New steroidal glycosides from the rhizome of Anemarrhena asphodeloides

Article abstract of DOI:10.1080/10286020.2014.942658

Two new steroidal saponins, timosaponin X (1) and timosaponin Y (2), and one new pregnane glycoside, timopregnane B (3), were isolated from the rhizomes of Anemarrhena asphodeloides, as well as three known compounds 25S-timosaponin BII (4), protodesgalactotigonin (5), and timosaponin BII-a (6) isolated from this plant for the first time. By the detailed analysis of 1D, 2D NMR, MS spectra, and chemical evidences, the structures of new compounds were elucidated as 26-O-β-d-glucopyranosyl-(25S)-5β-22-methoxy-furost-3β,26-diol 3-O-β-d-glucopyranosyl-(1 → 2)-α-l-arabinopyranoside (1), 5β-pseudo-spirost-3β,15α,23α-triol 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-galactopyranoside (2), (5β,17α)-Δ¹⁶⁽¹⁷⁾-20-one-pregn-2β,3β-diol 3-O-β-d-glucopyranosyl-(1 → 2)-β-d-galactopyranoside (3).

Full text of DOI:10.1080/10286020.2014.942658

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New steroidal glycosides from the
rhizome of Anemarrhena asphodeloides
ab
a
ab
a
Jian-Chao Yuan , Jie Zhang , Fang-Xu Wang , Xu Pang , Yang
a
a
a
Zhao , Cheng-Qi Xiong & Bai-Ping Ma
a
Department of Biotechnology, Beijing Institute of Radiation
Medicine, Beijing 100850, China
b
Graduate School, Tianjin University of Traditional Chinese
Medicine, Tianjin 300193, China
Published online: 16 Sep 2014.
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To cite this article: Jian-Chao Yuan, Jie Zhang, Fang-Xu Wang, Xu Pang, Yang Zhao, Cheng-Qi Xiong
Bai-Ping Ma (2014) New steroidal glycosides from the rhizome of Anemarrhena asphodeloides,
&
Journal of Asian Natural Products Research, 16:9, 901-909, DOI: 10.1080/10286020.2014.942658
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 9, 901–909, http://dx.doi.org/10.1080/10286020.2014.942658
New steroidal glycosides from the rhizome of Anemarrhena
asphodeloides
ab
Jian-Chao Yuan , Jie Zhang , Fang-Xu Wang , Xu Pang , Yang Zhao ,
a
ab
a
a
a
Cheng-Qi Xiong and Bai-Ping Ma *
a
a
Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850, China;
Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
b
(
Received 1 April 2014; final version received 3 July 2014)
Two new steroidal saponins, timosaponin X (1) and timosaponin Y (2), and one new
pregnane glycoside, timopregnane B (3), were isolated from the rhizomes of
Anemarrhena asphodeloides, as well as three known compounds 25S-timosaponin BII
(4), protodesgalactotigonin (5), and timosaponin BII-a (6) isolated from this plant for
the first time. By the detailed analysis of 1D, 2D NMR, MS spectra, and chemical
evidences, the structures of new compounds were elucidated as 26-O-b-D-
glucopyranosyl-(25S)-5b-22-methoxy-furost-3b,26-diol 3-O-b-D-glucopyranosyl-
(
1 ! 2)-a-L-arabinopyranoside (1), 5b-pseudo-spirost-3b,15a,23a-triol 3-O-b-D-gluco-
1
6(17)
pyranosyl-(1 ! 2)-b-D-galactopyranoside (2), (5b,17a)-D
-20-one-pregn-2b,
3
b-diol 3-O-b-D-glucopyranosyl-(1 ! 2)-b-D-galactopyranoside (3).
Keywords: Anemarrhena asphodeloides; steroidal saponin; pregnane glycoside; NMR
1
.
Introduction
6) obtained from this plant for the first
time. This paper mainly reports the
isolation and the structural elucidation of
new compounds (Figure 1).
The Anemarrhena asphodeloides Bunge.
Liliaceae) is a famous pharmaceutical
herb medicine of China, known as
zhimu” in Chinese. Pharmacological
(
“
studies showed that it had a lot of
biological activities, such as improving
senile dementia and anti-dementia [1–3],
inhibition of platelet aggregation [4,5],
lowering blood glucose level [6], anti-
inflammation [7], anti-oxidation [8,9],
anti-tumor [10,11], and anti-osteoporosis
2
.
Results and discussion
Compound 1 was obtained as a white
amorphous powder and showed positive
reaction in Lieberman–Burchard and Ehr-
lich reagent tests. The HR-ESI-MS showed
the ion peak at m/z 873.4852 [M þ H–
þ
CH OH] , and its molecular formula was
3
[12]. Steroidal saponins are the main
determined to be C H O . ESI-MS
5 76 18
4
active constituents of A. asphodeloides,
and our previous phytochemical studies
revealed a series of steroidal saponins
from its rhizome [5,13–15]. Recently, a
further phytochemical study on A. aspho-
deloides rhizome resulted in the isolation
of two new steroidal saponins (1 and 2),
and one new pregnane glycoside (3), as
well as three known steroidal saponins (4–
showed the ion peaks at m/z 889
[
2 2
M 2 H–14] , 727 [M 2 H–14–162] ,
2
65 [M 2 H–14–162–162] , and 433
5
2
M 2 H–14–162–162–132] , attribu-
[
table to the loss of two hexoses and one
pentose. That was also supported by the
anomeric proton signals at d 4.98 (1H, d,
J ¼ 5.4 Hz), 5.20 (1H, d, J ¼ 7.8 Hz), and
4.84 (1H, d, J ¼ 7.8 Hz), together with the
*Corresponding author. Email: mabaiping@sina.com
q 2014 Taylor & Francis

