Synthesis of carbohydrate-based natural products from leonurus japonicus and their biological evaluation as anti-oxidants

Article abstract of DOI:10.1071/CH13621

Natural products are important materials that have found a wide variety of uses, especially in medicine. Traditional Chinese medicine (TCM) has especially taken advantage of natural products and compounds found in Leonurus, a species of herb used extensiv

Full text of DOI:10.1071/CH13621

CSIRO PUBLISHING
Aust. J. Chem. 2014, 67, 1461–1470
Full Paper
http://dx.doi.org/10.1071/CH13621
Synthesis of Carbohydrate-based Natural Products
from Leonurus japonicus and their Biological
Evaluation as Anti-oxidants
A
A
A
Evette Clayton, Mitchell Hattie, Aleksandra W. Debowski,
A B -
, ,
and Keith A. Stubbs
A
School of Chemistry and Biochemistry, The University of Western Australia,
35 Stirling Highway, Crawley, WA 6009, Australia.
B
Corresponding author. Email: keith.stubbs@uwa.edu.au
y
The author was invited to contribute to the ‘RACI Series’ following receipt of the Rennie
Memorial Medal by the Royal Australian Chemical Institute.
Natural products are important materials that have found a wide variety of uses, especially in medicine. Traditional
Chinese medicine (TCM) has especially taken advantage of natural products and compounds found in Leonurus, a species
of herb used extensively in TCM to treat various ailments. Herein we describe the synthesis of three natural products from
Leonurus japonicus and our investigation of their hepatoprotective properties.
Manuscript received: 11 November 2013.
Manuscript accepted: 5 December 2013.
Published online: 17 January 2014.
Introduction
carbohydrate component of Leonurus japonicus and reported
that four compounds Cistanoside E 1, Leonoside F 2, Leonoside
E 3, and Verbascoside 4 (Fig. 1) exhibited hepatoprotective
properties.[6] Cistanoside E 1 and Verbascoside 4 have been
previously isolated [7,8] from other plant materials; the synthesis
of Verbascoside 4 has been reported.[7] However, despite the
biological activity of Cistanoside E 1, its chemical synthesis has
yet to be reported. In the present study, Cistanoside E 1,
Leonoside F 2, and Leonoside E 3 were selected as candidates
for synthesis to study their biological properties further. Recent-
ly, the synthesis of Leonoside F 2 and Leonoside E 3 has been
reported,[9] however, no rigorous studies relating to the bio-
logical activity of these compounds were discussed. Ggiven the
importance of access to these compounds for biological studies,
it seemed to us that a route for the preparation of them was
warranted.
Natural products are important both historically and currently as
people rely on nature as a means to every end, from food and
shelter to clothing and tools. Furthermore, nature was once the
only source of treatments for a wide variety of diseases through
the use of traditional medical systems.[1] Although there are now
many techniques in modern medicine, nature continues to
represent a valuable resource of materials for medical research.
Traditional medical systems use methods of preparing and
testing therapeutics, which differ greatly from modern techni-
ques. Even though traditional therapeutics may not be imme-
diately translatable to modern medicines, they still may
represent very useful starting points for drug discovery.[2] Tra-
ditional Chinese Medicine (TCM) dates back to 1100 BCE and is
an example of a traditional medical system based on the sys-
tematic testing of natural materials, which has been well docu-
mented for thousands of years.[3] As such, TCM has been used as
a starting point for investigating natural products that may be
useful for drug development.
Leonurus is a specific group of herbs that are highly impor-
tant in TCM.[4] This genus contains numerous species that can
be found throughout Europe and Asia, as well as in some regions
of America and Africa, where these species have been incorpo-
rated in traditional medicine systems.[4] The history and copious
use of Leonurus herbs in traditional medicine have made them
interesting candidates for modern scientific research. Numerous
species of Leonurus have been used to study the effects of the
intrinsic natural products, and extracts of various species have
shown that Leonurus contains biologically active labdane-type
diterpenoids, iridoids, flavonoids and flavonoid glycosides,
cyclic peptides, alkaloids, and phenylethyl glycosides.[5,6] Of
interest to us was a recent study by Li et al., who examined the
Results and Discussion
Our attention was first directed to the synthesis of 1 and 2, and
we identified the disaccharide 5 was as the key intermediate in
the synthesis of both molecules. The disaccharide 5[10] was
easily prepared from the allyl glycoside 6[11,12] and tri-
chloroacetimidate 7[13] using the procedures reported in the
literature (Scheme 1).
The disaccharide 5 was then treated with glacial acetic acid to
yield the diol 8 in good yield. For the second glycosylation
(towards the synthesis of Leonoside F 2), the diol was treated
with tert-butyldimethylsilyl chloride, producing the silyl ether 9
in excellent yield. Acetylation of 9 using acetic anhydride in
pyridine gave the fully protected disaccharide 10. Finally,
removal of the silyl ether with tetrabutylammonium fluoride
from 10 gave the alcohol 11 in excellent yield. For the synthesis
Journal compilation Ó CSIRO 2014
www.publish.csiro.au/journals/ajc

1462
E. Clayton et al.
OH
O
OH
O
HO
O
HO
HO
OMe
OH
O
O
O
O
HO
HO
O
HO
O
O
OMe
OH
HO
HO
HO
OH
HO
1
HO
OH
2
O
OH
O
OH
O
HO
HO
HO
O
OMe
O
O
O
OMe
O
HO
HO
O
O
O
OH
HO
HO
OH
HO
O
HO
OH
HO
HO
3
4
OH
Fig. 1. Carbohydrate-based hepatoprotective compounds from Leonurus japonicus.
O
Ph
O
O
HO
BzO
OAll
OR1
O
OAc
O
O
Ph
O
6
R2O
O
AcO
O
O
(a)
(b)
O
O
O
(d)
ϩ
BzO
BzO
BzO
O
71 %
OAll
OAll
OAll
84 %
AcO
AcO
AcO
AcO
O
CCl3
NH
AcO
AcO
AcO
OAc
OAc
R1
8
9
10
11
OAc
O
AcO
5
12
R2
AcO
OAc
H
H
H
98 % (c)
85 % (d)
86 % (e)
OAc
O
TBDMS
7
TBDMS Ac
(f)
40 %
O
AcO
H
Ac
AcO
AcO
O
OAc
O
O
AcO
AcO
BzO
AcO
CCl3
NH
OAll
O
O
AcO
13
AcO
OAc
14
Scheme 1. (a) TMSOTf, CH2Cl2. (b) CH3COOH/H2O (4 : 1). (c) TBDMSCl, DMAP, CH2Cl2. (d) Ac2O, C5H5N. (e) TBAF, CH3COOH, THF. (f) TMSOTf,
CH2Cl2.
of Cistanoside E 1, the diol 8 was acetylated directly to give the
benzoate 12. The alcohol 11 was then treated with the trichlor-
oacetimidate 13[14] under the conditions employed for glycosyl-
ation to give the trisaccharide 14 in good yield. With both the
desired compounds 12 and 14 in hand, attention was directed
towards the removal of their respective allyl glycosides. A
procedure involving palladium(II) chloride in methanol, a
standard method for the removal of allyl ethers,[12] was first
attempted using 12. However, this method resulted in the
formation of several products, as identified by thin layer
chromatography (TLC). A significant decrease in pH was also
observed. Based on this observation, it was rationalised that both
deacetylation and allyl glycoside removal were occurring.
Because it was necessary to perform the reaction on compounds
in the presence of acetyl groups, a different method was
investigated. It was hoped that the conditions of Ban and
Mrksich,[15] in which a solution of sodium acetate and acetic
acid is used as a buffer, would aid in the formation of the desired
hemiacetal. Gratifyingly, the hemiacetal 15 was obtained in
good yield in the absence of by-products; hemiacetal 16 was also
successfully synthesized from 14 (Scheme 2). Hemiacetals 15
and 16 were then converted to their corresponding
trichloroacetimidates. The latter were then used in situ as
glycosyl donors for the glycosylation of the protected homo-
vanillyl alcohol derivative 17[16] to yield 18 and 19, respective-
ly. Finally, the protecting groups were removed using sodium
methoxide in methanol to give Cistanoside E 1 and Leonoside F
2 in good yields (85 % and 50 %, respectively). The 1H and 13
C
NMR spectra for the prepared compounds were consistent with
those observed from natural sources of 1[8] and 2,[6] and
synthetic sources for 2.[9]
We then turned our attention to the synthesis of Leonoside E
3, which differs from the synthesis of Cistanoside E 1 and
Leonoside F 2. We felt that a suitably prepared L-rhamnose
derivative 20 could be used as a linker molecule to allow for
glycosylation to the parent glucose moiety of Leonoside E 3, and
the chloroacetyl moiety at O-2 of 20 could be selectively
removed, with the resultant alcohol, in turn, used as a glycosyl
acceptor for the arabinose portion of the molecule. Thus, using
procedures described in the literature,[17] the alcohol 21 was
prepared from L-rhamnose in good yield (Scheme 3). Treatment
of 21 with chloroacetyl chloride under basic conditions gave the
desired allyl glycoside 20 in good yield. Using the conditions
described above, the hemiacetal 22 was prepared from 20 in

Synthesis of natural products from Leonurus japonicus
1463
OAc
O
OAc
O
OR1
O
AcO
AcO
O
R1O
O
O
(a)
O
(b)
(c)
O
OMe
OR1
O
BzO
BzO
R2O
OH
33 %
79 %
O
OAll
AcO
AcO
R1O
OMe
HO
AcO
AcO
R1O
OAc
R1
Ac
H
R2
Bz
H
OAc
OR1
OAc
18
1
15
17
12
(d)
85 %
OR1
O
OAc
O
OAc
O
R1O
AcO
AcO
AcO
AcO
O
O
O
O
O
O
R1O
AcO
AcO
R1O
R1O
AcO
AcO
O
O
O
BzO
(b)
(c)
(a)
OMe
OR1
O
87 %
15 %
OMe
R2O
BzO
OH
OAll
O
O
O
R1O
AcO
AcO
HO
R1O
AcO
AcO
OR1
OAc
OAc
OAc
17
R1
Ac
H
R2
Bz
H
19
2
14
16
50 % (d)
Scheme 2. (a) PdCl2, CH3COOH, NaOAc, H2O. (b) CCl3CN, DBU, CH2Cl2. (c) TMSOTf, CH2Cl2. (d) NaOMe, MeOH.
