
eter. HREIMS were obtained on a Finnigan-MAT 95 instrument.
HRESITOFMS were obtained on a Mariner spectrometer, and
LRESIMS on a Finnigan LCQ-DECA spectrometer. The GC was
performed on a GC-14C gas chromatograph (Shimadzu, Japan) with a
DB-17 fused silica capillary column (30 m × 0.25 mm × 0.25 µm) (J
& W Scientific); detection, FID; carrier gas, N2; temperature for injector
and detector, 230 °C; temperature gradient system for the oven, 150
°C for 1 min and then raised to 230 °C at the rate of 5 °C/min. Silica
gel plates for TLC and silica gel for column chromatography were
produced by Qingdao Marine Chemical Company, Qingdao, People’s
Republic of China. Solvents and chemicals were of analytical grade.
Plant Material. The rhizomes of Abacopteris penangiana (Hook.)
Ching were collected in October 2004 from Wufeng County of Hubei
Province, People’s Republic of China. The rhizomes were air-dried at
room temperature. A specimen (PZX0310) was deposited in the College
of Pharmacy, Tongji Medical Center, Huazhong University of Science
and Technology.
3.76 s
105.3 d
73.9 d
76.2 d
70.1 d
76.7 d
61.0 t
4.44 d (7.6)
3.22-3.26 m
3.22 m
3.11 m
3.16 m
6′′
3.43 dd (6.6, 11.5)
3.64 brd (11.5)
O-glc-7
1′′′
2′′′
3′′′
4′′′
102.2 d
85.8 d
73.9 d
69.6 d
78.0 d
60.5 t
4.33 d (7.2)
3.18 m
3.22-3.26 m
3.20 m
3.21 m
3.50 dd (2.9, 11.7)
3.68 brd (11.7)
5′′′
6′′′
of the protons and carbons in the sugar moiety from the HSQC,
1H-1H COSY, and HMBC spectra, it was found that the chemical
shift of C-2′′′ of the sugar unit shifted downfield about 12 ppm, as
observed for C-2′′ of the sugar moiety of compound 1. This
suggested that C-2′′′ might be the binding site to the aglycon. The
correlation of H-11a with C-2′′′ (δ 85.8) in the HMBC spectrum
(Figure 1) confirmed that C-2′′′ of the sugar (located at C-7) formed
an ether linkage to C-11. Thus, between the sugar and the aglycon,
an unusual heptacyclic ring was formed. A ROESY experiment
was performed to determine the configurations at C-2 and C-4, in
which correlations of H-2 with H-3 and H-4 with H-3 were observed
(Figure 1), while a correlation between H-2 and H-4 was not
observed. The results of the ROESY experiment were not enough
to establish the configurations at C-2 and C-4. Therefore, the CD
spectrum and chemical shifts and coupling constants of H-2, H-3,
and H-4 were compared with those of 5 to further confirm the
configurations at C-2 and C-4. A negative Cotton effect at 224 nm
(∆ꢀ -1.80) and positive Cotton effects at 239 (∆ꢀ +0.89) and 282
nm (∆ꢀ +1.32) were observed in the CD spectrum of 4, which
were quite comparable to those of 5, and the H-2 (δ 5.25, dd,
J ) 1.6 Hz, J ) 12.0 Hz), H-3 (δ 2.06, m; δ 1.86, brt, J ) 12.0
Hz), and H-4 (δ 4.95, brs) signals of 4 were also consistent with
those of 5. The results showed that 4 has the same configuration
(2S,4R) as 5. Thus, the structure of 4 was established as (2S,4R)-
4-hydroxy-4′-methoxy-8-methyl-11,2′′′-oxidoflavan-5,7-di-O-â-D-
glucopyranoside.
Extraction and Isolation. The air-dried rhizomes (5.0 kg) were
ground and extracted with MeOH (5 × 10 L) at room temperature.
The MeOH extract was concentrated under vacuum to leave a residue,
which was suspended in H2O (3 L) and extracted with CHCl3 (3 L ×
3), EtOAc (3 L × 3), and n-BuOH (3 L × 3), sequentially. A part of
the EtOAc extract (20 g) was chromatographed on a silica gel (200-
300 mesh, 800 g) column, eluting with a CHCl3-MeOH gradient
system (50:1 f 20:1 f 15:1 f 10:1 f 5:1), to yield 80 fractions.