9
02
J.-C. Yuan et al.
OR2
OCH3
O
O
O
O
OH
HO
OH
R O
1
R O
R O
1
H
1
H
2
R1=S2
3 R1=S2
1
R1=S1, R2=S3
OR2
OR2
OR2
OH
O
OH
HO
O
O
R O
R O
1
1
R O
1
H
H
H
6
R1=S4, R2=S3
4
R1=S2, R2=S3
5 R1=S5, R2=S3
OH
O
S1=
OH
O
HO
OH
H
O
S3=
S4=
O
HO
HO
HO
HO
HO
S5=
OH
OH
OH
O
HO
O
O
OH
O
O
OH
O
HO
HO
OH
HO
OH O HO
O
OH
HO
HO
S2=
OH OH
OH
O
O
OH
O
OH
HO
HO
OH
OH
Figure 1. The structures of compounds 1–6.
corresponding anomeric carbon signals at d
1
those of timosaponin BII [13], except for
the significant difference in C-22 and an
additional carbon signal at d 47.4. The C-22
chemical shift of 1 was 112.7 ppm, about
2 ppm higher than that of timosaponin BII,
which suggested that the position of
methoxy group in 1 was at C-22. That was
confirmed by HMBC correlation between
1
01.7, 106.0, and 105.2 observed in the H
1
3
and C NMR spectra. Acid hydrolysis of 1
followed by GC analysis of its derivative
enabled the identification of the three sugar
units to be two D-glucoses and one L-
1
arabinose. However, in the H NMR
spectrum of 1, except for four characteristic
methyl proton signals of steroidal saponin
at d 0.93 (3H, s), 0.80 (3H, s), 1.04 (3H, d,
J ¼ 7.2 Hz), and 1.18 (3H, d, J ¼ 7.2 Hz),
an additional signal at d 3.26 (3H, s) was
observed, suggesting the existence of a
the proton signal at d 3.26 (CH O-22) and
3
C-22 at d 112.7 (Figure 2). By use of
COSY, HSQC, and HMBC experiments,
the proton and carbon signals for the
aglycone of 1 were assigned (Tables 1 and
2). In addition, starting from the anomeric
proton of each sugar, all proton and carbon
signals of the sugar units were fully
1
3
C
methoxyl group in the molecule. The
NMR data of 1 suggested that it had almost
identical agylcone carbon signals with