O
Ph
O
BzO
OAll
O
O
OH
Ph
O
O
O
(d)
(b)
(c)
(f)
O
O
O
BzO
BzO
O
OAll
BzO
46 %
91 %
30 %
BzO
AcO
AcO
OAll
O
OR
OAcCl
AcO
O
BzO
AcO
AcO
Ph
O
O
O
O
O
AcO
HO
22
R
H
AcCl
AcO
AcO
26
BzO
OAll
OR
AcO
CCl3
O
21
20
OAc
65 % (a)
6
R
25NH
23 AcCl
24
(e)
74 %
H
Scheme 3. (a) ClCH2COCl, CH2Cl2, C5H5N. (b) PdCl2, CH3COOH, NaOAc, H2O. (c) CCl3CN, DBU, CH2Cl2. (d) TMSOTf, CH2Cl2. (e) SC(NH2)2,
2,6-lutidine, MeOH, CH2Cl2. (f) TMSOTf, CH2Cl2.
OAc
O
OR1
O
Ph
O
O
AcO
O
R1O
O
O
O
O
OMe
OR1
O
BzO
BzO
R2O
OAll
O
(a)
OR
(c)
(d)
O
O
BzO
BzO
R2O
82 %
AcO
O
AcO
O
R1O
O
O
O
R1
[29] Ac
R2
Bz
H
AcO
AcO
AcO
AcO
81 % (b)
26
OMe
OAc
R1O
R1O
HO
OAc
OAc
10 % (e)
over three
steps
OR1
3
H
17
R
All
H
27
28
Scheme 4. (a) i. CH3COOH/H2O (4 : 1); ii. Ac2O, C5H5N. (b) PdCl2, CH3COOH, NaOAc, H2O. (c) CCl3CN, DBU, CH2Cl2. (d) TMSOTf, CH2Cl2.
(e) NaOMe, MeOH.
excellent yield. The hemiacetal 22 was then converted to its
corresponding trichloroacetimidate and used in situ as a glyco-
syl donor for the glycosylation of 6 to give the disaccharide 23.
Selective removal of the chloroacetyl group using a procedure
involving thiourea and 2,6-lutidine[18] gave the alcohol 24,
which was then treated with the trichloroacetimidate 25[19]
under conditions of glycosylation to give the trisaccharide 26
in good yield.
The benzylidene acetal was subsequently removed from 26,
followed by in situ acetylation to give the diacetate 27. Using the
conditions described above, the hemiacetal 28 was prepared
from 27 in excellent yield (Scheme 4). The hemiacetal 28 was
then converted to its corresponding trichloroacetimidate and
used in situ as a glycosyl donor for the glycosylation of the
protected homovanillyl alcohol derivative 17[16] to give the
trisaccharide 29. The latter compound was slightly conta-
minated with residual 17 (based on the RF value) following
purification. Finally, the protecting groups were removed using
sodium methoxide in methanol, generating Leonoside E 3 in
good yield. The 1H and 13C NMR spectra for the compound were
consistent with those observed for the naturally occurring[6] and
synthetic[9] compounds.
With the desired compounds in hand, we then directed our
attention to studying in more detail the hepatoprotective nature
of these compounds. Hepatoprotection is the ability (of a
material) to prevent damage to the liver. One pathway
that leads to damage within cells, particularly in the liver, is
mediated by oxidative stress.[20] Oxygen-free radicals and
other reactive oxygen species (ROS) are released as by-products
of numerous physiological processes.[21,22] However, excess
ROS can result in oxidative stress, as evidenced by DNA
degradation, lipid peroxidation, and protein damage.[23] Under
conditions of disease, the liver is particularly vulnerable to
oxidative stress and recent investigations have shown that
oxidant-induced liver injuries are mediated by the direct effects
of reactive oxygen species on signal transduction pathways.[24]
Therefore, we assessed the effects of the three compounds
1–3, at various concentrations, on indicators of oxidative stress

1464
E. Clayton et al.
(a) 10
7.5
(c)
8
6
4
2
0
∗
∗
5.0
∗
∗
2.5
0
Control
1
10
100
1000
1
3
Concentration of compound [µM]
Time [h]
(b) 35
30
No treatment
25
Treatment with H2O2 only
α-tocopherol
20
∗
Cistanoside E 1
Leonoside F 2
15
10
5
∗
Leonoside E 3
0
Control
1
10
100
1000
Concentration of compound [µM]
Fig. 2. Effect of compounds 1–3 at various concentrations on intracellular ROS production in HepG2 cells in the
presence of H2O2 at (a) 60 min and (b) 3 h. (c) Effect of compound 2 (100 mM) on catalase activity in the presence of
H2O2. Results are shown as (mean ꢀ standard deviation) of three replicates; significance of the results relating to
treatment with H2O2 was determined by ANOVA and Bonferroni–Dunn post hoc tests (p # 0.05).
in HepG2 cells (Fig. 2a, b). The oxidative stress agent H2O2,
which has been demonstrated previously as a good model for
oxidative stress in liver cells, was used.[25] Upon treatment
with H2O2, ROS production increased compared with cells that
were subjected to no stress at both time points studied (Fig. 2a,
b). Cells that had been pretreated with compound 1–3 or
a-tocopherol, a known inhibitor of ROS generation,[26] were also
stressed with H2O2 (250mM), and the amount of ROS was
measured. Interestingly, there was a dose-dependent effect for
each compound 1–3 studied; compound 2 gave the best result not
only at both time points, but also at the lowest effective concen-
tration (100 mM). Importantly, there was no loss in cell viability
for the compounds tested at the highest concentration (1mM).
To determine the effect of compound 2 on the activity of
endogenous anti-oxidants, the activity of catalase was mea-
sured. Catalase is an endogenous anti-oxidant enzyme that plays
a pivotal role in preventing cellular damage caused by ROS.[27]
We assessed the effects of compound 2 (100 mM) on catalase
activity in cells stressed with H2O2 over a 3-h period. Relative to
untreated controls, enzyme activity decreased in cells exposed
to compound 2 (Fig. 2c). These observations are supported by
the decrease in ROS formation observed in earlier experiments.
It is likely that over the 3-h time period, the amount of enzyme
produced in response to H2O2 stress is reduced because of a
reduction in the concentrations of ROS present, with Leonoside
F 2 found to have the best effect in controlling ROS generation.
Conclusion
In conclusion, we have demonstrated new syntheses for
three important carbohydrate-based natural products found in
Leonurus herbs, with the ease of preparation making this route
particularly attractive. Using oxidative stress assays we have
putatively found reasons for the hepatoprotective properties of
these compounds. All compounds were able to reduce oxidative
stress, and these results set the stage for further investigations
into the biological roles of these molecules, something our
laboratory is actively pursuing.
Experimental
1H and 13C NMR spectra were obtained on a Bruker ARX500
(500 MHz for H and 125.7 MHz for 13C) or a Bruker AV600
1
(600 MHz for 1H and 150.8 MHz for 13C) spectrometer. Unless
stated otherwise, deuterated chloroform (CDCl3) was used as
the solvent with CHCl3 (dH 7.26) or CDCl3 (dC 77.16) employed
as internal standards. For deuteriomethanol (CD3OD),
CD2HOD (1H, d 3.30), or CD3OD (13C, d 49.0) were employed
as internal standards. Mass spectra were recorded on a Waters
LCT Premier XE spectrometer, in W-mode, using the ESI
method, with CH3CN/H2O (9 : 1) as a matrix. IR spectra were
obtained using a PerkinElmer Spectrum One FT-IR spectrom-
eter fitted with a PerkinElmer Universal ATR sampling acces-
sory. Elemental analyses were performed at the Robertson

Synthesis of natural products from Leonurus japonicus
1465
Microanalytical Facility. Flash chromatography was performed
on silica gel (BDH) with the specified solvents. Thin layer
chromatography (TLC) was conducted on silica gel 60 F254
(Merck) and aluminium-backed plates that were stained by
heating (.2008C) with 5 % sulfuric acid in ethanol. Percentage
yields for chemical reactions as described are quoted only for
compounds that were purified by recrystallization or by column
chromatography, and purity was assessed by TLC or 1H NMR
spectroscopy.
81.0, 73.0, 71.2, 71.2, 71.0, 70.0, 68.7, 68.5, 67.4, 63.8 (C2, C20,
C3, C30, C4, C40, C5, C50, C6, CH2), 26.1 (SiC(CH3)3), 20.9
(C(O)CH3), 20.8 (C(O)CH3), 20.7 (C(O)CH3), 18.5 (SiC
(CH3)3), 17.4 (CH3), ꢂ5.2 (Si(CH3)2), ꢂ5.3 (Si(CH3)2). m/z
(HR-MS) (ESI) 711.3073; [MþH]þ requires 711.3048.
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-4-O-acetyl-
2-O-benzoyl-6-O-tert-butyldimethylsilyl-a-D-glucoside 10
Acetic anhydride (0.33 mL, 3.5 mmol) was added dropwise to a
solution of 9 (0.51 g, 0.72 mmol) in pyridine (5 mL) and stirred
at 408C for 24 h. After the reaction was complete (as assessed by
TLC), the reaction mixture was quenched with methanol
(MeOH, 10 mL) and concentrated. The residue was suspended
in EtOAc (30 mL) and washed with hydrochloric acid (1 M,
2 ꢁ 30 mL), saturated NaHCO3 solution (3 ꢁ 30 mL), and brine
(2 ꢁ 30 mL), dried over MgSO4, filtered, and concentrated.
Flash chromatography of the resulting oil (2 : 3 EtOAc : hexane
eluent) afforded, after concentration of the appropriate fractions
(RF 0.5), 10 as a colourless oil (0.46 g, 85 %). nmax (neat)/cmꢂ1
2930 (w), 2857 (w), 1748 (s). dH (500 MHz, CDCl3) 8.06–8.03
(m, 2H, Ar), 7.57–7.53 (m, 1H, Ar), 7.45–7.41 (m, 2H, Ar),
5.84–5.77 (m, 1H, CH¼CH2), 5.28–5.22 (m, 1H, CH¼CH2),
5.20 (d, J 3.7, 1H, H1), 5.14–4.93 (m, 7H, H10, H2, H20, H30, H4,
H40, CH¼CH2), 4.35 (dd, J 11.3, 11.3, 1H, H3), 4.21–4.14 (m,
1H, CH2), 4.00–3.94 (m, 1H, CH2), 3.89–3.82, 3.67–3.65 (2 m,
4H, H5, H50, H6, H6), 2.10 (s, 3H, C(O)CH3), 1.97 (s, 3H, C(O)
CH3), 1.92 (s, 3H, C(O)CH3), 1.77 (s, 3H, C(O)CH3), 1.15
(d, J 7.5, 3H, CH3), 0.90 (s, 9H, C(CH3)3), 0.05 (s, 6H, Si
(CH3)2). dC (125.7 MHz, CDCl3) 170.2 (C¼O), 169.6 (C¼O),
169.5 (C¼O), 169.4 (C¼O), 165.5 (C¼O), 133.5, 133.3
(CH¼CH2, Ar), 130.1 (Ar), 129.4 (Ar), 128.5 (Ar), 117.8
(CH¼CH2), 99.4, 94.8 (C1, C10), 77.7, 73.5, 71.1, 70.9, 70.1,
69.9, 68.6, 68.5, 67.4, 62.9 (C2, C20, C3, C30, C4, C40, C5, C50,
C6, CH2), 26.0 (SiC(CH3)3), 21.2 (C(O)CH3), 20.9 (C(O)CH3),
20.8 (C(O)CH3), 20.5 (C(O)CH3), 18.5 (SiC(CH3)3), 17.5
(CH3), ꢂ5.2 (Si(CH3)2), ꢂ5.3(Si(CH3)2). m/z (HR-MS) (ESI)
791.2711; [MþK]þ requires 791.2713.
