N-Substituted acetamide glycosides from the stems of Ephedra sinica

Article abstract of DOI:10.1016/j.phytol.2015.04.014

Abstract Five new N-mono-/bis-substituted acetamide glycosides, N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (1), N-methyl-N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (2), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (3), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (4), and N-methyl-N-{4-O-[3-O-(6-O-trans-cinnamoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (5), along with one known acetamide derivative, N-methyl-N-(4-hydroxyphenethyl)-acetamide, the shared aglycone of 2-5, were isolated from the ethanol extract of the stems of Ephedra sinica. The structures of these new compounds were elucidated on the basis of extensive spectroscopic examination, mainly including multiple 1D and 2D NMR and HRESIMS examinations, and qualitative chemical tests. All N,N-bissubstituted acetamide glycosides were found to show the obvious rotamerism, as in the case of the isolated known N-methyl-N-(4-hydroxyphenethyl)-acetamide, under the experimental NMR conditions, with the ratios of integrated intensities between anti- and syn-rotamers always being found to be about 4 to 3.

Full text of DOI:10.1016/j.phytol.2015.04.014

G Model
PHYTOL 933 1–8
Phytochemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
1
2
Short communication
N-Substituted acetamide glycosides from the stems of Ephedra sinica
Dan Zhang a, An-Jun Deng a, Lin Ma a, Xue-Fu You b, Zhi-Hui Zhang a, Zhi-Hong Li a,
3
4
Q1
Q2
a,
a,b,**
*
Jian-Dong Jiang
, Hai-Lin Qin
5
6
7
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, PR China
b
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Five new N-mono-/bis-substituted acetamide glycosides, N-{4-O-[3-O-(4-O-
a
-
L
-rhamnopyranosyl-
b
a
-
-
Received 26 January 2015
Received in revised form 16 April 2015
Accepted 24 April 2015
Available online xxx
D
-glucopyranosyl)-
-rhamnopyranosyl-
N-{4-O-[3-O-(6-O-benzoyl-4-O-
phenethyl}-acetamide (3), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-
nosyl]-phenethyl}-acetamide (4), and N-methyl-N-{4-O-[3-O-(6-O-trans-cinnamoyl-4-O-
pyranosyl- -glucopyranosyl)- -rhamnopyranosyl]-phenethyl}-acetamide (5), along with one
a
-
L
-rhamnopyranosyl]-phenethyl}-acetamide (1), N-methyl-N-{4-O-[3-O-(4-O-
-glucopyranosyl)-
-rhamnopyranosyl-
L
b
-D
a
-L
-rhamnopyranosyl]-phenethyl}-acetamide (2), N-methyl-
-glucopyranosyl)- -rhamnopyranosyl]-
-glucopyranosyl)- -rhamnopyra-
-rhamno-
a
-
L
b
-
D
a-L
b
-D
a-L
a-L
Keywords:
Ephedra sinica
b
-D
a-L
known acetamide derivative, N-methyl-N-(4-hydroxyphenethyl)-acetamide, the shared aglycone of
2–5, were isolated from the ethanol extract of the stems of Ephedra sinica. The structures of these new
compounds were elucidated on the basis of extensive spectroscopic examination, mainly including
multiple 1D and 2D NMR and HRESIMS examinations, and qualitative chemical tests. All
N,N-bissubstituted acetamide glycosides were found to show the obvious rotamerism, as in the case
of the isolated known N-methyl-N-(4-hydroxyphenethyl)-acetamide, under the experimental NMR
conditions, with the ratios of integrated intensities between anti- and syn-rotamers always being found
to be about 4 to 3.
Ephedraceae
N-mono-/bis-sssubstituted acetamide
glycosides
Rotamerism
ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
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1. Introduction
norephedrine, and the like, are the most important and
characteristic chemical constituents of Ma Huang (Ding et al.,
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Ephedra Herb, popularly known as the Chinese name
“Ma Huang” in China, has been used to treat rheums, asthma,
and cough with dyspnea, inter alia, in traditional Chinese medicine
(TCM) for thousands of years (Ding et al., 2006). According to the
Chinese Pharmacopia, Ephedra Herb derives from the dried
herbaceous stems of three Ephedra species from the plant family
of Ephedraceae, i.e., Ephedra sinica Stapf, Ephedra intermedia
Schrenk et C.A. Mey. and Ephedra equisetina Bge (Committee of the
Chinese Pharmacopia, 2010). It is well known in the fields of
medicinal chemistry and chemotaxonomy that the amphetamine-
type alkaloids, such as l-ephedrine, d-pseudoephedrine, and
2006 Zhao et al., 2009). In addition, some flavonoids (Amakura
et al., 2013), esters of organic acids (Zhao et al., 2009),
polysaccharides (Konno et al., 1985), and proanthocyanidins (Zang
et al., 2013) were also reported to be isolated from Ephedra species
in previous publications. But from the viewpoint of phytochemis-
try, the investigation on the chemical constituents of Ephedra
species is still very inadequate up to now while considering the
great progress of the related science and technologies. Thus, our
ongoing study aims at isolating and elucidating more constituents,
especially new and bioactive compounds or valuable ones full of
other academic significances, from the Ephedra species. Described
herein is a full account of the isolation and structure elucidation of
five new acetamide glycosides (1–5) from the stems of the
title plant, along with one known acetamide derivative, N-methyl-
N-(4-hydroxyphenethyl)-acetamide. All the new acetamide
glycosides contained the acetyl group as their acyl moieties and the
15 Q4
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* Corresponding author. Tel.: +86 10 83172503; fax: +86 10 63017757.
** Corresponding author at: Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, PR China.
Tel.: +86 10 83160005; fax: +86 10 63017757.
E-mail addresses: zhangdanid@163.com (D. Zhang), denganjun@imm.ac.cn
(A.-J. Deng), malin@imm.ac.cn (L. Ma), xuefuyou@hotmail.com (X.-F. You),
zhangzhihui@imm.ac.cn (Z.-H. Zhang), zhl@imm.ac.cn (Z.-H. Li),
jiang.jdong@163.com (J.-D. Jiang), qinhailin@imm.ac.cn (H.-L. Qin).
Q3
N-
(4-hydroxylphenyl)-ethylamine as their amine moieties, with
the hydroxyl group of the -(4-hydroxylphenyl)-ethyl group being
glycosidated by a sugar chain containing two or three nonacylated
b-(4-hydroxylphenyl)-ethylamine or the N-methyl-N-b-
b
http://dx.doi.org/10.1016/j.phytol.2015.04.014
1874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

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PHYTOL 933 1–8
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D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
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or acylated monosaccharides. As tertiary N,N-bissubstituted
acetamide derivatives, compounds 2–5 were found to exhibit
the obvious rotamerism under the experimental NMR conditions,
with the ratios of integrated intensities between anti- and
syn-rotamers always being found to be about 4 to 3, the same
situation as the isolated and known N-methyl-N-(4-hydroxyphe-
nethyl)-acetamide of their shared aglycone (Wright et al., 2009).
