Triterpenoid glycosides from Ladenbergia hexandra Klotzsch

Article abstract of DOI:10.1016/j.phytochem.2017.01.014

From the bark of Ladenbergia hexandra Klotzsch, ten triterpenoid glycosides were isolated along with five known compounds, and their structures were determined based on extensive NMR and mass spectroscopic, GC and HPLC analyses. Some triterpenoid glycosides contained 6-deoxy-D-allose or D-allose as a sugar moiety. The absolute stereochemical assignment of the sugars was determined by comparison with synthetic samples, as well as by GC and HPLC analysis.

Full text of DOI:10.1016/j.phytochem.2017.01.014

Phytochemistry xxx (2017) 1e9
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Triterpenoid glycosides from Ladenbergia hexandra Klotzsch
Megumi Furukawa, Satoshi Kamo, Mitsuko Makino, Masahiro Kurita, Keiichi Tabata,
Keiichi Matsuzaki, Takashi Suzuki, Taketo Uchiyama*
School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
From the bark of Ladenbergia hexandra Klotzsch, ten triterpenoid glycosides were isolated along with five
known compounds, and their structures were determined based on extensive NMR and mass spectro-
scopic, GC and HPLC analyses. Some triterpenoid glycosides contained 6-deoxy-D-allose or D-allose as a
sugar moiety. The absolute stereochemical assignment of the sugars was determined by comparison with
synthetic samples, as well as by GC and HPLC analysis.
Received 30 August 2016
Received in revised form
21 January 2017
Accepted 21 January 2017
Available online xxx
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Ladenbergia hexandra
Rubiaceae
Triterpene glycoside
b
-D-allose
6-Deoxy-
-D-quinovose
b-D-allose
b
Glycosyl iodide
1. Introduction
2. Results and discussion
Ladenbergia hexandra Klotzsch (Rubiaceae) is distributed in the
mountain forests of eastern Brazil where it is commonly called
“quina-vermelha.” Its plant height can be from 6 to 10 m and the
diameter of the stem can be up to 45 cm. Its bark and roots have
been used as a folk medicine by decocting in sthenic, stomachic,
antipyretic and antimalarial tonics in Brazil. The genus Ladenbergia
includes about 30 species, distributed in tropical America
(Hashimoto, 1996). Although alkaloidal constituents of the genus
Ladenbergia plant have been reported (Holker et al., 1964; Okunade
et al., 2001), there are currently no reports of triterpenoid
glycosides.
2.1. Structural elucidation
Investigation of the bark of L. hexandra, extracted with EtOH,
afforded fifteen tritenpenoid glycosides (1-15) (Fig. 1). The struc-
tures of the known compounds were determined to be quinovic
acid 3-O-
novic acid 3-O-
(12) (Zhao et al., 1995), quinovic acid 3-O-
27-O- -D-glucopyranoside (13) (Elgamal et al., 1995), cincholic acid
3-O- -D-quinovopyranoside (14) (Sanchez and Vazquez 1991), and
cincholic acid 3-O- -D-6-deoxyglucopyranosyl-28-O- -D-gluco-
b
-D-quinovopyranoside (11) (Elgamal et al., 1995), qui-
-D-glucopyranosyl-28-O- -D-glucopyranoside
-D-quinovopyranosyl-
b
b
b
b
b
b
b
In the present paper, the isolation and structural elucidation of
ten new triterpenoid glycosides are reported by spectroscopic
means. In addition, the facile preparation of D-quinovose and 6-
deoxy-D-allose from D-glucose and D-allose was established,
respectively, which allowed determination of the absolute config-
uration of the unique 6-deoxy sugar moieties in the isolated gly-
cosides by gas chromatography (GC) and high-performance liquid
chromatography (HPLC).
pyranoside (15) (Arriaga et al., 1990), respectively, by comparing
their spectroscopic data with those reported in the literature.
Compounds 1-10 were isolated as white amorphous powders.
Acid hydrolysis of 1-4, 5-6 and 7-10 yielded the aglycones identified
as quinovic acid, cincholic acid (Cheng et al., 2004) and urs-
12,20(30)-diene-27,28-dioic acid (Bila et al., 1991) by comparison of
their 1D NMR spectra with those in the literature. The mono-
saccharides obtained by acid hydrolysis of 1-10 were also identified
by comparison on TLC with authentic samples as 6-deoxy-allose for
1, 3, 5, 6, 8; allose for 2, 4, 10; glucose for 3, 4, 6, 9, 10 and quinovose
for 7, 9. The absolute configurations of the sugars were determined
to be D by gas chromatographic (GC) analysis of its trimethylsilyl
* Corresponding author.
E-mail address: uchiyama.taketo@nihon-u.ac.jp (T. Uchiyama).
http://dx.doi.org/10.1016/j.phytochem.2017.01.014
0031-9422/© 2017 Elsevier Ltd. All rights reserved.
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2
M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
Fig. 1. Structures of compounds 1-15.
thiazolidine derivatives (Hara et al., 1987) and HPLC analysis using
an optical rotation detector (Matsuzaki et al., 2010).
H-6;
d
1.63, 3H, d, J ¼ 6.0 Hz). In addition, from the COSY correlation
of sugar moiety, the HMBC of H-10 for 6-deoxy-D-allose and C-3,
and the NOESY correlation of H-10 and H-3, it was deduced that the
sugar was linked at C-3 of the aglycone (Fig. 2). Thus, structure 1
The molecular formula of compound 1 was established as
C
36H56O9, based on the negative-ion HR FAB-MS data [MꢀH]ꢀ at m/
z 631.3844 (calcd. for C36H55O9, 631.3846), suggesting that 1 was
quinovic acid 6-deoxy-D-alloside. The 1H and 13C NMR spectra of
the sugar moiety of 1 were consistent with data for a “rare” 6-
deoxy-allopyranoside moiety (Zuo et al., 2013) (Tables 1 and 3).
was determined to be quinovic acid
allopyranoside.
The negative-ion HR FAB-MS data of compound 2 (m/z 647.3793
[MꢀH]ꢀ calcd. for C36H55O10, 647.3795) supported a molecular
formula of C36H56O10, suggesting that 2 was quinovic acid D-allo-
side. Its 1H and 13C NMR spectra were similar to 1, except for the
3b-O-6-deoxy-b-D-
The 6-deoxyhexose unit was identified as 6-deoxy-
side by the spin-spin coupling constants (H-1;
J ¼ 8.0 Hz, H-2; 3.92, 1H, dd, J ¼ 8.0, 3.0 Hz, H-3;
J ¼ 3.0, 3.0 Hz, H-4; 4.67, 1H, dd, J ¼ 9.5, 3.0 Hz, H-5;
b
d
-allopyrano-
5.11, 1H, d,
4.61, 1H, dd,
d
d
presence of a hydroxylmethyl group (
rather than the methyl group (
d
4.45(2H, m) and
d
63.3)
19.1)
d
d
4.32, 1H, m,
d
1.63 (3H, d, J ¼ 6.0 Hz) and
d
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M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
3
Table 1
1H NMR chemical shifts for compounds 1-5 (in pyridine-d5, 500 MHz,
d
).
