A general synthetic strategy and the anti-proliferation properties on prostate cancer cell lines for natural phenylethanoid glycosides

Article abstract of DOI:10.1039/c3ob42503g

A general strategy for the synthesis of phenylethanoid glycosides (PhG) including echinacoside 1, acteoside 2, calceolarioside-A 3 and calceolarioside-B 4 is reported. The strategy features the application of low substrate concentration glycosylation and N-formyl morpholine modulated glycosylation methods for the construction of 1,2-trans β- and α-glycosidic bonds. The reported strategy does not invoke the use of the participatory acyl protecting function, which is incompatible with the ester function present in target PhG compounds. A preliminary study of the anti-proliferation properties of the PhG compounds 1-4 was performed; the acteoside 2 exhibited the best inhibition on the prostatic cancer cell proliferation. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42503g

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
A general synthetic strategy and the
anti-proliferation properties on prostate cancer
cell lines for natural phenylethanoid glycosides†
Cite this: Org. Biomol. Chem., 2014,
12, 2926
Shaheen K. Mulani,a Jih-Hwa Guhb and Kwok-Kong Tony Mong*a
A general strategy for the synthesis of phenylethanoid glycosides (PhG) including echinacoside 1, acteo-
side 2, calceolarioside-A 3 and calceolarioside-B 4 is reported. The strategy features the application of
low substrate concentration glycosylation and N-formyl morpholine modulated glycosylation methods
for the construction of 1,2-trans β- and α-glycosidic bonds. The reported strategy does not invoke the
use of the participatory acyl protecting function, which is incompatible with the ester function present in
target PhG compounds. A preliminary study of the anti-proliferation properties of the PhG compounds
1–4 was performed; the acteoside 2 exhibited the best inhibition on the prostatic cancer cell proliferation.
Received 17th December 2013,
Accepted 14th February 2014
DOI: 10.1039/c3ob42503g
www.rsc.org/obc
neighbouring group participation mechanism (NGP).11 However,
the removal of the acetyl functions in the final stage of the syn-
1 Introduction
Phenylethanoid glycosides (PhGs) are secondary metabolites thesis was complicated by side reactions such as the hydrolysis
widely distributed in the plant kingdom.1 A number of these or migration of the phenylpropenic ester group.10e,f In
plant species have long been used as herbal medicines or addition, the removal of the benzyl ether function by hydroge-
remedies for upper respiratory tract infections, common colds, nolysis has the risk to reduce the double bond in the phenyl-
diabetes, hepato-protection, etc.2 Such medicinal properties propenic ester function.10a,d,i The situation would have been
are believed to associate with the radical-scavenging and anti-
oxidant properties of the PhG metabolites.3 Given the poten-
tial in cosmetic and pharmaceutical applications, the chemical
synthesis of PhG metabolites and their analogues has attracted
the interest of synthetic chemists.4,5
Up to the now, a couple of hundred PhG compounds have
been identified and characterized.2,6–8 The core structure of
these compounds contain a 2-phenylethyl β-D-glucoside, which
is conjugated to a substituted phenylpropenic acid via an ester
bond at the C-4 hydroxyl of the glucoside.2,9 More complex
PhG metabolites contain additional saccharide units.
The major barrier to the total synthesis of PhG compounds
is the incompatibility of common hydroxyl protecting groups
with the phenylpropenic ester group.10 In previous syntheses
of PhG, the acetyl and benzyl ether functions were exploited
for the protection of the hydroxyl functions. The additional
function of the acetyl group at the C2 position is to direct
the formation of the 1,2-trans glycosidic bond through a
aApplied Chemistry Department, National Chiao Tung University of Taiwan, 1001,
Ta Hsueh Road, Hsinchu, Taiwan, Republic of China.