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-2-O-
benzoyl-a-D-glucopyranoside 8
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-2-O-benzoyl-4,
6-O-benzylidene-a-D-glucoside 5[10] (0.50 g, 0.73 mmol) was
treated with CH3COOH/H2O (4 : 1, 20 mL) and the resulting
solution was stirred at 708C for 2 h. After the reaction was
complete (as assessed by TLC), the reaction mixture was con-
centrated and the residue co-evaporated with toluene
(3 ꢁ 25 mL). Flash chromatography of the resulting oil (13 : 7
ethyl acetate (EtOAc) : hexane eluent) afforded, after concen-
tration of the appropriate fractions (RF 0.55), 8 as a white foam
(0.31 g, 71 %). nmax (neat)/cmꢂ1 3475 (w), 2935 (w), 1745 (s),
1724 (s). dH (600 MHz, CDCl3) 8.07–8.04 (m, 2H, Ar), 7.59–
7.55 (m, 1H, Ar), 7.46–7.43 (m, 2H, Ar), 5.84–5.77 (m, 1H,
CH¼CH2), 5.27–5.21, 5.05–4.99 (2 m, 7H, CH¼CH2, H1, H10,
H2, H20, H30, H40), 5.14–5.11 (m, 1H, CH¼CH2), 4.19–4.12,
3.92–3.86, 3.80–3.70 (3 m, 7H, H3, H4, H5, H50, H6, H6, CH2),
3.99 (dddd, J 1.5, 1.5, 6.0, 13, 1H, CH2), 3.56 (d, J 3, 1H, OH),
2.09 (dd, J 5.5, 5.5, 1H, OH), 2.02 (s, 3H, C(O)CH3), 1.98 (s, 3H,
C(O)CH3), 1.89 (s, 3H, C(O)CH3), 1.25 (d, J 6.5, 3H, CH3). dC
(150.8 MHz, CDCl3) 170.0 (C¼O), 169.7 (C¼O), 166.0 (C¼O),
133.5 (CH¼CH2), 133.4 (Ar), 130.0 (Ar), 129.5 (Ar), 128.6
(Ar), 117.9 (CH¼CH2), 99.3, 95.4 (C1, C10), 81.9, 72.7, 71.3,
71.1, 70.1, 69.9, 68.5, 67.8 (C2, C20, C3, C30, C4, C40, C5, C50),
68.8 (CH2), 62.4 (C6), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.7
(C(O)CH3), 17.6 (CH3). m/z (HR-MS) (ESI) 597.2186;
[MþH]þ requires 597.2183.
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-2-O-
benzoyl-6-O-tert-butyldimethylsilyl-a-D-glucopyranoside 9
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-4-O-acetyl-
2-O-benzoyl-a-D-glucoside 11
tert-Butyldimethylsilyl chloride (0.22 g, 1.5 mmol), 4-dime-
thylaminopyridine (DMAP; 15 mg) and triethylamine (0.70 mL,
5.0 mmol) were added to a solution of 8 (0.49 g, 0.82 mmol) in
CH2Cl2 (5 mL) at 08C, and then stirred at room temperature for
14 h. After the reaction was complete (as assessed by TLC), the
reaction mixture was diluted in CH2Cl2 (30 mL) and washed
with water (20 mL), saturated NaHCO3 solution (3 ꢁ 20 mL),
and brine (20 mL), dried over MgSO4, filtered, and concen-
trated. Flash chromatography of the resulting oil (1 : 1 EtOAc :
hexane eluent) afforded, after concentration of the appropriate
fractions (RF 0.5, 2 : 3 EtOAc : hexane eluent), 9 as a white
foam (0.57 g, 98 %). nmax (neat)/cmꢂ1 2934 (w), 1748 (s).
dH (500 MHz, CDCl3) 8.05–8.03 (m, 2H, Ar), 7.57–7.54 (m, 1H,
Ar), 7.45–7.41 (m, 2H, Ar), 5.84–5.76 (m, 1H, CH¼CH2),
5.27–5.20, 5.05–4.99 (2 m, 6H, H10, H2, H20, H30, H40,
CH¼CH2), 5.18 (d, J 3.5, 1H, H1), 5.12–5.10 (m, 1H,
CH¼CH2), 4.23–4.14, 3.98–3.95, 3.73–3.69 (3 m, 8H, H3, H4,
H5, H50, H6, H6, CH2), 3.48 (d, J 2.5, 1H, OH), 2.02 (s, 3H, C(O)
CH3), 1.97 (s, 3H, C(O)CH3), 1.89 (s, 3H, C(O)CH3), 1.22 (d,
J 6.5, 3H, CH3), 0.93 (s, 9H, C(CH3)3), 0.12 (s, 6H, Si(CH3)2).
dC (125.7 MHz, CDCl3) 170.1 (C¼O), 169.7 (C¼O), 169.6
(C¼O), 165.9 (C¼O), 133.7, 133.3 (CH¼CH2, Ar), 130.0 (Ar),
129.6 (Ar), 128.5 (Ar), 117.7 (CH¼CH2), 99.2, 95.3 (C1, C10),
A solution of tetra-N-butylammonium fluoride (TBAF) in THF
(1 M, 0.87mL, 0.87mmol) and CH3COOH (0.87 mL) were
added dropwise to a solution of 10 (0.58 g, 0.81mmol) in THF
(2 mL) at 08C, and stirred at room temperature overnight. The
reaction mixture was then concentrated and suspended in EtOAc
(30 mL) and washed with water/brine (30 mL, 1 : 1), saturated
NaHCO3 solution (30 mL), and brine (30 mL), dried over
MgSO4, filtered, and concentrated. Flash chromatography of the
resulting oil (1 : 1 EtOAc : hexane eluent) afforded, after con-
centration of the appropriate fractions (RF 0.27), 11 as a white
foam (0.45 g, 86%). nmax (neat)/cmꢂ1 3524 (w), 2938 (w), 1746
(s). dH (600 MHz, CDCl3) 8.04–8.02 (m, 2H, Ar), 7.56–7.54
(m, 1H, Ar), 7.43–7.41 (m, 2H, Ar), 5.82–5.76 (m, 1H,
CH¼CH2), 5.26–5.23 (m, 2H, H1, CH¼CH2), 5.13–4.99 (m, 6H,
H10, H2, H20, H30, H4, CH¼CH2), 4.94 (dd, J 9.6, 9.6, 1H, H40),
4.40 (dd, J 9.6, 9.6, 1H, H3), 4.16 (ddd, J 1.2, 5.4, 13.2, 1H, CH2),
3.98 (ddd, J 1.8, 6.0, 13.2, 1H, CH2), 3.91–3.86 (m, 1H, H50),
3.78 (ddd, J 2.4, 4.2, 10.2, 1H, H5), 3.71–3.67 (m, 1H, H6), 3.59
(ddd, J 4.8, 4.8, 13.2, 1H, H6), 2.46 (dd, J 5.4, 8.4, 1H, OH), 2.13
(s, 3H, C(O)CH3), 1.98(s, 3H, C(O)CH3), 1.93(s, 3H, C(O)CH3),
1.78 (s, 3H, C(O)CH3), 1.16 (d, J 6.0, 3H, CH3). dC (150.8 MHz,
CDCl3) 170.8 (C¼O), 170.1 (C¼O), 169.5 (C¼O), 169.4
(C¼O), 165.5 (C¼O), 133.4 (CH¼CH2), 133.3 (Ar), 130.0 (Ar),

1466
E. Clayton et al.
129.3 (Ar), 128.5 (Ar), 118.0 (CH¼CH2), 99.1, 95.3 (C1, C10),
76.7 (C3), 73.3, 71.0, 70.0, 69.9, 69.8, 68.5 (C2, C20, C30, C4,
C40, C5), 68.9 (CH2), 67.3 (C50), 61.3 (C6), 21.0 (C(O)CH3), 20.8
(C(O)CH3), 20.7 (C(O)CH3), 20.5 (C(O)CH3), 17.4 (CH3). m/z
(HR-MS) (ESI) 677.1811; [MþK]þ requires 677.1848.
(C¼O), 170.1 (C¼O), 169.9 (C¼O), 169.6 (C¼O), 169.5
(C¼O), 169.4 (C¼O), 169.3 (C¼O), 165.5 (C¼O), 133.4, 133.3
(CH¼CH2, Ar), 130.1 (Ar), 129.3 (Ar), 128.5 (Ar), 118.0
(CH¼CH2), 101.1, 99.3, 94.7 (C1, C10, C100), 77.3, 73.3, 72.8,
72.0, 71.2, 71.0, 70.0, 69.8, 69.0, 68.5, 67.4 (C2, C20, C200, C3,
C30, C300, C4, C40, C400, C5, C50, C500), 68.8, 68.4, 62.0 (C6, C600,
CH2) 21.1 (C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.7
(C(O)CH3), 20.7 (C(O)CH3), 20.5 (C(O)CH3), 17.5 (CH3). m/z
(HR-MS) (ESI) 991.3113; [MþNa]þ requires 991.3059. Anal.
Calc. for C44H56O24: C 54.54, H 5.83. Found: C 54.42, H 5.95 %.
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-4,6-di-
O-acetyl-2-O-benzoyl-a-D-glucoside 12
Acetic anhydride (0.27 mL, 2.9 mmol) was added to a solution
of the diol 8 (0.55 g, 0.92 mmol) in pyridine (2 mL) and CH2Cl2
(4 mL) at 08C, and the resulting solution was stirred at room
temperature for 1 h. After the reaction was complete (as assessed
by TLC), the reaction mixture was quenched with MeOH
(10 mL) and concentrated. The residue was suspended in EtOAc
(30 mL) and washed with hydrochloric acid (1 M, 3 ꢁ 20 mL),
water (20 mL), saturated NaHCO3 solution (3 ꢁ 20 mL), and
brine (20 mL), dried over MgSO4, filtered, and concentrated.
