Tupichinins B-D, three new spirostanol saponins from Tupistra chinensis rhizomes

Article abstract of DOI:10.1080/14786419.2013.838240

Three new spirostanol saponins were isolated from the EtOAc fraction of methanol extract from Tupistra chinensis rhizomes. Based on the detailed analysis of their 1D and 2D NMR spectra and chemical evidence, their structures were determined as 1β-O-acetyl

Full text of DOI:10.1080/14786419.2013.838240

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Tupichinins B–D, three new spirostanol
saponins from Tupistra chinensis
rhizomes
a
bc
d
e
Li-Mei Wei , Yang-Chang Wu , Chin-Chau Chen , Pei-Wen Hsieh &
a
Wen-Bin Pan
a
Department of Applied Chemistry and Materials Science, Fooyin
University, Kaohsiung 831, Taiwan, Republic of China
b
Graduate Institute of Integrated Medicine, College of Chinese
Medicine, China Medical University, Taichung 40402, Taiwan,
Republic of China
c
Natural Medicinal Products Research Center, China Medical
University Hospital, Taichung 40402, Taiwan, Republic of China
d
Department of Chemistry, National Sun Yat-sen University,
Kaohsiung 804, Taiwan, Republic of China
e
Graduate Institute of Natural Products, Chang Gung University,
Tao-Yuan333, Taiwan, Republic of China
Published online: 26 Nov 2013.
To cite this article: Li-Mei Wei, Yang-Chang Wu, Chin-Chau Chen, Pei-Wen Hsieh & Wen-
Bin Pan (2014) Tupichinins B–D, three new spirostanol saponins from Tupistra chinensis
rhizomes, Natural Product Research: Formerly Natural Product Letters, 28:2, 74-80, DOI:
10.1080/14786419.2013.838240
To link to this article: http://dx.doi.org/10.1080/14786419.2013.838240
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Natural Product Research, 2014
Vol. 28, No. 2, 74–80, http://dx.doi.org/10.1080/14786419.2013.838240
Tupichinins B–D, three new spirostanol saponins from Tupistra chinensis
rhizomes
a
bc
d
e
Li-Mei Wei , Yang-Chang Wu , Chin-Chau Chen , Pei-Wen Hsieh and Wen-Bin Pan *
a
a
Department of Applied Chemistry and Materials Science, Fooyin University, Kaohsiung 831, Taiwan,
Republic of China; Graduate Institute of Integrated Medicine, College of Chinese Medicine, China
b
c
Medical University, Taichung 40402, Taiwan, Republic of China; Natural Medicinal Products Research
d
Center, China Medical University Hospital, Taichung 40402, Taiwan, Republic of China; Department of
e
Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan, Republic of China; Graduate
Institute of Natural Products, Chang Gung University, Tao-Yuan 333, Taiwan, Republic of China
(
Received 1 May 2013; final version received 19 July 2013)
Three new spirostanol saponins were isolated from the EtOAc fraction of methanol
extract from Tupistra chinensis rhizomes. Based on the detailed analysis of their 1D
and 2D NMR spectra and chemical evidence, their structures were determined as 1b-O-
acetyl-spirost-5,25(27)-dien-3a-yl-O-b-D-glucopyranoside (1), (25S)-1b,2b,5b-trihy-
droxy-spirostane-3b-yl-O-b-D-glucopyranoside (2) and (25S)-1b,2b-dihydroxy-5b-
spirostane-3b-yl-O-b-D-xylopyranoside (3), respectively.
Keywords: Tupistra chinensis; Liliaceae; spirostanol saponins; tupichinins B, C and D
1
. Introduction
The genus Tupistra (Liliaceae) comprising about 26 species is mainly distributed in Asia, of
which about 17 species are growing in the People’s Republic of China, particularly in the south-
western region (Hang & Li 1990; Yang et al. 2005). Kai-Kou-Jian, the Chinese name of
rhizomes of Tupistra chinensis BAK., is used for the treatment of rheumatic diseases and
snakebite in Chinese folk medicine (Jiangsu New Medical College 1985). Kai-Kou-Jian is a
reputed folk medicine because of its power to markedly reduce carbuncles and to ameliorate
pharyngitis (Zhan 1994). Some furostanol saponins isolated from T. chinensis rhizomes showed
inhibition action against NO production (Xu et al. 2007) and against COX-2 production (Zou,
Wang et al. 2007; Zou, Wu et al. 2007).