Journal of Asian Natural Products Research
903
OH
O
O
HO
HO
OH
H
OCH3
O
HO
HO
H
O
O
OH
O
H
O
HO
HO
OH
H
Figure 2. Key HMBC correlations of compound 1.
assigned by combination of COSY, HSQC,
and HMBC experiments. The b-anomeric
configurations for two glucoses were
indicated its molecular formula to be
C H O . The ion peaks in ESI-MS
3
9 64 15
þ
(positive mode) at m/z 795 [M þ Na] ,
þ
determined due to their coupling constants
3
611 [M þ H–162] , and 449 [M þ H–
þ
(
ation for the arabinose was determined due
J1,2 . 7 Hz) and a-anomeric configur-
162–162] attributed to the sequential
loss of two hexoses. Two signals at d
4.86 (1H, d, J ¼ 7.2 Hz), 5.25 (1H, d,
J ¼ 7.8 Hz) and two carbon signals at d
3
to the coupling constant ( J , 7 Hz)
1
,2
[16]. The C-25 configuration of 1 was
deduced to be S due to the chemical shift
1
13
102.5 and d 106.2 in the H and C NMR
spectra of 2 also deduced the existence of
two sugar units in the molecule. GC
analysis of derivative of acid hydrolysis
product of 2 identified two sugar units to
be D-galactose and D-glucose. Comparison
of the NMR data of 2 and timosaponin F
revealed that they had the same partial
structure of rings A–E as well as the sugar
moiety at C-3 [19]. Unlike timosaponin F,
except for the characteristic methyl proton
difference of two protons H -26 at d 4.40
a
and H -26 at 3.52 (Dab ¼ 0.88 . 0.57)
b
[17,18]. In HMBC spectrum, the corre-
lations between H-1 at d 4.98 and C-3 at d
0
0
0
0
7
8
5.3, between H-1 at d 5.20 and C-2 at d
1.1, and between H-1 at d 4.84 and C-26
000
at d 75.0 determined the connectivities
of the sugar moieties as well as the
sequence of sugar chain (Figure 2). Accord-
ingly, the structure of 1 was elucidated to be
2
6-O-b-D-glucopyranosyl-(25S)-5b-22-
at d 1.02 (3H, s, CH -18), 0.65 (3H, s,
3
methoxy-furost-3b,26-diol 3-O-b-D-gluco-
pyranosyl-(1 ! 2)-a-L-arabinopyranoside,
named timosaponin X.
CH -19), and 1.10 (3H, d, J ¼ 7.2 Hz,
3
CH -21), another two methyl protons at d
3
1.32 (3H, s), 1.19 (3H, s) were observed in
1
the H NMR spectrum of 2. In the HMBC
Compound 2, a white amorphous
powder, showed positive reaction in
Lieberman–Burchard and anisaldehyde
reagent tests, which suggested that 2 was
a spirostanol glycoside. In the HR-ESI-MS
spectrum of 2, the correlations between H-
26 and C-27, C-23, C-24, C-25, between
H-27 and C-26, C-23, C-24, C-25, and
between H-20 and C-23 determined the
skeleton of ring F portion (Figure 3).
2
of 2, the [M 2 H] ion at m/z 771.4167

9
04
J.-C. Yuan et al.

Journal of Asian Natural Products Research
905
1
3
Table 2.
No.
C NMR spectral data (150 MHz, pyridine-d ) of compounds 1–3.
5
1
2
3
No.
1
2
3
0
0
0
1
2
3
4
5
6
7
8
9
30.9
27.0
75.3
31.1
37.2
27.0
26.8
35.5
40.2
35.3
21.1
40.2
41.3
56.4
32.2
81.5
64.5
16.5
24.0
40.5
16.4
112.7
31.1
28.2
34.5
75.0
17.6
47.4
30.9
27.2
75.5
31.1
36.3
27.0
26.8
37.0
40.4
35.4
21.2
41.2
41.5
60.9
79.0
91.4
60.5
18.0
24.2
35.7
15.1
117.1
72.3
45.1
77.8
31.2
29.8
40.4
67.2
81.9
31.9
36.7
26.6
26.2
34.2
41.9
37.2
21.3
35.4
46.6
56.3
32.2
144.6
155.5
16.2
23.8
196.3
27.1
3-Ara
3-Gal
3-Gal
0
1
2
3
4
5
6
101.7
81.1
73.4
68.3
65.2
102.5
81.9
75.2
69.9
76.6
62.2
103.3
81.8
75.2
69.8
76.9
62.0
0
0
0
0
0
0
00
0
00
0
00
2 -Glc
2 -Glc
2 -Glc
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
106.0
75.0
78.2
71.7
78.5
62.8
106.2
76.9
78.0
71.8
78.4
62.8
106.2
77.0
78.0
71.8
78.6
62.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0
00
26-Glc
105.2
76.4
0
00
00
00
00
00
00
1
0
0
0
0
0
2
3
4
5
6
78.8
71.8
78.5
63.0
ZOCH
3
According to the carbon chemical shift of
C-23 (d 72.3) and the molecule of 2, a
hydroxyl group was proved at C-23. The
H-20 was confirmed as b-configuration by
the correlations with H-18 at d 1.02,
then the a-configuration of 23-OH was
HMBC
H
H
C
H
O
ROESY
H
H
O
OH
OH
OH
OH
OH
O
O
HO
O
O
H
HO
HO
OH
H
Figure 3. Key HMBC and ROESY correlations of compound 2.

