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Furospirostanol and spirostanol saponins from the rhizome of Tupistra chinensis and their cytotoxic and anti-inflammatory activities
Tetrahedron xxx (2015) 1e8
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Furospirostanol and spirostanol saponins from the rhizome of Tupistra chinensis and their cytotoxic and anti-inflammatory activities Limin Xiang a, Yihai Wang b, Xiaomin Yi b, Jianying Feng b, Xiangjiu He a, b, * a b
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2015 Received in revised form 31 October 2015 Accepted 9 November 2015 Available online xxx
Five new furospirostanol saponins (1e5), four new spirostanol saponins (6e9), along with one known spirostanol saponin were isolated from the rhizome of Tupistra chinensis. The chemical structures were determined by spectroscopic and chemical methods, including IR, NMR, MS, and GC analyses. The antiproliferative effects against five human cancer cell lines were assayed for all the isolated compounds. Compounds 6e10 showed potent cytotoxic activities against the five cancer cell lines. The isolated compounds were evaluated the inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in a macrophage cell line RAW 264.7. Compounds 6e10 showed significant inhibition on NO production with IC50 values between 3.1 and 4.4 mM. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: Tupistra chinensis Furospirostanol saponins Spirostanol saponins Cytotoxicity NO inhibitory activity
1. Introduction The genus Tupistra has 12 species in southwestern China, possessing similar morphologic characteristics, and some can be substituted for each other as a folk medicine for the treatment of pharyngolaryngitis, rheumatic diseases and snake-bite.1 Previous phytochemical investigations on this genus have led to the isolation of a series of structurally diverse compounds, including steroidal sapogenins,2 saponins,3 cardenolides4 and flavans.5,6 These components exhibit diverse biological activities, such as anti-inflammatory,7 cytotoxicity8 and anti-fungal properties.9 Tupistra chinensis is distributed in Southwest China, and its dried rhizome is a reputed folk medicine to reduce carbuncles and ameliorate pharyngitis in the Shennongjia Forest District of Hubei Province in China.10 Steroidal saponins were believed to be the main active ingredients in this plant. As part of our research project to explore more diversity bioactive leading compounds from the medicinal herbs, we investigated the 60% EtOH extract of the dried rhizome of T. chinensis and obtained five new furospirostanes (1e5), four new steroid saponins (6e9), along with one known spirostanol saponin. Herein we report the isolation and structural elucidation of these compounds and their antiproliferative activity
* Corresponding author. Tel./fax: þ86 20 3935 2132; e-mail address: hexiangjiu@ 163.com (X. He).
against five human cancer cell lines, as well as the inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in a macrophage cell line RAW 264.7.
2. Results and discussion 2.1. Structural elucidation The 60% ethanol extract of the dried underground parts of T. chinensis was successively chromatographed on D101 macroporous resin, silica gel, ODS, and finally purified by semi-preparative HPLC to afford nine new steroidal saponins, along with one known compound. The structures of all the isolated compounds (1e10) (Fig. 1) were elucidated on the basis of spectroscopic data and chemical methods, including IR, NMR, MS, and GC analyses. The known compound was identified as 5b-spirost-25(27)-en-1b,3bdiol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-a1 13 L-rhamnopyranoside (10) by comparison of the H and C NMR data with those previously reported.11 Compound 1 was isolated as white amorphous powder, [a]29 D 50 (c 0.50, MeOH). The molecular formula was inferred as C39H64O16 according to the positive-ion HRESIMS peak at m/z 788.4294 [MþH]þ (calcd for C39H65O16, 788.4273). The IR spectrum showed strong absorption bands at 3400 cm1, ascribable to hydroxyl functionalities. The 1H NMR of 1 showed singlets at dH
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
Fig. 1. Structures of isolated compounds 1e10.
0.81 and 1.51 which had been assigned to the C18- and C19-tertiary methyl groups, respectively. Instead of the two doublets of the secondary methyl groups (C21 and C27), a doublet at dH 1.08 and a singlet at dH 1.39 were observed. Signals from a primary alcohol group at dH 3.89 (1H, d, J¼10.0 Hz, H-26) and 4.17 (1H, d, J¼10.0 Hz, H-26), and two methane protons indicative of secondary alcoholic functions at dH 4.14 (1H, brs, H-1) and 4.61 (1H, brs, H-3) were observed. The 13C NMR spectrum exhibited characteristic furospirostanol carbon signal at C-22 (dC 120.5) and C-25 (dC 84.2), diagnostic of C-26 hydroxylated F-ring ‘furanose’ sapogenin. The corresponding values for F-ring ‘pyranose’ sapogenin with C-25 hydroxylation were dC 109.0 and 67.0.12 The NMR spectrum of 1 showed two anomeric proton signals at dH 4.95 (1H, d, J¼7.8 Hz) and 5.00 (1H, d, J¼7.8 Hz), corresponding to two anomeric carbons at dC 105.8 and 103.2, respectively, in the HSQC spectrum, indicated the presence of two sugar moieties. The sugars were identified as D-glucose by GC analysis of their chiral derivatives after acid hydrolysis. The b anomeric configuration of the glucose was determined by its coupling constants (J¼7.8 Hz). From the results of 1H and 13C NMR (Table 1), it suggested that 1 was a furospirostanol glucoside. Compared the NMR data with those of convallagenin A 3-O-b-D-glucopyranoside,13 the NMR features of those two compounds were almost same except for the signals of the F-ring. The NMR spectroscopic data attributed to the F-ring of 1 were consistent with those reported in the literatures.14,15 Thus compound 1 was inferred as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-Ob-D-glucopyranoside from above information. These were further verified by 1He1H COSY, HSQC, HMBC and NOESY spectra. The 1 He1H COSY spectrum showed that two methylene protons at dH 2.02 (H-2a) and 2.55 (H-2b) were coupled to dH 4.14 (1H, brs, H-1) and 4.61 (1H, brs, H-3), and the oxymethine proton dH 4.61 (H-3) was coupled to two methylene protons at dH 2.20 (H-4b) and 2.33 (H-4a). These findings supported the location of the hydroxyl groups at C-1, C-3, C-5, together with the long-range correlations observed in the HMBC spectrum (Fig. 2). The linkage of the sugar
residues were further confirmed from the following HMBC correlations: H-1 (dH 5.00) of glucose with C-3 (dC 75.7) of the aglycon, and H-1 (dH 4.95) of glucose’ with C-26 (dC 77.8) of the aglycon. The stereochemistry of the ring junctions and substituents of the aglycon of 1 were determined via cross-peaks observed in a NOESY spectrum. The key correlations between H4a and H-7a/H-9a, and between H-2a/H-9a, supported the A/B cis ring junction pattern. Thus, the hydroxyl group at C-5 has a borientation. Additional NOE correlations between H-1a and Me-19/ H-11, and between H-3a and H-2a/H-4a, were indicative of b-orientations for OH-1 and OH-3. Consequently, the structure of 1 was deduced to be 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-O-b-D-glucopyranoside. Compound 2 was isolated as white amorphous powder. The molecular formula of C39H64O15 was determined by the positiveion HRESIMS peak at m/z 773.4389 [MþH]þ (calcd for C39H65O15, 773.4323). The IR spectrum showed strong absorption bands at 3423 cm1, ascribable to hydroxyl functionalities. Unambiguous complete assignments for the 1H and 13C NMR signals were made by combination of DEPT, 1He1H COSY, HSQC, HMBC, and NOESY spectra. Compared to 1, the NMR signals indicated the loss of a hydroxyl at C-5 in 2, which was supported by HMBC correlations of Me-19 (dH 1.21) with C-5 (dC 31.3). Thus, compound 2 was elucidated as 26-O-b-D-glucopyranosyl-(22S, 25S)-5b-furostan-22,25epoxy-1b, 3b, 26-triol-3-O-b-D-glucopyranoside. Compound 3 was isolated as white amorphous powder, and positive-ion HRESIMS provided an ion at m/z 795.4164 [MþNa]þ, which corresponded to a molecular formula of C39H64O15 as same as 2. The 1H and 13C NMR data of 3 were similar to those of 2 with differences apparent only in the chemical shifts at positions 2, 3, 4 and 5 in the A ring. The NOESY spectrum (Fig. 3) indicated correlation between H-3b and H-2b, with no evidence of any correlation between H-3b and H-2a/H-4a, suggesting that the hydroxyl group located at C-3 of the aglycon of 3 was a-orientation rather than borientation of 2. On the basis of the above spectroscopic evidence, the structure of 3 was deduced to be 26-O-b-D-glucopyranosyl-
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3
Table 1 NMR spectroscopic data for compounds 1e5 (pyridine-d5) Position
1
2
dC
o
dH (J in Hz)
3
dC
dH (J in Hz)
4
dC
dH (J in Hz)
5
dC
dH (J in Hz)
dC
dH (J in Hz) 2.35, m 1.28, m 1.70, m 1.55, m 4.22o 2.13o
1
73.3
4.14, brs
72.7
3.87o
72.8
4.13, brs
75.4
4.22, brs
25.7
2
33.3
32.3
2.71, 1.97, 5.18, 2.50, 2.31,
d (12.7) m m m m
28.6
5 6
75.5 36.9
7
29.1
8 9 10 11 12
35.1 45.7 44.2 21.8 40.2
2.57, d (12.7) 1.88, m 4.87, m 2.15, m 1.88, m 2.14, m 1.72, m 1.20o 1.20o 0.95, m 1.54, m 1.32, m
35.2
75.7 35.6
2.32o 1.82o 4.59, brs 1.87o
35.3
3 4
2.55, d (13.7) 2.02o 4.61, brs 2.33, m 2.20o
1.90, 1.47, 1.31, 0.85, 1.55, 1.23,
m d (13.0) m m dd (11.1, 3.8) m
13 14 15
41.0 56.3 32.4
16 17 18 19 20 21 22 23 24
81.2 62.8 16.8 13.8 38.9 15.4 120.5 33.4 34.2
25 26
84.2 77.8
27
24.7