Flash chromatography of the resulting oil (EtOAc : hexane ¼
2 : 3) afforded, after concentration of the appropriate fractions
(RF 0.45), 12 as a colourless oil (0.53 g, 84 %). nmax (neat)/cmꢂ1
2983 (w), 1742 (s). dH (500 MHz, CDCl3) 8.06–8.04 (m, 2H,
Ar), 7.58–7.54 (m, 1H, Ar), 7.45–7.42 (m, 2H, Ar), 5.84–5.76
(m, 1H, CH¼CH2), 5.30–5.09, 5.02–4.91 (2 m, 9H, H1, H10, H2,
H20, H30, H4, H40, CH¼CH2), 4.35 (dd, J 9.5, 9.5, 1H, H3), 4.24
(dd, J 4.5, 12.5, 1H, H6), 4.18–4.08, 4.01–3.97 (2 m, 4H, H5, H6,
CH2), 3.90–3.82 (m, 1H, H50), 2.12 (s, 3H, C(O)CH3), 2.11
(s, 3H, C(O)CH3), 1.98 (s, 3H, C(O)CH3), 1.94 (s, 3H, C(O)
CH3), 1.78 (s, 3H, CH3), 1.16 (d, J 6.0, 3H). dC (125.7 MHz,
CDCl3) 171.0 (C¼O), 170.1 (C¼O), 169.6 (C¼O), 169.5
(C¼O), 169.4 (C¼O), 165.5 (C¼O), 133.4, 133.2 (CH¼CH2,
Ar), 130.1 (Ar), 129.2 (Ar), 128.5 (Ar), 118.2 (CH¼CH2), 99.4,
95.3 (C1, C10), 77.3, 73.2, 71.0, 69.9, 69.2, 69.1, 68.5, 67.9, 67.4,
62.2, (C2, C20, C3, C30, C4, C40, C5, C50, C6, CH2), 21.1 (C(O)
CH3), 21.0 (C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.5
(C(O)CH3), 17.5 (CH3). m/z (HR-MS) (ESI) 681.2389;
[MþH]þ requires 681.2395. Anal. Calc. for C32H40O16: C
56.47, H 5.92. Found: C 56.22, H 5.98 %.
2,3,4-Tri-O-acetyl-a-L-rhamnosyl-(1-3)-4,6-di-O-acetyl-
2-O-benzoyl-a/b-D-glucose 15
Palladium(II) chloride (80 mg, 0.45 mmol) was added to a
solution of the disaccharide 12 (0.29 g, 0.43 mmol) in a solution
of CH3COOH, NaOAc, and H2O (35 mL, 1.4 g, and 1.9 mL,
respectively), and stirred at room temperature for 30 h. After the
reaction was complete (as assessed by TLC), the reaction mix-
ture was filtered through celite and concentrated. The residue
was suspended in CH2Cl2 (50 mL) and washed with water,
hydrochloric acid (1M, 3 ꢁ 20 mL), water (20 mL), saturated
NaHCO3 (2 ꢁ 20 mL), and brine (30 mL), dried over MgSO4,
filtered, and concentrated. Flash chromatography of the result-
ing oil (2 : 3 EtOAc: hexane eluent) afforded, after concentration
of the appropriate fractions (RF 0.22), 15 as a colourless
glass (0.22 g, 79 %). nmax (neat)/cmꢂ1 2984 (w), 1747 (s).
dH (600 MHz, CDCl3) (major anomer) 8.08–8.03 (m, 2H, Ar),
7.60–7.50 (m, 1H, Ar), 7.46–7.40 (m, 2H, Ar), 5.60 (dd, J 3.6,
3.6, 1H, H1), 5.24–5.08, 5.03–4.92 (2 m, 6H, H10, H2, H20, H30,
H4, H40), 4.39 (dd, J 9.6, 9.6, 1H, H3), 4.28–4.19, 4.17–4.07,
3.92–3.82 (3 m, 4H, H5, H50, H6, H6) 3.19 (d, J 3.6, 1H, OH),
2.12 (s, 3H, C(O)CH3), 2.11 (s, 6H, C(O)CH3), 1.98 (s, 3H, C(O)
CH3), 1.95 (s, 3H, C(O)CH3), 1.78 (s, 3H, C(O)CH3), 1.17 (d, J
6.0, 3H, CH3). dC (150.8 MHz, CDCl3) (major anomer) 171.0
(C¼O), 170.1 (C¼O), 169.6 (C¼O), 169.6 (C¼O), 169.4
(C¼O), 165.5 (C¼O), 133.5 (Ar), 130.1, (Ar), 129.1 (Ar), 128.5
(Ar), 99.3, 90.4 (C1, C10), 76.8, 73.5, 71.0, 69.9, 69.2, 68.5, 67.8,
67.5 (C2, C20, C3, C30, C4, C40, C5, C50), 62.3 (C6), 21.0 (C(O)
CH3), 20.9 (C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.5
(C(O)CH3), 17.5 (CH3). m/z (HR-MS) (ESI) 679.1653;
[MþK]þ requires 679.1640.
Allyl 2,3,4-tri-O-acetyl-a-L-rhamnosyl-(1-3)-[2,3,4,6-
tetra-O-acetyl-b-D-glucosyl-(1-6)-]-4-O-acetyl-2-O-
benzoyl-a-D-glucoside 14
A mixture of 11 (0.40 g, 0.63 mmol), 13[14] (0.39 g, 0.80 mmol),
2,3,4-Tri-O-acetyl-a-L-rhamnosyl-(1-3)-[2,3,4,6-tetra-
O-acetyl-b-D-glucosyl-(1-6)-]-4-O-acetyl-2-
O-benzoyl-a/b-D-glucose 16
˚
and dry 4-A molecular sieves (0.80 g) in CH2Cl2 (12 mL) were
stirred under argon for 15 min, then cooled to ꢂ308C. Tri-
methylsilyl trifluoromethanesulfonate (TMSOTf; 75 mL) was
then added, and after 5 min at ꢂ308C, stirred at room tempera-
ture for 2 h. After the reaction was complete (as assessed by
TLC), the reaction mixture was cooled to 08C, quenched with
triethylamine (100 mL), filtered through celite, and concen-
trated. Flash chromatography of the resulting oil (EtOAc :
hexane ¼ 1 : 1) afforded, after concentration of the appropriate
fractions (RF 0.44, 3 : 2 EtOAc : hexane eluent), 14 as a white
foam (0.24 g, 40 %). nmax (neat)/cmꢂ1 2941 (w), 1745 (s).
dH (600 MHz, CDCl3) 8.04–8.02 (m, 2H, Ar), 7.57–7.54 (m, 1H,
Ar), 7.44–7.41 (m, 2H, Ar), 5.82–5.75 (m, 1H, CH¼CH2), 5.30–
4.92 (m, 12H, H1, H10, H2, H20, H200, H30, H300, H4, H40, H400,
CH¼CH2), 4.56 (d, J 7.8, 1H, H100), 4.34 (dd, J 9.6, 9.6, 1H, H3),
4.31–4.27, 4.17–4.11, 3.97–3.89, 3.86–3.81, 3.72–3.68, 3.55–
3.51 (6 m, 9H, H5, H50, H500, H6, H6, H600, H600, CH2), 2.11
(s, 3H, C(O)CH3), 2.09 (s, 3H, C(O)CH3), 2.04 (s, 3H, C(O)
CH3), 2.02 (s, 3H, C(O)CH3), 1.99 (s, 3H, C(O)CH3), 1.98 (s,
3H, C(O)CH3), 1.93 (s, 3H, C(O)CH3), 1.77 (s, 3H, C(O)CH3),
1.15 (d, J 6.6, 3H). dC (150.8 MHz, CDCl3) 170.8 (C¼O), 170.3
Palladium(II) chloride (42 mg, 0.24 mmol) was added to a
solution of 14 (0.21 g, 0.22 mmol) in a mixture of CH3COOH,
NaOAc, and H2O (25 mL, 0.98 g, and 1.3 mL, respectively), and
stirred at room temperature for 48 h. After the reaction was
complete (as assessed by TLC), the reaction mixture was filtered
through celite and concentrated. The residue was suspended in
EtOAc (20 mL) and washed with water (2 ꢁ 20 mL), saturated
NaHCO3 solution (20 mL), and brine (20 mL), dried over
MgSO4, filtered, and concentrated. Flash chromatography of the
resulting oil (EtOAc : hexane ¼ 13 : 7) afforded, after concen-
tration of the appropriate fractions (RF 0.20, 3 : 2 EtOAc : hexane
eluent), 16 as a white foam (0.18 g, 87 %). nmax (neat)/cmꢂ1
3473 (w), 1744 (s). dH (600 MHz, CDCl3) (major anomer) 8.00–
8.03 (m, 2H, Ar), 7.58–7.53 (m, 1H, Ar), 7.45–7.41 (m, 2H, Ar),
5.55 (dd, J 3.6, 3.6, 1H, H1), 5.23–4.91 (m, 9H, H10, H2, H20,
H200, H30, H300, H4, H40, H400), 4.60 (d, J 8.4, 1H, H100), 4.39
(dd, J 8.0, 8.0, 1H, H3), 4.24–4.19, 3.89–3.82, 3.73–3.69,
3.62–3.59 (4 m, 7H, H5, H50, H500, H6, H6, H600, H600), 2.12

Synthesis of natural products from Leonurus japonicus
1467
(s, 3H, C(O)CH3), 2.09 (s, 3H, C(O)CH3), 2.03 (s, 3H, C(O)
CH3), 2.02 (s, 3H, C(O)CH3), 1.98 (s, 3H, C(O)CH3), 1.95 (s,
3H, C(O)CH3), 1.77 (s, 3H, C(O)CH3), 1.74 (s, 3H, C(O)CH3),
1.15 (d, J 6.0, 3H, CH3). dC (150.8 MHz, CDCl3) (major
anomer) 170.9 (C¼O), 170.4 (C¼O), 170.1 (C¼O), 170.0
(C¼O), 169.9 (C¼O), 169.6 (C¼O), 169.4 (C¼O), 165.5
(C¼O), 133.7 (Ar), 133.5 (Ar), 130.1 (Ar), 128.5 (Ar), 101.3,
99.2, 90.1 (C1, C10, C100), 76.7, 73.6, 72.7, 71.5, 71.0, 70.1, 69.9,
69.8, 68.7, 68.5, 68.5, 67.4 (C2, C20, C200, C3, C30, C300, C4, C40,
C400, C5, C50, C500), 69.3, 61.9 (C6, C600), 21.1 (C(O)CH3), 20.9
(C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.7 (C(O)CH3),
20.5 (C(O)CH3), 17.4 (CH3). m/z (HR-MS) (ESI) 951.2750;
[MþNa]þ requires 951.2746.