1
2
3
4
5
3
3.16 (1H, dd, 11.7, 4.5)
6.00 (1H, brs)
2.80 (1H, d, 11.5)
1.10 (3H, s)
0.96 (3H, s)
0.88 (3H, s)
1.10 (3H, s)
1.23 (3H, d, 6.0)
0.81 (3H, d, 6.0)
3.16 (1H, dd, 11.7, 4.1)
5.98 (1H, brs)
2.78 (1H, d, 11.5)
1.08 (3H, s)
0.96 (3H, s)
0.85 (3H, s)
1.08 (3H, s)
1.22 (3H, d, 6.0)
0.81 (3H, d, 6.0)
3.16 (1H, dd, 11.7, 4.3)
5.99 (1H, brs)
2.68 (1H, d, 11.5)
1.08 (3H, s)
0.95 (3H, s)
0.92 (3H, s)
1.22 (3H, s)
1.17 (3H, d, 6.0)
0.76 (3H, d, 6.0)
3.17 (1H, dd, 11.7, 4.2)
5.98 (1H, brs)
2.68 (1H, d, 11.0)
1.07 (3H, s)
0.95 (3H, s)
0.89 (3H, s)
1.21 (3H, s)
1.17 (3H, d, 6.0)
0.76 (3H, d, 6.0)
3.10 (1H, dd, 11.7, 4.2)
6.00 (1H, brs)
3.32 (1H, d, 10.0)
1.10 (3H, s)
0.92 (3H, s)
0.88 (3H, s)
1.21 (3H, s)
0.73 (3H, s)
0.87 (3H, s)
12
18
23
24
25
26
29
30
3-O-glycosyl moiety
6d-allo
5.11 (1H, d, 8.0)
3.92 (1H, dd, 8.0, 3.0)
4.61 (1H, dd, 3.0, 3.0)
3.64 (1H, dd, 9.5, 3.0)
4.32 (1H, m)
allo
6d-allo
allo
6d-allo
10
20
30
40
50
60
5.19 (1H, d, 8.0)
3.92 (1H, dd, 8.0, 2.5)
4.40 (1H, m)
4.67 (1H, brs)
4.16 (1H, dd, 9.5, 2.5)
4.45 (2H, m)
5.10 (1H, d, 8.0)
3.93 (1H, dd, 8.0, 3.0)
4.59 (1H, dd, 3.0, 3.0)
3.63 (1H, dd, 9.5, 3.0)
4.30 (1H, m)
5.19 (1H, d, 8.0)
3.93 (1H, brd, 8.0)
4.44e4.56a (1H)
4.65 (1H, brs)
4.18 (1H, brd, 9.5)
4.44e4.56a (2H)
5.08 (1H, d, 8.0)
3.90 (1H, dd, 8.0, 3.0)
4.59 (1H, brs)
3.62 (1H, dd, 9.0, 3.0)
4.27 (1H, m)
1.63 (3H, d, 6.0)
1.63 (3H, d, 6.0)
1.58 (3H, d, 6.0)
27-O-glycosyl moiety
glc
glc
100
200
300
400
500
600
6.36 (1H, d, 8.0)
4.22 (1H, dd, 9.0, 8.0)
4.28 (1H, t, 9.0)
4.35 (1H, t, 9.0)
4.04 (1H, m)
6.34 (1H,d, 8.0)
4.22 (1H, 9.0, 8.0)
4.30 (1H, t, 9.0)
4.35 (1H, t, 9.0)
4.04 (1H, m)
4.44e4.56a (2H)
4.41 (1H, dd, 12.5, 6.0)
4.52 (1H, dd, 12.5, 3.0)
The values in parentheses represent the coupling constants in Hz. The
d values are in ppm downfield from TMS.
6d-all: 6-deoxy-allopyranosyl, all: allopyranosyl, quino; quinovopyranosyl, glc; glucopyranosyl.
a
Overrapped signals.
(Tables 1 and 3). Careful analysis of the COSY, HMBC and NOESY
spectra indicated that the sugar was linked at C-3 of quinovic acid
2D NMR spectra enabled determination of the position of glyco-
sylation at C-3 with allose (anomeric-H;
5.19, 1H, d, J ¼ 8.0 Hz,
anomeric-C; 104.2) and C-27 with glucose (anomeric-H; 6.34,
1H, d, J ¼ 8.0 Hz, anomeric-C; 95.6). Thus, structure 4 was quinovic
acid 3 -O- -D-allopyranosyl-27-O- -D-glucopyranoside.
d
as an O-
structure of
b
-glycoside (anomeric-H;
d
5.19, 1H, d, J ¼ 8.0 Hz). The
d
d
2
was assigned as quinovic acid
3
b-O-
b-D-
d
allopyranoside.
b
b
b
From negative-ion HR FAB-MS of compound 3,
molecular ion peak at m/z 793.4373 (calcd for
a
quasi-
The negative-ion HR FAB-MS of compound 5 showed a quasi-
molecular ion peak at m/z 631.3841 [MꢀH]ꢀ (calcd. for C36H55O9,
631.3846) ascribable to the molecular formula C36H56O9, suggesting
that 5 was cincholic acid 6-deoxy-D-alloside. Its 1H and 13C NMR
spectra were similar to 1, except for those of an aglycone unit
(Tables 1 and 3). Detailed NMR studies allowed identification that
C
42H65O14,
793.4373) [MꢀH]ꢀ was observed, this being consistent with the
molecular formula C42H66O14, suggesting that 3 was quinovic acid
having 6-deoxy-D-allose and D-glucose as sugar moieties. Its 1H
and 13C NMR spectra were similar to 1, except for an additional
sugar moiety (Tables 1 and 3). Two anomeric proton signals at
(J ¼ 8.0 Hz) and 6.36 (J ¼ 8.0 Hz) indicated the presence of two
monosaccharides, a glycoside ( 5.10) and the other as a glycosyl
ester (
6.36) at C-27 or C-28. In the 13C NMR spectrum of 3, the
d
5.10
the sugar (anomeric-H;
104.1) was linked at C-3 of the aglycone. The structure of 5 was
determined to be cincholic acid 3 -O-6-deoxy- -D-allopyranoside.
The molecular formula of compound 6 was established as
C42H66O14, based on the negative-ion HR FAB-MS data at m/z
d
5.08, 1H, d, J ¼ 8.0 Hz, anomeric-C;
d
d
d
b
b
d
resonance for C-12 was shifted downfield (Dd þ0.6) and the signal
of C-13 was shifted upfield (Dd ꢀ0.9), as compared to quinovic acid.