1.95o 1.46 1.37o 0.91o 1.58o 1.08o 1.33o 1.64o 0.93o 0.91o 1.94o 1.32o 4.75, dd (15.2, 7.2) 1.76, dd (8.4, 6.9) 0.81, s 1.51, s 2.18o 1.08, d (6.9) 2.02o 2.21o 1.63o 4.17, d (10.0) 3.89, d (10.0) 1.39, s
75.1 29.6 31.3 26.8 26.7 36.0 42.4 40.7 21.4 40.5 41.0 56.5 32.5 81.3 63.0 16.9 19.4 38.9 15.5 120.6 33.4 34.3 84.2 77.8 24.7
2.34o 1.69o 1.15o 1.15o 0.92o 1.50o 1.12o 1.23o 1.62o 0.97o 0.96o 1.95o 1.31o 4.75, dd (15.2, 7.2) 1.79o 0.79, s 1.21, s 2.16o 1.08, d (6.9) 2.01o 2.18o 1.62o 4.16, d (10.0) 3.87o 1.37o
74.8 34.8 36.2 27.3 26.8 36.0 42.5 39.9 21.2 40.5 41.0 56.5 32.5 81.3 63.1 16.9 19.2 38.9 15.5 120.6 33.4 34.2 84.2 77.9 24.7
72.5 41.5 77.2 36.6 29.0 35.3 45.7 43.2 21.6 40.2
1.30, m 1.63, m 0.94, m 0.95, 1.98, 1.33, 4.83, 1.83, 0.82, 1.25, 2.18, 1.11,
41.1 56.3 32.5
m m m dd (14.6, 7.4) m s s m d (6.9)
81.3 62.8 16.8 13.6 39.0 15.5 120.6 33.4 34.3
2.00o 2.20, m 1.64, m
84.3 77.9
4.18, d (10.0) 3.92, d (10.0) 1.40, s
24.7
67.1 35.5
1.27, m 1.62, m 0.88, m 0.83, 1.95, 1.34, 4.83, 1.82, 0.82, 1.42, 2.19, 1.12,
m m m dd (15.2, 7.1) dd (8.5, 6.7) s s m d (6.9)
2.04, m 2.19, m 1.64, m
81.4 31.6
2.04, m 1.79, m 1.45, m 0.77, m 1.50, m 1.15o
29.5 34.5 43.2 41.5 21.5 40.3
1.35, m 1.69, m 1.06, m
41.1 56.5 32.5
0.96, m 1.96, m 1.35, m 4.81, dd (14.9, 7.1) 1.83o 0.81, s 1.22, s 2.22, m 1.14, d (6.9)
81.3 62.9 16.7 17.9 39.0 15.5 120.9 33.3 30.0
2.08, m 2.21, m 2.10, m
87.0 74.8
4.19, d (9.9) 3.93, d (10.0) 1.40, s
65.1
3-O-Glc 1 2 3 4 5 6
103.2 75.8 79.1 71.6 78.7 62.9
5.00, d (7.8) 3.96o 4.26o 4.25o 3.96o 4.55, m 4.40, m
101.7 75.4 79.1 71.9 79.0 63.0
4.98, d (7.8) 3.96o 4.25o 4.24o 3.94o 4.54, m 4.37, m
103.0 75.8 78.9 71.9 78.7 63.0
5.02, 4.07, 4.26, 4.27, 3.85, 4.50, 4.40,
d (7.7) t (8.2) m m ddd (9.3, 5.0, 2.5) dd (11.8, 2.3) m
103.2 75.8 78.9 71.8 78.8 63.0
5.09, d (7.8) 4.09o 4.27o 4.33, t (9.2) 3.85, ddd (9.5, 4.6, 2.6) 4.44o 4.40o
26-O-Glc 1 2 3 4 5 6
105.8 75.6 78.6 71.9 78.8 63.0
4.95, d (7.8) 4.03o 4.26o 4.25o 3.96o 4.55, m 4.40, m
105.8 75.7 78.8 71.9 78.9 63.1
4.93, d (7.7) 4.03, m 4.25o 4.20o 3.98o 4.54, m 4.37, m
105.8 75.7 78.6 71.9 78.9 63.1
4.97, 4.07, 4.26, 4.27, 3.96, 4.55, 4.40,
d (7.8) t (8.2) m m m dd (11.8, 2.2) m
105.8 75.7 78.7 72.0 78.9 63.0
4.98, d (7.8) 4.06o 4.27o 4.27o 3.96, m 4.55, d (10.3) 4.40o
5-O-Glc 97.6 75.7 78.9 71.9 79.2 63.3
105.9 75.5 78.7 72.1 78.9 62.9
4.45, 4.24, 4.16, 3.96,
d d d d
(10.2) (10.2) (10.9) (10.9)
5.14, 3.94, 4.24, 4.09, 4.03, 4.55, 4.25,
d (7.8) m m m m m m
5.04, 4.03, 4.24, 4.24, 3.96, 4.55, 4.38,
d (7.8) m m m m m m
Overlapped with other signals.
HMBC COSY
NOESY
O OH
O HO
HO HO HO
O
O
H
HO HO
OH
O
H H
O OH
H
H
H H H
H
H H
H H
RO A
Fig. 2. Selected 1He1H COSY (A), HMBC (A) and NOESY (B) correlations of 1.
HO
CH3
B
Fig. 3. Selected 1He1H COSY (A), HMBC (A) and NOESY (B) correlations of 3.
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(22S, 25S)-5b-furostan-22,25-epoxy-1b, 3a, 26-triol-3-O-b-Dglucopyranoside. Compound 4 was obtained as white amorphous powder, with the same molecular formula of C39H64O16 as 1 on the basis of HRESIMS (m/z 789.4258 [MþH]þ). The close similarity of the 13C shifts of 4 and 1 (Table 1), with the exception of C-1 (DdC 2.1), C-2 (DdC 1.9), C-3 (DdC 3.2), C-4 (DdC 5.9) and C-5 (DdC 1.7) of the aglycon, showed identical BeF ring substitution in the two compounds. The C-1, C-2, C-3, C-4 and C-5 differences in shifts indicated the a-orientation of the hydroxyl group at C-3. This was confirmed by the absence of any NOESY correlation between H-3b and H-2a/H4a. Therefore, the structure of 4 was identified as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3a, 5b, 26-tetrol-3O-b-D-glucopyranoside. Compound 5 was isolated as white amorphous powder. The HRESIMS of 5 showed the ion at m/z 789.4323 [MþH]þ, and its molecular formula was inferred as C39H64O16 on the basis of the analysis of 1H and 13C NMR and DEPT spectra. The IR spectrum showed strong absorption band at 3423 cm1, ascribable to hydroxyl functionalities. Acid hydrolysis and GC analysis of 5 gave Dglucose. The 1H NMR spectrum showed three typical steroidal methyl signals at dH 1.22 (3H, s, Me-19), 1.14 (3H, d, J¼6.9 Hz, Me21) and 0.81 (3H, s, Me-18). The lost signal of Me-27 was suggested some change on the substituent of C-25. The 13C NMR spectrum exhibited the C-22 carbon of the aglycon at dC 120.9, indicated a furanose F ring. On comparison of the 1H and 13C NMR spectra of 5 with those of 1e4, the NMR signals indicated the loss of methyl group at C-25 were replaced by an oxygenated primary alcohol group [dH 4.16 (1H, d, J¼10.9 Hz), 3.96 (1H, d, J¼10.9 Hz), dC 65.1] in 5. This was further evidenced by HMBC correlations of H-27 (dH 3.96, 4.16) with C-24 (dC 30.0), C-25 (dC 87.0), C-26 (dC 74.8). Thus, the aglycon of 5 was identified as a rare dihydroxylated F-ring furospirostane. Compared the NMR data with those of reinocarnoside B,16 the NMR features of those two compounds were almost same except for the signals of the F-ring, which was suggested the same AeE ring substitution in the two compounds. Hence, the structure of 5 was elucidated as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-3b, 5b, 26, 27-tetrol-5-Ob-D-glucopyranoside. Compound 6 was isolated as white amorphous powder. The HRESIMS of 6 showed the ion at m/z 857.4954 [MþH]þ, and its molecular formula was inferred as C44H72O16 on the basis of the analysis of 1H and 13C NMR and DEPT spectra. The IR spectrum showed the characteristic absorptions of hydroxyl group at 3421 cm1 and the four steroidal sapogenins absorption bands 981, 912, 899, 865 cm1. And the 899 band was stronger than the 912
band. It was suggested that 6 was a sapogenin with ‘iso’ configuration of the F ring.17 On acid hydrolysis, 6 liberated D-xylose and Lrhamnose, identified by GC analysis of their chiral derivatives, which were consistent with three anomeric protons at dH 6.44 (1H, d, J¼0.9 Hz), 5.42 (1H, s), and 5.08 (1H, d, J¼6.4 Hz) in the 1H NMR spectrum. The 1H NMR spectrum showed four typical steroidal methyls, including two angular methyl group at dH 1.31, 0.84, and two secondary methyl groups at dH 1.11 (1H, d, J¼6.9 Hz), 0.67 (1H, d, J¼5.3 Hz). In the 13C NMR spectrum, a typical quaternary carbon signal of spirostanol at dC 109.6 (C-22), and three anomeric carbon signals at dC 100.0, 100.3, and 102.0 were observed. Compared the 13 C NMR data of aglycon of 6 with those of isorhodeasapogenin,18 the downfield shifts of C-1 (þ5.8 ppm) and C-3 (þ2.9 ppm) indicated that 6 was a bidesmosidic saponin with glycosidic linkages at C-1 and C-3, respectively. The linkages of the sugar residues were further confirmed from the following HMBC correlations (Fig. 4): H1 (dH 6.44) of rhamnose with C-2 (dC 77.4) of xylose, H-1 (dH 5.08) of xylose with C-1 (dC 79.2) of the aglycon, and H-1 (dH 5.42) of rhamnose with C-3 (dC 71.1) of the aglycon. From above spectroscopic evidence, the structure of 6 was deduced to be (25R)-5bspirostan-1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside.
Fig. 4. Selected 1He1H COSY and HMBC correlations of 6.
Compound 7 was isolated as white amorphous powder, with the same molecular formula of C44H72O16 as 6 on the basis of HRESIMS (m/z 857.4948 [MþH]þ). The close similarity of the 13C shifts of 7 and 6 (Table 2), with the exception of F-ring of the aglycon, suggested that the configuration of C-25 of the aglycon was different. It was further evidenced by the IR spectrum: the bands occur at 984, 917, 897, 849 cm1, with the 917 band stronger than the 897 band.17 Thus the configuration of C-25 of the aglycon of 7 was S-form. Therefore, the structure of 7 was elucidated as (25S)-5b-spirostan1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido3-O-a-L-rhamnopyranoside.
Table 2 NMR spectroscopic data for compounds 6 and 7 (pyridine-d5) Position
6
7
dC 1 2 3 4
79.2 31.3 71.1 31.8
5 6
35.5 26.7
7
27.7
8 9 10 11
34.5 46.8 39.9 23.1
dH (J in Hz) 4.04, t (5.9) 2.27, m 4.17, m 1.79o 1.56o 2.00, m 1.54o 1.27o 1.37o 0.92o 1.51o 1.28o 2.01o 1.46o
6
dC 79.0 31.0 71.0 31.8 35.5 26.7 27.8 34.6 46.7 39.9 23.1
7
dH (J in Hz)
Position
4.04, m 2.25, m 4.15, m 1.78, m 1.55o 2.00o 1.55o 1.29o 1.38o 0.91o 1.49o 1.26o
23
32.2
1.66o
26.7
24
29.6
1.55o
26.5
25 26
30.9 67.2
o
27.7 65.4
27 1-O-Xyl 1 2 3 4 5
17.7
1.98o 1.44o
dC
100.3 77.4 79.7 71.7 67.3
dH (J in Hz)
1.55 3.57, m 3.48, m 0.67, d (5.3) 5.08, d (6.4) 4.26o 4.27o 4.14, m 4.32, m 3.66, m
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dC
16.6 100.2 77.4 79.6 71.7 67.3
dH (J in Hz) 1.90o 1.48o 2.11o 1.38o 1.55o 4.05, m 3.34, d (11.0) 1.03, d (7.0) 5.08, d (6.8) 4.26o 4.27o 4.14, m 4.32, m 3.65, t (10.6)
L. Xiang et al. / Tetrahedron xxx (2015) 1e8
5
Table 2 (continued ) Position
6
7
dC
o
12
40.8
13 14 15
40.9 57.3 32.6
16 17 18 19 20 21 22
81.5 63.4 17.0 17.4 42.3 15.4 109.6
dH (J in Hz) 1.69o 1.18o 1.07o 2.02o 1.42, m 4.55, dd (14.9, 7.3) 1.80, m 0.84, s 1.31, s 1.95, dt (13.9, 7.0) 1.11, d (6.9)
6
dC 40.8 40.9 57.3 32.5 81.5 63.2 17.0 17.4 42.7 15.2 110.1
dH (J in Hz)
Position
1.67o 1.15o
Rha(1/2) 1 2 3 4 5 6 3-O-Rha 1 2 3 4 5 6
1.03o 1.99o 1.41, m 4.51, dd (14.9, 7.3) 1.78, m 0.82, s 1.29, s 1.87, m 1.10, d (6.9)
7
dC
dH (J in Hz)
dC
dH (J in Hz)
102.0 72.8 72.7 74.4 69.8 19.4
6.44, d (0.9) 4.84, m 4.66o 4.32, m 4.78, dq (12.4, 6.1) 1.75, d (6.2)
102.0 72.8 72.7 74.4 69.8 19.4
6.42, s 4.84, m 4.66o 4.32, m 4.77, dq (12.3, 6.1) 1.72, d (6.2)
100.0 73.1 72.8 74.6 70.3 19.0
5.42, s 4.66o 4.64o 4.27,o 4.37, dd (10.8, 4.6) 1.68, d (6.2)
100.0 73.0 72.7 74.5 70.3 19.0
5.41o 4.66o 4.64o 4.27,o 4.37, m 1.66, d (6.1)
Overlapped with other signals.