Phytochemical investigation on T. chinensis in the past 15 years has resulted in the isolation
of some steroidal sapogenins and steroidal saponins (Cai et al. 2007; Guo et al. 2009; Liu, Guo,
Xue, Cheng et al. 2012; Liu, Guo, Xue, Zhang et al. 2012; Pan et al. 2000a, 2000b, 2003, 2006,
2012; Wu et al. 2005; Xu et al. 2007; Zou et al. 2005, 2006, 2007, 2009). Previously, we reported
the isolation and structural elucidation of several spirostanol sapogenins, flavonoids, lignans, a
pregnane glycoside, a pregnane genin and other chemical constituents from the CHCl fraction
3
of methanol extract from T. chinensis rhizomes (Pan et al. 2000a, 2000b, 2003, 2006). On
continuing the study of this plant, we have now isolated three new spirostanol saponins, namely
tupichinins B, C and D (Figure 1), from the EtOAc fraction of methanol extract from T. chinensis
rhizomes. This study reported the isolation and structural elucidation of three new spirostanol
saponins by detailed analysis of their 1D and 2D NMR spectra and acid hydrolysis.
*
Corresponding author. Email: sc008@fy.edu.tw
q 2013 Taylor & Francis

Natural Product Research
75
O
O
O
OH
O
O
HO
OH
R O
1
O
R2
O
OH
OH
2 R1=Glc R2=OH
3 R =Xyl R =H
OH
Figure 1. The chemical structures of compounds 1–3.
. Results and discussion
1
1
2
2
The EtOAc fraction of the methanol extract from the rhizomes of T. chinensis was
chromatographed successively on silica gel to afford 1, 2 and 3.
2
4
Tupichinin B (1), an amorphous white solid, ½aꢀ –188 (c 0.004, MeOH), showed in the
D
þ
HRFABMS (positive mode) a pseudo-molecular [M þ Na] peak at m/z 655.3563 (calcd
6
skeleton with 10 degrees of unsaturation.
55.3560), consistent with the molecular formula C H O , suggesting a spirostanol saponin
35 52 10
1
Unambiguous assignments for the H and C NMR signals were based on an analysis of the
13
1
1
combination of distortionless enhancement by polarization transfer (DEPT), H– H COSY,
nuclear overhauser effect spectroscopy (NOESY), heteronuclear multiple quantum coherence
and heteronuclear multiple bond correlation (HMBC) spectral data.
1
In the H NMR spectrum in pyridine-d of 1, signals characteristic of the spirostanol saponin
5
1
skeleton were observed. The H NMR spectrum showed signals for two tertiary methyl groups at
d 0.88 (3H, s, Me-18) and 1.22 (3H, s, Me-19), a secondary methyl group at d 1.14 (3H, d,
J ¼ 7.0 Hz, Me-21) and an anomeric proton at d 4.98 (1H, d, J ¼ 7.5 Hz). Two oxymethine
proton resonances at d 5.53 (1H, d, J ¼ 4.8 Hz) and 4.42 (1H, br s) were assigned to H-1a and H-
3
b, respectively. Evidence for the presence of two double bonds at C-5 and C-25(27) came from
an olefinic proton signal at d 5.67 (1H, d, J ¼ 4.8 Hz, H-6) and an exomethylene group at d 4.86
and 4.89 (each 1H, s, H-27), respectively. Furthermore, an oxymethine proton resonance at d
.55 (1H, q-like, J ¼ 7.0 Hz) was assigned to H-16. Two oxymethine proton resonances at d 4.11
4
(
respectively. The presence of an acetyl group in 1 was confirmed by H NMR [d 2.08 (3H, s)]
1H, d, J ¼ 12.0 Hz) and 4.50 (1H, d, J ¼ 12.0 Hz) were assigned to H-26b and H-26a,
1
1
3
13
and C NMR [d 171.5 (CvO) and 22.6 (Me)] spectra. The fully decoupled C and DEPT
nuclear magnetic resonance (NMR) spectra of 1 exhibited 35 carbon signals, which consisted of
4
1
1
methyls, 11 methylenes, 14 methines and 6 quaternary carbons. One carbonyl carbon at d
71.5 (1-O-Ac); two vinylic carbons at d 138.3 (C-5) and 126.7 (C-6); an anomeric carbon at d
0
04.2 (C-1 of glucose) and three methyl groups at d 17.2 (C-18), 14.9 (C-19) and 15.8 (C-21)
1
3
were confirmed in C NMR spectra. Moreover, two oxygen-bearing carbon signals at d 82.2
CH) and 65.8 (CH ) were assigned to the C-16 and C-26, respectively. Exomethylene carbon
signals at d 145.0 (C) and 109.9 (CH ) were assigned to the C-25 and C-27, respectively.