9
06
J.-C. Yuan et al.
determined by correlations between b-
oriented protons H-20 at d 2.62 and H-23
at d 4.37 in ROESY spectrum (Figure 3).
By combined analysis of COSY, HSQC,
and HMBC spectra, all the proton and
carbon signals of 2 were fully assigned
and galactose moieties have b-configur-
ation [16]. Finally, by combined analysis
of COSY, HSQC, and HMBC spectra, all
the proton and carbon signals were fully
assigned (Tables 1 and 2) and the structure
of 3 was elucidated as (5b,17)-20-one-
pregn-16-ene-2b,3b-diol 3-O-b-D-gluco-
pyranosyl-(1 ! 2)-b-D-galactopyrano-
side, named timopregnane B.
(
Tables 1 and 2). Thus, the structure of 2
was elucidated as 5b-pseudo-spirost-
b,15a,23a-triol 3-O-b-D-glucopyrano-
3
syl-(1 ! 2)-b-D-galactopyranoside,
The structures of three known com-
pounds 4–6 isolated from this plant for the
first time were determined as 25R-
timosaponin BII [21], protodesgalactoti-
gonin [22], and timosaponin BII-a [23],
respectively, by comparison of their NMR
spectral data with those reported in the
literature.
named timosaponin Y.
Compound 3 was isolated as a white
amorphous powder. Its molecular formula
was determined to be C H O by HR-
3
3 52 13
2
ESI-MS at m/z 655.3533 [M 2 H] . ESI-
MS (negative mode) showed the ion peaks
2
at m/z 655 [M 2 H] , 493 [M 2 H–
2
62] , and 331 [M 2 H–162–162] ,
2
1
which attributed to loss of two hexoses.
Acid hydrolysis of 3 followed by GC
analysis of its derivative enabled the
identification of the two sugar units to be
3
.
Experimental
3.1 General experimental procedures
Optical rotations were measured with a
JASCO J-810 polarimeter (JASCO Corpor-
ation, Tokyo, Japan). The measurements of
IR were performed using Bruker Vertex 70
(Bruker Corporation, Karlsruhe, Germany).
1D and 2D NMR spectra in pyridine-d5
1
D-galactose and D-glucose. The H NMR
spectrum of 3 showed three singlet methyl
proton signals at d 0.96 (3H, s), 0.88 (3H,
s), and 2.21 (3H, s), one olefinic proton
signal at d 6.60 (1H, s), in addition to two
anomeric protons at d 4.98 (1H, d,
J ¼ 7.8 Hz) and 5.27 (1H, d, J ¼ 7.8 Hz).
UNITY-
solution were recorded on Varian
1
INOVA (600 MHz for H NMR and
13
150 MHz for C NMR) (Palo Alto, CA,
1
3
In the C NMR spectrum, a carbon signal
at d 196.3 (C-20) and two olefinic carbon
signals at d 144.6 and 155.5 were
observed, suggesting the existence of a
ketone group and one olefinic bond in the
molecule. The NMR data of 3 suggested
that it had the almost identical structure
with timopregnane A [20], except for the
ring A proportion. Compound 3 had one
more oxygen atom than timoprengane A
by comparing their molecular formula, and
the chemical shifts of C-1 (d 40.4), C-2 (d
USA), and the chemical shifts are expressed
in d (ppm) scale with TMS as an internal
reference. The HR-ESI-MS and ESI-MS
were recorded on Synapt Q/TOF MS
(Waters Corporation, Milford, MA, USA).
HPLC was undertaken with an Agilent 1100
(Agilent Technologies, Santa Clara, CA,
USA) equipped with Alltech 2000 ES
evaporative light-scattering detector (tem-
perature: 1108C, gas: 2.4l/min; Alltech
Corporation, Chicago, IL, USA) and Venu-
sil XBP C18 analytical column (4.6mm
£ 250 mm, 5 mm; Bonna-Agela, Tianjin,
China). Semi-preparativeHPLCseparations
were performed using the NP7000 module
(Hanbon Co. Ltd, Huaian, China) equipped
with a Shodex RID 102 detector (Showa
Denko Group, Tokyo, Japan) and a Vensiul
C18 column (8.0 mm £ 250 mm, ODS,
6
7.2), C-3 (d 81.9), C-9 (d 41.9), and C-10
d 37.2) inferred that 3 had a hydroxyl
(
group at C-2. The structure of ring A
proportion of 3 was further certified by
comparing its NMR data with those of
1
timosaponin N [14]. H NMR coupling
3
constants ( J . 7 Hz) for anomeric
protons revealed that both the glucose
1
,2