resulting oil (1 : 1EtOAc : hexane eluent) afforded a yellow oil
(58 mg). The yellow oil was then suspended in CH2Cl2 (3 mL)
and stirred with 17[16] (12 mg, 0.057 mmol) and dry 4-A
˚
molecular sieves (0.10 g) for 15 min. The mixture was then
cooled to ꢂ308C and treated with TMSOTf (30 mL). The reac-
tion mixture was stirred at room temperature and once complete
(as assessed by TLC, 1 h) was cooled to 08C and quenched with
triethylamine (100 mL), filtered through celite, and concentrat-
ed. Flash chromatography of the resulting oil (3 : 2 EtOAc :
hexane eluent) afforded, after concentration of the appropriate
fractions (RF 0.24), 19 as a colourless glass (30 mg, 15 % over
two steps). nmax (neat)/cmꢂ1 1751 (s). dH (600 MHz, CDCl3)
7.98–7.96 (m, 2H, Ar), 7.56–7.53 (m, 1H, Ar), 7.43–7.40
(m, 2H, Ar), 6.76 (d, J 1.8, 1H, Ar), 6.68–6.64 (m, 2H, Ar), 5.25
(dd, J 7.8, 9.0, 1H, H2), 5.16 (dd, J 9.0, 9.0, 1H, H4), 5.10–5.07,
4.98–4.86 (2 m, 6H, H20, H200, H30, H300, H40, H400), 4.82 (d, J
1.8, 1H, H10), 4.56, 4.52 (2d, 2H, H1, H100), 4.27 (dd, J 4.8, 12.6,
1H, H6), 4.12–4.05, 3.84–3.80, 3.68–3.61 (3 m, 8H, H5, H50,
H500, H6, H600, H600, CH2), 3.99 (dd, J 9.0, 9.0, 1H, H3), 3.75
(s, 3H, OCH3), 2.77 (t, J 6.6, 2H, CH2), 2.25 (s, 3H, C(O)CH3),
2.08 (s, 6H, C(O)CH3), 2.02 (s, 3H, C(O)CH3), 1.98 (s, 6H, C(O)
CH3), 1.91 (s, 3H, C(O)CH3), 1.87 (s, 3H, C(O)CH3), 1.79
(s, 3H, C(O)CH3), 1.13 (d, J 6.6, 3H, CH3). dC (150.8 MHz,
CDCl3) 170.7 (C¼O), 170.3 (C¼O), 170.1 (C¼O), 169.8
(C¼O), 169.6 (C¼O), 169.5 (C¼O), 169.4 (C¼O), 169.3
(C¼O), 169.1 (C¼O), 164.7 (C¼O), 150.8 (Ar), 138.2 (Ar),
137.5 (Ar), 133.3 (Ar), 130.0 (Ar), 129.4 (Ar), 128.4 (Ar), 122.5
(Ar), 121.0 (Ar), 113.4 (Ar), 100.9, 100.7, 99.0 (C1, C10, C100),
79.8, 73.9, 73.0, 72.9, 72.0, 71.3, 71.0, 70.2, 69.9, 68.4, 68.3,
67.4, 55.9, (C2, C20, C200, C3, C30, C300, C4, C40, C400, C5, C50,
C500, OCH3), 70.4, 68.7, 61.9 (C6, C600, CH2), 36.0 (CH2), 21.1
(C(O)CH3), 20.8 (C(O)CH3), 20.8 (C(O)CH3), 20.7 (C(O)CH3),
20.7 (C(O)CH3), 20.5 (C(O)CH3), 17.4 (CH3). m/z (HR-MS)
(ESI) 1143.3586; [MþNa]þ requires 1143.3533.
2-(4-Acetoxy-3-methoxyphenyl)ethyl 2,3,4-tri-O-acetyl-
a-L-rhamnosyl-(1-3)-4,6-di-O-acetyl-2-O-benzoyl-
b-D-glucoside 18
Trichloroacetonitrile (65 mL, 0.65 mmol) and 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU; 50 mL) were added to a solution of
the hemiacetal 15 (0.21 g, 0.33 mmol) in CH2Cl2 (5 mL) at 08C,
and the resulting solution was left to stand at room temperature
for 15 h. After the reaction was complete (as assessed by TLC),
the reaction mixture was concentrated. Flash chromatography of
the resulting oil (2 : 3 EtOAc : hexane eluent) afforded a white
foam (0.14 g). The foam was then suspended in CH2Cl2 (6 mL)
and stirred at ꢂ308C with 17[16] (30 mg, 0.14 mmol) and dry 4-A
˚
molecular sieves (0.25 g) for 30 min. The resulting mixture was
cooled to ꢂ308C and treated with TMSOTf (50 mL), and after
5 min, stirred at room temperature for 1 h. After the reaction was
complete (as assessed by TLC), the reaction mixture was cooled
to 08C and quenched with triethylamine (100 mL), and filtered
through celite and concentrated. Flash chromatography of the
resulting oil (1 : 1 EtOAc : hexane eluent) afforded, after con-
centration of the appropriate fractions (RF 0.14), 18 as a col-
ourless glass (89 mg, 33 % over two steps). nmax (neat)/cmꢂ1
2962 (w), 1741 (s). dH (600 MHz, CDCl3) 8.00–7.94 (m, 2H,
Ar), 7.57–7.51 (m, 1H, Ar), 7.44–7.39 (m, 2H, Ar), 6.72 (d, J 1.2,
1H, Ar), 6.65 (d, J 7.8, 1H, Ar), 6.62 (dd, J 1.8, 7.8, 1H, Ar), 5.30
(dd, J 7.8, 9.6, 1H, H2), 5.16 (dd, J 9.6, 9.6, 1H, H4), 5.10 (dd, J
3.6, 10.2, 1H, H30), 4.90–4.86 (m, 2H, H20, H40), 4.83 (d, J 1.8,
1H, H10), 4.57 (d, J 7.8, 1H, H1), 4.24 (dd, J 4.8, 12, 1H, H6),
4.14–4.04 (m, 2H, H5, H6), 4.00 (dd, J 9.0, 9.0, 1H, H3), 3.84–
3.81 (m, 1H, H50), 3.72 (s, 3H, OCH3), 3.65–3.60 (m, 2H, CH2),
2.77 (dd, J 6.6, 6.6, 2H, CH2), 2.25 (s, 3H, C(O)CH3), 2.09
(s, 6H, C(O)CH3), 2.08 (s, 3H, C(O)CH3), 1.97 (s, 3H, C(O)
CH3), 1.78 (s, 3H, C(O)CH3), 1.13 (d, J 6.6, 3H, CH3).
dC (150.8 MHz, CDCl3) 170.9 (C¼O), 170.2 (C¼O), 169.6
(C¼O), 169.5 (C¼O), 169.3 (C¼O), 164.7 (C¼O), 150.8 (Ar),
138.2 (Ar), 137.4 (Ar), 133.3 (Ar), 130.1 (Ar), 129.4 (Ar), 128.4
(Ar), 122.5 (Ar), 121.0 (Ar), 113.3 (Ar), 101.1, 99.2 (C1, C10),
80.2, 73.0, 72.3, 71.1, 70.6, 70.0, 69.4, 68.4, 67.4, 62.3, 55.9 (C2,
C20, C3, C30, C4, C40, C5, C50, C6, CH2, OCH3), 36.1 (CH2),
21.0 (C(O)CH3), 20.9 (C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)
CH3), 20.7 (C(O)CH3), 20.5 (C(O)CH3), 17.5 (CH3). m/z
(HR-MS) (ESI) 871.1653; [MþK]þ requires 871.2427.
2-(4-Hydroxy-3-methoxyphenyl)ethyl a-L-
rhamnopyranosyl-(1-3)-b-D-glucopyranoside
(Cistanoside E) 1
Sodium methoxide in MeOH (0.50 mL, 0.2 M) was added to a
solution of 18 (84 mg, 0.10 mmol) in MeOH (5 mL) at 08C and
the resulting solution was stirred at room temperature for 4 days.
After the reaction was complete (as assessed by TLC), the
reaction mixture was treated with Amberlite IR-120 (Hþ) resin
(100 mg), filtered, and concentrated. Flash chromatography of
the resulting oil (1 : 4 MeOH : CHCl3 eluent) afforded, after
concentration of the appropriate fractions (RF 0.26), 1 as a col-
ourless glass (41 mg, 85 %). nmax (neat)/cmꢂ1 2969 (w), 1736
(s), 1512 (w), 1429 (w), 1368 (w), 1217 (s), 1143 (w), 1029 (s).
1H and 13C NMR data were consistent with that found in the
literature.[6] m/z (HR-MS) (ESI) 499.1791; [MþNa]þ requires
499.1791. Anal. Calc. for C21H32O12: C 52.94, H 6.77. Found:
C 52.78, H 6.84 %.
2-(4-Hydroxy-3-methoxyphenyl)ethyl a-L-
rhamnopyranosyl-(1-3)-[b-D-glucopyranosyl-
(1-6)-]-b-D-glucopyranoside (Leonoside F) 2
2-(4-Acetoxy-3-methoxyphenyl)ethyl 2,3,4-tri-O-acetyl-
a-L-rhamnosyl-(1-3)-[2,3,4,6-tetra-O-acetyl-b-D-glucosyl-
(1-6)-]-4-O-acetyl-2-O-benzoyl-b-D-glucoside 19
Sodium methoxide (10 mg, 0.19 mmol) was added to a solution
of 19 (28 mg, 0.025 mmol) in MeOH (3 mL) and the resulting
solution was stirred at room temperature for 5 days. After the
reaction was complete (as assessed by TLC), the reaction mix-
ture was treated with Amberlite IR-120 (Hþ) resin (100 mg),
filtered, and concentrated. Flash chromatography of the result-
ing oil (39 : 11 : 4 EtOAc : MeOH : H2O eluent) afforded, after
Trichloroacetonitrile (36 mL, 0.36 mmol) and DBU (0.07 mL)
were added to a solution of 16 (0.17 g, 0.18 mmol) in CH2Cl2
(4 mL) and the resulting solution was left to stand overnight. The
reaction mixture was concentrated. Flash chromatography of the

1468
E. Clayton et al.
concentration of the appropriate fractions (RF 0.32), 2 as a col-
ourless glass (8 mg, 50 %). nmax (neat)/cmꢂ1 3365 (s), 2928 (w),
room temperature for 15 h. After the reaction was complete
(as assessed by TLC), the reaction mixture was concentrated.