(
2
2
In the HMBC spectrum (Figure S1), there were correlations between H-1a and C-2, C-9, C-
0, C-19 and CvO (1-O-Ac). These findings supported the placement of an acetyl group on C-1
1
position. In the NOESY spectrum, the anomeric proton signal at d 4.98 (H-1 of glucose) showed
0
0
correlations with the proton signals at 4.11 (1H, d, J ¼ 9.6 Hz, H-2 of glucose) and 4.42 (H-3b
of aglycone). These findings support that glucose was attached to the C-3 position. The
a-configuration of the anomeric carbon of glucopyranosyl unit was determined by JH1-H2 value

7
6
L.-M. Wei et al.
(
.7.0 Hz). Upon acid hydrolysis of 1 with 2.0 N HCl, glucose was detected on thin layer
chromatography in the hydrolysed product. Accordingly, 1 was established as 1 b-O-acetyl-
spirost-5,25(27)-dien-3a-yl-O-b-D-glucopyranoside, namely tupichinin B.
2
4
Tupichinin C (2) was obtained as colourless oil, [a] -228 (c ¼ 0.004, MeOH), and showed
D
þ
in the HRFABMS (positive mode) a pseudomolecular [M þ Na] peak at m/z 649.3668 (calcd
6
skeleton with seven degrees of unsaturation.
49.3666), consistent with the molecular formula C H O , suggesting a spirostanol saponin
33 54 11
1
Unambiguous assignments for the H and C NMR signals were based on an analysis of the
13
1
combination of DEPT, H– H COSY, NOESY and HETCOR spectral data.
1
1
In the H NMR spectrum in CD OD of 2, signals characteristic of the spirostanol saponin
3
1
skeleton were observed. The H NMR spectrum showed signals for two tertiary methyl groups at
d 0.79 (3H, s, Me-18) and d 1.31 (3H, s, Me-19), a secondary methyl group at d 0.98 (3H, d,
J ¼ 6.8 Hz, Me-21) and a secondary methyl group at d 1.08 (3H, d, J ¼ 7.2 Hz, Me-27). Three
oxymethine proton resonances at d 3.76 (1H, br s), 3.70 (1H, br s) and 4.15 (1H, br s) were
assigned to H-1a, H-2a and H-3a, respectively. Two methylene proton resonances at d 2.46
(
1H, dd, J ¼ 16.0, 3.0 Hz) and 2.29 (1H, d, J ¼ 16.0 Hz) were assigned to H-4a and H-4b,
respectively. Besides, an oxymethine proton resonance at d 4.42 (1H, q, J ¼ 7.2 Hz) was
assigned to H-16. Two oxymethine proton resonances at d 3.28 (1H, d, J ¼ 10.8 Hz) and 3.92
(
1H, dd, J ¼ 10.8, 2.4 Hz) were assigned to H-26b-eq and H-26a-ax, respectively. The anomeric
1
3
proton resonance appeared at d 4.75 (1H, d, J ¼ 7.6 Hz). The fully decoupled C and DEPT
NMR spectra of 2 exhibited 33 carbon signals, which consisted of 4 methyls, 10 methylenes, 15
1
3
methines and 4 quaternary carbons. When the C signals of 2 were compared with those of
tupichigenin F (Pan et al. 2000a, 2000b), a set of additional six signals corresponding to a b-D-
glucopyranosyl unit appeared. Four methyl groups at d 16.8 (C-18), 13.6 (C-19), 14.7 (C-21) and
1
upper field gave evidence for the 25S configuration of 2. Three signals at d 56.9 (CH), 82.2 (CH)
1
3
13
6.4 (C-27) were confirmed in C NMR spectra, while the C signal of C-27 resonance at the
and 63.4 (CH) were assigned to the C-14, C-16 and C-17 positions, respectively. Two signals at
d 111.1 (C) and 66.1 (CH ) were the characteristic resonances of C-22 and C-26, respectively.