793.4377 [MꢀH]ꢀ (calcd. for C42H65O14, 793.4373), suggesting that
6 was cincholic acid having 6-deoxy-D-allose and D-glucose as
sugar moieties. Its 1H and 13C NMR spectra were similar to 5, except
for an additional sugar moiety, and these were also similar to 3,
except for the aglycone unit (Tables 2 and 3). In addition, the COSY
correlation of sugar moiety, the cross-peaks in the HMBC spectra
[corresponding to long-range coupling between H-10 for 6-
deoxyallose and C-3; H-3 and C-10 for 6-deoxyallose; H-18 and C-
28; H-100 for glucose and C-28], as well as the NOESY correlation of
H-10 and H-3 enabled determination of the position of glycosylation
The chemical shifts of the C-12 (d 129.6) and C-13 (d 133.2) reso-
nances were in agreement with glycosylation at C-27 (Elgamal
et al., 1995). In addition, the COSY correlation of the sugar moiety,
the cross-peaks in the HMBC spectra [corresponding to long-range
coupling between H-10 for 6-deoxyallose and C-3; H-3 and C-10 for
6-deoxyallose; H-18 and C-28; and H-100 for glucose and C-27], as
well as the NOESY correlation of H-10 and H-3, enabled us to
determine the position of glycosylation at C-3 with 6-deoxyallose
and C-27 with glucose, respectively (Fig. 2). On the basis of the
above results, the structure of 3 was provided to be quinovic acid
to be at C-3 to 6-deoxyallose (anomeric-H;
anomeric-C; 104.0) and C-28 to glucose (anomeric-H;
d, J ¼ 8.0 Hz, anomeric-C;
95.7) (Fig. 2). Furthermore, in the 13C
NMR spectrum of 6, it was observed that the C-28 esterified
d
5.08, 1H, d, J ¼ 8.0 Hz,
3
b
-O-6-deoxy-
b
-D-allopyranosyl-27-O-
b
-D-glucopyranoside.
d
d
6.39, 1H,
Compound 4 was shown to have a molecular formula was
d
established as C42H66O15 on the basis of the negative-ion HR FAB-
MS data [MꢀH]ꢀ at m/z 809.4338 (calcd. for C42H65O15
,
carboxyl group appeared at
d
176.5, shifted upfield (glycosylation
809.4323), suggesting that 4 was quinovic acid having a D-allose
and a D-glucose. Its 1H and 13C NMR spectra were similar to 2,
except for an additional sugar moiety and these were also similar to
those of 3 except for the sugar moiety (Tables 1 and 3). Analysis of
shift) in respect to the C-28 (
d
180.8) in the aglycone. According to
the above results, the structure of 6 was formulated as cincholic
acid 3 -O- -D-6-deoxy-allopyranosyl-28-O- -D-glucopyranoside.
Compounds 7 and 8 were shown to have the same molecular
b
b
b
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M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
Fig. 2. Key COSY, HMBC and NOESY of 1, 3, 6 and 9.
formula. These were established as C36H54O9, on the basis of the
negative-ion HR FAB-MS data [MꢀH]ꢀ at m/z 629.3696 and
629.3686 (calcd. for C36H53O9, 629.3689), suggesting that 7 and 8
were, respectively, urs-12,20(30)-diene-27,28-dioic acid D-quino-
voside and urs-12,20(30)-diene-27,28-dioic acid 6-deoxy-D-allo-
side. Their 1H and 13C NMR spectra were similar to those of 11
expect for those of the aglycone unit (Tables 2 and 3). Careful
analysis of the COSY, HMBC and NOESY spectra enabled determi-
nation of the position of glycosylation at C-3 with quinovose
(anomeric-H;
6-deoxy-D-allopyranose (anomeric-H;
anomeric-C; 104.1) for 8. Consequently, the structures of 7 and 8
were characterized as urs-12,20(30)-diene-27,28-dioic acid 3 -O-
-D-quinovopyranoside and urs-12,20(30)-diene-27,28-dioic acid
d
4.68,1H, d, J ¼ 7.5 Hz, anomeric-C;
d 106.6) for 7 and
5.10, 1H, d, J ¼ 8.0 Hz,
d
d
b
b
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M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
5
Table 2
1H NMR chemical shifts for compounds 6-10 (in pyridine-d5, 500 MHz,
d
).
6
7
8
9
10
3
3.11 (1H, dd, 11.7, 4.4)
6.02 (1H, brs)
3.35 (1H, brd, 10.2)
1.10 (3H, s)
0.95 (3H, s)
0.92 (3H, s)
1.21 (3H, s)
0.73 (3H, s)
0.87 (3H, s)
3.20 (1H, dd, 11.4, 4.1)
6.00 (1H, brs)
2.94 (1H, d, 11.7)
1.16 (3H, s)
0.96 (3H, s)
0.89 (3H, s)
1.11 (3H, s)
1.45 (3H, d, 5.9)
4.76 (1H, s)
3.16 (1H, dd, 11.7, 4.1)
6.00 (1H, brs)
2.95 (1H, d, 11.7)
1.10 (3H, s)
0.96 (3H, s)
0.89 (3H, s)
1.10 (3H, s)
1.45 (3H, d, 6.2)
4.76 (1H, s)
3.19 (1H, dd, 11.4, 4.4)
5.99 (1H, brs)
2.83 (1H, d, 11.7)
1.15 (3H, s)
0.95 (3H, s)
0.92 (3H, s)
1.22 (3H, s)
1.39 (3H, d, 6.2)
4.68 (1H, s)
3.19 (1H, dd, 11.4, 4.4)
5.98 (1H, brs)
2.83 (1H, d, 11.5)
1.07 (3H, s)
0.96 (3H, s)
0.89 (3H, s)
1.21 (3H, s)
1.39 (3H, d, 6.3)
4.69 (1H, s)
12
18
23
24
25
26
29
30
4.79 (1H, s)
4.78 (1H, s)
4.73 (1H, s)
4.73 (1H, s)
3-O-glycosyl moiety
6d-allo
5.08 (1H, d, 8.0)
3.90 (1H, dd, 8.0, 3.0)
4.59 (1H, brs)
3.62 (1H, dd, 9.0, 3.0)
4.27 (1H, m)
quino
6d-all
quino
all
10
20
30
40
50
60
4.68 (1H, d, 7.5)
3.97 (1H, dd, 8.5, 8.0)
4.10 (1H, dd, 8.5, 8.5)
3.73 (1H, dd, 8.5, 8.5)
3.77 (1H, m)
5.10 (1H, d, 8.0)
3.91 (1H, dd, 8.0, 3.0)
4.59 (1H, brs)
3.63 (1H, dd, 8.0, 3.0)
4.29 (1H, m)
4.67 (1H, d, 7.5)
4.00 (1H, dd, 8.5, 8.0)
4.10 (1H, dd, 8.5, 8.5)
3.76 (1H, dd, 8.5, 8.5)
3.77 (1H, m)
5.21 (1H, d, 8.0)
3.92 (1H, dd, 8.0, 2.5)
4.31e4.46a (1H)
4.66 (1H, brs)
4.16 (1H, dd, 9.4, 2.6)
1.58 (3H, d, 6.0)
1.66 (3H, d, 6.0)
1.62 (3H, d, 6.0)
1.66 (3H, d, 6.0)
4.31e4.46a (2H)
28-O-glycosyl moiety
glc
glc
glc
100
200
300
400
500
600
6.39 (1H, d, 8.0)
4.22 (1H, dd, 9.0, 8.0)
4.29 (1H, t, 9.0)
4.35 (1H, t, 9.0)
4.06 (1H, m)
6.35 (1H, d, 8.0)
4.33 (1H, dd, 9.0, 8.0)
4.38(1H, t, 9.0)
4.21 (1H, t, 9.0.0)
4.08 (1H, m)
6.38 (1H, d, 8.0)
4.33 (1H, dd, 9.0, 8.0)
4.40a (1H)
4.21 (1H, t, 9.0)
4.05 (1H, m)
4.31e4.46a (2H)
4.42 (1H, dd, 15.0, 5.5)
4.49 (1H, dd, 15.0, 3.0)
4.50 (1H, dd, 11.5, 2.0)
4.57 (1H, dd, 11.5, 4.0)
The values in parentheses represent the coupling constants in Hz. The
d values are in ppm downfield from TMS.