All the isolated acetamide derivatives did not show cytotoxicity
against several human cancer cell lines and antibacterial activity
against some organisms, meaning that these compounds are safe
for the traditional application of the title plant.
conjunction with the 13C and DEPT NMR and HSQC experiment
which showed direct correlations between the above two 1H NMR
43
signals and the 13C NMR signal at
d
60.1 (t, C-600). In addition, one
44
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labile proton at dH 7.89 (1H, t, J = 6.0 Hz), assignable to an secondary
amide group according to its chemical shift, was also exhibited in
the 1H NMR spectrum of 1, which was confirmed by HSQC
experiment, with no 13C NMR signal being correlated with
the1H NMR signal. In addition to the above NMR information,
there are twelve additional 13C NMR signals being left over in all in
the 13C NMR spectrum, which were all classified into methineoxy
by DEPT spectrum. Taken together, they make the presence of two
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50
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Q6 93
94
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a
-rhamnopyranosyl groups and one
b
-glucopyranosyl group in 1
-glucose and -rhamnose,
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2. Results and discussion
very clear. Acid hydrolysis of 1 afforded
D
L
97
which were detected by derivatization reaction and GC analysis as
described in Section ‘3.5’ (Yu et al., 2013). Furthermore, in the
HMBC spectrum, the long range 1H–13C correlations from
H-7 to C-1, C-2/6, and C-8, from H-2/6 to C-7, C-3/5, and C-4,
from H-3/5 to C-1, C-2/6, and C-4, from H-8 to C-1, C-7, and C-10,
from NH to C-8 and C-10, and from H-11 to C-10, coupled with
chemical shifts of these protons and carbons, confirmed the
presence of N-phenethylacetamide with the para-position of
ethylene in the benzene ring being substituted by oxygen atom.
The HMBC correlations from H-10 to C-4, from H-100 to C-30, and
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60
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The neutral n-BuOH-soluble section of the ethanol extract of
the stems of E. sinica was subjected to multiple column
chromatography (CC) and further purified by preparative
HPLC procedure, affording five new N-mono-/bis-substituted
acetamide glycosides (1–5) (Fig. 1), along with the known
N-methyl-N-(4-hydroxyphenethyl)-acetamide.
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103
104
105
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107
108
109
110
111
112
113
114
115
116
Compound 1 was isolated as a white amorphous powder, with
½
a D
20-82.7 (c 0.071, MeOH) being determined by optical rotation
ꢀ
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measurement and calculation. The positive HRESIMS experiment
gave a [M+H]+ quasi-molecular ion peak at m/z 634.2701 and a [M
+Na]+ quasi-molecular ion peak at m/z 656.2525, indicative of a
molecular formula of C28H43NO15. The IR spectrum suggested the
from H-1000 to C-400 indicated that the 3-O-(4-O-
anosyl- -glucopyranosyl)- -rhamnopyranosyl group was
a-L-rhamnopyr-
b
-D
a-L
linked to C-4 position of the benzene ring by glucosidic bond
(Fig. 2). Full assignments of the proton and carbon resonances of 1
were achieved by comprehensive examination of multiple 1D and
2D NMR experiments. Therefore the structure of 1 was determined
existence of hydroxyl (3323 cmꢁ1
)
and amide carbonyl
(1640 cmꢁ1) functional groups, as well as aromatic (1511 cmꢁ1
)
moiety in 1. Together, the 1H and 13C NMR spectra of 1 showed up
the characteristic signals of a para-bissubstituted benzene ring,
with the AA0BB0-type aromatic coupling system being exhibited by
resonances at dH 7.13 and 6.97 (4H, AA0BB0-q, J = 8.4 Hz, H-2, 3, 5, 6)
and at dC 133.0 (C-1), 129.6 (C-2, 6), 116.6 (C-3, 5), and 154.3 (C-4),
and an acetyl group at dH 1.78 (3H, s, H-11) and dC 22.6 (C-11) and
169.0 (C-10). The 1H NMR spectrum also displayed the diagnostic
to be N-{4-O-[3-O-(4-O-a-L-rhamnopyranosyl-b-D-glucopyrano-
syl)- -rhamnopyranosyl]-phenethyl}-acetamide on the basis of
a-L
systematic nomenclature.
Compound 2 was obtained as a white amorphous powder, and it
a D
20-104.1 (c 0.18, MeOH) by optical rotation
ꢀ
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118
119
120
121
122
123
124
125
126
127
takes on
½
measurement and calculation. The HRESIMS experiment of 2
showed up the [M+H]+ quasi-molecular ion peak at m/z
648.2866 and the [M+Na]+ quasi-molecular ion peak at m/z
signals of three anomeric protons at
4.51(1H, d, J = 7.8 Hz, H-100), and 4.71(1H, d, J = 1.2 Hz, H-1000) and two
secondary methyl groups at
1.109 (3H, d, J = 6.0 Hz, H-60) and 1.107
(3H, d, J = 6.0 Hz, H-6000) from the sugar moiety and two methylene
signals at 2.63 (2H, t, J = 7.8 Hz, H-7) and 3.20 (2H, ov, H-8) from
d
5.33 (1H, d, J = 1.8 Hz, H-10),
d
670.2677, indicative of the molecular formula of C29H45NO15
,
meaning one carbon atom and two hydrogen atoms more than that
of the above described 1. The IR absorptions at 3397, 1613, and
d
1510 cmꢁ1
, etc., suggested the existence of hydroxyl, amide
one 1,2-ethylene unit, all of which were further correlated to
their corresponding carbon signals by the HSQC spectrum
(Tables 1 and 2). One methyleneoxy was also indicated by the
carbonyl, and aromatic functional groups in 2. A scrutiny into
the 1H NMR spectrum of 2 found that some of the well-resolved
signals came forth in pairs. Especially, two methyl signals typically
1H NMR signals at 3.61 (1H, m, H-600a) and 3.47 (1H, m, H-600b) in
d
Fig. 1. Structures of compounds 1–5.
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

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PHYTOL 933 1–8
D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
3
Table 1
1H NMR spectroscopic data of compounds 1–5 (in DMSO-d6).
No.