2
Furthermore, four signals at d 78.6 (CH), 68.4 (CH), 71.4 (CH) and 84.1 (C) were assigned to the
C-1, C-2, C-3 and C-5 positions, respectively. In the NOESY spectrum (Figure S2), the anomeric
0
proton signal at d 4.75 (H-1 of glucose) showed correlations with the proton signal at d 2.29
(
H-4b of aglycone). The oxymethine proton signal at d 3.76 (H-1 of aglycone) showed
correlations with the proton signals at d 1.31 (Me-19 of aglycone) and 1.45 (H-11a of
aglycone). The oxymethine proton signal at d 4.15 (H-3 of aglycone) showed correlations
with the proton signals at d 3.70 (H-2 of aglycone), 2.46 (H-4a of aglycone) and 2.29 (H-4b
of aglycone). Furthermore, there were correlations between signals at d 3.28 (H-26b of
aglycone) and signals at d 3.92 (H-26a of aglycone) and 1.08 (Me-27 of aglycone),
respectively, in the NOESY spectrum. Upon acid hydrolysis of 2 with 2.0 N HCl, glucose
was detected on thin layer chromatography in the hydrolysed mixture. Based on the above
spectroscopic evidence, the structure of 2 was identified as (25S)-1b,2b,5b-trihydroxy-
spirostane-3b-yl-O-b-D-glucopyranoside, namely tupichinin C.
2
4
Tupichinin D (3) was obtained as colourless plate, [a ]D þ 598 (c 0.002, CHCl ), and
3
þ
showed in the HRFABMS (positive mode) a pseudomolecular [M þ Na] peak at m/z 603.3512
(
saponin skeleton with seven degrees of unsaturation.
calcd 603.3509), consistent with the molecular formula C H O , suggesting a spirostanol
32 52 9
1
13
Unambiguous assignments for the H and C NMR signals were based on an analysis of the
1
combination of DEPT, H– H COSY, NOESY and HETCOR spectral data.
1
1
In the H NMR spectrum in pyridine-d of 3, signals characteristic of the spirostanol saponin
5
1
skeleton were observed. The H NMR spectrum showed signals for two tertiary methyl groups
at d 0.79 (3H, s, Me-18) and d 1.12 (3H, s, Me-19), a secondary methyl group at d 0.98

Natural Product Research
77
(
3H, d, J ¼ 7.2 Hz, Me-21) and a secondary methyl group at d 1.08 (3H, d, J ¼ 7.2 Hz, Me-27).
Three oxymethine proton resonances at d 3.86 (1H, br s), 3.74 (1H, t, J ¼ 2.4 Hz) and 4.20 (1H,
br s) were assigned to H-1a, H-2a and H-3a, respectively. The oxymethine proton resonance at
d 4.38 (1H, m) was assigned to H-16. Two oxymethine proton resonances at d 3.20 (1H, t,
J ¼ 11.6 Hz) and 3.84 (1H, dd, J ¼ 11.6, 5.6 Hz) were assigned to H-26b-eq and H-26a-ax,
respectively. The anomeric proton resonance appeared at d 4.41 (1H, d, J ¼ 7.2 Hz). The fully
1
3
decoupled C and DEPT NMR spectra of 3 exhibited 32 carbon signals, which consisted of 4
methyls, 10 methylenes, 15 methines and 3 quaternary carbons.