Compound 8 was isolated as white amorphous powder. The molecular formula of C44H70O17 was determined by the positiveion HRESIMS peak at m/z 895.4703 [MþNa]þ (calcd for C44H72O17Na, 895.4667). The IR spectrum showed the characteristic absorptions of hydroxyl group at 3420 cm1 and the four steroidal sapogenins absorption bands 982, 917, 899, 863 cm1. And the 899 band was stronger than the 917 band. It was suggested that 8 was a sapogenin with ‘iso’ configuration of the F ring.17 On acid hydrolysis, 8 liberated D-xylose, L-rhamnose, and D-glucose, identified by GC analysis of their chiral derivatives. From a comparison of 1H and 13C NMR data of 8 (Table 3) with those of 6, it was apparent that 8 contained the same aglycon as 6, except for a little different in the saccharide chains. The linkage of the sugar units was established from the following HMBC correlations (Fig. 5): H-1 (dH 6.39) of
Fig. 5. Selected 1He1H COSY and HMBC correlations of 8.
Table 3 NMR spectroscopic data for compounds 8 and 9 (pyridine-d5) Position
8
9
dC
o
1 2
79.0 30.6
3 4
73.1 31.6
5 6o
35.1 26.9
7
27.4
8 9 10 11
35.1 46.6 39.9 22.7
12
40.8
13 14 15
40.9 57.2 32.5
16 17 18 19 20 21 22 23
81.4 63.4 17.0 17.5 42.3 15.4 109.6 32.1
dH (J in Hz) 3.91, dd (8.8, 2.9) 2.29, m 2.15o 4.35o 1.83o 1.72o 1.94, m 1.47o 1.19o 1.30o 0.90, m 1.47o 1.20o 2.01o 1.41o 1.69o 1.17o 1.07o 2.02o 1.41o 4.55, m 1.81o 0.84, s 1.24, s 1.93, m 1.11, d (7.0) 1.65o
8
dC 79.1 30.7 73.1 31.6 35.1 27.0 27.5 35.1 46.7 39.9 22.8 40.8 40.9 57.2 32.5 81.8 63.4 17.0 17.5 42.2 15.3 109.8 33.5
9
dH (J in Hz)
Position
3.90, dd (8.9, 2.7) 2.30, m 2.17o 4.35o 1.84o 1.70o 1.94, m 1.46o 1.20o 1.30, m 0.90, m 1.47o 1.21o
24
29.6
1.55o
25 26
30.9 67.2
27
17.6
1.55o 3.56, d (3.0) 3.49, t (10.6) 0.67, d (5.6)
2.06o 1.43o 1.68o 1.17o
Rha(1/2) 1 2 3 4 5 6 3-O-Glc 1 2 3 4 5 6
1.06o 2.01o 1.41o 4.53, m 1.80o 0.84, s 1.24, s 1.94, m 1.06, d (6.9)
1-O-Xyl 1 2 3 4 5
dC
dH (J in Hz)
dC 29.3 144.8 65.3 109.0
dH (J in Hz) 2.70, m 2.23o 4.45, 4.02, 4.80, 4.77,
d (12.0) d (12.0) s s
100.0 76.7 78.4 71.5 66.8
4.97, d (6.6) 4.31o 4.18, m 4.12o 4.34o 3.61, m
100.0 76.7 78.5 71.5 66.8
4.95, d (6.6) 4.31o 4.18, m 4.13o 4.34o 3.60, m
101.7 72.6 72.9 74.4 70.0 19.4
6.39, 4.78, 4.66, 4.34, 4.74, 1.76,
101.7 72.7 72.9 74.4 70.0 19.4
6.41, s 4.79o 4.67, dd (9.3, 3.3) 4.35, m 4.77o 1.76, d (6.1)
103.1 75.3 78.8 72.1 78.9 63.2
4.92, d (7.7) 4.13o 4.28, m 4.24o 3.97, m 4.56, m 4.36, m
103.1 75.3 78.8 72.1 78.9 63.2
4.93, d (7.7) 4.13o 4.28, m 4.26o 3.97, m 4.58, dd (11.9, 2.2) 4.37o
d (1.1) dd (3.3, 1.5) dd (9.3, 3.3) m dd (10.8, 4.7) d (6.2)
1.80o 1.76o
Overlapped with other signals.
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
rhamnose with C-2 (dC 76.7) of xylose, H-1 (dH 4.97) of xylose with C-1 (dC 79.0) of the aglycon, and H-1 (dH 4.92) of glucose with C-3 (dC 73.1) of the aglycon. Thus, compound 8 was elucidated as (25R)5b-spirostan-1b, 3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-Dxylopyranosido-3-O-b-D-glucopyranoside. Compound 9 was isolated as white amorphous powder, and positive-ion HRESIMS provided an ion at m/z 893.4498 [MþNa]þ, which corresponded to a molecular formula of C44H70O17. The 1H and 13C NMR data of 9 were similar to those of 8 with differences apparent only in the chemical shifts at positions 23, 24, 25, 26 and 27 in the F ring of the aglycon. The signals due to the methyl of C-27 in 8 were replaced by the signals assigned to the C-25(27) exomethylene group at dH 4.77 (1H, s) and 4.80 (1H, s), and dC 144.8 (C-25) and 109.0 (C-27) in 9. Hence, the structure of 9 was elucidated as 5b-spirost-25(27)-en-1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside. Two biosynthetic routes to the relatively rare 3a-OH sapogenin (compounds 3, 4) were therefore considered possible. The first involves oxidation of the 3b-OH followed by reduction to given the 3aOH derivative. The second route oxidation of the 5b-spirost-2-en.19
exhibited moderate inhibition with IC50 values of 23.3 mM. Indomethacin was selected as a positive control (IC50 47.4 mM). Cell viability was determined by the MTT method to find whether inhibition of NO production was due to the cytotoxicity of the compounds 5e10. These six compounds exhibited no cytotoxicity against RAW 264.7 macrophage cells at their effective concentrations. 3. Conclusion Five new furospirostanol and four new spirostanol saponins, along with one known spirostanol saponin were isolated from the rhizome of T. chinensis. The chemical structures were determined by spectroscopic and chemical methods. Compounds 1e5 were uncommon polyhydroxylated furospirostanol glycosides and compounds 6e9 were unusual spirostanol saponins with glycosidation both at C-1 and C-3 of the aglycon. Some compounds showed potent cytotoxic activities against the five cancer cell lines and significant inhibition on NO production. 4. Experimental section
2.2. Cytotoxicity of isolated compounds 4.1. General experimental procedures Compounds 1e10 were evaluated for cytotoxicity against five human tumor cell lines including HepG2 (human hepatocellular carcinoma), CNE-1 (high differentiation human nasopharyngeal carcinoma), CNE-2 (low differentiation human nasopharyngeal carcinoma), K562 (human chronic leukemia), SPC-A-1 (human lung adenocarcinoma) with a modified MTT method according to reported protocols,20 and cis-dichlorodiamineplatinum (II) was used as a positive control. Compounds 7 and 9 exhibited significant cytotoxic effects against all of the human cancer cell lines tested with IC50 values smaller than 12 mM (Table 4). Compounds 6 and 8 exhibited moderate cytotoxic effects against all of the human cancer cell lines tested. Compound 10 showed inhibitory activities against four out of five human cancer cell lines tested except K562. The other compounds exhibited IC50 values larger than 50.0 mM against the five tested cell lines and were considered to be inactive. Table 4 Cytotoxicities of compounds 1e10 to human cancer cell lines Compound
6 7 8 9 10 CISPb
IC50 (mM)a HepG2
CNE-1
CNE-2
K562
SPC-A-1
18.10.1 6.10.5 8.50.3 6.10.2 7.50.1 6.50.3
16.91.0 8.30.2 16.80.7 8.41.0 15.00.4 10.80.4
14.82.1 9.10.1 3.40.3 10.61.7 13.50.4 8.01.1
15.00.5 10.30.9 17.50.3 11.60.5 >50 13.50.8
12.10.8 8.30.1 10.00.3 8.70.6 11.00.4 3.80.3
Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were obtained on a PerkinElmer 100 IR spectrometer with KBr. NMR spectra were recorded on Ultrashield 500 Plus instrument and were recorded in pyridine-d5. Chemical shifts (d) are stated in ppm from the internal standard, tetramethylsilane (TMS). HRESIMS were measured on a Waters AQUITY UPLC/Q-TOF micro spectrometer. Semi-preparative HPLC was performed on a RAININ pump equipped with a Gilson 133 refractive index detector and a COSMOSIL packed column (5C18-AR-Ⅱ, 10ID250 mm). D101 macroporus resin (Xi’an Lanxiao Resin Corporation Ltd., Xi’an, China), Silica gel (200e300 mesh, Anhui Liangchen Silicon Material Co. Ltd., Lu’an, China) and ODS (40e60 mm, Merck KGaA, Darastadt, Germany) were used for column chromatography. HPLC-grade methanol was purchased from Oceanpak Chemical Co. (Gothenburg, Sweden). All solvents used for column chromatography were of analytical grade (Shanghai Chemical Reagents Company, Ltd., Shanghai, China). Sugar reagents (Sigma, St. Louis, MO, USA) were used for GC analysis. 4.2. Plant material
Results are presented as means SD (n¼3). Positive control. Compounds 1e5 with IC50 values >50.0 mM for all the tested cell lines.
The rhizomes of T. chinensis Baker was purchased from Shennongjia Forest District (Shennongjia, China), and identified by Prof. Xiangjiu He, the School of Pharmacy, Guangdong Pharmaceutical University. A voucher specimen (No. GDPU-NPR-2013002) was deposited in the Department of Medicinal Chemistry, Guangdong Pharmaceutical University, Guangzhou, China.