E-mail: tmong@mail.ncut.edu.tw; Fax: +886-3-5723764
bSchool of Pharmacy, National Taiwan University, Taipei, Taiwan, Republic of China
†Electronic supplementary information (ESI) available: 1H NMR, 13C NMR
spectra of final products and intermediates, procedures for the preparation of
building blocks 5, 6, 7, 11, 12, 13 and 14 are given. See DOI: 10.1039/c3ob42503g
Fig. 1 The PhG compounds 1–4.
2926 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
worse when the number of saccharide units increases. Thus protected thioglucoside 5, NAP-protected glucosyl phosphate
far, only the synthesis of monosaccharide and disaccharide 6, NAP-protected thiorhamnoside 7, t-butyldimethylsilyl (TBS)
PhG metabolites have been reported, but the total synthesis of protected phenylethyl alcohol 8 and TBS-protected caffeic
trisaccharide PhG has not met with success.10g
acid 9.
Herein, we describe a general synthetic route for the prepa-
ration of PhG compounds including echinacoside 1 (trisac-
2.2 The preparation of the building blocks for echinacoside 1
and other PhG targets
charide PhG),12 acteoside
2
(disaccharide PhG),13
calceolarioside-A 3 and calceolarioside-B 4 (monosaccharide
PhG) (Fig. 1).14 Furthermore, a preliminary study was per-
formed to screen the anti-proliferation activities of the syn-
thesized metabolites on human prostatic cancer cell lines.15
The key thioglucosyl building block 5 was derived from a
known thioglucoside 1019 via thioglucosyl intermediates 11
and 12 in 72% yield over three reaction steps including (i) the
formation of the naphthylidene acetal (Napth) function at the
C-4 and C-6 hydroxyl functions, (ii) the alkylation of the
C-2 hydroxyl function with 2-naphthylmethyl bromide
(NAPBr), and (iii) the removal of the 4,6-O-acetal function by
acid treatment (Scheme 1a). It was noted that the NAP ether
protection remained viable in acidic conditions (pTSA in
deprotection of the acetal function). For the synthesis of other
PhG targets 2, 3 and 4, 4-hydroxyl unprotected thioglucoside
13 and 6-hydroxyl unprotected thioglucoside 14 were needed.
They were prepared from the thioglucoside 12 by selective
acetal reduction methods.20,21
2. Results and discussion
2.1. General consideration
The PhG targets 1 and 2 contain 1,2-trans β- and α-glycosidic
linkages, which are usually constructed under the neighbour-
ing group participation (NGP) effect of the C-2 acyl protecting
function.11 As explained above, the acyl protecting function is
incompatible with the synthesis of the PhG compounds.
Therefore, we applied the low concentration glycosylation
(LCG)16 and formamide-modulated glycosylation methods17
for the construction of the 1,2-trans β- and α-glycosidic bonds,
respectively. Both methods control the stereochemistry of
glycosylation through an external factor; therefore, the stereo-
directing acyl protecting function is not required. Further-
more, to prevent the reduction of the double bond in the PhG
targets, 2-naphthylmethyl (NAP) ether is explored as the per-
manent protection function, which can be removed by an oxi-
dation method.18
The synthesis of NAP-protected glucosyl phosphate
6
started with a known thioglucoside 6a.22 Thus 6a was alkylated
with NAPBr, which was followed by the cleavage of thioacetal
function to furnish the hemiacetal 6b.23 The phosphorylation
of 6b with diphenylphosphoryl chloride [ClP(O)(OPh)2] and
The proposed synthetic route for the echinacoside 1 is laid
out in Chart 1. Target echinacoside 1 is derived from a trisac-
charide glycoside 1a, which in turn is assembled from a 2,3-O-
Scheme 1 (a) The preparation of thioglucoside building blocks 5, 13
and 14; (b) the preparation of NAP-protected glucosyl phosphate 6. All =
allyl; Napth = naphthylidene; NAP = 2-naphthylmethyl; STol = p-tolue-
nyl thioacetal.