1
2
3
4
5
2,6
3,5
7
8
11
7.13 d (8.4)
6.97 d (8.4)
2.63 t (7.2)
3.20 m
7.15 d (8.4), 7.13 d (8.4)
6.97 d (8.4), 6.96 d (8.4)
2.76 t (7.2), 2.68 t (7.2)
ꢂ3.43 ov, ꢂ3.40 ov
1.76 s, 1.94 s
2.79 s, 2.89 s
5.33 br s, 5.32 br s
4.08 br s
7.13 d (8.5), 7.11 d (8.5)
6.87 d (8.5), 6.86 d (8.5)
2.76 t (7.5), 2.67 t (7.5)
ꢂ3.45 m, ꢂ3.43 m
1.74 s, 1.95 s
2.80 s, 2.91 s
5.26 br s, 5.25 br s
4.01 m
3.76 m
3.48 m
3.46 m
1.08 d (5.5), 1.09 d (5.5)
4.59 d (8.5)
3.19 ov
3.37 m
3.45 ov
3.72 m
4.57 ov
4.27 dd (12.0, 6.5)
4.71 br s
3.65 m
3.47 m
3.19 ov
3.87 m
1.12 d (6.5)
7.97 d (8.5)
7.37 t (7.5)
7.56 m
7.13 d (8.4), 7.11 d (8.4)
6.86 d (8.4), 6.84 d (8.4)
2.76 t (7.2), 2.67 t (7.2)
ꢂ3.46 m, ꢂ3.45 m
1.73 s, 1.95 s
2.80 s, 2.91 s
5.25 d (1.8), 5.22 d (1.8)
4.05 m
3.76 dd (9.0, 3.0)
3.50 ov
3.50 ov
1.08 d (5.4), 1.09 d (5.4)
4.54 d (7.8)
3.16 m
3.26 m
3.21 m
3.63 m
4.63 m
4.25 m
–
6.94 d (8.4), 6.93 d (8.4)
6.77 d (8.4), 6.76 d (8.4)
2.65 t (7.2), 2.57 t (7.2)
ꢂ3.37 m, ꢂ3.33 m
1.64 s, 1.91 s
2.73 s, 2.83 s
5.20 d (1.8), 5.18 d (1.8)
3.99 m
3.75 m
3.44 m
3.65 m
1.03 d (6.0), 1.04 d (6.0)
4.518 d (7.8), 4.515 d (7.8)
3.16 m
3.36 m
3.37 m
1.78 s
H-NH
7.89 t (6.0)
5.33 d (1.8)
4.07 m
3.75 dd (9.0, 3.0)
3.61 m
10
20
30
3.78 dd (9.0, 3.0)
3.51 ddd (9.6, 9.0, 4.2)
3.40ꢂ3.43 ov
40
50
3.55 m
60
1.109 d (6.0)
4.51 d (7.8)
3.14 m
3.25 m
3.40 m
3.15 m
3.61 m
3.47 m
4.71 d (1.2)
3.50 m
1.13 d (6.0), 1.12 (6.0)
4.52 d (7.8)
100
200
3.12 dd (8.4, 8.4)
3.27 ddd (9.0, 9.0, 4.2)
3.39 dd (9.0, 9.0)
3.28 m
3.62 ov
3.49 ov
300
400
500
3.19 m
600a
600b
1000
4.36 br d (12.0)
4.21 dd (12.0, 7.2)
4.64 d (2.4)
3.65 m
3.43 m
3.20 m
3.80 m
1.09 d (6.6)
7.52 d (7.8), 7.50 d (7.8)
7.31 t (7.8), 7.29 t (7.8)
7.37 m, 7.40 m
7.609 d (15.6), 7.614 d (15.6)
6.52 d (15.6), 6.51d (15.6)
4.75 br s
3.65 br s
2000
7.955 br d (7.8), 7.952 br d (7.8)
7.33 br t (7.8), 7.32 br t (7.8)
7.53 br t (7.8), 7.52 br t (7.8)
7.33 br t (7.8), 7.32 br t (7.8)
7.955 br d (7.8), 7.952 br d (7.8)
3000
3.42 m
3.19 m
3.88 m
1.107 d (6.0)
3.40ꢂ3.43 ov
4000
3.22 ddd (9.6, 9.6, 5.4)
3.84 dq (9.6, 6.6)
1.13 d (6.0)
5000
6000
20000,60000
30000, 50000
40000
70000
80000
Table 2
13C NMR spectroscopic data for compounds 1–5 (in DMSO-d6).
No.
1
2
3
4
5
1
133.0
129.6
116.6
154.3
34.3
40.2
169.0
22.6
–
132.4
129.6
116.8
154.32
33.0
51.7
169.4
20.7
132.8
129.9
116.7
154.28
32.2
48.5
169.3
21.7
35.9
98.5
69.35
80.95
68.79
132.4
129.7
116.8
154.4
33.0
51.8
169.4
20.9
32.6
98.3
69.3
81.3
68.9
70.4
17.90
104.6
74.0
74.3
77.6
72.4
63.9
100.9
70.6
70.5
71.8
132.9
129.6
116.7
132.3
129.9
116.6
154.2
33.0
51.6
169.3
20.8
32.5
98.1
69.5
81.4
132.7
129.6
133.0
130.6
117.4
154.75
33.4
52.4
171.3
21.1
33.5
98.8
70.2
81.01
69.6
133.4
2, 6
3, 5
4
7
8
130.4
117.3
154.73
32.6
49.3
171.4
22.1
32.3
48.7
169.5
21.8
36.0
98.5
69.4
32.2
48.5
169.4
21.7
35.9
98.3
69.6
81.3
10
11
H-Me
32.5
98.4
36.7
98.9
10
98.6
69.3
81.0
70.6
68.9
17.8
104.4
74.5
75.3
76.2
74.2
60.1
100.5
70.7
70.6
71.8
68.6
17.7
20
69.30
80.98
68.82
70.62
17.8
104.4
74.2
74.6
76.3
75.3
60.1
100.5
70.70
70.62
71.9
68.6
17.7
30
80.96
40
68.8
70.3
70.4
68.86
17.71
50
68.81
17.68
104.8
73.8
75.9
70.3
71.1
60
17.86
18.36
104.6
74.4
74.9
78.3
73.1
63.9
101.3
71.1
70.9
72.3
69.7
18.2
134.29
128.95
129.73
131.4
145.9
117.95
167.0
18.31
104.7
100
200
300
400
70.4
78.4
500
73.7
64.6
600
1000
2000
3000
4000
5000
6000
10000
20000, 60000
30000, 50000
40000
70000
80000
90000
129.6
129.1
128.6
133.2
128.6
129.1
165.6
69.0
17.8
130.0
129.2
128.7
133.4
165.6
134.26
128.92
129.71
117.97
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

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no NOE correlation of N-Me/Me-11 from anti-form (2a) as more
major component was obtained but significant NOE correlation of
N-Me/Me-11 from syn-form (2b) as more minor component was
observed in the nuclear Overhauser effect difference spectrum
(Fig. 3). Moreover, the 1H NMR signal of N-Me exhibited well-
resolved long-range 1H–13C cross peaks with the acetyl carbonyl in
the HMBC spectrum for both anti- and syn-rotamers. For the sugar
moiety, the 1H NMR spectrum also exhibited the diagnostic signals
O
N
H
O
O
HO
HO
O
O
O
OH
of three anomeric protons at
d
5.33 and 5.32 (1H in total, 2 ꢃ br s,
HO
OH
H-10), 4.52 (1H, d, J = 7.8 Hz, H-100), and 4.75 (1H, br s, H-1000) and two
O
HO
secondary methyl groups at
d
1.13 and 1.12 (3H in total, 2 ꢃ d,
2 ꢃ J = 6.0 Hz, H-60) and 1.13 (3H, d, J = 6.0 Hz, H-6000), all of which
were further correlated to their corresponding carbon signals by
HSQC spectrum (Tables 1 and 2). With the aid of 13C NMR and HSQC
experiments, one methyleneoxy of hexopyranose was also
HO
OH
Fig. 2. HMBC correlations of compound 1.
indicated by the 1H NMR signals at
d
3.62 (1H, ov, H-600a) and
3.49 (1H, ov, H-600b), which showed direct cross-peaks with the 13C
128
129
130
131
132
133
134
135
136
137
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144
145
146
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153
154
155
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assignable to one acetyl group and one N-methyl group,
respectively, by the chemical shifts and comparing to those of
compound 1, were detected in the form of pairs at dH 1.76 and 1.94
(3H, 2 ꢃ s) and dH 2.79 and 2.89 (3H, 2 ꢃ s), respectively, in the
relatively high field region. The ratios of the relative integrated
intensities of the two signals in each pair were indicated to be
around the same 4–3, with the total integrated intensities of both
the former pair and the latter one amounting to a methyl group.