2.3. Inhibitory effects on NO production
4.3. Extraction and isolation
Compounds 1e10 were tested for their inhibitory effects on NO production induced by LPS in a macrophage cell line RAW 264.7. Compounds 6e10 showed significant inhibition on NO production with IC50 values between 3.1 and 4.4 mM (Table 5). Compound 5
The air-dried underground parts of T. chinensis (17.0 Kg) were extracted four times with 60% EtOH (70 L) for 4 h at reflux. The combined EtOH extracts were evaporated to 20 L, and partitioned between H2O and EtOAc to give an EtOAc fraction (218.0 g). The
a
b
Table 5 Inhibitory effects of compounds 1e10 on NO production induced by LPS in macrophagesa Compoundb
5
6
7
8
9
10
Indomethacina
IC50 (mM)
23.35.1
3.10.3
3.20.03
4.40.3
3.20.9
3.10.02
47.44.5
a b
Positive control. Compounds 1e4 with IC50 >50.0 mM.
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
aqueous fraction was applied to a D101 macroporous resin column, eluted with H2O (50 L), 20% EtOH (50 L), 60% EtOH (50 L), 80% EtOH (50 L) to give four fractions (AeD). A part of fraction B (100.0 g) was subjected to silica gel column chromatography eluted with a gradient of CHCl3/MeOH (20:1 to 1:1) to yield 22 fractions (B1eB22). A half part of fraction B18 (6.5 g) was separated over a column of ODS silica gel eluted with a gradient of MeOHeH2O (1:9 to 3:7) to afford six fractions (B18-1 to B186). Subfraction B18-4 (2.44 g) was further purified by semipreparative HPLC (3.0 mL/min, 50% MeOH in H2O isocratic elution) to yield compound 1 (109.0 mg). One-fifth of fraction B19 (5.0 g) was separated by an ODS column eluted with a gradient of MeOHeH2O (1:9 to 2:8) to afford seven fractions (B19-1 to B19-7). Subfraction B19-3 (875.8 mg) was further purified by semipreparative HPLC (2.7 mL/min, 45% MeOH in H2O isocratic elution) to yield compound 5 (23.1 mg). Subfraction B19-4 (849.5 mg) was further separated by semi-preparative HPLC (2.7 mL/min, 50% MeOH in H2O isocratic elution) to yield compound 4 (18.4 mg). A half part of fraction C (200.0 g) was subjected to silica gel column chromatography eluted with a gradient of CHCl3/MeOH (20:1 to 2:1, followed by MeOH) to yield 18 fractions (C1eC18). A half part of fraction C14 (5.2 g) was further separated by ODS eluted with a gradient of MeOH/H2O (7:3 to 2:8) to afford nine fractions (C14-1 to C14-9). Subfraction C14-4 was further purified by semipreparative HPLC (2.8 mL/min, 60% MeOH in H2O isocratic elution) to yield compound 3 (40.0 mg). Fraction D (83.3 g) was separated by silica gel column chromatography eluted with a gradient of CHCl3/MeOH (100:1 to 1:1) to obtain 21 fractions (D1eD21). Fraction D18 (11.4 g) was subjected to ODS to obtain seventeen fractions (D18-1 to D18-17). Subfraction D18-9 (1.2 g) was further purified by semi preparative HPLC (3.0 mL/min, 65% MeOH in H2O isocratic elution) to yield compound 2 (32.3 mg). Fraction D19 (3.6 g) was purified by ODS silica gel to obtain eight fractions (D19-1 to D19-8). Subfraction D19-7 was further purified by semi preparative HPLC to yield compound 6. (23.9 mg), 7 (10.7 mg), and 10 (27.4 mg). Fraction D 20 (5.0 g) was subjected to ODS to obtain ten fractions (D 20-1 to D 20-10). Subfraction D 20-8 (612.9 mg) was further purified by semi preparative HPLC to yield compound 8 (58.4 mg) and 9 (58.7 mg). 4.3.1. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-O-b-D-glucopyranoside (1). White amorphous powder; [a]29 D 50 (c 0.50, MeOH); IR (KBr) nmax 3394, 2918, 1636, 1456, 1094 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 788.4294 [MþH]þ (calcd for C39H65O16, 788.4273). 4.3.2. 26-O- b - D -Glucopyranosyl-(22S, 25S)-5 b -furostan-22,25epoxy-1b, 3b, 26-triol-3-O-b-D-glucopyranoside (2). White amorphous powder; [a]29 D 62 (c 0.50, MeOH); IR (KBr) nmax 3423, 2926, 1635, 1452, 1046 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 773.4389 [MþH]þ (calcd for C39H65O15, 773.4323). 4.3.3. 26-O- b - D -Glucopyranosyl-(22S, 25S)-5 b -furostan-22,25epoxy-1b, 3a, 26-triol-3-O-b-D-glucopyranoside (3). White amorphous powder; [a]29 D þ9 (c 0.50, MeOH); IR (KBr) nmax 3400, 2918, 1637, 1452, 1036 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 795.4164 [MþNa]þ (calcd for C39H64O15Na, 795.4143). 4.3.4. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy1b, 3a, 5b, 26-tetro L-3-O-b-D-glucopyranoside (4). White amorphous powder; [a]27 D 40 (c 0.25, MeOH); IR (KBr) nmax 3406, 2931, 1637, 1452, 1036 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 789.4258 [MþH]þ (calcd for C39H65O16, 789.4273). 4.3.5. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy3b, 5b, 26, 27-tetrol-5-O-b-D-glucopyranoside (5). White
7
amorphous powder; [a]28 D 32 (c 0.25, MeOH); IR (KBr) nmax 3423, 2928, 1634, 1453, 1046 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 789.4323 [MþH]þ (calcd for C39H65O16, 789.4273). 4.3.6. (25R)-5 b -Spirostan-1 b ,3 b -diol-1-O- a -L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside (6). White amorphous powder; [a]29 D 105 (c 0.5, MeOH); IR (KBr) nmax 3421, 2929, 1642, 1452, 1046, 981, 912, 899, 865 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [MþH]þ 857.4954 (calcd for C44H73O16 [MþH]þ, 857.4899). 4.3.7. (25S)-5 b -Spirostan-1 b ,3 b -diol-1-O- a- L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside (7). White amorphous powder; [a]29 D 99 (c 0.5, MeOH); IR (KBr) nmax 3423, 2933, 1642, 1451, 1047, 984, 917, 897, 849 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [MþH]þ 857.4948(calcd for C44H73O16 [MþH]þ, 857.4899). 4.3.8. (25R)-5 b -Spirostan-1 b ,3 b -diol-1-O- a -L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside (8). White amorphous powder; [a]29 D 86 (c 0.5, MeOH); IR (KBr) nmax 3420, 2928, 1645, 1453, 1049, 982, 917, 899, 863 cm1; 1H and 13C NMR data, see Table 3; HRESIMS m/z [MþNa]þ 895.4703(calcd for C44H72O17Na [MþNa]þ, 895.4667). 4.3.9. 5b-Spirost-25(27)-en-1b,3b-diol-1-O-a-L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside (9). White amorphous powder; [a]29 D 94 (c 0.5, MeOH); IR (KBr) nmax 3411, 2928, 1650, 1452, 1045, 983, 918, 897, 838 cm1; 1H and 13C NMR data, see Table 3; HRESIMS m/z [MþNa]þ 893.4498 (calcd for C44H70O17Na [MþNa]þ, 893.4511). 4.4. Acid hydrolysis and GC analysis The hydrolysis and GC analysis of the chiral derivatives of the sugars of new compounds were done as previously described.21 Compounds 1e9 (1e2 mg) were heated in an ampoule with 5 mL 2 M HCl at 90 C for 2e8 h. The aglycon was extracted with EtOAc three times and the aqueous residue was evaporated under reduced pressure at 60 C. Then 600 mL pyridine and 5 mg NH2OH$HCl were added to the residue and the mixtures were heated at 90 C for 30 min. After cooling, 300 mL Ac2O was added to the mixtures. After homogenized, the mixtures were heated at 90 C for 1 h. After cooling, the reaction mixtures were analyzed by GC using standard aldononitrile peracetates as reference samples. 4.5. Cytotoxicity assays Compounds 1e10 were evaluated for cytotoxicity against five tumor cell lines including HepG2, CNE-1, CNE-2, K562, SPC-A-1 with a modified MTT method according to reported protocols in triplicate independent experiments. cis-Dichlorodiamineplatinum (II) was used as a positive control. 4.6. NO production assay The NO production’s inhibition, and viability assay were done as previously described. All experiments were performed in three independent replicates. Indomethacin was selected as a positive control. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 81573303), the Natural Science Foundation of Guangdong province (No. 2014A030313588), and the
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starting fund of Guangdong Pharmaceutical University. The authors were also great acknowledgments to Prof. Dr. Hao Gao, Jinan University, for his generous help in the measurement of optical rotation. Supplementary data Supplementary data (The original spectra of new compounds 1e9, including 1H, 13C NMR, 2D-NMR (HSQC, HMBC, 1He1H COSY, NOESY), HRESIMS, and IR, were given as supplementary data.) associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2015.11.012. References and notes 1. Y. W. Z.; Hua, X.; Cao, B.; Yao, G. Shanghai Publishing House of Science and Techniques, 1991. 2. Pan, W. B.; Chang, F. R.; Wu, Y. C. J. Nat. Prod. 2000, 63, 861e863. 3. Liu, C. X.; Guo, Z. Y.; Xue, Y. H.; Cheng, J.; Huang, N. Y.; Zhou, Y.; Cheng, F.; Zou, K. Fitoterapia 2012, 83, 323e328. 4. Pan, Z. H.; Li, Y.; Liu, J. L.; Ning, D. S.; Li, D. P.; Wu, X. D.; Wen, Y. X. Fitoterapia 2012, 83, 1489e1493.
5. Pan, W. B.; Chang, F. R.; Wei, L. M.; Wu, Y. C. J. Nat. Prod. 2003, 66, 161e168. 6. Pan, W. B.; Wei, L. M.; Wei, L. L.; Wu, Y. C. Chem. Pharm. Bull. 2006, 54, 954e958. 7. Zou, K.; Wang, J. Z.; Wu, J.; Zhou, Y.; Liu, C.; Dan, F. J.; Zhang, Y. X.; Yang, J. Chin. Chem. Lett. 2007, 18, 1239e1242. 8. Luo, H. J.; Wang, J. Z.; Deng, W. Q.; Zou, K. Med. Chem. Res. 2013, 22, 4970e4979. 9. Wu, G. X.; Liu, A. Y.; Wei, X. Y.; Chen, W. X. Wuhan. Zhiwuxue Yanjiu 2007, 25, 89e92. 10. Guo, Z.; Zou, K.; Wang, J.; Liu, C.; Tang, Z.; Yang, C. Magn. Reson. Chem. 2009, 47, 613e616. 11. Iwagoe, K.; Konishi, T.; Kiyosawa, S. Yakugaku Zasshi 1987, 107, 140e149. 12. Yang, M.-H.; Blunden, G.; Patel, A.; Crabb, T. A.; Griffin, W. J. Phytochemistry 1989, 28, 3171e3173. 13. Wu, G. X.; Wei, X. Y.; Chen, W. X. Chin. Chem. Lett. 2005, 16, 911e914. 14. Yang, Q. X.; Zhang, Y. J.; Li, H. Z.; Yang, C. R. Steroids 2005, 70, 732e737. 15. Hong, X. X.; Luo, J. G.; Guo, C.; Kong, L. Y. Carbohydr. Res. 2012, 361, 19e26. 16. Wang, Q.; Hou, Q. N.; Guo, Z. Y.; Zou, K.; Xue, Y. H.; Fluang, N. Y.; Cheng, F.; Zhou, Y. Nat. Prod. Res. 2013, 27, 85e92. 17. Eddy, C. R.; Wall, M. E.; Scott, M. K. Anal. Chem. 1953, 25, 266e271. 18. Agrawal, P. K.; Jain, D. C.; Gupta, R. K.; Thakur, R. S. Phytochemistry 1985, 24, 2479e2496. 19. Takeda, K.; Minato, H.; Shimaoka, A.; Nagasaki, T. J. Chem. Soc., Perkin Trans. 1972, 957e962. 20. Septisetyani, E. P.; Ningrum, R. A.; Romadhani, Y.; Wisnuwardhani, P. H.; Santoso, A. Indones. J. Pharm. 2014, 25, 245e254. 21. Chen, H.; Ou, W.; Wang, G.; Wang, N.; Zhang, L.; Yao, X. Molecules 2010, 15, 4589e4598.