Chart 1 The synthetic route for echinacoside 1 and the required build-
ing blocks 5–9. All = allyl; NAP = 2-naphthylmethyl; TBS = t-butyldi-
methylsilyl; STol = p-toluenyl thioacetal.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2927

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 2 (a) The preparation of NAP-protected thiorhamnoside 7; (b)
the preparation of TBS-protected caffeic acid 9. NAP = 2-naphthyl-
methyl; TBS = t-butyldimethylsilyl; STol = p-toluenyl thioacetal.
4-dimethyl-aminopyridine (DMAP) furnished the desired glu-
cosyl phosphate 6 in 67% overall yield (Scheme 1b).24 To
reduce the formation of α-glucosyl chloride 6c in phosphoryl-
ation, a low reaction temperature (−10 °C), a shorter reaction
time (<2 h), and excess amounts (2.0 to 3.0 equiv.) of ClP(vO)-
(OPh)2 and DMAP reagents were needed.
The NAP-protected thiorhamnoside 7 was prepared from a
known thiorhamnoside 7a25 in 92% yield through alkylation
with NAPBr (Scheme 2a). In the synthesis of TBS-protected
caffeic acid 9, 3,4-dihydroxyl substituted cinnamic acid 9a was
treated with an excess amount of tributylsilyl trifluoromethane
triflate (TBSOTf) and pyridine to furnish a TBS-protected
caffeic acid silyl ester 9b (Scheme 2b).26 The silyl ester 9b was
treated with K2CO3 in a THF–water mixture to afford the TBS-
protected caffeic acid 9 in good 80% yield over two steps. The
preparation of TBS-protected phenylethyl alcohol 8 began with
a commercially available 3,4-dihydroxy-phenyl acetic acid by
applying a literature procedure.10f
Scheme 3 (a) The synthesis of phenylethyl disaccharide 16 from build-
ing blocks 5, 6 and 8. (b) The synthesis of the echinacoside 1 from build-
ing blocks 7, 9 and 16. All = Allyl ether; NAP = naphthylmethyl; Napth =
naphthylidene acetal; TBS = t-butyldimethylsilyl; NFM = N-formyl mor-
pholine; STol = p-toluenyl thioacetal.
2.3 The assemblage of echinacoside 1
After preparing the required building blocks 5 to 9, we next
attempted the total synthesis of target echinacoside 1. In the
beginning, the thioglucosyl acceptor 5 was coupled with the
NAP-protected glucosyl phosphate 6 under the LCG conditions
to produce the disaccharide thioglycoside 15 in 65% as a
single isolable anomer (Scheme 3a). In this reaction, we did
not obtain any α-anomer of 15, therefore an estimated α : β
ratio of >1 : 19 was assigned. Though there are two hydroxyl
groups in the acceptor 5, only the primary 6-hydroxyl group
was glycosylated due to the higher reactivity. The β-configur-
increasing the proportion of CH2Cl2 and the reaction tempera-
ture. Thus, a 2 : 1 : 1 CH2Cl2–CH3CN–EtCN solvent mixture and
−50 to −40 °C were applied; and the β-anomer of the phenyl-
ethyl disaccharide 16 was obtained in 60% yield (from 15) as
a single isolable anomer. Some hydrolysis product from 15 (ca.