Four methyl groups at d 16.9 (C-18), 19.2 (C-19), 14.7 (C-21) and 16.4 (C-27) were
1
3
confirmed in C NMR spectra. Three signals at d 57.2 (CH), 82.3 (CH) and 63.7 (CH) were
assigned to the C-14, C-16 and C-17 positions, respectively. Two signals at d 111.1 (C) and 67.0
(
signals at d 78.5 (CH), 75.2 (CH) and 71.1 (CH) were assigned to the C-1, C-2 and C-3 positions,
CH ) were the characteristic resonances of C-22 and C-26, respectively. Furthermore, three
2
0
respectively. In the NOESY spectrum (Figure S3), the anomeric proton signal at d 4.41 (H-1 of
xylose) showed correlations with the proton signals at d 3.74 (H-2 of aglycone) and 4.20 (H-3 of
aglycone). The oxymethine proton signal at d 3.74 (H-2 of aglycone) showed correlations with
the proton signals at d 3.86 (H-1 of aglycone) and 4.20 (H-3 of aglycone). Moreover, there was
correlation between signal at d 3.20 (H-26b of aglycone) and signal at d 1.08 (Me-27 of
aglycone). When 3 was hydrolysed with 2.0 N HCl, only xylose was detected in the hydrolysate
on TLC. Thus, 3 was identified as (25S)-1b,2b-dihydroxy-5b-spirostane-3b-yl-O-b-D-
xylopyranoside, namely tupichinin D.
3
. Experimental
3
.1. General
1
13
H NMR (400 MHz), C NMR (100 MHz), DEPT, HETCOR, COSY, NOESY and long-range
HETCOR spectra were obtained on a Varian NMR spectrometer (Unity Plus). Chemical shifts (d)
1
were reported in ppm relative to residual solvent signals. The multiplicities of H signals are
designated by the following abbreviations: s ¼ singlet; d ¼ doublet; t ¼ triplet; q ¼ quartet;
1
3
br ¼ broad and m ¼ multiplet. All coupling constants, J, are reported in Hertz. C NMR spectra
were acquired on a broad band decoupled mode, and the multiplicities were obtained using DEPT
sequences. Optical rotations were measured with a JASCO DIP-370 digital polarimeter (Japan).
Low-resolution FABMS was collected on a JEOL JMS-SX/SX 102A mass spectrometer (Tokyo,
Japan) or a Quattro GC/MS spectrometer (USA) with a direct inlet system. High-resolution
FABMS were measured on a JEOL JMS-HX 110 mass spectrometer (Tokyo, Japan). Silica gel 60
(
Merck, 230–400 mesh, Darmstadt, Germany) was used for column chromatography, precoated
silica gel plates (Merck, Kieselgel60 F-254, 0.20 mm) were used for analytical TLC and precoated
silica gel plates (Merck, Kieselgel60 F-254, 0.50 mm) were used for preparative TLC. The spots
were detected by spraying with 50% H SO followed by heating on a hot plate.
2
4
3
.2. Plant material
The rhizomes of T. chinensis were purchased in Tainan, Taiwan. A voucher specimen
No. 20120209) was deposited in the Department of Applied Chemistry and Materials Science,
Fooyin University, Kaohsiung, Taiwan.
(
3
.3. Extraction and isolation
The air-dried rhizomes of T. chinensis (3 kg) were extracted repeatedly with MeOH at room
temperature. After evaporation of the methanol under vacuum, the residue was suspended in

7
8
L.-M. Wei et al.
water and then partitioned successively to yield n-hexane (64.6 g), CHCl (19.5 g) and EtOAc
3
(11.4 g) fractions. The extraction and the isolation processes were carried out under the neutral
conditions. The EtOAc fraction (11.4 g) of T. chinensis was loaded onto the silica gel column
and subjected to gradient elution with CHCl –MeOH mixtures of increasing polarity to yield 12
3
fractions. The fraction obtained by elution with CHCl :MeOH (10:1) was subjected to
3
rechromatography on silica gel elution with CHCl :MeOH (100:8) to afford compound 1
3
(
rechromatography on silica gel elution with CHCl :MeOH (100:15) to afford compound 2
25 mg) and the fraction obtained by elution with CHCl :MeOH (10:2) was subjected to
3
3
(
elution with CHCl :MeOH (100:4) to afford compound 3 (15 mg).