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Furospirostanol and spirostanol saponins from the rhizome of Tupistra chinensis and their cytotoxic and anti-inflammatory activities Limin Xiang a, Yihai Wang b, Xiaomin Yi b, Jianying Feng b, Xiangjiu He a, b, * a b
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 September 2015 Received in revised form 31 October 2015 Accepted 9 November 2015 Available online xxx
Five new furospirostanol saponins (1e5), four new spirostanol saponins (6e9), along with one known spirostanol saponin were isolated from the rhizome of Tupistra chinensis. The chemical structures were determined by spectroscopic and chemical methods, including IR, NMR, MS, and GC analyses. The antiproliferative effects against five human cancer cell lines were assayed for all the isolated compounds. Compounds 6e10 showed potent cytotoxic activities against the five cancer cell lines. The isolated compounds were evaluated the inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in a macrophage cell line RAW 264.7. Compounds 6e10 showed significant inhibition on NO production with IC50 values between 3.1 and 4.4 mM. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: Tupistra chinensis Furospirostanol saponins Spirostanol saponins Cytotoxicity NO inhibitory activity
1. Introduction The genus Tupistra has 12 species in southwestern China, possessing similar morphologic characteristics, and some can be substituted for each other as a folk medicine for the treatment of pharyngolaryngitis, rheumatic diseases and snake-bite.1 Previous phytochemical investigations on this genus have led to the isolation of a series of structurally diverse compounds, including steroidal sapogenins,2 saponins,3 cardenolides4 and flavans.5,6 These components exhibit diverse biological activities, such as anti-inflammatory,7 cytotoxicity8 and anti-fungal properties.9 Tupistra chinensis is distributed in Southwest China, and its dried rhizome is a reputed folk medicine to reduce carbuncles and ameliorate pharyngitis in the Shennongjia Forest District of Hubei Province in China.10 Steroidal saponins were believed to be the main active ingredients in this plant. As part of our research project to explore more diversity bioactive leading compounds from the medicinal herbs, we investigated the 60% EtOH extract of the dried rhizome of T. chinensis and obtained five new furospirostanes (1e5), four new steroid saponins (6e9), along with one known spirostanol saponin. Herein we report the isolation and structural elucidation of these compounds and their antiproliferative activity
* Corresponding author. Tel./fax: þ86 20 3935 2132; e-mail address: hexiangjiu@ 163.com (X. He).
against five human cancer cell lines, as well as the inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in a macrophage cell line RAW 264.7.
2. Results and discussion 2.1. Structural elucidation The 60% ethanol extract of the dried underground parts of T. chinensis was successively chromatographed on D101 macroporous resin, silica gel, ODS, and finally purified by semi-preparative HPLC to afford nine new steroidal saponins, along with one known compound. The structures of all the isolated compounds (1e10) (Fig. 1) were elucidated on the basis of spectroscopic data and chemical methods, including IR, NMR, MS, and GC analyses. The known compound was identified as 5b-spirost-25(27)-en-1b,3bdiol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-a1 13 L-rhamnopyranoside (10) by comparison of the H and C NMR data with those previously reported.11 Compound 1 was isolated as white amorphous powder, [a]29 D 50 (c 0.50, MeOH). The molecular formula was inferred as C39H64O16 according to the positive-ion HRESIMS peak at m/z 788.4294 [MþH]þ (calcd for C39H65O16, 788.4273). The IR spectrum showed strong absorption bands at 3400 cm1, ascribable to hydroxyl functionalities. The 1H NMR of 1 showed singlets at dH
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2
L. Xiang et al. / Tetrahedron xxx (2015) 1e8
Fig. 1. Structures of isolated compounds 1e10.
0.81 and 1.51 which had been assigned to the C18- and C19-tertiary methyl groups, respectively. Instead of the two doublets of the secondary methyl groups (C21 and C27), a doublet at dH 1.08 and a singlet at dH 1.39 were observed. Signals from a primary alcohol group at dH 3.89 (1H, d, J¼10.0 Hz, H-26) and 4.17 (1H, d, J¼10.0 Hz, H-26), and two methane protons indicative of secondary alcoholic functions at dH 4.14 (1H, brs, H-1) and 4.61 (1H, brs, H-3) were observed. The 13C NMR spectrum exhibited characteristic furospirostanol carbon signal at C-22 (dC 120.5) and C-25 (dC 84.2), diagnostic of C-26 hydroxylated F-ring ‘furanose’ sapogenin. The corresponding values for F-ring ‘pyranose’ sapogenin with C-25 hydroxylation were dC 109.0 and 67.0.12 The NMR spectrum of 1 showed two anomeric proton signals at dH 4.95 (1H, d, J¼7.8 Hz) and 5.00 (1H, d, J¼7.8 Hz), corresponding to two anomeric carbons at dC 105.8 and 103.2, respectively, in the HSQC spectrum, indicated the presence of two sugar moieties. The sugars were identified as D-glucose by GC analysis of their chiral derivatives after acid hydrolysis. The b anomeric configuration of the glucose was determined by its coupling constants (J¼7.8 Hz). From the results of 1H and 13C NMR (Table 1), it suggested that 1 was a furospirostanol glucoside. Compared the NMR data with those of convallagenin A 3-O-b-D-glucopyranoside,13 the NMR features of those two compounds were almost same except for the signals of the F-ring. The NMR spectroscopic data attributed to the F-ring of 1 were consistent with those reported in the literatures.14,15 Thus compound 1 was inferred as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-Ob-D-glucopyranoside from above information. These were further verified by 1He1H COSY, HSQC, HMBC and NOESY spectra. The 1 He1H COSY spectrum showed that two methylene protons at dH 2.02 (H-2a) and 2.55 (H-2b) were coupled to dH 4.14 (1H, brs, H-1) and 4.61 (1H, brs, H-3), and the oxymethine proton dH 4.61 (H-3) was coupled to two methylene protons at dH 2.20 (H-4b) and 2.33 (H-4a). These findings supported the location of the hydroxyl groups at C-1, C-3, C-5, together with the long-range correlations observed in the HMBC spectrum (Fig. 2). The linkage of the sugar
residues were further confirmed from the following HMBC correlations: H-1 (dH 5.00) of glucose with C-3 (dC 75.7) of the aglycon, and H-1 (dH 4.95) of glucose’ with C-26 (dC 77.8) of the aglycon. The stereochemistry of the ring junctions and substituents of the aglycon of 1 were determined via cross-peaks observed in a NOESY spectrum. The key correlations between H4a and H-7a/H-9a, and between H-2a/H-9a, supported the A/B cis ring junction pattern. Thus, the hydroxyl group at C-5 has a borientation. Additional NOE correlations between H-1a and Me-19/ H-11, and between H-3a and H-2a/H-4a, were indicative of b-orientations for OH-1 and OH-3. Consequently, the structure of 1 was deduced to be 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-O-b-D-glucopyranoside. Compound 2 was isolated as white amorphous powder. The molecular formula of C39H64O15 was determined by the positiveion HRESIMS peak at m/z 773.4389 [MþH]þ (calcd for C39H65O15, 773.4323). The IR spectrum showed strong absorption bands at 3423 cm1, ascribable to hydroxyl functionalities. Unambiguous complete assignments for the 1H and 13C NMR signals were made by combination of DEPT, 1He1H COSY, HSQC, HMBC, and NOESY spectra. Compared to 1, the NMR signals indicated the loss of a hydroxyl at C-5 in 2, which was supported by HMBC correlations of Me-19 (dH 1.21) with C-5 (dC 31.3). Thus, compound 2 was elucidated as 26-O-b-D-glucopyranosyl-(22S, 25S)-5b-furostan-22,25epoxy-1b, 3b, 26-triol-3-O-b-D-glucopyranoside. Compound 3 was isolated as white amorphous powder, and positive-ion HRESIMS provided an ion at m/z 795.4164 [MþNa]þ, which corresponded to a molecular formula of C39H64O15 as same as 2. The 1H and 13C NMR data of 3 were similar to those of 2 with differences apparent only in the chemical shifts at positions 2, 3, 4 and 5 in the A ring. The NOESY spectrum (Fig. 3) indicated correlation between H-3b and H-2b, with no evidence of any correlation between H-3b and H-2a/H-4a, suggesting that the hydroxyl group located at C-3 of the aglycon of 3 was a-orientation rather than borientation of 2. On the basis of the above spectroscopic evidence, the structure of 3 was deduced to be 26-O-b-D-glucopyranosyl-