6d-all: 6-deoxy-allopyranosyl, all: allopyranosyl, quino; quinovopyranosyl, glc; glucopyranosyl.
a
Overrapped signals.
3
b-O-6-deoxy-D-allopyranoside, respectively.
D-allopyranosyl-28-O-b-D-glucopyranoside.
The negative-ion HR FAB-MS spectrum of compound 9 (m/z
791.4215 [MꢀH]ꢀ, calcd. for C42H63O14, 791.4217) supported a
molecular formula of C42H64O14, suggesting that 9 was urs-
12,20(30)-diene-27,28-dioic acid having a D-quinovose and a D-
glucose as its sugar units. Its 1H and 13C NMR spectra were similar
to 7, except for an additional sugar moiety, and these were also
similar to 15 except for the aglycone unit (Tables 2 and 3). In
addition, the COSY correlation of sugar moiety, the cross-peaks in
the HMBC spectra [corresponding to long-range coupling between
H-10 for quinovose and C-3; H-3 and C-1'; H-18 and C-28; and H-100
for glucose and C-28], as well as the NOESY correlation of H-10 and
H-3 enabled determination of the position of glycosylation at C-3
2.2. Synthesis of 6-deoxy sugars 22 (quinovose) and 23 (6-deoxy-
allose)
To determine the absolute configuration of the 6-deoxy sugar
moieties in the new glycosides by GC and HPLC analysis, the 6-
deoxy sugars needed for standard samples were synthesized.
Although there have been a few reports on the synthesis of 6-deoxy
sugars (Durrwacher et al.,1986; Garegg et al.,1987; Kobayashi et al.,
1992), a new synthetic route using glycosyl iodide (Uchiyama and
Hindsgaul, 1996; Uchiyama et al., 2016) was developed for the
preparation (Fig. 3). D-quinovose and 6-deoxy-D-allose were pre-
pared in 32% yield from D-glucose and 16% yield from D-allose,
respectively. These synthetic 6-deoxy sugars were used for GC and
HPLC analysis of standard samples.
with quinovose (anomeric-H;
d
d
4.67, 1H, d, J ¼ 7.5 Hz, anomeric-C;
106.7) and C-28 with glucose (anomeric-H; 6.35, 1H, d,
95.8), respectively (Fig. 2). Furthermore, it
d
J ¼ 8.0 Hz, anomeric-C;
d
was deduced that the C-28 esterified carboxyl group, which
appeared at
180.2) in the aglycone. Accordingly, the structure of 9 was eluci-
dated as urs-12,20(30)-diene-27,28-dioic acid 3 -O- -D-quinovo-
pyranosyl-28-O- -D-glucopyranoside.
d 175.9, was shifted upfield in respect to the C-28 (d
2.3. Cytotoxic activity of compounds
b
b
b
Neuroblastoma is among the most fatal of solid tumors in pe-
diatric age groups, even when treated aggressively. Therefore, an
effective therapeutic drug for neuroblastoma is urgently needed,
including the need for continuing examining the cytotoxic and
mitogen/invasion-inhibitory effects of compounds against neuro-
blastoma cells (Tabata et al., 2015). Many triterpenoid glycosides
show cytotoxic activity (Horo et al., 2015; Li et al., 2015), and qui-
novic acid and its glycosides show cytotoxic activity (Tapondjou
et al., 2002).
The negative-ion HR FAB-MS spectra of compound 10 gave a
quasi-molecular ion peak at m/z 807.4182 ([MꢀH]ꢀ, calcd. for
C
C
42H63O15, 807.4167) corresponding to the molecular formula of
42H64O15, suggesting that 10 was urs-12,20(30)-diene-27,28-dioic
acid having a D-quinovose and a D-glucose as sugar units. Its 1H and
13C NMR spectra were similar to 4, except for the aglycone unit and
these were also found to be almost identical to those of 9 except for
the sugar moiety (Tables 2 and 3). Careful analysis of the 2D NMR
spectra enabled determination of the position of glycosylation at C-
All isolated compounds were tested for cytotoxic activity against
the tumor cell line IMR-32 (human neuroblastoma). However, all
3 with allose (anomeric-H;
d
d
5.21, 1H, d, J ¼ 8.0 Hz, anomeric-C;
104.3) and C-28 with glucose (anomeric-H; 6.38, 1H, d,
95.8), respectively. Therefore, structure of
-O-b-
d
tested samples were less active (IC50 > 50
11.6 M) and doxorubicin (IC50 2.01 M), which were used as
positive controls.
mM) than cisplatin (IC50
J ¼ 8.0 Hz, anomeric-C;
d
m
m
10 was established as urs-12,20(30)-diene-27,28-dioic acid 3
b
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http://dx.doi.org/10.1016/j.phytochem.2017.01.014

Table 3
13C NMR chemical shifts for compounds 1-10 (in pyridine-d5, 125 MHz,
d).