Considering that no labile proton of secondary amide group like
that in the case of 1 was observed, but the appearance of the
abovementioned N-methyl group was evident in the 1H NMR
spectrum, 2 was determined to be a pair of anti- and syn-rotamers
of N,N-bissubstitued acetamide in the conditions of NMR
measurement. And when examined as anti- and syn-rotamers, it
was demonstrated that the 1H and 13C NMR spectra of 2 were very
similar to those of 1, with the main difference being the
aforementioned disappearance of the labile proton of amide
group and the appearance of the N-methyl group in 2. The category
of N,N-bissubstitued acetamide for 2 was confirmed by the
following spectroscopic data. The 1H and 13C NMR spectra of 2,
in conjunction with the consecutive correlations detected in 1H-1H
COSY spectrum and the one bond 1H–13C correlations in HSQC
spectrum, showed the characteristic signals assignable to a
para-bissubstituted benzene ring at dH 7.15 and 7.13 (2H in total,
2 ꢃ d, 2 ꢃ J = 8.4 Hz, H-2, 6) and 6.97 and 6.96 (2H in total, 2 ꢃ d,
2 ꢃ J = 8.4 Hz, H-3, 5) and at dC 132.8 and 132.4 (C-1), 129.9 and
129.6 (C-2, 6),116.8 and 116.7 (C-3, 5), and 154.32 and 154.28 (C-4),
an 1, 2-ethylene unit at dH 2.76 and 2.68 (2H in total, 2 ꢃ t,
2 ꢃ J = 7.2 Hz, H-7) and ꢂ3.43 and ꢂ3.40 (2H in total, 2 ꢃ ov, H-8)
and at dC 33.0 and 32.2 (C-7) and 51.7 and 48.5 (C-8), an acetyl
group at dH 1.94 and 1.76 (3H in total, 2 ꢃ s, H-11) and at dC 21.7 and
20.7 (C-11) and 169.3 and 169.4 (C-10), and a N-methyl group at dH
2.89 and 2.79 (3H in total, 2 ꢃ s, N-Me) and at dC 35.9 and 32.5
(N-Me). The rotamerism of 2 was further illustrated by the fact that
NMR signal at d
60.1 (C-600) in the HSQC spectrum. In addition to the
above NMR information, twelve additional 13C NMR signals of
methineoxy of sugar moiety were left over in total in the 13C NMR
spectrum. Taken together, it makes the presence of two
a
-rhamnopyranosyls and one
b
D
-glucopyranosyl in 2 very clear.
-glucose and -rhamnose, which
Acid hydrolysis of 2 also afforded
L
were detected by derivatization reaction and GC analysis, as
described in Section ‘3.5’ (Yu et al., 2013). Based on the assignment
of 1H NMR signals of sugar moiety made by 1D TOCSY and 1H–1H
COSY experiments, the HMBC correlations from H-10 to C-4, from
H-100 to C-30, and from H-1000 to C-400 indicated that the 3-O-(4-O-
a-
L-rhamnopyranosyl-b-D-glucopyranosyl)-a-L-rhamnopyranosyl
group was linked to C-4 position of the benzene ring by glucosidic
bond. Full assignments of the proton and carbon resonances of 2
were accomplished by comprehensive examination of multiple 1D
and 2D NMR experiments. According to the structure features, it
seems to be that when the temperature of 1H NMR detection is set
at above 70 ꢄC, the rotamerism of N,N-bissubstitued acetamide
could be eliminated (Chen et al., 2009). But it was not the case in
terms of 2. At a temperature of 80 ꢄC in the NMR experiment, the
rotamerism still exists in our study. Obviously, some
barriers causing rotamerism in 2 are very strong, which was
similar to the case obtained in the NMR measurement of N-methyl-
N-(4-hydroxyphenethyl)-acetamide (Wright et al., 2009), a known
compound also isolated in the present study. Based on
the above evidences, the structure of 2 was determined as
N-methyl-N-{4-O-[3-O-(4-O-a-L-rhamnopyranosyl-b-D-glucopyr-
anosyl)- -rhamnopyranosyl]-phenethyl}-acetamide on the
a-L
basis of systematic nomenclature, with its manifestation being
anti- and syn-rotamers in DMSO solution.
Compound 3 was obtained as a white amorphous powder,
½
aꢀD
20-87.9 (c 0.14, MeOH). The HRESIMS experiment showed up the
210
O
N
O
N
O
O
O
O
HO
HO
HO
O
HO
HO
O
O
O
O
OH
OH
O
HO
OH
OH
O
O
HO
HO
HO
HO
OH
OH
2b
2a
Fig. 3. Key HMBC and NOE correlations of compound 2.
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

G Model
PHYTOL 933 1–8
D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
5
O
N
O
N
O
O
O
O
O
O
HO
HO
O
O
O
O
O
O
OH
OH
O
O
HO
OH
HO
OH
O
O
HO
HO
HO
HO
OH
OH
3a
3b
Fig. 4. Key HMBC and NOE correlations of compound 3.
quasi-molecular ion peaks of 3 at m/z 752.3121 [M+H]+ and
774.2946 [M+Na]+, indicative of the molecular formula of
C36H49NO16, i.e., seven carbon atoms, five hydrogen atoms, and
one oxygen atom more than those of 2 which match a benzoyl
group when one proton in 2 is substituted. The IR absorptions at
3395, 1720, 1613, and 1510 cmꢁ1, etc., suggested the existence of
hydroxyl, ester carbonyl, amide carbonyl, and aromatic groups in 3.
A detailed examination into the NMR spectra of 3 and a comparison
to those of 2 found that 3 was very similar to 2, with the
same rotamerism as in the case of 2 from the aglycone of N-methyl-
N-(4-oxyphenethyl)-acetamide being exhibited by the signals in
and 50000), 133.4 (C-40000), and 165.6 (C-70000), which matched the
aforementioned benzoyl group when explored from their 1D and
2D NMR data. The benzoyl group was determined, on the basis of
observed esterification shifts of +0.95 and +0.78 from H-600a and
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1
H-6600b in the H NMR spectrum and +3.8 from C-6600 in the
13C NMR spectrum, respectively, as compared with 2, to be linked
to C-6600position through ester linkage. This conclusion was also
confirmed by the long-range 1H–13C correlations between H-6600a/
H-6600b and C-70000
(dC 165.6) in the HMBC spectrum. And the HMBC
experiment also confirmed through the long-range 1H–13C
correlation between H-10 and C-4, etc., that the 3-O-(6-O-
the 1H NMR spectrum at
d
1.74 and 1.95 (3H in total, 2 ꢃ s, H-11),
benzoyl-4-O-a-L-rhamnopyranosyl-b-D-glucopyranosyl)-a-L-
rhamnopyranosyl was linked to C-4 by glucosidic bond (Fig. 4).