28 mg). The fraction eluted from CHCl :MeOH (100:5) was rechromatographed on silica gel
3
3
3
.4. Tupichinin B (1)
2
4
þ
1
White solid, ½aꢀ –188 (c 0.004, MeOH). FAB-MS (positive mode) m/z: 655 [M þ Na] . H
D
NMR (400 MHz, pyridine-d ) d: 0.88 (3H, s, H-18), 1.14 (3H, d, J ¼ 7.0 Hz, H-21), 1.22 (3H, s,
5
H-19), 2.08 (3H, s, 1-O-Ac), 4.11 (1H, d, J ¼ 12.0 Hz, H-26b), 4.42 (1H, br s, H-3), 4.50 (1H, d,
J ¼ 12.0 Hz, H-26 a), 4.55 (1H, q-like, J ¼ 7.0 Hz, H-16), 4.86 (1H, s, H-27 ), 4.89 (1H, s, H-
A
0
2
and C NMR (100 MHz, pyridine-d ) d: 78.9 (d, C-1), 33.9 (t, C-2), 75.2 (d, C-3), 39.0 (t, C-4),
7 ), 4.98 (1H, d, J ¼ 7.5 Hz,H-1 ), 5.53 (1H, d, J ¼ 4.8 Hz,H-1), 5.67 (1H, d, J ¼ 4.8 Hz, H-6)
B
1
3
5
1
1
1
38.3 (s, C-5), 126.7 (d, C-6), 32.5 (t, C-7), 33.0 (d, C-8), 50.0 (d, C-9), 43.6 (s, C-10), 24.4 (t, C-
1), 40.8 (t, C-12), 40.7 (s, C-13), 57.2 (d, C-14), 32.9 (t, C-15), 82.2 (d, C-16), 63.7(d, C-17),
7.2 (q, C-18), 14.9 (q, C-19), 42.6 (d, C-20), 15.8 (q, C-21), 110.5 (s, C-22), 33.9 (t, C-23), 29.7
0
0
(t, C-24), 145.0 (s, C-25), 65.8 (t, C-26), 109.9 (t, C-27), 104.2 (d, C-1 ), 75.7 (d, C-2 ), 79.1 (d,
0
C-3 ), 72.4 (d, C-4 ), 79.1 (d, C-5 ), 63.5 (t, C-6 ), 171.5 (s, 1-O-COCH ), 22.6 (q, 1-O-COCH ).
0
0
0
3
3
þ
HRFABMS m/z 655.3563 [M þ Na] (calcd for C H O Na 655.3560).
3
5 52 10
3
.5. Tupichinin C (2)
2
4
þ 1
Colourless oil, [a ]D 2228(c 0.004, MeOH). FAB-MS (positive mode) m/z 649 [M þ Na] . H
NMR (400 MHz,CD OD) d: 0.79 (3H, s, Me-18),0.98 (3H, d, J ¼ 6.8 Hz, Me-21), 1.08 (3H, d,
3
J ¼ 7.2 Hz, Me-27), 1.31 (3H, s, Me-19), 1.45 (2H, m, H-11), 2.29 (1H, d, J ¼ 16.0 Hz, H-4b),
2
2
6
.46 (1H, dd, J ¼ 16.0, 3.0 Hz, H-4a), 3.28 (1H, d, J ¼ 10.8 Hz, H-26b-eq), 3.70 (1H, br s, H-
0
a), 3.71 (1H, dd, J ¼ 11.6, 4.0 Hz, H-6 ), 3.76 (1H, br s, H-1a), 3.85 (1H, d, J ¼ 11.6 Hz, H-
B
0
), 3.92 (1H, dd, J ¼ 10.8, 2.4 Hz, H-26a-ax), 4.15 (1H, br s, H-3a), 4.42 (1H, q, J ¼ 7.2 Hz,
A
0
13
H-16), 4.75 (1H, d, J ¼ 7.6 Hz, H-1 ) and C NMR (100 MHz, CD OD) spectral data d: 78.6 (d,
3
C-1), 68.4 (d, C-2), 71.4 (d, C-3), 36.4 (t, C-4), 84.1 (s, C-5), 31.1 (t, C-6), 29.7 (t, C-7), 35.5 (d,
C-8), 46.4 (d, C-9), 47.2 (s, C-10), 22.1 (t, C-11), 40.8 (t, C-12), 41.3 (s, C-13), 56.9 (d, C-14),
3
2.7 (t, C-15), 82.2 (d, C-16), 63.4(d, C-17), 16.9 (q, C-18), 13.6 (q, C-19), 43.4 (d, C-20), 14.7
q, C-21), 111.1 (s, C-22), 27.0 (t, C-23), 26.7 (t, C-24), 28.5 (d, C-25), 66.1 (t, C-26), 16.4 (q, C-
(
0
0
0
0
0
0
2
7), 97.2 (d, C-1 ), 75.4 (d, C-2 ), 78.2 (d, C-3 ), 70.9 (d, C-4 ), 77.9 (d, C-5 ), 62.0 (t, C-6 ).