20%) occurred that accounted for the moderate yield of the
disaccharide 16. The β-anomeric configuration of the glycosi-
1
1
dic bonds in 16 was confirmed by the JC–H coupling constant
ation of 15 was confirmed by the JC–H coupling constant of
of ca. 161.9 Hz.27
the anomeric C–H bond of 161.9 Hz.27
As the phenylethyl disaccharide 16 contained an unpro-
tected hydroxyl function at the C-4 position, it was directly
coupled with the TBS-protected caffeic acid 9 using a modified
Steglich procedure (Scheme 3b).28 The reaction went smoothly
to afford the caffeic acid conjugated glycoside 17. A search in
the literature revealed that the E/Z-isomerisation of the double
The disaccharide thioglycoside 15 was coupled with the
phenylethyl alcohol 8 using the LCG method. Due to the poor
solubility of 15, the initial LCG reaction was sluggish in a
1 : 2 : 1 CH2Cl2–CH3CN–EtCN solvent mixture; even though
such a solvent system has been used in previous LCG
methods. Fortunately, the solubility issue was resolved by
2928 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
bond in the PhG compound is common in solution.10f,29 In
the present reaction, only trace amounts of the Z-isomer of 17
1
were detected in the H NMR spectrum. As such, a >19 : 1 E : Z
ratio was given to 17. To monitor the isomerization, the E : Z
ratios of the subsequent intermediates and final PhG product
were closely examined.30
The treatment of the caffeic acid conjugated glycoside 17
with PdCl2 in a buffered solution removed the allyl ether func-
tion to afford the disaccharide glycoside 18 with a C-3 hydroxyl
function.31 The disaccharide 18 was then glycosylated with the
NAP-protected thiorhamnosyl donor 7. Due to the hindered
position of the C-3 hydroxyl group, the DMF-modulated glyco-
sylation was not productive.17a Accordingly, we employed the
N-formyl morpholine (NFM) modulated glycosylation method,
which has been shown to be effective for less reactive glycosyl
substrates. To our delight, the desired PhG target 1a was pro-
duced in
a reasonable 60% yield as a single isolable
anomer.17b
In global deprotection, the protected PhG 1a was treated (i)
with DDQ to remove the NAP ether functions and (ii) with
hydrogen fluoride-triethylamine (3HF·Et3N) reagent to remove
the TBS ether protections. By using the standard and size-
exclusion chromatography purification, the target echinaco-
side 1 was obtained as a 10 : 1 E : Z mixture in 41% yield (over
two steps).30 Accordingly, we completed the first total synthesis
of echinacoside 1 from building blocks 5–9 over seven steps in
4.5% overall yield. The NMR spectroscopic data and optical
rotation ([α]2D7 = −71.1) of the synthesized echinacoside 1 are in
perfect agreement with the literature data ([α]2D7 = −69.9).32
Scheme 4 (a) The synthesis of the disaccharide intermediate 21 for
acteoside synthesis. (b) The assembly of acteoside 2 from building
blocks 7 and 21. All = allyl ether; NAP = naphthylmethyl ether; Napth =
naphthylidene acetal; NFM = N-formyl formamide; TBS = tert-butyldi-
methylsilyl ether.
2.4. The assemblage of acteoside 2, calceolarioside-A 3 and
calceolarioside-B 4
in a 68% yield as a 12 : 1 E : Z mixture (based on 1H NMR
spectrum).30
Encouraged by the success of the synthesis of the echinacoside
1, we applied the same strategy to prepare other PhG meta-
bolites, namely acteoside 2,10e,f calceolarioside-A 333 and calceo-
larioside-B 4.33 The synthesis of the acteoside 2 required the
same set of building blocks 7, 8 and 9 as used for the syn-
thesis of echinacoside 1 with the exception that the thiogluco-
syl building block 13 having a C-4 unprotected hydroxyl group
was employed instead of 5.
The monosaccharide PhG targets, namely calceolarioside-A
3 and calceolarioside-B 4, are truncated structures of the acteo-
side 2. Both of the PhG compounds lack a rhamnose residue
and the caffeic acid component is conjugated to different
hydroxyl functions of the glucosyl core. Accordingly, the syn-
thesis of 3 and 4 could easily be achieved by the same set of
building blocks and the same glycosylation strategy.
For the synthesis of calceolarioside-A 3, the phenylethyl
4-caffeoyl-glucoside 21 undertook DDQ oxidation to remove
the NAP protecting functions at the C-2 and C-6 positions.