Thus, the structure of 3 was determined as N-methyl-N-{4-O-[3-O-
2.80 and 2.91 (3H in total, 2 ꢃ s, N-Me), 7.13 and 7.11 (2H in total, 2
ꢃ d, 2 ꢃ J = 8.5, H-2, 6), 6.87 and 6.86 (2H in total, 2 ꢃ d, 2 ꢃ J = 8.5,
H-3, 5), 2.76 and 2.67 (2H in total, 2 ꢃ t, 2 ꢃ J = 7.5 Hz, H-7), and
3.45 and 3.43 (2H in total, 2 ꢃ m, H-8), and with the same three
anomeric protons, two secondary methyl groups, and one
methyleneoxy of hexopyranoses from sugar moiety as in 2 being
shown up by the resonances at dH 5.26 and 5.25 (1H in total, 2 ꢃ br
s, H-10), 4.59 (1H, d, J = 8.5 Hz, H-100), 4.71 (1H, br s, H-1000), 1.08 and
1.09 (3H in total, 2 ꢃ d, 2ꢃJ = 5.5 Hz, H-6'), 1.12 (3H, d, J = 6.5 Hz,
H-6000), and 4.57 (1H, m, H-600a) and 4.27 (1H, dd, J = 12.0, 6.5 Hz,
H-600b). The findings that no NOE correlation of N-Me/Me-11 of
anti-form (3a) as more major component was observed but
significant NOE correlation of N-Me/Me-11 of syn-form (3b) as
more minor component was obtained in the nuclear Overhauser
effect difference spectrum further illustrated the rotamerism
(6-O-benzoyl-4-O-a-L-rhamnopyranosyl-b-D-glucopyranosyl)-
a-L
-rhamnopyranosyl]-phenethyl}-acetamide on the basis of
nomenclature, with the manifestation being anti- and
syn-rotamers in DMSO solution. Full assignments of the proton
and carbon resonances of 3 were achieved by comprehensive
examination of multiple 1D and 2D NMR experiments.
Compound 4 was obtained as a white amorphous powder,
a D
20-85.1 (c 0.098, MeOH). The HRESIMS experiment of 4 showed
ꢀ
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267
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269
270
271
272
273
274
275
276
277
278
½
up the quasi-molecular ion peaks at m/z 606.2566 [M+H]+ and
628.2372 [M+Na]+, indicative of the molecular formula of
C30H39NO12, i.e., six carbon atoms, ten hydrogen atoms, and four
oxygen atoms less than those of 3 which matches a rhamnopyr-
anosyl unit when it is substituted by a hydrogen atom. The IR
absorptions at 3373, 1719, 1615, and 1510 cmꢁ1, etc., suggested the
existence of hydroxyl, ester carbonyl, amide carbonyl, and aromatic
groups in 4. An in-depth analysis of the NMR spectra of 4 and a
comparison to those of 3 found that 4 was very similar to 3, with
the same rotamerism from the aglycone of N-methyl-N-
(4-oxyphenethyl)-acetamide as in the case of 3 being exhibited
(Fig. 4). Acid hydrolysis of
3 also afforded D-glucose and
L-rhamnose, which were detected by derivatization reaction and
GC analysis, as described in Section ‘3.5’ (Yu et al., 2013). The main
difference between 3 and 2 lay in the appearance of a set of
additional 1H and 13C NMR signals in 3 at dH 7.97 (2H, d, J = 8.5 Hz,
H-20000 and 60000), 7.56 (1H, m, H-40000), and 7.37 (2H, t, J = 8.5 Hz, H-30000
and 50000) and at dC 130.0 (C-10000), 129.2 (C-20000 and 60000), 128.7 (C-30000
by the signals in the 1H NMR spectrum at
d 1.73 and 1.95 (3H in
O
N
O
N
O
O
O
O
O
O
HO
HO
O
O
O
O
O
O
OH
OH
HO
HO
HO
OH
HO
OH
4a
4b
Fig. 5. Key HMBC and NOE correlations of compound 4.
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

G Model
PHYTOL 933 1–8
6
D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
O
N
O
N
O
O
O
O
O
O
HO
O
HO
O
O
O
O
OH
O
OH
O
O
HO
OH
HO
OH
O
HO
O
HO
HO
HO
OH
OH
5a
5b
Fig. 6. Key HMBC and NOE correlations of compound 5.
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total, 2 ꢃ s, H-11), 2.80 and 2.91 (3H in total, 2 ꢃ s, N-Me), 7.11 and
7.13 (2H in total, 2 ꢃ d, 2 ꢃ J = 8.4 Hz, H-2, 6), 6.84 and 6.86 (2H in
total, 2 ꢃ d, 2 ꢃ J = 8.4 Hz, H-3, 5), 2.76 and 2.67 (2H in total, 2 ꢃ t,
2 ꢃ J = 7.2 Hz, H-7), and ꢂ3.46 and ꢂ3.45 (2H in total, 2 ꢃ m, H-8).