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
39.6
27.1
88.6
39.6
56.0
18.9
37.8
40.3
47.4
37.2
23.6
128.9
134.0
57.0
22.5
26.7
49.0
55.1
38.0
39.3
30.8
37.4
28.3
17.4
16.9
19.2
177.8
180.0
18.6
21.6
39.5
27.0
88.9
39.5
55.9
18.8
37.7
40.2
47.3
37.2
23.6
128.9
133.9
56.9
25.8
26.6
49.0
55.1
37.9
39.2
30.8
37.3
28.3
17.5
16.8
19.2
177.8
180.0
18.6
21.6
39.1
26.8
88.5
39.4
55.9
18.6
37.5
40.2
47.3
37.0
23.4
129.6
133.2
56.7
25.5
26.1
48.9
54.7
37.5
39.1
30.3
36.4
28.0
17.1
16.6
19.2
176.5
178.0
18.1
21.2
39.0
26.7
88.7
39.3
55.8
18.5
37.5
40.2
47.2
37.0
23.3
129.5
133.2
56.7
25.5
26.1
48.9
54.7
37.5
39.0
30.3
36.4
28.1
17.1
16.6
19.1
176.4
178.0
18.1
21.2
39.2
27.1
88.5
39.6
56.1
18.9
37.6
40.1
47.7
37.4
23.8
125.9
138.0
56.8
25.6
25.3
48.0
44.5
44.3
31.2
34.4
33.1
28.3
17.5
16.8
19.1
178.3
180.0
33.5
24.1
39.0
26.8
88.4
39.4
55.9
18.6
37.7
40.1
47.6
37.1
23.5
126.5
137.4
56.6
25.3
24.8
48.0
44.1
43.8
30.7
33.9
32.2
28.1
17.1
16.5
19.0
178.5
176.5
33.1
23.7
39.2
27.0
88.5
39.6
55.9
18.8
37.6
40.3
47.3
37.2
23.6
129.4
133.6
56.9
25.7
26.7
49.0
57.2
36.5
153.4
32.6
39.6
28.2
17.3
16.7
19.1
177.7
179.2
16.9
105.9
39.3
27.1
88.5
39.6
56.0
18.9
37.7
40.4
47.3
37.3
23.7
129.4
133.6
56.9
25.8
26.8
49.1
57.3
36.5
153.5
32.8
39.6
28.3
17.4
16.8
19.1
177.7
179.3
17.0
105.9
39.4
27.1
88.6
39.7
56.0
18.9
37.7
40.5
47.4
37.3
23.7
130.1
132.8
56.9
25.8
26.6
49.2
57.0
36.3
153.1
32.4
38.9
28.3
17.4
16.9
19.5
178.0
175.9
16.8
106.2
39.0
26.7
88.8
39.4
55.8
18.6
37.5
40.3
47.1
37.0
23.5
130.1
133.0
56.8
25.5
26.4
49.1
56.9
36.1
153.1
32.2
38.7
28.1
17.1
16.6
19.1
178.0
175.9
16.6
106.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-O-glycosyl moiety
6d-all
all
6d-all
104.1
72.8
73.1
74.4
70.0
18.8
all
6d-all
104.1
72.9
73.2
74.5
70.1
19.1
6d-all
104.0
72.8
73.1
74.4
70.0
18.8
quino
106.6
75.9
78.4
76.9
72.7
19.0
6d-allo
104.1
72.9
73.2
74.5
quino
106.7
76.0
78.5
77.0
72.8
19.1
all
10
20
30
40
50
60
104.1
72.9
73.2
74.6
70.1
19.1
104.2
72.8
73.4
69.3
75.6
63.3
104.2
72.7
73.2
69.2
75.5
63.3
104.3
72.8
73.3
69.3
75.6
63.3
70.1
19.1
28-O-glycosyl moiety
glc
glc
glc
glc
glc
100
200
300
400
500
600
95.7
74.1
49.2
71.3
78.9
62.4
95.6
74.1
79.2
71.2
78.8
62.4
95.7
74.2
79.3
71.2
79.0
62.4
95.8
74.2
79.0
71.3
79.4
62.5
95.8
74.1
79.3
71.3
78.9
62.5
The values in parentheses represent the coupling constants in Hz. The
d values are in ppm downfield from TMS.
6d-all: 6-deoxy-allopyranosyl, all: allopyranosyl, quino; quinovopyranosyl, glc; glucopyranosyl.
Fig. 3. Preparation of D-quinovose and 6-deoxy-D-allose from D-glucose and D-allose.
Please cite this article in press as: Furukawa, M., et al., Triterpenoid glycosides from Ladenbergia hexandra Klotzsch, Phytochemistry (2017),
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M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
7
3. Conclusions
CHCl3:MeOH:H2O (6:4:0.5, 1 L) and MeOH (3 L) to give fractions I-
1eI-13. Fraction I-5 (4765 mg) was purified by HPLC (Kaseisorb LC
ODS-PH Super column, 20 ꢁ 250 mm, flow rate 8 ml/min, mobile
phase H2O:MeOH (38:62, v/v)) to give 3 (900 mg, tR ¼ 65 min), 13
(700 mg, tR ¼ 54 min) and fractions II-1eII-7. Fraction II-3 was
purified by HPLC (Kaseisorb LC ODS-PH Super column,
10 ꢁ 250 mm, flow rate 3 ml/min, mobile phase H2O:MeOH (45:55,
v/v)) to give 4 (135 mg, tR ¼ 46 min), 10 (12 mg, tR ¼ 36 min), and 12
(15 mg, tR ¼ 42 min). Fraction II-5 was purified by HPLC (Senshu Pak
PEGASIL ODS column, 10 ꢁ 250 mm, flow rate 4 ml/min, mobile
phase H2O:CH3CN (73:27, v/v)) to give 9 (45 mg, tR ¼ 27 min) and 15
(84 mg, tR ¼ 25 min). Fraction II-6 was purified by HPLC (Senshu
Pak PEGASIL ODS column, 10 ꢁ 250 mm, flow rate 4 ml/min, mobile
phase H2O:CH3CN (70:30, v/v)) to give 6 (69 mg, tR ¼ 26 min). The
MeOH fraction was applied to a silica gel column, that was eluted
successively with CHCl3:MeOH (50:1, 5 L, 30:1, 2 L, 20:1, 4 L, 15:1,
2.5 L, 10:1, 1 L, 5:1, 1 L), CHCl3:MeOH:H2O (6:4:0.5, 1.5 L) and MeOH
(1 L) to give fractions III-1eIII-12. Fraction III-4 (3613 mg) was
purified by HPLC (Shiseido CAPCELL PAK ODS column,
20 ꢁ 250 mm, flow rate 8 ml/min, mobile phase H2O:MeOH (30:70,
v/v)) to give 1 (2600 mg, tR ¼ 43 min), 5 (343 mg, tR ¼ 37 min), and
8 (45 mg, tR ¼ 35 min). Fraction III-5 (3216 mg) was purified by
HPLC (Shiseido CAPCELL PAK ODS column, 20 ꢁ 250 mm, flow rate
8 ml/min, mobile phase H2O:MeOH (30:70, v/v)) to give 11
(1800 mg, tR ¼ 40 min). Fraction III-6 (1358 mg) was purified by
HPLC (Shiseido CAPCELL PAK ODS column, 20 ꢁ 250 mm, flow rate
8 ml/min, mobile phase H2O:MeOH (30:70, v/v)) to give 2 (460 mg,
tR ¼ 25 min), 7 (68 mg, tR ¼ 33 min), and 14 (61 mg, tR ¼ 36 min).
Ten new triterpenoid glycosides, together with five known
compounds, were isolated from an EtOH extract of bark of
L. hexandra Klotzsch. Their structures were elucidated by extensive
spectroscopic examination. This is the first report of triterpene
glycosides in the genus Ladenbergia, which was previously known
to contain alkaloids. Interestingly, some of these new triterpenoid
glycosides contained a rare 6-deoxy-D-allose moiety. To our
knowledge, this is the first example of the presence of a triterpe-
noid 6-deoxy-D-allopyranoside in natural products.