þ
HRFABMS m/z 649. 3668 [M þ Na] (calcd for C H O Na 649. 3666).
3
3 54 11
3
.6. Tupichinin D (3)
2
4
þ
Colourless plate, [a ] þ 598 (c 0.002, CHCl ). FAB-MS (positive mode) m/z 603 [M þ Na] .
D
3
1
H NMR (400 MHz, pyridine-d ) d: 0.79 (3H, s, Me-18), 0.98 (3H, d, J ¼ 7.2 Hz, Me-21), 1.08
5
(
3H, d, J ¼ 7.2 Hz, Me-27), 1.12 (3H, s, Me-19), 1.98 (1H, m, H-5), 3.20 (1H, t, J ¼ 11.6 Hz, H-
0
0
0
2
3
5
6b-eq), 3.27 (1H, d, J ¼ 8.8 Hz, H-5 ), 3.28 (1H, m, H-2 ), 3.34 (1H, t, J ¼ 10.2 Hz, H-3 ),
B
0
.50 (1H, td, J ¼ 10.4, 5.6 Hz, H-4 ), 3.74 (1H, t, J ¼ 2.4 Hz, H-2a), 3.84 (1H, dd, J ¼ 11.6,
0
.6 Hz, H-26a-ax), 3.86 (1H, br s, H-1a), 3.92 (1H, dd, J ¼ 10.8, 2.4 Hz, H-5 ), 4.20 (1H, br s,
A

Natural Product Research
79
0
13
H-3a), 4.38 (1H, m, H-16), 4.41 (1H, d, J ¼ 7.2 Hz, H-1 ) and C NMR (100 MHz, pyridine-d5)
d: 78.5 (d, C-1), 75.2 (d, C-2), 71.1 (d, C-3), 34.0 (t, C-4), 31.6 (d, C-5), 27.3 (t, C-6), 26.5 (t, C-
7
), 36.8 (d, C-8), 43.0 (d, C-9), 42.5 (s, C-10), 21.8 (t, C-11), 41.1 (t, C-12), 41.5 (s, C-13), 57.2
d, C-14), 32.7 (t, C-15), 82.3 (d, C-16), 63.7 (d, C-17), 16.9 (q, C-18), 19.2 (q, C-19), 43.4 (d, C-
(
2
1
0), 14.7 (q, C-21), 111.1 (s, C-22), 27.0 (t, C-23), 26.7 (t, C-24), 28.5 (d, C-25), 67.0 (t, C-26),
0
0
0
0
0
6.4 (q, C-27), 103.8 (d, C-1 ), 75.0 (d, C-2 ), 77.6 (d, C-3 ), 71.2 (d, C-4 ), 66.1 (t, C-5 ).
þ
HRFABMS m/z 603.3512 [M þ Na] (calcd for C H O Na 603.3509).
3
2 52 9
3
.7. Acid hydrolysis
Compounds 1 (5 mg), 2 (4 mg) and 3 (4 mg) were hydrolysed with 2.0 mol/L HCl. The
hydrolysis and the detection of sugars in hydrolysed products were carried out according to the
procedures described in the literature (Zou et al. 2005).
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S21.
Acknowledgements
The authors gratefully acknowledge the Department of Applied Chemistry and Materials Science, Fooyin
University, Taiwan for financial support.
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