Each fraction (500 mL) was examined by TLC, and fractions with
similar TLC patterns were combined to yield 10 major fractions
(A1-A10). Fraction A3 (0.8 g) was separated over a Sephadex LH 20
column eluted with MeOH-CHCl3 (3:2), then further purified on a
silica gel (300-400 mesh, 80 g) column, eluted with a petroleum-
acetone gradient (4:1 f 2:1 f 1:1), to yield 1 (62 mg) and 2 (15 mg).
Purification of fraction A4 (1.2 g) was carried out on a Sephadex LH
20 column, eluted with MeOH-CHCl3 (3:2), to yield 15 subfractions
designated as A4.1-A4.15. Subfraction A4.8 (0.4 g) was further purified
by RP C18 (230-400 mesh, 100 g) column chromatography, eluted
with MeOH-H2O (4:3), to yield 3 (40 mg). Compound 6 (25 mg) was
isolated from fraction A8 by chromatography on silica gel columns,
eluted with MeOH-CHCl3 (6:1). A part of the n-BuOH extract (15 g)
was chromatographed on silica gel (200-300 mesh, 750 g), using
CHCl3-MeOH-H2O (4:1:0.1) for elution, to yield 20 fractions,
B1-B20 (each 500 mL). Compound 5 (200 mg) was obtained from
fraction B15-17 (1.0 g) by chromatography on a Sephadex LH-20 column
(MeOH-H2O, 2:1). Fraction B9-11 (0.5 g) was further separated over
a Sephadex LH-20 column (MeOH-H2O, 2:1) and finally purified on
RP C18 (230-400 mesh, 100 g) (MeOH-H2O, 1:2) to yield 4 (22 mg).
Compound 5 was identified as triphyllin A and compound 6 as
6,8-dimethyl-7-hydroxy-4′-methoxyanthocyanidin-5-O-â-D-glu-
copyranoside, respectively, by comparison with their spectroscopic
data reported in the literature.4,5
In compounds 1-4, besides the glycosidic bond of the anomeric
carbon, the C-2 of the sugar also formed an ether linkage to the
aglycon, and thus an unusual seven-membered ring was formed
between the sugar and the aglycon. Due to the ring strain of the
Abacopterin A (1): white needles, mp 188-190 °C; [R]25 -15
D
(c 0.25, MeOH); CD (c 0.0032, MeOH), λ (∆ꢀ) 226 (-6.83), 279
(-1.69) nm; UV (MeOH) λmax (log ꢀ) 227 (4.35), 274 (3.45), 280 (3.42)

268 Journal of Natural Products, 2006, Vol. 69, No. 2
Notes
nm; IR (KBr) νmax 3420, 2879, 1724, 1613, 1516, 1466, 1251, 1136,
Weak Acid Hydrolysis of 3. Compound 3 (1 mg) was treated with
90% HOAc (1 mL) in 90 °C for 4 h, and then the reaction mixture
was freeze-dried. The reaction residue was finally dissolved in MeOH
(0.5 mL) and examined by co-TLC (CHCl3-MeOH, 5:1), in which
compound 6 was detected (Rf 0.50).
1
824 cm-1; H (400 MHz in acetone-d6) and 13C NMR (100 MHz in
acetone-d6), see Table 1; EIMS m/z 502 [M]+ (5), 299 (100), 267 (4),
207 (2), 191 (6), 180 (6), 145 (12), 121 (15), 91 (2); HREIMS m/z
502.1854 [M]+ (calcd for C26H30O10, 502.1839).
Abacopterin B (2): white needles, mp 160-163 °C; [R]25D -18 (c
0.24, MeOH); CD (c 0.0070, MeOH), λ (∆ꢀ) 222 (-6.17), 278 (-1.30)
nm; UV (MeOH) λmax (log ꢀ) 227 (4.32), 274 (3.46), 280 (3.43) nm;
IR (KBr) νmax 3436, 2927, 1726, 1614, 1517, 1466, 1256, 1134, 828
cm-1; 1H (400 MHz in acetone-d6) and 13C NMR (100 MHz in acetone-
d6), see Table 1; EIMS m/z 502 [M]+ (6), 299 (100), 267 (4), 207 (3),
191 (7), 180 (5), 145 (12), 121 (13), 91 (2); ESITOFMS m/z 503.1928
[M + H]+ (calcd for C26H31O10, 503.1917).