The isolated compounds can be classified into four groups. The
first is composed of triterpenoid glycosides 1, 3, 5, 6 and 8 with a 6-
deoxy-allose moiety at C-3. The second group is composed of tri-
terpenoid glycosides 2, 4 and 10 with an allose moiety at C-3. The
third group is composed of triterpenoid glycosides 7, 9, 11, 13, 14
and 15 with quinovose moiety at C-3. The other group is composed
of the sole triterpenoid glycoside 12 moiety with a glucose at C-3.
Furthermore, a facile preparation of D-quinovose and 6-deoxy-
D-allose from D-glucose and D-allose was established, respectively,
which allowed determination of the absolute configurations of the
unique 6-deoxy sugar moieties in the isolated glycosides by GC and
HPLC analysis.
4. Experimental
4.1. General experimental procedures
1H and 13C NMR spectra were measured on a JEOL GSX-500
spectrometer (1H 500 MHz; 13C, 125 MHz, JEOL Co., Tokyo), a JEOL
GSX-400 spectrometer (1H 400 MHz; 13C,100 MHz, JEOL Co., Tokyo)
or a Varian Mercury 300BB NMR spectrometer (1H, 300 MHz; 13C,
75 MHz, Varian Co., Tokyo) in CDCl3, D2O, MeOH-d4, pyridine-d5
containing TMS as an internal standard. Mass spectra were recor-
ded on a Hitachi RMU-6M instrument. GC was conducted using a
Shimadzu GC-14B chromatograph. Optical rotations were
measured on a JASCO DIP-360 polarimeter. Column chromatog-
raphy (CC) was carried out using either silica gel (Wakogel C-200)
or Diaion HP-20 resin (Nippon Rensui Co., Tokyo). HPLC was con-
ducted with a Spectra Physics SP 8800 pump and a Sensyu SSC-
3160 pump equipped with either an ERC-7520 (ERMA)-RI or Hita-
chi L-400-UV detector. Silica gel 60 F254 (Merck) TLC plates used a
mobile phase of CHCl3:MeOH (4:1), with detection of compounds
by spraying with a 5% H2SO4 solution and heating.
4.3.1. Compound 1 (quinovic acid 3b-O-6-deoxy-b-D-
allopyranoside)
White amorphous powder; [
a
]
þ40.8 (c 2.0, MeOH); HR-FAB-
D
MS m/z 631.3844 [MꢀH]ꢀ (calcd for C36H55O9, 631.3846); FAB-MS
m/z 631 [MꢀH]ꢀ, 587 [MꢀHꢀCO2]ꢀ, 483 [MꢀHꢀ(6d-allꢀH2O)]ꢀ,
439 [MꢀHꢀ CO2ꢀ(6d-allꢀH2O)]ꢀ; For 1H NMR and 13C NMR
spectroscopic data, see Tables 1 and 3
4.3.2. Compound 2 (quinovic acid 3-O-b-D-allopyranoside)
White amorphous powder; [
a
]
þ32.3 (c 1.0, MeOH); HR-FAB-
D
MS m/z 647.3793 [MꢀH]ꢀ (calcd for C36H55O10, 647.3795); FAB-
MS m/z 647 [MꢀH]ꢀ, 603 [MꢀHꢀCO2]ꢀ; For 1H NMR and 13C
NMR spectroscopic data, see Tables 1 and 3
4.3.3. Compound 3 (quinovic acid 3
allopyranosyl-27-O- -D-glucopyranoside)
White amorphous powder; [
b-O-6-deoxy-b-D-
b
4.2. Plant material
a
]
þ28.8 (c 1.0, MeOH); HR-FAB-
D
MS m/z 793.4373 [MꢀH]ꢀ (calcd for C42H65O14, 793.4373); FAB-
MS m/z 793 [MꢀH]ꢀ, 587 [MꢀHꢀCO2ꢀ(glcꢀH2O)]ꢀ, 439
[MꢀHꢀCO2ꢀ(glcꢀH2O)ꢀ(6d-allꢀH2O)]ꢀ; For 1H NMR and 13C NMR
spectroscopic data, see Tables 1 and 3
Ladenbergia hexandra was purchased from and identified by
~
Farmaervas Ltd. in Sao Paulo, Brazil, 1999.
4.3. Extraction and isolation
4.3.4. Compound 4 (quinovic acid 3
b-O-
b
-D-allopyranosyl-27-O-
b-
L. hexandra dried bark (1 kg) was powdered and extracted with
EtOH (4 L ꢁ 4) by ultrasonication at room temperature. The EtOH
extract was then concentrated under reduced pressure to give a
powder (154 g). This was after dissolved it in H2O:MeOH (60:40, v/
v) and applied to a Diaion HP-20 resin (Nippon Rensui Co., Tokyo)
column, which was eluted successively with H2O and stepwise
gradients of H2O:MeOH [60:40 v/v, 3 L; 30:70 v/v, 3 L], MeOH (3 L)
and acetone (4 L). The H2O:MeOH (30:70, v/v) fraction (70 g) was
concentrated in vacuo and CHCl3:MeOH (2:1, 1 L) was added to the
residue to afford a precipitate (40 g) and a supernatant (30 g). The
supernatant was concentrated in vacuo and the residue was sub-
jected to silica gel CC, this being eluted successively with
CHCl3:MeOH (10:1, 2.5 L, 5:1, 3 L, 4:1, 1 L, 3:1, 1 L, 2:1, 2 L)
D-glucopyranoside)
White amorphous powder; [
a
]
þ25.5 (c 1.0, MeOH); HR-FAB-
D
MS m/z 809.4338 [MꢀH]ꢀ (calcd for C42H65O15, 809.4323); FAB-
MS m/z 809 [MꢀH]ꢀ, 603 [MꢀHꢀCO2ꢀ(glcꢀH2O)]ꢀ, 439
[MꢀHꢀCO2ꢀ(glcꢀH2O) ꢀ(allꢀH2O)]ꢀ; For 1H NMR and 13C NMR
spectroscopic data, see Tables 1 and 3
4.3.5. Compound 5 (cincholic acid 3b-O-6-deoxy-b-D-
allopyranoside)
White amorphous powder; [
a]
þ55.8 (c 0.6, MeOH); HR-FAB-
D
MS m/z 631.3841 [MꢀH]ꢀ (calcd for C36H55O9, 631.3846); FAB-MS
m/z 631 [MꢀH]ꢀ, 587 [MꢀHꢀCO2]ꢀ; For 1H NMR and 13C NMR
spectroscopic data, see Tables 1 and 3
Please cite this article in press as: Furukawa, M., et al., Triterpenoid glycosides from Ladenbergia hexandra Klotzsch, Phytochemistry (2017),
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8
M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
4.3.6. Compound 6 (cincholic acid 3
b-O-
b-D-6-deoxy-
concentrated and purified by silica gel CC (CHCl3:MeOH (50:1)) to
allopyranosyl-28-O-
b
-D-glucopyranoside)
afford the benzyl 6-iodo-glycoside (20, 94 mg, 67%; 21, 79 mg, 56%).