Flash chromatography of the resulting oil (3 : 17 EtOAc : hexane
eluent) afforded a white foam (0.24 g). The foam was then
suspended in CH2Cl2 (10 mL) and stirred with 6[11] (0.14 g,
1585 (w), 1518 (w), 1375 (w), 1272 (w), 1037 (s). 1H and 13
C
NMR data were consistent with that found in the literature.[6,9]
m/z (HR-MS) (ESI) 677.2052; [MþK]þ requires 677.2059.
˚
0.34 mmol) and dry 4-A molecular sieves (0.4 g) for 30 min. The
Allyl 3-O-acetyl-4-O-benzoyl-2-O-chloroacetyl-
a-L-rhamnoside 20
mixture was then cooled to ꢂ308C and treated with TMSOTf
(50 mL) and after 5 min, stirred at room temperature for 30 min.
After reaction was complete (as assessed by TLC), the reaction
mixture was cooled to 08C, quenched with triethylamine
(100 mL), filtered through celite, and concentrated. Flash chro-
matography of the resulting oil (1 : 3 EtOAc : hexane eluent)
afforded, after concentration of the appropriate fractions
(RF 0.58, 3 : 7 EtOAc : hexane eluent), 23 as a white foam
(0.26 g, 30 % over two steps). nmax (neat)/cmꢂ1 3370 (w), 2968
(w), 1751 (w), 1725 (s), 1695 (w). dH (600 MHz, CDCl3)
8.03–7.98 (m, 2H, Ar), 7.89–7.85 (m, 2H, Ar), 7.64–7.57 (m,
2H, Ar), 7.53–7.40 (m, 6H, Ar), 7.22–7.13 (m, 3H, Ar),
5.85–5.78 (m, 1H, CH¼CH2), 5.64 (s, 1H, PhCH), 5.46 (dd, J
3.6, 10.2, 1H, H30), 5.31–5.25 (m, 1H, CH¼CH2), 5.24 (d, J 3.6,
1H, H1), 5.18–5.10 (m, 4H, H2, H20, H40, CH¼CH2), 5.07 (d, J
1.8, 1H, H10), 4.50 (dd, J 9.6, 9.6, 1H, H3), 4.37–4.32,
4.06–3.97, 3.85–3.83 (3 m, 6H, H5, H50, H6, H6, CH2, C(O)
CH2Cl), 4.24–4.18 (m, 1H, CH2), 3.91 (A part of an ABq, J 15.0,
1H, C(O)CH2Cl), 3.78 (dd, J 9.6, 9.6, 1H, H4), 1.82 (s, 3H, C(O)
CH3), 0.81 (d, J 6.6, 3H, CH3). dC (150.8 MHz, CDCl3) 170.0
(C¼O), 166.1 (C¼O), 165.8 (C¼O), 165.7 (C¼O), 137.2 (Ar),
133.7 (Ar), 133.5, 133.4 (Ar, CH¼CH2), 129.9 (Ar), 129.8 (Ar),
129.4 (Ar), 129.3 (Ar), 129.2 (Ar), 128.7 (Ar), 128.6 (Ar), 128.3
(Ar), 126.4 (Ar), 118.0 (CH¼CH2), 102.2, 97.8, 96.2 (C1, C10,
PhCH), 79.7, 74.7, 73.5, 71.5, 71.4, 69.1, 69.0, 68.8, 66.7, 63.2
(C2, C20, C3, C30, C4, C40, C5, C50, C6, CH2), 40.5 (C(O)
CH2Cl), 20.7 (C(O)CH3), 16.8 (CH3). m/z (HR-MS) (ESI)
781.2246; [MþH]þ requires 781.2263.
Chloroacetyl chloride (0.45 mL, 5.7 mmol) was added to a
solution of 21[17] (1.4 g, 4.0 mmol) in pyridine (3 mL) and
CH2Cl2 (15 mL) at 08C, and the solution was stirred at room
temperature for 2 h. After the reaction was complete (as assessed
by TLC), the reaction mixture was quenched with MeOH
(10 mL) and concentrated. The residue was suspended in EtOAc
(50 mL) and washed with hydrochloric acid (1 M, 30 mL), sat-
urated NaHCO3 solution (30 mL), and brine (30 mL), dried over
MgSO4, filtered, and concentrated. Flash chromatography of the
resulting oil (1 : 9 EtOAc : hexane eluent) afforded, after con-
centration of the appropriate fractions (RF 0.25), 20 as a white
foam (1.1 g, 65 %). nmax (neat)/cmꢂ1 2983 (w), 1753 (s), 1728
(s). dH (600 MHz, CDCl3) 8.00–7.99 (m, 2H, Ar), 7.59–7.56
(m, 1H, Ar), 7.46–7.43 (m, 2H, Ar), 5.95–5.88 (m, 1H,
CH¼CH2), 5.55 (dd, J 3.6, 10.2, 1H, H3), 5.37–5.29 (m, 3H, H2,
H4, CH¼CH2), 5.27–5.24 (m, 1H, CH¼CH2), 4.86 (d, J 1.2, 1H,
H1), 4.26–4.16, 4.08–4.02 (2 m, 5H, H5, CH2, C(O)CH2Cl), 1.89
(s, 3H, C(O)CH3), 1.27 (d, J 6.6, 3H, CH3). dC (150.8 MHz,
CDCl3) 170.0 (C¼O), 166.9 (C¼O), 165.8 (C¼O), 133.6, 133.2
(CH¼CH2, Ar), 129.9 (Ar), 129.4 (Ar), 128.7 (Ar), 118.5
(CH¼CH2), 96.3 (C1), 72.0, 71.6, 69.0, 68.7, 66.9 (C2, C3, C4,
C5, CH2) 40.9 (C(O)CH2Cl), 20.8 (C(O)CH3), 17.6 (CH3). m/z
(HR-MS) (ESI) 465.0743; [MþK]þ requires 465.0719. Anal.
Calc. for C20H23ClO8: C56.28, H 5.43. Found:C 56.14, H 5.61 %.
3-O-Acetyl-4-O-benzoyl-2-O-chloroacetyl-
a/b-L-rhamnose 22
Allyl 3-O-acetyl-4-O-benzoyl-a-L-rhamnosyl-(1-3)-
2-O-benzoyl-4,6-O-benzylidene-a-D-glucoside 24
Palladium(II) chloride (0.28 g, 1.6 mmol) was added to a solu-
tion of 20 (0.64 g, 1.5 mmol) in a mixture of CH3COOH,
NaOAc, and H2O (70 mL, 2.8 g, and 3.8 mL, respectively), and
stirred at room temperature for 20 h. After the reaction was
complete (as assessed by TLC), the reaction mixture was filtered
through celite and concentrated. The residue was suspended in
CH2Cl2 (100 mL) and washed with water (50 mL), saturated
NaHCO3 solution (3 ꢁ 50 mL), and brine (2 ꢁ 50 mL), dried
over MgSO4, filtered, and concentrated. Flash chromatography
of the resulting oil (3 : 7 EtOAc : hexane eluent) afforded, after
concentration of the appropriate fractions (RF 0.32), 22 as a
white foam (0.53 g, 91 %). nmax (neat)/cmꢂ1 3462 (w), 2985 (w),
1726 (w). dH (600 MHz, CDCl3) (major anomer) 8.02–7.97
(m, 2H, Ar), 7.60–7.57 (m, 1H, Ar), 7.46–7.43 (m, 2H, Ar), 5.61
(dd, J 3.0, 10.0, 1H, H3), 5.40–5.25 (m, 3H, H1, H2, H4),
4.34–4.15 (m, 3H, H5, C(O)CH2Cl), 3.42 (d, J 3.0, 1H, OH),
1.91 (s, 3H, C(O)CH3), 1.26 (d, J 4.5, 3H, CH3). dC (150.8 MHz,
CDCl3) (major anomer) 170.2 (C¼O), 167.0 (C¼O), 165.8
(C¼O), 133.6 (Ar), 129.9 (Ar), 129.3 (Ar), 128.7 (Ar), 92.0
(C1), 72.4, 71.6, 68.7, 66.8 (C2, C3, C4, C5), 40.9 (C(O)CH2Cl),
20.8 (C(O)CH3), 17.7 (CH3). m/z (HR-MS) (ESI) 425.0406;
[MþK]þ requires 425.0406.
Thiourea (0.27 g, 3.5 mmol) and 2,6-lutidine (46 mL, 0.40 mmol)
were added to a solution of 23 (0.28 g, 0.36 mmol) in MeOH/
CH2Cl2 (1 : 1, 20 mL), and the solution was stirred at 358C for
24 h. After the reaction was complete (as assessed by TLC),
the reaction mixture was concentrated. The residue was sus-
pended in CH2Cl2 (30 mL) and washed with water (15 mL),
saturated NaHCO3 (2 ꢁ 15 mL), water (15 mL), and brine
(20 mL), dried over MgSO4, filtered, and concentrated. Flash
chromatography of the resulting oil (7 : 13 EtOAc : hexane
eluent) afforded, after concentration of the appropriate fractions
(RF 0.23), 24 as a colourless oil (0.19 g, 74 %). nmax (neat)/cmꢂ1
3420 (w), 2929 (w), 1724 (s). dH (600 MHz, CDCl3) 8.07–8.04
(m, 2H, Ar), 7.89–7.85 (m, 2H, Ar), 7.62–7.55 (m, 2H, Ar),
7.52–7.40 (m, 6H, Ar), 7.20–7.15 (m, 3H, Ar), 5.85–5.78
(m, 1H, CH¼CH2), 5.64 (s, 1H, PhCH), 5.38 (dd, J 3.0, 9.6, 1H,
H30), 5.29–5.23 (m, 2H, H1, CH¼CH2), 5.20 (dd, J 10.2, 10.2,
1H, H40), 5.16–5.12 (m, 2H, H10, CH¼CH2), 5.10 (dd, J 3.6, 9.6,
1H, H2), 4.53 (dd, J 9.6, 9.6 1H, H3), 4.38–4.32, 4.17–3.98 (2 m,
4H, H5, H50, H6, CH2), 4.23–4.18 (m, 1H, CH2), 3.90–3.87
(m, 1H, H20), 3.83 (t, J 10.2, 10.2, 1H, H6), 3.76 (dd, J 9.6,9.6
1H, H4), 2.00 (d, J 4.2, 1H, OH), 1.90 (s, 3H, C(O)CH3), 0.81
(d, J 6.6, 3H, CH3). dC (150.8 MHz, CDCl3) 170.0 (C¼O), 165.9
(C¼O), 165.8 (C¼O), 137.2 (Ar), 133.8 (Ar), 133.5, 133.4
(Ar, CH¼CH2), 129.9 (Ar), 129.8 (Ar), 129.6 (Ar), 129.3 (Ar),
128.9 (Ar), 128.6 (Ar), 128.3 (Ar), 126.4 (Ar), 118.0
(CH¼CH2), 102.2, 100.0, 96.1 (C1, C10, PhCH), 79.9, 75.1,
73.3, 72.0, 71.5, 69.8, 66.4, 63.1 (C2, C20, C3, C30, C4, C40, C5,
Allyl 3-O-acetyl-4-O-benzoyl-2-O-chloroacetyl-
a-L-rhamnosyl-(1-3)-2-O-benzoyl-4,6-O-benzylidene-
a-D-glucoside 23
Trichloroacetonitrile (0.23 mL, 2.3 mmol) and DBU (0.1 mL)
were added to a solution of 22 (0.44 g, 1.1 mmol) in CH2Cl2
(5 mL) at 08C, and the resulting solution was left to stand at

Synthesis of natural products from Leonurus japonicus
1469
C50), 69.1, 69.0 (C6, CH2), 21.0 (C(O)CH3), 16.9 (CH3). m/z
(HR-MS) (ESI) 743.2107; [MþK]þ requires 743.2106. Anal.