Journal of Asian Natural Products Research
907
5
mm; Bonna-Agela). Sephadex LH-20
20–100 mm; Pharmacia Fine Chemical
rate: 4.0 ml/min) with CH CN–H O
3
2
(
(21:79) as mobile phase to afford 3
(40 mg; t : 13.8 min), 4 (40 mg; t :
15.1 min), and 5 (38 mg; t : 20.1 min).
Co. Ltd, Piscataway, NJ, USA), Macropor-
ous resin SP825 (Mitsubishi-Kasei, Tokyo,
Japan), and silica gel (Qingdao Marine
Chemical, Qingdao, China) were used for
column chromatography (CC). TLC was
performed on precoated kieselgel GF254
plates (Qingdao Marine Chemical). Gas
chromatographic analyses were carried out
using an Agilent 6890N Network GC
system equipped with an FID detector and
an HP-5 capillary column (30 m
R
R
R
Fr. 27–29 (2.0 g) was divided into
supernatant and precipitation in the pro-
cess of evaporation under reduced press-
ure. The precipitation was dissolved in
(CH ) CO–H O (60:40) and purified by
3
2
2
crystallizing repeatedly to yield
6
(30.3 mg). The supernatant was separated
on silica gel CC (10 cm £ 30 cm) with a
solvent system of CHCl –MeOH–H O
3
2
£
0.25 mm £ 0.25 mm; Agilent Technol-
(70:15:2) to produce 100 fractions (Fr. s1–
s100). The mixed solution of Fr. s –s
ogies).
44
47
was separated by CC on Sephadex LH-20
eluted with MeOH to obtain 1 (27.4 mg)
eventually. Another mixed solution of Fr.
s –s was purified by semi-preparative
3
.2 Plant material
Plant material used in this research was
collected from Anguo, Hebei Province of
China, and was identified as rhizomes of A.
asphodeloides Bunge by Prof. Li-juan
Zhang (Tianjin University of Traditional
Chinese Medicine). A voucher specimen
5
8
66
RP-HPLC (column: 10 mm £ 250 mm,
Venusil XBP C18, 5 mm, flow rate:
4.0 ml/min) with MeOH–H O (75:25) as
2
mobile phase to obtain 2 (25.3 mg).
(
No. 040912) was deposited in the
3
.3.1 Timosaponin X (1)
herbarium of Beijing Institute of Radiation
Medicine, Beijing.
White amorphous powder, positive reac-
tion to Liebermann–Burchard test and
2
D
5
Ehrlich reagent tests; ½aꢀ
227.3
2
1
3
.3 Extraction and isolation
(
c ¼ 0.862, MeOH); IR (KBr, cm ):
Dried slices of rhizome of A. asphode-
loides (5 kg) were extracted with 40 l
water, for three times (3 £ 2 h). The crude
extract was concentrated under reduced
pressure and then subjected to macropor-
ous resin SP825 CC (10 cm £ 85 cm)
eluted with a gradient mixture of EtOH–
n
1174, 1077, 918, 785, 639; for H and
3400, 2931, 1638, 1451, 1379,
max
1
1
3
C NMR spectral data, see Tables 1 and 2;
ESI-MS (negative mode): m/z 889
2
2
[M 2 H–14] , 727 [M 2 H–14–162] ,
2
565 [M 2 H–14–162–162] , 433
2
[M 2 H–14–162–162–132] ; HR-ESI-
þ
H O (0:100, 20:80, 40:60, and 80:20) to
2
MS: m/z 873.4852 [M þ H–CH OH]
3
yield four fractions (Fr. A–D). Fr. C
(50.0 g) was further purified by DAC-150
(calcd for C H O –CH OH, 873.4848).
3
4
5
77 18
filled with ODS silica gel with a gradient
mixture (CH ) CO–H O (30:70 to 80:20)
3.3.2 Timosaponin Y (2)
3
2
2
to give four pooled fractions (F1–F4). F4
8.0 g) was separated on DAC-150 ODS
with (CH ) CO–H O (30:70) again to
White amorphous powder, positive reac-
tion to Liebermann–Burchard test and
(
2
5
anisaldehyde reagent tests. ½aꢀ 225.3
3
2
2
D
2
1
yield Fr. 1–37. Integrated Fr. 16–17
600 mg) was further purified by semi-
preparative RP-HPLC (column: 10 mm
150 mm; Venusil XBP C18, 5 mm, flow
(c ¼ 0.838, MeOH). IR (KBr, cm ): nmax
(
3412, 2931, 1638, 1453, 1383, 1070, 893,
1
13
769, 598; for H and C NMR spectral
data, see Tables 1 and 2; ESI-MS (positive
£