The main difference between 4 and 3 lay in the disappearance in 4
of a set of 1H and 13C NMR signals of a rhamnopyranosyl which
exists in 3, with the evidence being indicated by the findings that
only two anomeric protons, one secondary methyl group, one
methyleneoxy from glucopyranose, and one benzoyl were shown
and a comparison to those of 3 found that 5 was very similar to 3,
with the same rotamerism from the aglycone of N-methyl-N-
(4-oxyphenethyl)-acetamide as in the case of 3 being exhibited by
the signals in the 1H NMR spectrum at
d 1.64 and 1.91 (3H in total, 2
ꢃ s, H-11), 2.73 and 2.83 (3H in total, 2 ꢃ s, N-Me), 6.94 and 6.93
(2H in total, 2 ꢃ d, 2 ꢃ J = 8.4 Hz, H-2, 6), 6.77 and 6.76 (2H in total,
2 ꢃ d, 2 ꢃ J = 8.4 Hz, H-3, 5), 2.65 and 2.57 (2H in total, 2 ꢃ t,
2 ꢃ J = 7.2 Hz, H-7), and ꢂ3.37 and ꢂ3.33 (2H in total, 2 ꢃ m, H-8),
and with the same three anomeric protons, two secondary methyl
groups, and one methyleneoxy of hexopyranoses from sugar
moiety being shown by the signals at dH 5.20 and 5.18 (1H in total,
2 ꢃ d, 2 ꢃ J = 1.8 Hz, H-10), 4.518 and 4.515 (1H in total, 2 ꢃ d,
2 ꢃ J = 7.8 Hz, H-100), 4.64 (1H, d, J = 2.4 Hz, H-1000), 1.03 and 1.04
(3H in total, 2 ꢃ d, 2 ꢃ J = 6.0 Hz, H-60), 1.09 (3H, d, J = 6.6 Hz, H-6000),
and 4.36 (1H, br d, J = 12.0 Hz, H-600a) and 4.21 (1H, dd, J = 12.0 and
7.2 Hz, H-600b). The anti- and syn-rotamers were determined by the
nuclear Overhauser effect difference spectrum, with the anti form
as more major component (5a) exhibiting no NOE correlation
between H-11 (dH 1.64) and N-Me (dH 2.73), while the syn form (5b)
as more minor component showing significant NOE correlation
between H-11 (dH 1.91) and N-Me (dH 2.83) (Fig. 6). All the direct
linkages between every proton and their corresponding carbon
were detected by HSQC experiment (Tables 1 and 2). Moreover, a
up at
d
5.25 and 5.22 (1H in total, 2 ꢃ d, 2 ꢃ J = 1.8 Hz, H-10), 4.54
(1H, d, J = 7.8 Hz, H-100), 1.09 and 1.08 (3H, in total, 2 ꢃ d,
2 ꢃ J = 5.4 Hz, H-60), 4.63 (1H, m, H-600a) and 4.25 (1H, m, H-600b),
7.955 and 7.952 (2H in total, 2 ꢃ br d, 2 ꢃ J = 7.8 Hz, H-2000 and 6000),
7.53 and 7.52 (1H in total, 2 ꢃ br t, 2 ꢃ J = 7.8 Hz, H-4000), and 7.33 and
7.32 (2H in total, 2 ꢃ br t, 2 ꢃ J = 7.8 Hz, H-3000 and 5000). All the direct
linkages between every proton and their corresponding carbon
were detected by HSQC experiment (Tables
1 and 2). The
aforementioned missed rhamnopyranosyl was also confirmed by
the deglycosidation shifts of ꢁ0.24 from H-400 in the 1H NMR
spectrum and ꢁ7.3 from C-400 in the 13C NMR spectrum,
respectively, as compared to 3. Acid hydrolysis of 4 also afforded
D
-glucose and L-rhamnose, which were detected by derivatization
reaction and GC analysis, as described in Section ‘3.5’ (Yu et al.,
2013). In addition, the long-range 1H–13C correlations observed in
HMBC experiment unambiguously confirmed the constitution of 4
determined by the above information (Fig. 5), and the anti- and
syn-rotamers were determined by the nuclear Overhauser effect
difference spectrum, with the anti form (4a) as more major
component showing no NOE correlation between H-11 (dH 1.73)
and N-Me (dH 2.80), while the syn form as more minor component
(4b) exhibiting significant NOE correlation between H-11(dH 1.95)
and N-Me (dH 2.91) (Fig. 5). Thus, the structure of 4 was determined
monosubstituted benzene ring was also displayed by the 1H/13
C
NMR signals at dH
/
dC 7.52 and 7.50 (2H in total, 2 ꢃ d, 2 ꢃ J = 7.8 Hz,
H-20000 and 60000)/128.95 and 128.92 (C-20000 and 60000), 7.37 and 7.40 (1H
in total, 2 ꢃ m, H-40000)/131.4 (C-40000), and 7.31 and 7.29 (2H in total,
2 ꢃ t, 2 ꢃ J = 7.8 Hz, H-30000 and 50000)/129.73 and 129.71 (C-30000 and
50000). Acid hydrolysis of 5 also afforded
D-glucose and L-rhamnose,
which were detected by derivatization reaction and GC analysis, as
described in Section ‘3.5’ (Yu et al., 2013). The main difference
between 5 and 3 lay in the appearance in 5 of the additional 1H/13
C
NMR signals assignable to
E-configuration at dH
a 1,2-disubstituted ethylene of
dC 7.609 and 7.614 (1H in total, 2 ꢃ d,
as
N-methyl-N-{4-O-[3-O-(6-O-benzoyl-
b-D-glucopyranosyl)-
a
-L-rhamnopyranosyl]-phenethyl}-acetamide.
/
Compound 5 was obtained as a white amorphous powder,
20-114.8 (c 0.099, MeOH). The HRESIMS experiment of 5 showed
2 ꢃ J = 15.6 Hz, H-70000)/145.9 (C-70000) and 6.52 and 6.51 (1H in total,
2 ꢃ d, 2 ꢃ J = 15.6 Hz, H-80000)/117.95 and 117.97 (C-80000), which was
compatible with the aforementioned molecular formula. This
disubstituted ethylene moiety was substantiated to be inserted
between the monosubstituted benzene ring and the ester carbonyl
314
315
316
317
318
319
320
321
½aꢀD
up the quasi-molecular ion peaks at m/z 778.3281 [M+H]+ and
800.3104 [M+Na]+, indicative of the molecular formula of
C38H51NO16, i.e., two carbon atoms and two hydrogen atoms more
than that of 3 which matches a disubstituted ethylene. The IR
absorptions at 3337, 1731, 1646, and 1447 cmꢁ1, etc., suggested the
existence of hydroxyl, ester carbonyl, amide carbonyl, and aromatic
and olefinic groups in 5. Careful inspection of the NMR data of 5
by the long-range 1H–13C correlations fromH-70000 to C-10000, C-20000/60000
,
and C-80000 and from H-80000 to C-10000, C-70000, and C-90000 observed in
HMBC experiment, and thus, taken together, a trans-cinnamoyl was
ascertained (Fig. 6). With the aid of detailed multiple 2D NMR
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

G Model
PHYTOL 933 1–8
D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
7
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analysis, especially in the identification for the linkages of the
sugar moiety, the structure of 5 was finalized as N-methyl-N-{4-O-
pre-coated silica gel GF254. Spots were visualized under UV light
and by spraying with 10% H2SO4 in 95% EtOH, followed by heating.
Acetonitrile used in preparative HPLC procedure was of
chromatographic grade, and other solvents were of analytical
[3-O-(6-O-trans-cinnamoyl-4-O-
a-L-rhamnopyranosyl-b-D-gluco-
pyranosyl)- -rhamnopyranosyl]-phenethyl}-acetamide on the
a-L
grade.
L-Cysteine methyl ester hydrochloride, N-trimethylsilylimi-
basis of nomenclature.
dazole, and authentic sugars, including
D
-glucose and -rhamnose,
L
The known congener of compounds 2–5 isolated for the
first time from the title plant in this study was identified as
N-methyl-N-(4-hydroxyphenethyl)-acetamide, i.e., the shared
aglycone of 2–5, by comparison of the spectroscopic data with
those reported in literature (Wright et al., 2009). Because of the
similar N,N-bissubstituted acetamide structure as compounds 2–5,
the 1H NMR spectrum of N-methyl-N-(4-hydroxyphenethyl)-
acetamide was also found to exhibit the same rotamerism as
reported in publication, with the acetyl, N-methyl, and
1,2-ethylene, and the AA0BB0-type aromatic coupling system being
were all products from Sigma–Aldrich Chemical Co.