The benzyl 6-iodo-glycoside (50 mg, 0.13 mmol) was catalytically
reduced using Pd/C in 50%MeOH in H2O (25 mL) for 8 h, and the
product was purified by silica gel CC (CHCl3:MeOH (20:1)) to afford
the 6-deoxygenated carbohydrate (22, D-quinovose, 16 mg, 75%;
23, 6-deoxy-D-allose, 14 mg, 65%). The structures of quinovose
(Durrwachter et al., 1986) and 6-deoxy-D-allose (Kobayashi et al.,
1992) were determined by comparing spectroscopic data with
those reported in the literature, respectively.
White amorphous powder; [
a]
þ33.2 (c 1.0, MeOH); HR-FAB-
D
MS m/z 793.4377 [MꢀH]ꢀ (calcd for C42H65O14, 793.4373); FAB-
MS m/z 793 [MꢀH]ꢀ, 587 [MꢀHꢀCO2ꢀ(glcꢀH2O)]ꢀ; For 1H NMR
and 13C NMR spectroscopic data, see Tables 2 and 3
4.3.7. Compound 7 (urs-12,20(30)-diene-27,28-dioic acid 3b-O-b-
D-quinovopyranoside)
White amorphous powder; [
a]
D þ101.4 (c 0.45, MeOH); HR-FAB-
MS m/z 629.3696 [MꢀH]ꢀ (calcd for C36H53O9, 629.3689); FAB-MS
m/z 629 [MꢀH]ꢀ, 585 [MꢀHꢀCO2]ꢀ; For 1H NMR and 13C NMR
spectroscopic data, see Tables 2 and 3
16: 1H NMR (300 MHz,
d
, CDCl3); 0.00e0.07 (TMS-O- ꢁ 5, each
12H, s), 3.20e3.31 (2H), 3.53e3.69 (4H), 4.89 (1H, d, J ¼ 2.9 Hz). 13
C
NMR (75 MHz,
d
, CDCl3); 0.00, 0.40, 0.69, 1.18, 1.50 (TMS-O- ꢁ 5),
62.3, 72.2, 72.4, 74.2, 93.8.
4.3.8. Compound 8 (urs-12,20(30)-diene-27,28-dioic acid 3
D-6-deoxy-allopyranoside)
b
-O-
b
-
17: 1H NMR (300 MHz,
d
, CDCl3); 0.00e0.05 (TMS-O- ꢁ 5, each
12H, s), 3.11 (1H, dd, J ¼ 7.3, 2.6 Hz, H-2), 3.38 (1H, dd, J ¼ 9.4, 2.3 Hz,
White amorphous powder; [
a]
þ87.3 (c 1.0, MeOH); HR-FAB-
H-4), 3.44e3.67 (3H, m, H-5, H-6), 3.75 (1H, dd, J ¼ 2.3, 2.3 Hz, H-3),
D
MS m/z 629.3686 [MꢀH]ꢀ (calcd for C36H53O9, 629.3689); FAB-
MS m/z 629 [MꢀH]ꢀ, 587 [MꢀHꢀCO2]ꢀ; For 1H NMR and 13C
NMR spectroscopic data, see Tables 2 and 3
4.71 (1H, d, J ¼ 7.3 Hz, H-1). 13C NMR (75 MHz,
d, CDCl3); 0.00, 0.63,
0.75, 0.92, 1.11 (TMS-O- ꢁ 5), 62.7, 69.3, 74.2, 74.4, 75.8 95.0.
18: FAB-MS m/z 269 [MꢀH]ꢀ; 1H NMR (300 MHz,
d, CD3OD);
3.27e3.43 (3H), 3.59e3.80 (3H), 4.54 (1H, d, J ¼ 11.7 Hz, eOe
CH2ePh), 4.87 (1H, d, J ¼ 3.5 Hz, eOeCH2ePh), 7.24e7.41 (5H,
4.3.9. Compound 9 (urs-12,20(30)-diene-27,28-dioic acid 3
D-quinovopyranosyl-28-O- -D-glucopyranoside)
White amorphous powder; [
b-O-b-
b
eOeCH2ePh). 13C NMR (75 MHz,
d, CD3OD); 61.6, 69.9, 70.7, 72.5,
a
]
D
þ65.9 (c 1.0, MeOH); HR-FAB-
72.8, 74.0, 98.1, 127.4, 128.0 ( ꢁ 4), 137.8.
MS m/z 791.4215 [MꢀH]ꢀ (calcd for C42H63O14, 791.4217); FAB-MS
m/z 791 [MꢀH]ꢀ, 587 [MꢀHꢀ44ꢀCO2ꢀ(glcꢀH2O)]ꢀ; For 1H NMR
and 13C NMR spectroscopic data, see Tables 2 and 3
19: FAB-MS m/z 269 [MꢀH]ꢀ; 1H NMR (300 MHz,
d, CD3OD);
3.47 (1H, dd, J ¼ 9.4, 3.2 Hz, H-4), 3.61 (1H, dd, J ¼ 3.5, 3.5 Hz, H-2),
3.67e3.84 (3H, m, H-5, H-6), 3.99 (1H, dd, J ¼ 3.2, 3.2 Hz, H-3) 4.56
(1H, d, J ¼ 11.7 Hz, eOeCH2ePh), 4.79 (1H, d, J ¼ 11.7 Hz, eOe
CH2ePh), 4.89 (1H, d, J ¼ 3.8 Hz, H-1), 7.23e7.42 (5H, eOeCH2ePh).
4.3.10. Compound 10 (urs-12,20(30)-diene-27,28-dioic acid 3
-D-allopyranosyl-28-O- -D-glucopyranoside)
White amorphous powder; [
b-O-
b
b
13C NMR (75 MHz,
d, CD3OD); 61.7, 67.3, 68.3, 69.7, 72.4, 98.4, 127.8,
a]
D þ42.5 (c 0.35, MeOH); HR-FAB-
128.1( ꢁ 2), 128.2 ( ꢁ 2), 137.5.
MS m/z 807.4182 [MꢀH]ꢀ (calcd for C42H63O15, 807.4167); FAB-MS
m/z 807 [MꢀH]ꢀ, 601 [MꢀHꢀCO2ꢀ(glcꢀH2O)]ꢀ; For 1H NMR and
13C NMR spectroscopic data, see Tables 2 and 3
20: FAB-MS m/z 381 [MþH]þ; 1H NMR (300 MHz,
d, CD3OD);
3.01e3.60 (6H), 4.47 (1H, d, J ¼ 11.4 Hz, eO-CH2-Ph), 4.73 (1H, d,
J ¼ 11.4 Hz, eO-CH2-Ph), 4.75 (1H, d, J ¼ 4.1 Hz), 7.13e7.33 (5H, eO-
CH2-Ph). 13C NMR (75 MHz,
d, CD3OD); 6.58 (eCH2-I), 69.3, 71.7,
4.4. Preparation of D-quinovose (22) and 6-deoxy-D-allose (23)
from D-glucose and D-allose
72.5, 73.5, 73.5, 74.7, 98.0, 127.6, 128.1 ( ꢁ 2), 128.2 ( ꢁ 2), 137.5.