Determination of the Absolute Configuration of Sugars.8 Com-
pounds 1-4 (3 mg) were each subjected to acid hydrolysis as described
for 1. The filtrate was examined by TLC with two solvent systems for
sugar analysis (A: CHCl3-MeOH-H2O, 16:9:2; B: EtOAc-n-
BuOH-H2O-HOAc, 4:4:1:1), with Rf values of D-glucose being 0.60
in solvent A and 0.35 in solvent B, respectively. Each remaining filtrate
was freeze-dried to give a residue and dissolved in 100 µL of dry
pyridine, to which was added 200 µL of L-cysteine methyl ester
hydrochloride (0.1 M). The mixture was stirred at 60 °C for 1 h, then
150 µL of hexamethyldisilazane-trimethylchlorosilane (2:1) was added,
and the mixture was stirred at 60 °C for another 30 min. After
centrifugation, the supernatant was directly subjected to GC analysis.
The sugar derivatives obtained from compounds 1-4 showed a single
peak (tR at 24.10 min) comparable with that of a D-glucose derivative,
and the retention time of the L-glucose derivative was 25.06 min.
Abacopterin C (3): white needles, 194-196 °C; [R]25D -16 (c 0.26,
MeOH); CD (c 0.0034, MeOH), λ (∆ꢀ) 226 (-5.09), 278 (-1.59) nm;
UV (MeOH) λmax (log ꢀ) 227 (4.24), 274 (3.52), 280 (3.50) nm; IR
(KBr) νmax 3411, 2929, 1613, 1517, 1467, 1250, 1133, 1080, 826 cm-1
;
1H (400 MHz in DMSO-d6) and 13C NMR (100 MHz in DMSO-d6),
see Table 1; EIMS m/z 460 [M]+ (7), 299 (100), 267 (5), 207 (2), 191
(7), 180 (6), 145 (11), 121 (15), 91 (2); HREIMS m/z 460.1725 [M]+
(calcd for C24H28O9, 460.1733).
Abacopterin D (4): white needles, mp 278-280 °C; [R]25 +18
Cytotoxicity Bioassay. Cytotoxic activity was determined against
a human hepatoblastoma cell line (HepG 2) using the MTT method,9
according to a previously described procedure.10 The OD value was
read on a plate reader at a wavelength of 570 nm. 5-Fluorouracil was
used as the positive control.
D
(c 0.20, MeOH); CD (c 0.0048, MeOH), λ (∆ꢀ) 224 (-1.80), 233 (0),
239 (+0.89), 282 (+1.32) nm; UV (MeOH) λmax (log ꢀ) 227 (4.32),
275 (3.42), 281 (3.40) nm; IR (KBr) νmax 3422, 2926, 1610, 1517, 1461,
1252, 1133, 1085, 834 cm-1; 1H (400 MHz in DMSO-d6) and 13C NMR
(100 MHz in DMSO-d6), see Table 2; EIMS m/z 638 [M]+ (1), 458
(18), 297 (100), 282 (20), 267 (7), 181 (30), 134(58), 121 (23), 91 (7),
Acknowledgment. The authors are grateful to the members of the
analytical group in Shanghai Institute of Materia Medica, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, for
measurements of the mass and NMR spectra.
83 (9); ESITOFMS m/z 661.2103 [M + Na]+ (calcd for C30H38O15
+
Na, 661.2108).