434
3.2. Plant material
435
436
437
438
439
440
441
442
443
444
445
446
The stems of E. sinica were obtained from Bozhou crude drug
market, Anhui Province, P. R. China, in December 2011, and was
identified by associate Prof. Lin Ma (a savant in plant systematics
from Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College) through careful
examination of the morphological features and according to the
description for E. sinica in the state Pharmacopoeia of China
(Committee of the Chinese Pharmacopia, 2010). All the
experimental material was carefully examined to ensure the
purity and a voucher specimen (ID-S-2555) was deposited in the
Herbarium of Institute of Materia Medica, Chinese Academy of
Medical Sciences, Beijing, P. R. China.
displayed, mainly in pairs, by the signals at dH/dC 1.94 and 1.74
(3H in total, 2 ꢃ s, H-11)/ 21.6 and 20.7 (C-11), 2.88 and 2.78 (3H in
total, 2 ꢃ s, N-Me)/32.9 and 32.1 (N-Me), 2.68 and 2.60 (2H in total,
2 ꢃ d, 2 ꢃ J = 7.5 Hz, H-7)/ 35.8 and 32.4 (C-7), 3.42 and 3.39 (2H in
total, 2 ꢃ d, 2 ꢃ J = 7.5 Hz, H-8)/51.8 and 48.7 (C-8), and 7.00 and
6.99 (2H in total, 2 ꢃ d, 2 ꢃ J = 8.5 Hz, H-2, 6)/129.7 and 129.4 (C-2,
6) and 6.68 and 6.67 (2H in total, 2 ꢃ d, 2 ꢃ J = 8.5 Hz, H-3, 5)/
115.0 and 115.0 (C-3, 5), respectively. And at a temperature of
measurement of 80 ꢄC, the rotamerism also exists.
Compounds 1–5 and the known N-methyl-N-(4-hydroxyphe-
nethyl)-acetamide were tested in this study for several
bioactivities in vitro, including the cytotoxicity examination
against several human cancer cell lines with 5-fluorouracil being
used as a positive control and the detection of antibacterial activity
with levofloxacin as a positive control against Staphylococcus
epidermidis, Staphylococcus aureus, Enterococcus faecalis,
Enterococcus faecium, Escherichia coli, Klebsiella pneumonia,
Pseudomonas aeruginosa, and Acinetobacter calcoacetious, and so
forth. None of the isolated compounds showed significant effects
against all the test cancer cell lines and all the test microbes.
However, the findings of this study also provided some meaningful
information that a compound with low cytotoxicity and low
antimicrobial activity may be very interesting as investigating its
other bioactivities, such as in the application of the title plant for
the treatment of rheums, asthma, and cough with dyspnea, inter
alia. At least, the noncytotoxic compounds were relatively safe for
the clinical application of the title plant in the traditional form.
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3.3. Extraction and isolation
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Dried and cut stems of E. sinica (19 kg) were extracted for three
times with 85% aq. EtOH (2 h, 1 h, 1 h) under reflux condition. After
evaporation of the aq. EtOH solvent under vacuum, a dark-brown
residue of 3.0 kg was obtained. The residue was suspended in 80%
EtOH in water (8000 mL) and extracted in a separatory funnel with
petroleum ether (8000 mL each) for several times until the upper
fraction was very transparent. The 80% aq. ethanol soluble section
was evaporated under vacuum condition to afford another dark-
brown residue, which was re-dissolved in water (7000 mL), and
then extracted in a separatory funnel with EtOAc for three times
(7000 mL each). Next, the water layer was further extracted in a
separatory funnel with n-BuOH (7000 mL each) for three times.
The combined n-BuOH solution was extracted in a separatory
funnel with a solution of 5% NaHCO3 in water for three times
(3 ꢃ 1500 mL) and then pure H2O (2 ꢃ 1000 mL), respectively, to pH
7.0. The neutral n-BuOH solution was evaporated under vacuum
condition to afford the corresponding brown residue (220 g) of
n-BuOH extract.
The n-BuOH extract was separated by CC over silica gel using a
gradient of CHCl3–MeOH (100:1–1:1) as eluent, yielding seven
fractions (designated as A–G) according to their TLC profiles.
Fraction C (21 g, CHCl3–MeOH = 10:1) was subjected to silica gel CC
and eluted with CHCl3–MeOH (50:1 to 20:1, gradient elution),
which yielded seven subfractions (C-1 to C-7). Fraction C-4 (7.5 g,
CHCl3–MeOH = 30:1) was chromatographed over silica gel CC
eluting with CH3Cl–MeOH (30:1, isocratic elution), giving four
subfractions again (C-4-1 to C-4-4) according to their TLC profiles.
Fraction C-4-2 (2.5 g) was separated into three subfractions (C-4-2-
1 to C-4-2-3) using silica gel CC (EtOAc–Me2CO = 10:1, isocratic
elution) according to their TLC profiles. Fraction C-4-2-1 (500 mg)
was chromatographed by Sephedax LH-20CC eluting with MeOH
to give three subfractions (C-4-2-1-1 to C-4-2-1-3) according to
their TLC profiles. Fraction C-4-2-1-1 (200 mg) was further purified
by preparative RP-HPLC (50% MeOH in H2O, isocratic elution)
to give 3 (60 mg) and 5 (30 mg). Compound 4 (6 mg) and N-methyl-
N-(4-hydroxyphenethyl)-acetamide (3 mg) were isolated from
C-4-2-1-2 (40 mg) by preparative RP-HPLC (50% MeOH in H2O,
isocratic elution). Fraction C-4-2-1-3 (150 mg) was purified by
preparative RP-HPLC using a mobile phase of MeCN-H2O (15:85) to
afford 1 (3 mg) and 2 (60 mg).
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3. Experimental
3.1. General experimental procedures
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Optical rotations were measured on a PerkinElmer 241 polar-
imeter at a temperature of 20 ꢄC. UV spectra were obtained on a
JASCO V-650 spectrophotometer, and IR (KBr) spectra on a Nicolet
5700 spectrophotometer. 1D and 2D NMR spectra were recorded
on either a Bruker AV-III-500 or a Bruker AV-IIIHD-600 NMR
spectrometers, respectively, using DMSO-d6 or chloroform-d as
solvents and tetramethylsilane (TMS) as internal standard. Both
ESIMS experiment in negative mode and HRESIMS experiment in
positive mode were performed using an Agilent 1100 series LC/
MSD Trap SL mass spectrometer. Preparative HPLC were carried
out on a Shimadzu LC-6AD system equipped with a SPD-10A
detector, and a reversed-phase C18 column (YMC-Pack ODS-A 20
ꢃ 250 mm, 5
mm, YMC CO., Kyoto, Japan) was employed. GC
analyses were undertaken on an Agilent 7890A instrument.
Column chromatography (CC) was conducted over silica gel
(200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, P. R.
China) or Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala,
Sweden), and thin layer chromatography (TLC) with glass plate
Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014

G Model
PHYTOL 933 1–8
8
D. Zhang et al. / Phytochemistry Letters xxx (2015) xxx–xxx
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3.3.1. N-{4-O-[3-O-(4-O-
a
-
L
-rhamnopyranosyl-
b
-
D
-
method (Yu et al., 2013). About 2 mg of each new compound was
dissolved in 2 M HCl (2 mL) and heated at 85 ꢄC for 10 h in oil bath.