Calc. for C38H40O13: C 64.76, H 5.72. Found: C 64.55, H 5.79 %.
cmꢂ1 2939 (w), 1735 (s). dH (600MHz, CDCl3) 8.10–8.05
(m, 2H, Ar), 7.96–7.92 (m, 2H, Ar), 7.59–7.53 (m, 2H, Ar), 7.46–
7.40 (m, 4H, Ar), 5.85–5.75 (m, 1H, CH¼CH2), 5.29–5.10
(m, 9H, H1, H10, H2, H200, H30, H4, H40, CH¼CH2), 5.07–5.03
(m, 1H, H400), 4.90–4.85 (m, 1H, H300), 4.40 (dd, J 9.6, 9.6, 1H,
H3), 4.27 (dd, J 2.4, 4.2, 1H, H6), 4.19–4.08, 4.03–3.92, (2m, 5H,
H5, H50, H6, CH2), 4.07, (d, J 7.2, 1H, H100), 3.80–3.78 (m, 1H,
H20), 3.20–3.07 (m, 2H, H500, H500), 2.14 (s, 3H, C(O)CH3), 2.13
(s, 3H, C(O)CH3), 2.12 (s, 3H, C(O)CH3), 2.12 (s, 3H, C(O)CH3),
2.04 (s, 3H, C(O)CH3), 1.81 (s, 3H, C(O)CH3), 1.20 (d, J 6.0, 3H,
CH3). dC (150.8MHz, CDCl3) 170.9 (C¼O), 170.4 (C¼O), 170.2
(C¼O), 169.7(C¼O), 169.6 (C¼O), 169.6 (C¼O), 169.5(C¼O),
169.4 (C¼O), 133.4, 133.4, 133.3 (Ar, CH¼CH2), 130.2 (Ar),
129.8 (Ar), 129.6 (Ar), 129.4 (Ar), 128.6 (Ar), 128.5 (Ar), 118.2
(CH¼CH2), 102.4, 100.3, 95.3 (C1, C10, C100), 77.0, 76.2, 73.5,
71.7, 70.5, 69.8, 69.2, 69.0, 68.0, 67.4, 67.7, (C2, C20, C200, C3,
C30, C300, C4, C40, C400, C5, C50), 68.8, 62.5, 62.2(C500, C6, CH2),
21.2 (C(O)CH3), 21.0 (C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)
CH3), 20.7 (C(O)CH3), 17.7 (CH3). m/z (HR-MS) (ESI)
997.2706; [MþK]þ requires 997.2744. Anal. Calc. for
C46H54O22: C 57.62, H 5.68. Found: C 57.49, H 5.78 %.
Allyl 2,3,4-tri-O-acetyl-a-L-arabinosyl-(1-2)-3-O-acetyl-
4-O-benzoyl-a-L-rhamnosyl-(1-3)-2-O-benzoyl-4,6-
O-benzylidene-a-D-glucopyranoside 26
A mixture of 24 (0.18 g, 0.26 mmol), 25[19] (0.29 g, 0.69 mmol),
˚
and dry 4-A molecular sieves (0.4 g) in CH2Cl2 (10 mL) were
stirred at room temperature for 15 min. The reaction mixture was
cooled to ꢂ308C and treated with TMSOTf (50 mL) and after
5 min, the mixture was stirred at room temperature for 1 h. After
the reaction was complete (as assessed by TLC), the reaction
mixture was cooled to 08C and treated with triethylamine
(100 mL), filtered through celite, and concentrated. Flash chro-
matography of the resulting oil (2 : 3 EtOAc : hexane eluent)
afforded, after concentration of the appropriate fractions
(RF 0.33), 26 as a white foam (0.12 g, 46 %). nmax (neat)/cmꢂ1
2940 (w), 1748 (s). dH (600 MHz, CDCl3) 8.11–8.05 (m, 2H,
Ar), 7.83–7.80 (m, 2H, Ar), 7.58–7.38 (m, 8H, Ar), 7.20–7.12
(m, 3H, Ar), 5.82–5.24 (m, 1H, CH¼CH2), 5.64 (s, 1H, PhCH),
5.31 (dd, J 3.6, 10.2, 1H, H30), 5.27–5.21 (m, 2H, H10,
CH¼CH2), 5.18 (d, J 4.2, 1H, H1), 5.15–5.05 (m, 4H, H2, H200,
H40, CH¼CH2), 4.97–4.94 (m, 1H, H400), 4.85 (dd, J 3.6, 9.6,
1H, H300), 4.52 (dd, J 9.6, 9.6, 1H, H3), 4.34–4.32 (m, 2H, H50,
H6), 4.21–4.16 (m, 1H, CH2), 4.11 (d, J 7.2, 1H, H100), 4.06–
3.99 (m, 1H, H5), 3.99–3.94 (m, 1H, CH2), 3.90–3.87 (m, 1H,
H20), 3.82 (dd, J 10.2, 10.2, 1H, H6), 3.76 (dd, J 9.6, 9.6, 1H,
H4), 2.94 (dd, J 1.8, 12.6, 1H, H500), 2.70–2.60 (m, 1H, H500),
2.15 (s, 3H, C(O)CH3), 2.14 (s, 3H, C(O)CH3), 2.02 (s, 3H, C(O)
CH3), 1.90 (s, 3H, C(O)CH3), 0.81 (d, J 6.0, 3H, CH3).
dC (150.8 MHz, CDCl3) 170.4 (C¼O), 170.2 (C¼O), 169.7
(C¼O), 165.8 (C¼O), 165.3 (C¼O), 137.2 (Ar), 133.4, 133.4,
133.2 (Ar, CH¼CH2) 130.2 (Ar), 129.8 (Ar), 129.7 (Ar), 129.3
(Ar), 129.3 (Ar), 128.6 (Ar), 128.5 (Ar), 128.2 (Ar), 126.4 (Ar),
117.8 (CH¼CH2), 102.4, 102.2, 99.2, 96.0 (C1, C10, C100,
PhCH), 79.9, 77.0, 74.9, 72.4, 71.9, 71.1, 69.8, 68.8, 67.4, 66.3,
63.0 (C2, C20, C200, C3, C30, C300, C4, C40, C400, C5, C50), 69.1,
68.9, 62.2 (C500, C6, CH2), 21.0 (C(O)CH3), 20.9 (C(O)CH3),
20.8 (C(O)CH3), 20.8 (C(O)CH3), 16.9 (CH3). m/z (HR-MS)
(ESI) 985.3155; [MþNa]þ requires 985.3106.
2,3,4-tri-O-acetyl-a-L-arabinosyl-(1-2)-3-O-acetyl-4-
O-benzoyl-a-L-rhamnosyl-(1-3)-4,6-di-O-acetyl-2-
O-benzoyl-a/b-D-glucose 28
Palladium(II) chloride (15 mg, 0.085 mmol) was added to a
solution of 27 (75 mg, 0.078 mmol) in a mixture of CH3COOH,
NaOAc, and H2O (17 mL, 0.7 g, and 0.9 mL, respectively), and
stirred at room temperature for 48 h. After the reaction was
complete (as assessed by TLC), the reaction mixture was filtered
through celite and concentrated. The residue was suspended in
CH2Cl2 (20 mL) and washed with water (2 ꢁ 10 mL), hydro-
chloric acid (1 M, 10 mL), water (2 ꢁ 10 mL), saturated
NaHCO3 solution (3 ꢁ 15 mL), and brine (15 mL), dried over
MgSO4, filtered, and concentrated. Flash chromatography of the
resulting oil (1 : 1 EtOAc : hexane eluent) afforded, after con-
centration of the appropriate fractions (RF 0.20), 28 as a col-
ourless glass (58 mg, 81 %). nmax (neat)/cmꢂ1 2971 (w), 1735 (s).
dH (600MHz, CDCl3) (major anomer) 8.07–8.03 (m, 2H, Ar),
7.93–7.91 (m, 2H, Ar), 7.56–7.51 (m, 2H, Ar), 7.44–7.39 (m, 4H,
Ar), 5.53–5.51 (m, 1H, H1), 5.25–5.08 (m, 6H, H10, H2, H200,
H30, H4, H40), 5.05–5.02 (m, 1H, H400), 4.87–4.85 (m, 1H, H300),
4.43 (dd, J 9.6, 9.6, 1H, H3), 4.24–4.21, 4.12–4.05, 3.95–3.92,
3.79–3.77 (4 m, 6H, H100, H20, H5, H50, H6, H6), 3.18–3.05
(m, 2H, H500, H500), 2.12 (s, 3H, C(O)CH3), 2.11 (s, 6H, C(O)
CH3), 2.10, (s, 3H, C(O)CH3), 2.01 (s, 3H, C(O)CH3), 1.79
(s, 3H, C(O)CH3), 1.18 (d, J 6.6, 3H, CH3). dC (150.8MHz,
CDCl3) (major anomer) 171.0 (C¼O), 170.3 (C¼O), 170.2
(C¼O), 169.7 (C¼O), 169.6 (C¼O), 169.5 (C¼O), 165.4
(C¼O), 165.4 (C¼O), 133.4 (Ar), 133.3 (Ar), 130.2 (Ar), 129.8
(Ar), 129.6 (Ar), 129.4 (Ar), 128.7 (Ar), 128.6 (Ar), 102.3, 100.2,
90.2, (C1, C10, C100) 77.1, 75.7, 73.8, 71.7, 70.4, 69.8, 69.2, 68.9,
67.7, 67.4, 67.3, 62.6, 62.1 (C2, C20, C200, C3, C30, C300, C4, C40,
C400, C5, C50, C500, C6), 21.0 (C(O)CH3), 20.9 (C(O)CH3), 20.8
(C(O)CH3), 20.8 (C(O)CH3), 20.7 (C(O)CH3), 17.5 (CH3). m/z
(HR-MS) (ESI) 941.2698; [MþNa]þ requires 941.2691.