Triphyllin A (5): colorless prisms, mp 197-200 °C; [R]25 +19
D
(c 0.26, MeOH); CD (c 0.0038, MeOH), λ (∆ꢀ) 224 (-3.22), 233 (0),
239 (+1.71), 281 (+1.43) nm; UV (MeOH) λmax (log ꢀ) 227 (4.33),
276 (3.36), 281 (3.36) nm; IR (KBr) νmax 3401, 2920, 1602, 1517, 1458,
1250, 1152, 1074, 835 cm-1; 1H NMR (400 MHz in DMSO-d6) δ 7.40
(2H, d, J ) 8.4 Hz, H-2′ and H-6′), 6.98 (2H, d, J ) 8.4 Hz, H-3′ and
H-5′), 5.25 (1H, brd, J ) 13.0 Hz, H-2), 5.08 (1H, d, J ) 7.6 Hz,
H-1′′), 4.95 (1H, brs, H-4), 4.70, 4.56 (each 1H, d, J ) 12.5 Hz,
CH2OH-6), 4.60 (1H, d, J ) 7.6 Hz, H-1′′′), 3.77 (3H, s, OCH3-4′),
2.14 (3H, s, CH3-8), 2.08 (1H, brd, J ) 13.0 Hz, H-3a), 1.86 (1H, brt,
J ) 12.0 Hz, H-3b); 13C NMR (100 MHz in DMSO-d6) δ 72.2 (CH,
C-2), 36.5 (CH2, C-3), 56.9 (CH, C-4), 152.0 (C, C-5), 120.9 (C, C-6),
154.1 (C, C-7), 115.7 (C, C-8), 152.8 (C, C-9), 115.5 (C, C-10), 53.0
(CH2, CH2OH-6), 9.5 (CH3, CH3-8), 133.3 (C, C-1′), 127.5 (CH, C-2′
and C-6′), 113.9 (CH, C-3′ and C-5′), 158.9 (C, C-4′), 55.1 (CH3, 4′-
OCH3), 104.1 (CH, C-1′′), 73.9 (CH, C-2′′), 76.2 (CH, C-3′′), 70.0
(CH, C-4′′), 77.0 (CH, C-5′′), 60.9 (CH2, C-6′′), 103.7 (CH, C-1′′′),
73.9 (CH, C-2′′′), 76.1 (CH, C-3′′′), 70.0 (CH, C-4′′′), 76.5 (CH, C-5′′′),
60.9 (CH2, C-6′′′); ESIMS m/z 679 [M + Na]+, 477 [M - Glc + H]+,
297 [M - 2Glc + H]+.
Acid Hydrolysis of 1. A solution of 1 (30 mg) in 9% HCl (2 mL)
was stirred at 90 °C for 5 h. After being cooled to 2-4 °C, the reaction
mixture was filtered. The product was chromatographed on a silica
gel (300-400 mesh, 10 g) column (CHCl3-MeOH, 15:1) to yield 1a
(5 mg).
Alkaline Hydrolysis of 1 and 2. Compounds 1 and 2 (0.5 mg) were
each hydrolyzed with 1% aqueous KOH (0.5 mL) for 1 h at room
temperature. The reaction mixture was adjusted to pH 6 with dilute
1% HCl and then extracted with EtOAc (3 × 0.5 mL). Evaportation of
the EtOAc gave compound 3, analyzed by co-TLC (petroleum-acetone,
1:1, Rf 0.30).
Supporting Information Available: CD spectra of compounds 1-5
and spectroscopic data of compound 6. These materials are available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
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(2) Ding, H. S. The Medicinal Spore Plants of China; Shanghai Scientific
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(3) New Medical Jiangsu College. Dictionary of Chinese Traditional
Medicine; Shanghai Scientific and Technical Publishers: Shanghai,
1977; p 1633.
(4) Tanaka, N.; Murakami, T.; Wada, H.; Gutierrez, A. B.; Saiki, Y.;
Chen, C. M. Chem. Pharm. Bull. 1985, 33, 5231-5238.
(5) Tanaka, N.; Sada, T.; Murakami, T.; Saiki, Y.; Chen, C. M. Chem.
Pharm. Bull. 1984, 32, 490-496.
(6) Tanaka, N.; Ushioda, T.; Fuchino, H.; Braggins, J. E. Aust. J. Chem.
1997, 50, 329-332.
(7) Pouget, C.; Fagnere, C.; Basly, J. P.; Leveque, H.; Chulia, A. J.
Tetrahedron 2000, 56, 6047-6052.
(8) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1986, 34,
1843-1845.
(9) Xu, S. J.; Bian, R. L.; Chen X. The Methodology of Pharmacological
Experiments, 3rd ed.; People’s Medical Publishing House: Beijing,
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(10) Tang, M. J.; Shen D. D.; Hu, Y. C.; Gao, S.; Yu, S. S. J. Nat. Prod.
2004, 67, 1969-1974.
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