The mixture was concentrated under reduced pressure to dryness.
The resulting residue was suspended in H2O and extracted in a
separatory funnel with CHCl3 for three times. The aqueous layer
was evaporated under vacuum, then diluted with H2O, and then re-
evaporated again under vacuum, and with the re-dilution and re-
evaporation procedure being repeated for several times, to
produce a neutral residue.
glucopyranosyl)-
White amorphous powder; ½a D
(MeOH) lmax (log ) 202.4 (3.89), 219.8 (3.78), 272.4 (2.86) nm; IR
(KBr) nmax 3323, 2934, 1640, 1511, 1380, 1230, 1027, 829, 815,
a
-L
-rhamnopyranosyl]-phenethyl}-acetamide (1)
ꢀ
20-82.7 (c 0.071, MeOH); UV
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491
492
493
494
495
496
e
764 cmꢁ1 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz,
;
DMSO-d6) data see Tables 1 and 2; ESIMS m/z 632.2 [MꢁH]ꢁ;
HRESIMS m/z 634.2701 [M+H]+ (calcd for C28H44NO15, 634.2705)
and 656.2525 [M+Na]+ (calcd for C28H43NNaO15, 656.2525).
Each residue of compounds 1–5 was dissolved in anhydrous
pyridine (1 mL), separately. L-Cysteine methyl ester hydrochloride
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3.3.2. N-methyl-N-{4-O-[3-O-(4-O-
glucopyranosyl)- -rhamnopyranosyl]-phenethyl}-acetamide (2)
White amorphous powder; ½a D
20-104.1 (c 0.18, MeOH); UV
(MeOH) lmax (log ) 203.4 (4.08), 221.6 (3.96), 272.4 (2.98) nm; IR
(KBr) nmax 3397, 2933, 1613, 1510, 1405, 1230, 1065, 1032, 837, 814,
a-L-rhamnopyranosyl-b-D-
(2 mg) was then added, and the reaction mixture was incubated at
60 ꢄC for 2 h. After the mixture was concentrated to dryness under
reduced pressure, 0.2 mL of N-trimethylsilylimidazole was added,
and the mixture was further incubated at 60 ꢄC for 2 h. Then H2O
(2 mL) was added, and the solution was extracted with hexane for
three times (2 mL each). The hexane extract was subjected to GC
under the following conditions: capillary column: HP-5
a-L
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500
501
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505
ꢀ
e
764 cmꢁ1 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz,
;
DMSO-d6) data see Tables 1 and 2; ESIMS m/z 646.2 [MꢁH]ꢁ;
HRESIMS m/z 648.2866 [M+H]+ (calcd for C29H46NO15, 648.2862)
and 670.2677 [M+Na]+ (calcd for C29H45NNaO15, 670.2681).
(30 m ꢃ 0.25 mm, with a 0.25
mm film, Dikma); detection: FID;
detector temperature: 280 ꢄC; injection temperature: 260 ꢄC;
initial temperature 16 ꢄC, which was raised to 280 ꢄC at the rate
of 5 ꢄC/min and the final temperature was maintained for 10 min;
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3.3.3. N-methyl-N-{4-O-[3-O-(6-O-benzoyl-4-O-
rhamnopyranosyl- -glucopyranosyl)- -rhamnopyranosyl]-
phenethyl}-acetamide (3)
White amorphous powder; ½a D
(MeOH) lmax (log ) 203.4 (4.22), 224.2 (4.14), 273.4 (3.11) nm; IR
(KBr) nmax 3395, 2933, 1720, 1616, 1510, 1451, 1405, 1275, 1064,
1024, 836, 812, 716 cmꢁ1 1H NMR (500 MHz, DMSO-d6) and 13C
a-L-
b-
D
a-L
Carrier: N2 gas. The authentic monosaccharides,
-rhamnose, were also treated by the same procedure as
compounds 1–5. From the acidic hydrolysates of 1–5, -glucose
and -rhamnose were all confirmed by comparing the retention
times of their derivatives with those of authentic sugars, with the
retention times of -glucose and -rhamnose being 27.96 and
D-glucose and
L
ꢀ
20-87.9 (c 0.14, MeOH); UV
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D
e
L
;
D
L
NMR (125 MHz, DMSO-d6) data see Tables 1 and 2; ESIMS m/z
750.2[MꢁH]ꢁ; HRESIMS m/z 752.3127[M+H]+ (calcd for
C36H50NO16, 752.3124) and 774.2946[M+Na]+ (calcd for
C36H49NNaO16, 774.2944).
22.31 min, respectively.
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Acknowledgements
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Q9 571
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3.3.4. N-methyl-N-{4-O-[3-O-(6-O-benzoyl-
-rhamnopyranosyl]-phenethyl}-acetamide (4)
White amorphous powder; ½a D
20-85.1 (c 0.098, MeOH); UV
(MeOH) lmax (log ) 203 (4.39), 223.6 (4.15), 272.8 (3.10) nm; IR
(KBr) nmax 3373, 2922,1719,1615,1510,1451,1405,1276,1064,1019,
834, 715 cmꢁ1 1H NMR (600 MHz, DMSO-d6) and 13C NMR
(150 MHz, DMSO-d6) data see Tables 1 and 2; ESIMS m/z 604.3
[MꢁH]ꢁ; HRESIMS m/z 606.2566[M+H]+ (calcd for C30H40NO12,
606.2545) and 628.2372[M+Na]+ (calcd for C30H39NNaO12,
628.2364).
b-D-glucopyranosyl)-
This work was supported by grants from NSFC
(No. 81361138020) and from the National Science and Technology
Project of China (2012ZX09301002001003).
a-L
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References
;
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rhamnopyranosyl- -glucopyranosyl)- -rhamnopyranosyl]-
phenethyl}-acetamide (5)
White amorphous powder; ½a D
(MeOH) lmax (log ) 203.4 (4.37), 217.8 (4.30), 278.2 (4.20) nm; IR
(KBr) nmax 3337, 2922, 1731, 1646, 1547, 1374, 1159, 1110, 1053, 898,
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ꢀ
20-114.8 (c 0.099, MeOH); UV
530
531
532
533
534
535
536
e
672 cmꢁ1 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz,
;
DMSO-d6) data see Tables 1 and 2; ESIMS m/z 776.2[MꢁH]ꢁ;
HRESIMS m/z 778.3281 [M+H]+ (calcd for C38H52NO16, 778.3281)
and 800.3104 [M+Na]+ (calcd for C38H51NNaO16, 800.3113).
537
538
3.4. Acid hydrolysis of 1–5 and determination of the absolute
configuration of sugars
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proanthocyanidins from the stems of Ephedra sinica (Ephedraceae) and their
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539 Q8
The acid hydrolysis of 1–5 and the determination of the
absolute configuration of sugars were performed using reported
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Please cite this article in press as: Zhang, D., et al., N-Substituted acetamide glycosides from the stems of Ephedra sinica. Phytochem. Lett.
(2015), http://dx.doi.org/10.1016/j.phytol.2015.04.014