Allyl 2,3,4-tri-O-acetyl-a-L-arabinosyl-(1-2)-3-O-acetyl-
4-O-benzoyl-a-L-rhamnosyl-(1-3)-4,6-di-O-acetyl-2-
O-benzoyl-a-D-glucoside 27
Allyl 2,3,4-tri-O-acetyl-a-L-arabinosyl-(1-2)-3-O-acetyl-4-O-
benzoyl-b-L-rhamnosyl-(1-3)-2-O-benzoyl-4,6-O-benzyli-
dene-a-D-glucoside 26 (0.10 g, 0.10 mmol) was treated with
CH3COOH/H2O (4: 1, 5 mL) and stirred at 808C for 6 h. After the
reaction was complete (as assessed by TLC), the reaction mixture
was concentrated and the residue was co-evaporated with toluene
(3ꢁ 3 mL). The residue was suspended in pyridine (5mL) and
the resulting mixture was treated with acetic anhydride (0.10 mL,
1.1 mmol) and DMAP (25 mg), and stirred at room temperature
for 12 h. After the reaction was complete (as assessed by TLC),
the reaction mixture was quenched with MeOH (5mL) and
concentrated. The residue was suspended in EtOAc (15 mL) and
washed with water (10 mL), hydrochloric acid (1M, 3 ꢁ 5 mL),
water (10 mL), saturated NaHCO3 solution (10 mL), and brine
(10 mL), dried over MgSO4, filtered, and concentrated. Flash
chromatography of the resulting oil (2: 3 EtOAc : hexane eluent)
afforded, after concentration of the appropriate fractions
(RF 0.22), 27 as a colourless glass (79 mg, 82%). nmax (neat)/
2-(4-Hydroxy-3-methoxyphenyl)ethyl a-L-
arabinopyranosyl-(1-2)-a-L-rhamnopyranosyl-(1-3)-b-D-
glucopyranoside (Leonoside E) 3
Trichloroacetonitrile (12 mL, 0.12 mmol) and DBU (50 mL)
were added to a solution of 28 (53 mg, 0.058 mmol) in CH2Cl2
(2.5 mL) at 08C, and the resulting solution was stirred at room

1470
E. Clayton et al.
temperature for 15 h. After the reaction was complete (as
assessed by TLC), the reaction mixture was concentrated. Flash
chromatography of the resulting oil (1 : 1 EtOAc : hexane elu-
ent) afforded a white foam (14 mg). The foam was then sus-
pended in CH2Cl2 (2 mL) and stirred with 17[16] (45 mg,
Acknowledgements
The authors thank the Centre for Microscopy, Characterisation and Analysis
at The University of Western Australia, which is supported financially by the
University, as well as the State and Federal Governments. KAS also thanks
the Australian Research Council for funding. MH is supported by an Aus-
tralian Postgraduate Award from the University of Western Australia and a
Jean Rogerson Postgraduate Scholarship.
˚
0.02 mmol) and dry 4-A molecular sieves (0.1 g) for 30 min. The
mixture was then cooled to ꢂ308C and treated with TMSOTf
(50 mL) and after 5 min, stirred at room temperature for 1 h.
After the reaction was complete (as assessed by TLC), the
reaction mixture was cooled to 08C and quenched with triethy-
lamine (100 mL), filtered through celite, and concentrated. Flash
chromatography of the resulting oil (1 : 1 EtOAc : hexane elu-
ent) afforded 29 a mixture with 17 (13 mg). Sodium methoxide
in MeOH (0.50 mL, 0.2 M) was added to the mixture (12 mg) in
MeOH (2 mL) at 08C, and the mixture was allowed to warm to
room temperature for 4 days. After the reaction was complete (as
assessed by TLC), the reaction mixture was treated with
Amberlite IR-120 (Hþ) resin (100 mg), filtered, and concen-
trated. Flash chromatography of the resulting oil (11 : 4 : 35
MeOH : H2O : EtOAc eluent) afforded, after concentration of
the appropriate fractions (RF 0.20), 3 as a colourless glass
(3.5 mg, 10 % over three steps). nmax (neat)/cmꢂ1 3366 (s), 2926
(w), 1639 (s), 1517 (w), 1063 (w). 1H and 13C NMR data were
consistent with that found in the literature.[6,9] m/z (HR-MS)
(ESI) 631.2206; [MþNa]þ requires 631.2214.
References
[1] D. J. Newman, G. M. Cragg, K. M. Snader, J. Nat. Prod. 2003, 66,
1022. doi:10.1021/NP030096L
[2] R. Graziose, M. A. Lila, I. Raskin, Curr. Drug Discov. Technol. 2010,
7, 2. doi:10.2174/157016310791162767
[3] B. K. H. Tan, J. Vanitha, Curr. Med. Chem. 2004, 11, 1423.
doi:10.2174/0929867043365161
[4] S. O. Oyedemi, M. T. Yakubu, A. J. Afolayan, J. Med. Plant. Res 2011,
5, 119.
[5] X. H. Liu, L. L. Pan, Y. Z. Zhu, Clin. Exp. Pharmacol. P. 2012, 39, 274.
doi:10.1111/J.1440-1681.2011.05630.X
[6] Y. Li, Z. Chen, Z. Feng, Y. Yang, J. Jiang, P. Zhang, Carbohyd. Res.
2012, 348, 42. doi:10.1016/J.CARRES.2011.10.034
[7] H. I. Duynstee, M. C. de Koning, H. Ovaa, G. A. van der Marel,
J. H. van Boom, Eur. J. Org. Chem. 1999, 2623. doi:10.1002/(SICI)
1099-0690(199910)1999:10,2623::AID-EJOC2623.3.0.CO;2-K
[8] H. Kobayashi, H. Karasawa, T. Miyase, S. Fukushima, Chem. Pharm.
Bull. 1985, 33, 1452. doi:10.1248/CPB.33.1452
[9] G. Gu, Y. Zhao, Z. Guo, Carbohyd. Res. 2013, 380, 174. doi:10.1016/
J.CARRES.2013.07.012
Intracellular Reactive Oxygen Species (ROS) Cell Study
[10] M. Ota, K. Takahashi, H. Kofujita, J. Wood Sci. 1998, 44, 320.
doi:10.1007/BF00581314
[11] M. Izumi, K. Fukase, S. Kusumoto, Biosci. Biotech. Bioch. 2002, 66,
211. doi:10.1271/BBB.66.211
[12] G. Huang, X. Mei, M. Liu, Carbohydr. Res. 2005, 340, 603.
doi:10.1016/J.CARRES.2005.01.015
[13] M. G. Donahue, J. N. Johnston, Bioorg. Med. Chem. Lett. 2006, 16,
5602. doi:10.1016/J.BMCL.2006.08.024
Intracellular accumulation of ROS was determined using a 20,70-
dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Cell
Biolabs). HepG2 cells were seeded in 96-well black, clear-
bottomed plates at a density of 104 cells per well and were grown
for 24 h before incubation with 1ꢁ DCFH-DA for 30 min at
378C (according to the manufacturer’s instructions), followed
by treatment with compounds 1 3 or a-tocopherol. H O
]
2
2
[14] R. R. Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984, 1343.
doi:10.1002/JLAC.198419840710
[15] L. Ban, M. Mrksich, Angew. Chem. Int. Ed. 2008, 47, 3396.
doi:10.1002/ANIE.200704998
[16] S. G. Mills, A. Ali, C. Smith, Prodrugs of Oxolidinone CETP
inhibitors 2010, WO2010/039474.
(250 mM, final concentration) was then added and the cells were
incubated at 378C for 60 min and 3 h. The cells were then washed
three times with Hank’s balanced salt solution (without phenol
red) at room temperature. Cell fluorescence was measured at
excitation and emission wavelengths of 480 and 530 nm,
respectively. The results are expressed as relative fluorescence
units per cell (RFU cellꢂ1).
[17] J. Zhang, F. Kong, Tetrahedron 2003, 59, 1429. doi:10.1016/S0040-
4020(03)00075-9
[18] R. K. Jain, C. F. Piskorz, B. Huang, R. D. Locke, H. Han, A. Koenig,
A. Varki, L. Khushi, Glycobiology 1998, 8, 707. doi:10.1093/
GLYCOB/8.7.707
[19] T. Murakami, H. Matsuda, M. Inadzuki, K. Hirano, M. Yoshikawa,
Chem. Pharm. Bull. 1999, 47, 1717. doi:10.1248/CPB.47.1717
[20] M. J. Czaja, Semin. Liver Dis. 2007, 27, 378. doi:10.1055/
S-2007-991514
[21] B. Halliwell, Lancet 1994, 344, 721. doi:10.1016/S0140-6736(94)
92211-X
[22] H. E. Poulsen, H. Prieme, S. Loft, Eur. J. Cancer Prev. 1998, 7, 9.
[23] O. Akyol, H. Herken, E. Uz, E. Fadillioglu, S. Unal, S. Sogut,
H. Ozyurt, H. A. Savas, Prog. Neuro.-Psychoph 2002, 26, 995.
doi:10.1016/S0278-5846(02)00220-8
Determination of Catalase Activity
An OxiSelect Catalase Activity assay kit (Cell Biolabs) was
used to determine catalase activity in HepG2 cells. Cells (104
cells per well) were grown for 24 h and pre-incubated with 2 or
a-tocopherol for 30 min. H2O2 (250 mM, final concentration)
was then added and the cells were incubated at 378C for 1 and
3 h. The cells were collected by treating them with cell disso-
ciation buffer (enzyme free). Cell pellets were homogenized in
ice-cold PBS and 1 mM EDTA and centrifuged at 10621g for
15 min at 48C. The catalase assay was then performed on 20-mL
aliquots of the supernatant according to the manufacturer’s
instructions and catalase activity was determined by comparison
against a catalase standard curve. Results are expressed relative
to protein concentration.
[24] M. J. Czaja, Antioxid. Redox Sign. 2002, 4, 759. doi:10.1089/
152308602760598909
[25] X. Chen, Z. Zhong, Z. Xu, L. Chen, Y. Wang, Pharmacol. Rep. 2011,
63, 724.
[26] G. E. Henry, R. A. Momin, M. G. Nair, D. L. Dewitt, J. Agric. Food
Chem. 2002, 50, 2231. doi:10.1021/JF0114381
[27] Z. Jia, H. Zhu, J. Li, X. Wang, H. Misra, Y. Li, Spinal Cord 2012, 50,
Supplementary Material
1H and 13C NMR spectra of the prepared compounds are
available on the Journal’s website.
264. doi:10.1038/SC.2011.111