21: FAB-MS m/z 381 [MþH]þ; 1H NMR (300 MHz,
d, CD3OD);
3.23e3.70 (5H, H-2, H-4, H-4, H-6), 3.96 (1H, d, J ¼ 3.2, 3.2 Hz), 4.60
(1H, d, J ¼ 11.7 Hz, eOeCH2ePh), 4.86 (1H, d, J ¼ 2.6 Hz, H-1), 4.87
To a stirred solution of each sugar [D-glucose, D-allose] (180 mg,
1 mmol) in dry pyridine (5 ml), was slowly added chloro-
(1H, d, J ¼ 11.7 Hz, eOeCH2ePh), 7.24e7.44 (5H, eOeCH2ePh). 13
C
trimethylsilane (TMS-Cl, 772
ml, 6.0 mmol) at room temperature
NMR (75 MHz, d, CD3OD); 6.85 (eCH2-I), 67.5, 68.6, 69.7, 71.3, 72.1,
under Ar atmosphere. The reaction mixture was then stirred for 4 h
at room temperature. n-Hexane (30 ml) and ice cold H2O
(10 ml ꢁ 1) were next added to the reaction mixture successively,
and the organic layer was extracted with H2O (10 ml ꢁ 3), dried
over anhydrous Na2SO4 and evaporated in vacuo to afford per-O-
trimethylsilylated carbohydrate (16, 17) as an oil in quantitative
yield (Uchiyama et al., 2016).
98.2, 127.7, 128.2 ( ꢁ 4), 137.4.
22, 6-Deoxy-D-glucose; D-quinovose (Durrwachter et al.,
1986): FAB-MS m/z 163 [MꢀH]ꢀ; 1H NMR (300 MHz,
d, D2O); a
form:1.15 (3H, d, J ¼ 6.2 Hz, H-6), 5.06 (1H, d, J ¼ 3.5 Hz, H-1),
3.30e3.83 (4H, overlapping, H-2, H-3, H-4, H-5).
J ¼ 6.2 Hz, H-6), 4.51 (1H, d, J ¼ 8.0 Hz, H-1), 3.30e3.83 (4H,
overlapping, H-2, H-3, H-4, H-5). 13C NMR (75 MHz,
, D2O); form:
form: 17.3, 72.2, 74.7, 75.2, 75.8,
b form: 1.18 (3H, d,
d
a
To solution of per-O-trimethylsilylated carbohydrate (540 mg,
17.3, 67.8, 72.1, 72.8, 75.5, 92.2.
96.0.
b
1 mmol) in dry CH2Cl2 (3 ml), iodotrimethylsilane (144
ml,
1.0 mmol) was added at room temperature under Ar atmosphere
and the reaction mixture was stirred for 30 min. To the reaction
23, 6-Deoxy-D-allose (Kobayashi et al., 1992): FAB-MS m/z 163
[MꢀH]ꢀ; 1H NMR (300 MHz,
, DMSO-d6); 1.09 (3H, d, J ¼ 6.2 Hz, H-
d
mixture, a solution of benzyl alcohol (207
ml, 2.0 mmol/1 mL) and
6), 2.97 (1H, brd, J ¼ 8.8 Hz, H-4), 3.03 (1H, dd, 7.6, 2.6 Hz, H-2), 3.55
(1H, m, H-5), 3.79 (1H, dd, J ¼ 2.6, 2.6 Hz, H-3), 4.57 (1H, d,
2,6-di-tert-butylpyridine (243 l, 2.0 mmol) in dry CH2Cl2 (2 ml)
m
was added, and the resulting mixture was stirred for 4 h at room
temperature. MeOH (4 ml) was next added (to remove the TMS
groups) to the reaction mixture, with the latter neutralized using
Amberlite IRA400 resin (OHꢀ form), then filtered, concentrated and
purified by silica gel CC (CHCl3:MeOH (10:1)) to afford the corre-
J ¼ 7.6 Hz, H-1). 13C NMR (75 MHz,
d, DMSO-d6); 19.0, 69.4, 72.2,
72.9, 73.7, 94.7 (b form).
4.5. Acid hydrolysis of compound 1-10
sponding benzyl glycoside (18, 173 mg, 64%,
a
:
b
¼ 8:1; 19, 121 mg,
Each sample [1 (10 mg), 2 (10 mg), 3 (4.5 mg), 4 (3.5 mg), 5
(5 mg), 6 (6.5 mg), 7 (1 mg), 8 (1 mg), 9 (1 mg), 10 (1 mg)] was
individually heated in 1 M HCl (1.0 ml) at 90 ꢂC for 3 h. After the
reaction, each reaction mixture was neutralized by passing each
through an ion-exchange resin (Amberlite IRA400, OHꢀ form)
column. Each filtrate was then transferred to a Sep-Pak C18
45%, form only) (Uchiyama and Hindsgaul, 1996).
a
To solution of the benzyl glycoside (100 mg, 0.37 mmol) in THF
(7 ml), Ph3P (490 mg, 3.0 mmol), imidazole (203 mg, 3.0 mmol) and
I2 (509 mg, 2.0 mmol) were added at 90 ꢂC, and the reaction
mixture was stirred for 1.0 h. The reaction mixture was filtered,
Please cite this article in press as: Furukawa, M., et al., Triterpenoid glycosides from Ladenbergia hexandra Klotzsch, Phytochemistry (2017),
http://dx.doi.org/10.1016/j.phytochem.2017.01.014

M. Furukawa et al. / Phytochemistry xxx (2017) 1e9
9
cartridge and eluted with H2O and MeOH. Each MeOH eluate was
Appendix A. Supplementary data
concentrated with each aglycone structurally elucidated by means
of 1D-NMR. On the other hand, each aqueous eluate was concen-
trated, and then analyzed by TLC over silica gel (CHCl3:MeOH:H2O
6:4:1) by comparison with authentic samples. Furthermore, the
sugar residue was dissolved in anhydrous pyridine (4 ml), and
treated with L-cysteine methyl ester hydrochloride (4 mg), with the
mixture kept at 60 ꢂC for 1 h. After each solvent (0.1 ml) was tri-
methylsilylated with hexamethyldisilazane/trimethylchlorosilane
(0.15 ml, Tokyo Kasei Co., Tokyo, Japan) at 40 ꢂC for 10 min, the TMS
derivative of each sugar was identified by GC analysis with a
standard (Hara et al., 1987). GC conditions: ULBON HR-1 column,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2017.01.014.
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Acknowledgments
This work was partly supported by a Grant-in-Aid for Scientific
Research from JSPS (No. 25460138, TU) and a Research Grant from
the Nihon University, School of Pharmacy Alumni Association (H28,
MF).
Please cite this article in press as: Furukawa, M., et al., Triterpenoid glycosides from Ladenbergia hexandra Klotzsch, Phytochemistry (2017),
http://dx.doi.org/10.1016/j.phytochem.2017.01.014