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
3
Table 1 NMR spectroscopic data for compounds 1e5 (pyridine-d5) Position
1
2
dC
o
dH (J in Hz)
3
dC
dH (J in Hz)
4
dC
dH (J in Hz)
5
dC
dH (J in Hz)
dC
dH (J in Hz) 2.35, m 1.28, m 1.70, m 1.55, m 4.22o 2.13o
1
73.3
4.14, brs
72.7
3.87o
72.8
4.13, brs
75.4
4.22, brs
25.7
2
33.3
32.3
2.71, 1.97, 5.18, 2.50, 2.31,
d (12.7) m m m m
28.6
5 6
75.5 36.9
7
29.1
8 9 10 11 12
35.1 45.7 44.2 21.8 40.2
2.57, d (12.7) 1.88, m 4.87, m 2.15, m 1.88, m 2.14, m 1.72, m 1.20o 1.20o 0.95, m 1.54, m 1.32, m
35.2
75.7 35.6
2.32o 1.82o 4.59, brs 1.87o
35.3
3 4
2.55, d (13.7) 2.02o 4.61, brs 2.33, m 2.20o
1.90, 1.47, 1.31, 0.85, 1.55, 1.23,
m d (13.0) m m dd (11.1, 3.8) m
13 14 15
41.0 56.3 32.4
16 17 18 19 20 21 22 23 24
81.2 62.8 16.8 13.8 38.9 15.4 120.5 33.4 34.2
25 26
84.2 77.8
27
24.7
1.95o 1.46 1.37o 0.91o 1.58o 1.08o 1.33o 1.64o 0.93o 0.91o 1.94o 1.32o 4.75, dd (15.2, 7.2) 1.76, dd (8.4, 6.9) 0.81, s 1.51, s 2.18o 1.08, d (6.9) 2.02o 2.21o 1.63o 4.17, d (10.0) 3.89, d (10.0) 1.39, s
75.1 29.6 31.3 26.8 26.7 36.0 42.4 40.7 21.4 40.5 41.0 56.5 32.5 81.3 63.0 16.9 19.4 38.9 15.5 120.6 33.4 34.3 84.2 77.8 24.7
2.34o 1.69o 1.15o 1.15o 0.92o 1.50o 1.12o 1.23o 1.62o 0.97o 0.96o 1.95o 1.31o 4.75, dd (15.2, 7.2) 1.79o 0.79, s 1.21, s 2.16o 1.08, d (6.9) 2.01o 2.18o 1.62o 4.16, d (10.0) 3.87o 1.37o
74.8 34.8 36.2 27.3 26.8 36.0 42.5 39.9 21.2 40.5 41.0 56.5 32.5 81.3 63.1 16.9 19.2 38.9 15.5 120.6 33.4 34.2 84.2 77.9 24.7
72.5 41.5 77.2 36.6 29.0 35.3 45.7 43.2 21.6 40.2
1.30, m 1.63, m 0.94, m 0.95, 1.98, 1.33, 4.83, 1.83, 0.82, 1.25, 2.18, 1.11,
41.1 56.3 32.5
m m m dd (14.6, 7.4) m s s m d (6.9)
81.3 62.8 16.8 13.6 39.0 15.5 120.6 33.4 34.3
2.00o 2.20, m 1.64, m
84.3 77.9
4.18, d (10.0) 3.92, d (10.0) 1.40, s
24.7
67.1 35.5
1.27, m 1.62, m 0.88, m 0.83, 1.95, 1.34, 4.83, 1.82, 0.82, 1.42, 2.19, 1.12,
m m m dd (15.2, 7.1) dd (8.5, 6.7) s s m d (6.9)
2.04, m 2.19, m 1.64, m
81.4 31.6
2.04, m 1.79, m 1.45, m 0.77, m 1.50, m 1.15o
29.5 34.5 43.2 41.5 21.5 40.3
1.35, m 1.69, m 1.06, m
41.1 56.5 32.5
0.96, m 1.96, m 1.35, m 4.81, dd (14.9, 7.1) 1.83o 0.81, s 1.22, s 2.22, m 1.14, d (6.9)
81.3 62.9 16.7 17.9 39.0 15.5 120.9 33.3 30.0
2.08, m 2.21, m 2.10, m
87.0 74.8
4.19, d (9.9) 3.93, d (10.0) 1.40, s
65.1
3-O-Glc 1 2 3 4 5 6
103.2 75.8 79.1 71.6 78.7 62.9
5.00, d (7.8) 3.96o 4.26o 4.25o 3.96o 4.55, m 4.40, m
101.7 75.4 79.1 71.9 79.0 63.0
4.98, d (7.8) 3.96o 4.25o 4.24o 3.94o 4.54, m 4.37, m
103.0 75.8 78.9 71.9 78.7 63.0
5.02, 4.07, 4.26, 4.27, 3.85, 4.50, 4.40,
d (7.7) t (8.2) m m ddd (9.3, 5.0, 2.5) dd (11.8, 2.3) m
103.2 75.8 78.9 71.8 78.8 63.0
5.09, d (7.8) 4.09o 4.27o 4.33, t (9.2) 3.85, ddd (9.5, 4.6, 2.6) 4.44o 4.40o
26-O-Glc 1 2 3 4 5 6
105.8 75.6 78.6 71.9 78.8 63.0
4.95, d (7.8) 4.03o 4.26o 4.25o 3.96o 4.55, m 4.40, m
105.8 75.7 78.8 71.9 78.9 63.1
4.93, d (7.7) 4.03, m 4.25o 4.20o 3.98o 4.54, m 4.37, m
105.8 75.7 78.6 71.9 78.9 63.1
4.97, 4.07, 4.26, 4.27, 3.96, 4.55, 4.40,
d (7.8) t (8.2) m m m dd (11.8, 2.2) m
105.8 75.7 78.7 72.0 78.9 63.0
4.98, d (7.8) 4.06o 4.27o 4.27o 3.96, m 4.55, d (10.3) 4.40o
5-O-Glc 97.6 75.7 78.9 71.9 79.2 63.3
105.9 75.5 78.7 72.1 78.9 62.9
4.45, 4.24, 4.16, 3.96,
d d d d
(10.2) (10.2) (10.9) (10.9)
5.14, 3.94, 4.24, 4.09, 4.03, 4.55, 4.25,
d (7.8) m m m m m m
5.04, 4.03, 4.24, 4.24, 3.96, 4.55, 4.38,
d (7.8) m m m m m m
Overlapped with other signals.
HMBC COSY
NOESY
O OH
O HO
HO HO HO
O
O
H
HO HO
OH
O
H H
O OH
H
H
H H H
H
H H
H H
RO A
Fig. 2. Selected 1He1H COSY (A), HMBC (A) and NOESY (B) correlations of 1.
HO
CH3
B
Fig. 3. Selected 1He1H COSY (A), HMBC (A) and NOESY (B) correlations of 3.
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(22S, 25S)-5b-furostan-22,25-epoxy-1b, 3a, 26-triol-3-O-b-Dglucopyranoside. Compound 4 was obtained as white amorphous powder, with the same molecular formula of C39H64O16 as 1 on the basis of HRESIMS (m/z 789.4258 [MþH]þ). The close similarity of the 13C shifts of 4 and 1 (Table 1), with the exception of C-1 (DdC 2.1), C-2 (DdC 1.9), C-3 (DdC 3.2), C-4 (DdC 5.9) and C-5 (DdC 1.7) of the aglycon, showed identical BeF ring substitution in the two compounds. The C-1, C-2, C-3, C-4 and C-5 differences in shifts indicated the a-orientation of the hydroxyl group at C-3. This was confirmed by the absence of any NOESY correlation between H-3b and H-2a/H4a. Therefore, the structure of 4 was identified as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3a, 5b, 26-tetrol-3O-b-D-glucopyranoside. Compound 5 was isolated as white amorphous powder. The HRESIMS of 5 showed the ion at m/z 789.4323 [MþH]þ, and its molecular formula was inferred as C39H64O16 on the basis of the analysis of 1H and 13C NMR and DEPT spectra. The IR spectrum showed strong absorption band at 3423 cm1, ascribable to hydroxyl functionalities. Acid hydrolysis and GC analysis of 5 gave Dglucose. The 1H NMR spectrum showed three typical steroidal methyl signals at dH 1.22 (3H, s, Me-19), 1.14 (3H, d, J¼6.9 Hz, Me21) and 0.81 (3H, s, Me-18). The lost signal of Me-27 was suggested some change on the substituent of C-25. The 13C NMR spectrum exhibited the C-22 carbon of the aglycon at dC 120.9, indicated a furanose F ring. On comparison of the 1H and 13C NMR spectra of 5 with those of 1e4, the NMR signals indicated the loss of methyl group at C-25 were replaced by an oxygenated primary alcohol group [dH 4.16 (1H, d, J¼10.9 Hz), 3.96 (1H, d, J¼10.9 Hz), dC 65.1] in 5. This was further evidenced by HMBC correlations of H-27 (dH 3.96, 4.16) with C-24 (dC 30.0), C-25 (dC 87.0), C-26 (dC 74.8). Thus, the aglycon of 5 was identified as a rare dihydroxylated F-ring furospirostane. Compared the NMR data with those of reinocarnoside B,16 the NMR features of those two compounds were almost same except for the signals of the F-ring, which was suggested the same AeE ring substitution in the two compounds. Hence, the structure of 5 was elucidated as 26-O-b-D-glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-3b, 5b, 26, 27-tetrol-5-Ob-D-glucopyranoside. Compound 6 was isolated as white amorphous powder. The HRESIMS of 6 showed the ion at m/z 857.4954 [MþH]þ, and its molecular formula was inferred as C44H72O16 on the basis of the analysis of 1H and 13C NMR and DEPT spectra. The IR spectrum showed the characteristic absorptions of hydroxyl group at 3421 cm1 and the four steroidal sapogenins absorption bands 981, 912, 899, 865 cm1. And the 899 band was stronger than the 912
band. It was suggested that 6 was a sapogenin with ‘iso’ configuration of the F ring.17 On acid hydrolysis, 6 liberated D-xylose and Lrhamnose, identified by GC analysis of their chiral derivatives, which were consistent with three anomeric protons at dH 6.44 (1H, d, J¼0.9 Hz), 5.42 (1H, s), and 5.08 (1H, d, J¼6.4 Hz) in the 1H NMR spectrum. The 1H NMR spectrum showed four typical steroidal methyls, including two angular methyl group at dH 1.31, 0.84, and two secondary methyl groups at dH 1.11 (1H, d, J¼6.9 Hz), 0.67 (1H, d, J¼5.3 Hz). In the 13C NMR spectrum, a typical quaternary carbon signal of spirostanol at dC 109.6 (C-22), and three anomeric carbon signals at dC 100.0, 100.3, and 102.0 were observed. Compared the 13 C NMR data of aglycon of 6 with those of isorhodeasapogenin,18 the downfield shifts of C-1 (þ5.8 ppm) and C-3 (þ2.9 ppm) indicated that 6 was a bidesmosidic saponin with glycosidic linkages at C-1 and C-3, respectively. The linkages of the sugar residues were further confirmed from the following HMBC correlations (Fig. 4): H1 (dH 6.44) of rhamnose with C-2 (dC 77.4) of xylose, H-1 (dH 5.08) of xylose with C-1 (dC 79.2) of the aglycon, and H-1 (dH 5.42) of rhamnose with C-3 (dC 71.1) of the aglycon. From above spectroscopic evidence, the structure of 6 was deduced to be (25R)-5bspirostan-1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside.
Fig. 4. Selected 1He1H COSY and HMBC correlations of 6.
Compound 7 was isolated as white amorphous powder, with the same molecular formula of C44H72O16 as 6 on the basis of HRESIMS (m/z 857.4948 [MþH]þ). The close similarity of the 13C shifts of 7 and 6 (Table 2), with the exception of F-ring of the aglycon, suggested that the configuration of C-25 of the aglycon was different. It was further evidenced by the IR spectrum: the bands occur at 984, 917, 897, 849 cm1, with the 917 band stronger than the 897 band.17 Thus the configuration of C-25 of the aglycon of 7 was S-form. Therefore, the structure of 7 was elucidated as (25S)-5b-spirostan1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido3-O-a-L-rhamnopyranoside.