9
08
J.-C. Yuan et al.
þ
mode): m/z 795 [M þ Na] , 611 [M þ H–
column (30 m £ 0.25 mm £ 0.25 mm). The
conditions are as follows: column tem-
perature, 1808C/2508C; programmed
increase, 38C/min; carrier gas, N (1 ml/
2
min); injection and detector temperature,
2508C; injection volume, 4.0 ml; split ratio,
þ
þ
1
62] , 449 [M þ H–162–162] , 431
þ
[
M þ H–162–162–18] , 413 [M þ H–
þ
1
7
62–162–18–18] ; HR-ESI-MS: m/z
71.4167
2
[M 2 H]
(calcd
for
C H O , 771.4167).
39 63 15
1:50. The absolute configurations were
identified to be D-glucose, D-galactose,
and L-arabinose by comparison of the
retention times of monosaccharide deriva-
tives with those of standard samples:
D-glucose (20.22 and 21.95 min), D-galac-
tose (20.81 and 22.51 min), and L-arabi-
nose (15.09 and 16.92 min).
3
.3.3 Timopregnane B (3)
White amorphous powder, positive reac-
tion to Liebermann–Burchard test. ½aꢀ
2
D
5
2
cm ): n
36.5 (c ¼ 0.946, MeOH). IR (KBr,
2
1
3392, 2933, 1660, 1440,
max
1
372, 1166, 1076, 934, 893, 604; for H
1
and C NMR spectral data, see Tables 1
and 2; ESI-MS (negative mode) m/z: 655
1
3
2
2
M 2 H] , 493 [M 2 H–162] , 331
[
[
2
M 2 H–162–162] ; HR-ESI-MS: m/z
Acknowledgments
2
The investigation was financially supported by
National Natural Science Foundation of China
6 [M 2 H]
C H O , 655.3330).
55.3353
(calcd
for
3
3 51 13
(
No. 81274053) and Major National Science
and Technology Projects (Grant Nos
009ZX09102-106 and 2011ZX09102-002-
2
3
.4 Acid hydrolysis of compounds 1–3
09). The NMR spectra were recorded at the
National Center of Biomedical Analysis. The
assistance of the staff is also gratefully
acknowledged.
Compounds 1–3 (each 2.0 mg) were
treated in 2 M trifluoroacetic acid (5 ml)
at 958C for 5 h, respectively. After cooling,
the hydrolyzed reaction mixture was
extracted with CH Cl and H O. Then, in
2
2
2
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