Table 2 NMR spectroscopic data for compounds 6 and 7 (pyridine-d5) Position
6
7
dC 1 2 3 4
79.2 31.3 71.1 31.8
5 6
35.5 26.7
7
27.7
8 9 10 11
34.5 46.8 39.9 23.1
dH (J in Hz) 4.04, t (5.9) 2.27, m 4.17, m 1.79o 1.56o 2.00, m 1.54o 1.27o 1.37o 0.92o 1.51o 1.28o 2.01o 1.46o
6
dC 79.0 31.0 71.0 31.8 35.5 26.7 27.8 34.6 46.7 39.9 23.1
7
dH (J in Hz)
Position
4.04, m 2.25, m 4.15, m 1.78, m 1.55o 2.00o 1.55o 1.29o 1.38o 0.91o 1.49o 1.26o
23
32.2
1.66o
26.7
24
29.6
1.55o
26.5
25 26
30.9 67.2
o
27.7 65.4
27 1-O-Xyl 1 2 3 4 5
17.7
1.98o 1.44o
dC
100.3 77.4 79.7 71.7 67.3
dH (J in Hz)
1.55 3.57, m 3.48, m 0.67, d (5.3) 5.08, d (6.4) 4.26o 4.27o 4.14, m 4.32, m 3.66, m
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dC
16.6 100.2 77.4 79.6 71.7 67.3
dH (J in Hz) 1.90o 1.48o 2.11o 1.38o 1.55o 4.05, m 3.34, d (11.0) 1.03, d (7.0) 5.08, d (6.8) 4.26o 4.27o 4.14, m 4.32, m 3.65, t (10.6)
L. Xiang et al. / Tetrahedron xxx (2015) 1e8
5
Table 2 (continued ) Position
6
7
dC
o
12
40.8
13 14 15
40.9 57.3 32.6
16 17 18 19 20 21 22
81.5 63.4 17.0 17.4 42.3 15.4 109.6
dH (J in Hz) 1.69o 1.18o 1.07o 2.02o 1.42, m 4.55, dd (14.9, 7.3) 1.80, m 0.84, s 1.31, s 1.95, dt (13.9, 7.0) 1.11, d (6.9)
6
dC 40.8 40.9 57.3 32.5 81.5 63.2 17.0 17.4 42.7 15.2 110.1
dH (J in Hz)
Position
1.67o 1.15o
Rha(1/2) 1 2 3 4 5 6 3-O-Rha 1 2 3 4 5 6
1.03o 1.99o 1.41, m 4.51, dd (14.9, 7.3) 1.78, m 0.82, s 1.29, s 1.87, m 1.10, d (6.9)
7
dC
dH (J in Hz)
dC
dH (J in Hz)
102.0 72.8 72.7 74.4 69.8 19.4
6.44, d (0.9) 4.84, m 4.66o 4.32, m 4.78, dq (12.4, 6.1) 1.75, d (6.2)
102.0 72.8 72.7 74.4 69.8 19.4
6.42, s 4.84, m 4.66o 4.32, m 4.77, dq (12.3, 6.1) 1.72, d (6.2)
100.0 73.1 72.8 74.6 70.3 19.0
5.42, s 4.66o 4.64o 4.27,o 4.37, dd (10.8, 4.6) 1.68, d (6.2)
100.0 73.0 72.7 74.5 70.3 19.0
5.41o 4.66o 4.64o 4.27,o 4.37, m 1.66, d (6.1)
Overlapped with other signals.
Compound 8 was isolated as white amorphous powder. The molecular formula of C44H70O17 was determined by the positiveion HRESIMS peak at m/z 895.4703 [MþNa]þ (calcd for C44H72O17Na, 895.4667). The IR spectrum showed the characteristic absorptions of hydroxyl group at 3420 cm1 and the four steroidal sapogenins absorption bands 982, 917, 899, 863 cm1. And the 899 band was stronger than the 917 band. It was suggested that 8 was a sapogenin with ‘iso’ configuration of the F ring.17 On acid hydrolysis, 8 liberated D-xylose, L-rhamnose, and D-glucose, identified by GC analysis of their chiral derivatives. From a comparison of 1H and 13C NMR data of 8 (Table 3) with those of 6, it was apparent that 8 contained the same aglycon as 6, except for a little different in the saccharide chains. The linkage of the sugar units was established from the following HMBC correlations (Fig. 5): H-1 (dH 6.39) of
Fig. 5. Selected 1He1H COSY and HMBC correlations of 8.
Table 3 NMR spectroscopic data for compounds 8 and 9 (pyridine-d5) Position
8
9
dC
o
1 2
79.0 30.6
3 4
73.1 31.6
5 6o
35.1 26.9
7
27.4
8 9 10 11
35.1 46.6 39.9 22.7
12
40.8
13 14 15
40.9 57.2 32.5
16 17 18 19 20 21 22 23
81.4 63.4 17.0 17.5 42.3 15.4 109.6 32.1
dH (J in Hz) 3.91, dd (8.8, 2.9) 2.29, m 2.15o 4.35o 1.83o 1.72o 1.94, m 1.47o 1.19o 1.30o 0.90, m 1.47o 1.20o 2.01o 1.41o 1.69o 1.17o 1.07o 2.02o 1.41o 4.55, m 1.81o 0.84, s 1.24, s 1.93, m 1.11, d (7.0) 1.65o
8
dC 79.1 30.7 73.1 31.6 35.1 27.0 27.5 35.1 46.7 39.9 22.8 40.8 40.9 57.2 32.5 81.8 63.4 17.0 17.5 42.2 15.3 109.8 33.5
9
dH (J in Hz)
Position
3.90, dd (8.9, 2.7) 2.30, m 2.17o 4.35o 1.84o 1.70o 1.94, m 1.46o 1.20o 1.30, m 0.90, m 1.47o 1.21o
24
29.6
1.55o
25 26
30.9 67.2
27
17.6
1.55o 3.56, d (3.0) 3.49, t (10.6) 0.67, d (5.6)
2.06o 1.43o 1.68o 1.17o
Rha(1/2) 1 2 3 4 5 6 3-O-Glc 1 2 3 4 5 6
1.06o 2.01o 1.41o 4.53, m 1.80o 0.84, s 1.24, s 1.94, m 1.06, d (6.9)
1-O-Xyl 1 2 3 4 5
dC
dH (J in Hz)
dC 29.3 144.8 65.3 109.0
dH (J in Hz) 2.70, m 2.23o 4.45, 4.02, 4.80, 4.77,
d (12.0) d (12.0) s s
100.0 76.7 78.4 71.5 66.8
4.97, d (6.6) 4.31o 4.18, m 4.12o 4.34o 3.61, m
100.0 76.7 78.5 71.5 66.8
4.95, d (6.6) 4.31o 4.18, m 4.13o 4.34o 3.60, m
101.7 72.6 72.9 74.4 70.0 19.4
6.39, 4.78, 4.66, 4.34, 4.74, 1.76,
101.7 72.7 72.9 74.4 70.0 19.4
6.41, s 4.79o 4.67, dd (9.3, 3.3) 4.35, m 4.77o 1.76, d (6.1)
103.1 75.3 78.8 72.1 78.9 63.2
4.92, d (7.7) 4.13o 4.28, m 4.24o 3.97, m 4.56, m 4.36, m
103.1 75.3 78.8 72.1 78.9 63.2
4.93, d (7.7) 4.13o 4.28, m 4.26o 3.97, m 4.58, dd (11.9, 2.2) 4.37o
d (1.1) dd (3.3, 1.5) dd (9.3, 3.3) m dd (10.8, 4.7) d (6.2)
1.80o 1.76o
Overlapped with other signals.
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
rhamnose with C-2 (dC 76.7) of xylose, H-1 (dH 4.97) of xylose with C-1 (dC 79.0) of the aglycon, and H-1 (dH 4.92) of glucose with C-3 (dC 73.1) of the aglycon. Thus, compound 8 was elucidated as (25R)5b-spirostan-1b, 3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-Dxylopyranosido-3-O-b-D-glucopyranoside. Compound 9 was isolated as white amorphous powder, and positive-ion HRESIMS provided an ion at m/z 893.4498 [MþNa]þ, which corresponded to a molecular formula of C44H70O17. The 1H and 13C NMR data of 9 were similar to those of 8 with differences apparent only in the chemical shifts at positions 23, 24, 25, 26 and 27 in the F ring of the aglycon. The signals due to the methyl of C-27 in 8 were replaced by the signals assigned to the C-25(27) exomethylene group at dH 4.77 (1H, s) and 4.80 (1H, s), and dC 144.8 (C-25) and 109.0 (C-27) in 9. Hence, the structure of 9 was elucidated as 5b-spirost-25(27)-en-1b,3b-diol-1-O-a-L-rhamnopyranosyl-(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside. Two biosynthetic routes to the relatively rare 3a-OH sapogenin (compounds 3, 4) were therefore considered possible. The first involves oxidation of the 3b-OH followed by reduction to given the 3aOH derivative. The second route oxidation of the 5b-spirost-2-en.19
exhibited moderate inhibition with IC50 values of 23.3 mM. Indomethacin was selected as a positive control (IC50 47.4 mM). Cell viability was determined by the MTT method to find whether inhibition of NO production was due to the cytotoxicity of the compounds 5e10. These six compounds exhibited no cytotoxicity against RAW 264.7 macrophage cells at their effective concentrations. 3. Conclusion Five new furospirostanol and four new spirostanol saponins, along with one known spirostanol saponin were isolated from the rhizome of T. chinensis. The chemical structures were determined by spectroscopic and chemical methods. Compounds 1e5 were uncommon polyhydroxylated furospirostanol glycosides and compounds 6e9 were unusual spirostanol saponins with glycosidation both at C-1 and C-3 of the aglycon. Some compounds showed potent cytotoxic activities against the five cancer cell lines and significant inhibition on NO production. 4. Experimental section
2.2. Cytotoxicity of isolated compounds 4.1. General experimental procedures Compounds 1e10 were evaluated for cytotoxicity against five human tumor cell lines including HepG2 (human hepatocellular carcinoma), CNE-1 (high differentiation human nasopharyngeal carcinoma), CNE-2 (low differentiation human nasopharyngeal carcinoma), K562 (human chronic leukemia), SPC-A-1 (human lung adenocarcinoma) with a modified MTT method according to reported protocols,20 and cis-dichlorodiamineplatinum (II) was used as a positive control. Compounds 7 and 9 exhibited significant cytotoxic effects against all of the human cancer cell lines tested with IC50 values smaller than 12 mM (Table 4). Compounds 6 and 8 exhibited moderate cytotoxic effects against all of the human cancer cell lines tested. Compound 10 showed inhibitory activities against four out of five human cancer cell lines tested except K562. The other compounds exhibited IC50 values larger than 50.0 mM against the five tested cell lines and were considered to be inactive. Table 4 Cytotoxicities of compounds 1e10 to human cancer cell lines Compound
6 7 8 9 10 CISPb
IC50 (mM)a HepG2
CNE-1
CNE-2
K562
SPC-A-1
18.10.1 6.10.5 8.50.3 6.10.2 7.50.1 6.50.3
16.91.0 8.30.2 16.80.7 8.41.0 15.00.4 10.80.4
14.82.1 9.10.1 3.40.3 10.61.7 13.50.4 8.01.1
15.00.5 10.30.9 17.50.3 11.60.5 >50 13.50.8
12.10.8 8.30.1 10.00.3 8.70.6 11.00.4 3.80.3
Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were obtained on a PerkinElmer 100 IR spectrometer with KBr. NMR spectra were recorded on Ultrashield 500 Plus instrument and were recorded in pyridine-d5. Chemical shifts (d) are stated in ppm from the internal standard, tetramethylsilane (TMS). HRESIMS were measured on a Waters AQUITY UPLC/Q-TOF micro spectrometer. Semi-preparative HPLC was performed on a RAININ pump equipped with a Gilson 133 refractive index detector and a COSMOSIL packed column (5C18-AR-Ⅱ, 10ID250 mm). D101 macroporus resin (Xi’an Lanxiao Resin Corporation Ltd., Xi’an, China), Silica gel (200e300 mesh, Anhui Liangchen Silicon Material Co. Ltd., Lu’an, China) and ODS (40e60 mm, Merck KGaA, Darastadt, Germany) were used for column chromatography. HPLC-grade methanol was purchased from Oceanpak Chemical Co. (Gothenburg, Sweden). All solvents used for column chromatography were of analytical grade (Shanghai Chemical Reagents Company, Ltd., Shanghai, China). Sugar reagents (Sigma, St. Louis, MO, USA) were used for GC analysis. 4.2. Plant material
Results are presented as means SD (n¼3). Positive control. Compounds 1e5 with IC50 values >50.0 mM for all the tested cell lines.
The rhizomes of T. chinensis Baker was purchased from Shennongjia Forest District (Shennongjia, China), and identified by Prof. Xiangjiu He, the School of Pharmacy, Guangdong Pharmaceutical University. A voucher specimen (No. GDPU-NPR-2013002) was deposited in the Department of Medicinal Chemistry, Guangdong Pharmaceutical University, Guangzhou, China.
2.3. Inhibitory effects on NO production
4.3. Extraction and isolation
Compounds 1e10 were tested for their inhibitory effects on NO production induced by LPS in a macrophage cell line RAW 264.7. Compounds 6e10 showed significant inhibition on NO production with IC50 values between 3.1 and 4.4 mM (Table 5). Compound 5
The air-dried underground parts of T. chinensis (17.0 Kg) were extracted four times with 60% EtOH (70 L) for 4 h at reflux. The combined EtOH extracts were evaporated to 20 L, and partitioned between H2O and EtOAc to give an EtOAc fraction (218.0 g). The
a
b
Table 5 Inhibitory effects of compounds 1e10 on NO production induced by LPS in macrophagesa Compoundb
5
6
7
8
9
10
Indomethacina
IC50 (mM)
23.35.1
3.10.3
3.20.03
4.40.3
3.20.9
3.10.02
47.44.5
a b
Positive control. Compounds 1e4 with IC50 >50.0 mM.
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L. Xiang et al. / Tetrahedron xxx (2015) 1e8
aqueous fraction was applied to a D101 macroporous resin column, eluted with H2O (50 L), 20% EtOH (50 L), 60% EtOH (50 L), 80% EtOH (50 L) to give four fractions (AeD). A part of fraction B (100.0 g) was subjected to silica gel column chromatography eluted with a gradient of CHCl3/MeOH (20:1 to 1:1) to yield 22 fractions (B1eB22). A half part of fraction B18 (6.5 g) was separated over a column of ODS silica gel eluted with a gradient of MeOHeH2O (1:9 to 3:7) to afford six fractions (B18-1 to B186). Subfraction B18-4 (2.44 g) was further purified by semipreparative HPLC (3.0 mL/min, 50% MeOH in H2O isocratic elution) to yield compound 1 (109.0 mg). One-fifth of fraction B19 (5.0 g) was separated by an ODS column eluted with a gradient of MeOHeH2O (1:9 to 2:8) to afford seven fractions (B19-1 to B19-7). Subfraction B19-3 (875.8 mg) was further purified by semipreparative HPLC (2.7 mL/min, 45% MeOH in H2O isocratic elution) to yield compound 5 (23.1 mg). Subfraction B19-4 (849.5 mg) was further separated by semi-preparative HPLC (2.7 mL/min, 50% MeOH in H2O isocratic elution) to yield compound 4 (18.4 mg). A half part of fraction C (200.0 g) was subjected to silica gel column chromatography eluted with a gradient of CHCl3/MeOH (20:1 to 2:1, followed by MeOH) to yield 18 fractions (C1eC18). A half part of fraction C14 (5.2 g) was further separated by ODS eluted with a gradient of MeOH/H2O (7:3 to 2:8) to afford nine fractions (C14-1 to C14-9). Subfraction C14-4 was further purified by semipreparative HPLC (2.8 mL/min, 60% MeOH in H2O isocratic elution) to yield compound 3 (40.0 mg). Fraction D (83.3 g) was separated by silica gel column chromatography eluted with a gradient of CHCl3/MeOH (100:1 to 1:1) to obtain 21 fractions (D1eD21). Fraction D18 (11.4 g) was subjected to ODS to obtain seventeen fractions (D18-1 to D18-17). Subfraction D18-9 (1.2 g) was further purified by semi preparative HPLC (3.0 mL/min, 65% MeOH in H2O isocratic elution) to yield compound 2 (32.3 mg). Fraction D19 (3.6 g) was purified by ODS silica gel to obtain eight fractions (D19-1 to D19-8). Subfraction D19-7 was further purified by semi preparative HPLC to yield compound 6. (23.9 mg), 7 (10.7 mg), and 10 (27.4 mg). Fraction D 20 (5.0 g) was subjected to ODS to obtain ten fractions (D 20-1 to D 20-10). Subfraction D 20-8 (612.9 mg) was further purified by semi preparative HPLC to yield compound 8 (58.4 mg) and 9 (58.7 mg). 4.3.1. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy-1b, 3b, 5b, 26-tetrol-3-O-b-D-glucopyranoside (1). White amorphous powder; [a]29 D 50 (c 0.50, MeOH); IR (KBr) nmax 3394, 2918, 1636, 1456, 1094 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 788.4294 [MþH]þ (calcd for C39H65O16, 788.4273). 4.3.2. 26-O- b - D -Glucopyranosyl-(22S, 25S)-5 b -furostan-22,25epoxy-1b, 3b, 26-triol-3-O-b-D-glucopyranoside (2). White amorphous powder; [a]29 D 62 (c 0.50, MeOH); IR (KBr) nmax 3423, 2926, 1635, 1452, 1046 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 773.4389 [MþH]þ (calcd for C39H65O15, 773.4323). 4.3.3. 26-O- b - D -Glucopyranosyl-(22S, 25S)-5 b -furostan-22,25epoxy-1b, 3a, 26-triol-3-O-b-D-glucopyranoside (3). White amorphous powder; [a]29 D þ9 (c 0.50, MeOH); IR (KBr) nmax 3400, 2918, 1637, 1452, 1036 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 795.4164 [MþNa]þ (calcd for C39H64O15Na, 795.4143). 4.3.4. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy1b, 3a, 5b, 26-tetro L-3-O-b-D-glucopyranoside (4). White amorphous powder; [a]27 D 40 (c 0.25, MeOH); IR (KBr) nmax 3406, 2931, 1637, 1452, 1036 cm1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 789.4258 [MþH]þ (calcd for C39H65O16, 789.4273). 4.3.5. 26-O-b-D-Glucopyranosyl-(22S, 25S)-furostan-22,25-epoxy3b, 5b, 26, 27-tetrol-5-O-b-D-glucopyranoside (5). White
7
amorphous powder; [a]28 D 32 (c 0.25, MeOH); IR (KBr) nmax 3423, 2928, 1634, 1453, 1046 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 789.4323 [MþH]þ (calcd for C39H65O16, 789.4273). 4.3.6. (25R)-5 b -Spirostan-1 b ,3 b -diol-1-O- a -L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside (6). White amorphous powder; [a]29 D 105 (c 0.5, MeOH); IR (KBr) nmax 3421, 2929, 1642, 1452, 1046, 981, 912, 899, 865 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [MþH]þ 857.4954 (calcd for C44H73O16 [MþH]þ, 857.4899). 4.3.7. (25S)-5 b -Spirostan-1 b ,3 b -diol-1-O- a- L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-a-L-rhamnopyranoside (7). White amorphous powder; [a]29 D 99 (c 0.5, MeOH); IR (KBr) nmax 3423, 2933, 1642, 1451, 1047, 984, 917, 897, 849 cm1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [MþH]þ 857.4948(calcd for C44H73O16 [MþH]þ, 857.4899). 4.3.8. (25R)-5 b -Spirostan-1 b ,3 b -diol-1-O- a -L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside (8). White amorphous powder; [a]29 D 86 (c 0.5, MeOH); IR (KBr) nmax 3420, 2928, 1645, 1453, 1049, 982, 917, 899, 863 cm1; 1H and 13C NMR data, see Table 3; HRESIMS m/z [MþNa]þ 895.4703(calcd for C44H72O17Na [MþNa]þ, 895.4667). 4.3.9. 5b-Spirost-25(27)-en-1b,3b-diol-1-O-a-L -rhamnopyranosyl(1/2)-b-D-xylopyranosido-3-O-b-D-glucopyranoside (9). White amorphous powder; [a]29 D 94 (c 0.5, MeOH); IR (KBr) nmax 3411, 2928, 1650, 1452, 1045, 983, 918, 897, 838 cm1; 1H and 13C NMR data, see Table 3; HRESIMS m/z [MþNa]þ 893.4498 (calcd for C44H70O17Na [MþNa]þ, 893.4511). 4.4. Acid hydrolysis and GC analysis The hydrolysis and GC analysis of the chiral derivatives of the sugars of new compounds were done as previously described.21 Compounds 1e9 (1e2 mg) were heated in an ampoule with 5 mL 2 M HCl at 90 C for 2e8 h. The aglycon was extracted with EtOAc three times and the aqueous residue was evaporated under reduced pressure at 60 C. Then 600 mL pyridine and 5 mg NH2OH$HCl were added to the residue and the mixtures were heated at 90 C for 30 min. After cooling, 300 mL Ac2O was added to the mixtures. After homogenized, the mixtures were heated at 90 C for 1 h. After cooling, the reaction mixtures were analyzed by GC using standard aldononitrile peracetates as reference samples. 4.5. Cytotoxicity assays Compounds 1e10 were evaluated for cytotoxicity against five tumor cell lines including HepG2, CNE-1, CNE-2, K562, SPC-A-1 with a modified MTT method according to reported protocols in triplicate independent experiments. cis-Dichlorodiamineplatinum (II) was used as a positive control. 4.6. NO production assay The NO production’s inhibition, and viability assay were done as previously described. All experiments were performed in three independent replicates. Indomethacin was selected as a positive control. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 81573303), the Natural Science Foundation of Guangdong province (No. 2014A030313588), and the
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starting fund of Guangdong Pharmaceutical University. The authors were also great acknowledgments to Prof. Dr. Hao Gao, Jinan University, for his generous help in the measurement of optical rotation. Supplementary data Supplementary data (The original spectra of new compounds 1e9, including 1H, 13C NMR, 2D-NMR (HSQC, HMBC, 1He1H COSY, NOESY), HRESIMS, and IR, were given as supplementary data.) associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2015.11.012. References and notes 1. Y. W. Z.; Hua, X.; Cao, B.; Yao, G. Shanghai Publishing House of Science and Techniques, 1991. 2. Pan, W. B.; Chang, F. R.; Wu, Y. C. J. Nat. Prod. 2000, 63, 861e863. 3. Liu, C. X.; Guo, Z. Y.; Xue, Y. H.; Cheng, J.; Huang, N. Y.; Zhou, Y.; Cheng, F.; Zou, K. Fitoterapia 2012, 83, 323e328. 4. Pan, Z. H.; Li, Y.; Liu, J. L.; Ning, D. S.; Li, D. P.; Wu, X. D.; Wen, Y. X. Fitoterapia 2012, 83, 1489e1493.
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