The resulting intermediate was subsequently treated with a
3HF·Et3N complex to cleave the TBS ether functions affording
the calceolarioside-A 3 in 75% yield with >19 : 1 E/Z ratio
(Scheme 5a).
The synthesis of the calceolarioside-B 4 commenced with
the coupling of the TBS-protected caffeic acid to the thiogluco-
side 14 to produce the 6-caffeoyl-thioglucoside 23 (Scheme 5b).
Subsequent coupling of the thioglucoside 23 with the phenyl-
ethyl alcohol 8 under the LCG conditions furnished the fully
protected calceolarioside-B 24. The global deprotection of 24
included: (i) PdCl2 catalysed deallylation, (ii) the DDQ oxi-
dative cleavage of the NAP ether function, and (iii) the cleavage
Thus, the thioglucoside 13 was coupled with the protected
phenylethyl alcohol 8 under the LCG conditions to afford the
phenylethyl β-glucoside 19 in 63% yield as a single isolable
anomer (estimated α : β = 1 : >19) (Scheme 4a). The subsequent
coupling of the β-glucoside 19 with the protected caffeic acid 9
furnished the caffeic acid conjugated β-glucoside 20 in 60%
yield (Z : E = >19 : 1). The removal of the C-3 allyl ether function
in 20 with PdCl2 catalyst in a buffered solution furnished the
phenylethyl β-glucoside 21 in 70% yield (Z : E = >19 : 1). The
glucoside intermediate 21 was then glycosylated with the NAP-
protected thiorhamnoside 7 using the NFM-modulated glyco-
sylation protocol to furnish the protected acteoside 22 in 60%
yield as a single isolable isomer (Scheme 4b). The deprotection
of the NAP and TBS ether functions in 22 with DDQ oxidation
and 3HF·Et3N complex afforded the desired acteoside 2
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2929

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 2 The anti-proliferation properties of the PhGs 1, 2, 3 and 4 on
human hormone-refractory prostate cancer PC-3 cell lines (see Experi-
mental for detail).
Among compounds 1–4, the disaccharide PhG acteoside 2
exhibits the best inhibition for the proliferation of the PC-3
cancer cell line. About 23%–30% of the proliferation activity of
the cancer cells is inhibited by 2, which is roughly double the
potency afforded by echinacoside 1 as well as calceolariosides
3 and 4 (about 10%–15% proliferation inhibition from 1, 3,
and 4). Empirically, the α-Rha-(1→3)-Glc disaccharide unit and
the 4-caffeoyl function in 2 are related to a higher anti-prolifer-
ation activity. The acteoside 2 has been shown to exhibit inhi-
bition on the proliferation of HL-60 leukaemia cells39 and
collectively the results implicate that the acteoside shows
general inhibition capacity on different cancer cell lines.
Scheme 5 (a) The synthesis of calceolarioside-A 3 and (b) the synthesis
of calceolarioside-B 4 from 8, 9, 14 and 21. All = allyl ether; NAP =
naphthylmethyl ether; Napth = naphthylidene acetal; TBS = tert-butyl-
dimethylsilyl ether.
3 Conclusions
of the TBS ether functions with the 3HF·Et3N complex. As
such, the desired calceolarioside-B 4 was produced in 60%
yield as a single E-isomer (based on 1H NMR spectrum).
A general strategy for the total synthesis of natural phenyletha-
noid glycosides (PhG) was described. The synthesis features
the use of the low concentration glycosylation and NFM-modu-
lated glycosylation methods for the construction of 1,2-trans
glycosidic bonds. Four related PhG metabolites including echi-
nacoside 1, acteoside 2, calceolarioside-A 3 and calceolario-
side-B 4 were prepared and their anti-proliferation properties
for the human prostate cancer PC-3 cell line were studied. The
acteoside 2 was found to exhibit the best anti-proliferation
capacity. Further biological studies such as IC-50 analysis,
anti-proliferation for other cancer cell lines, etc., are underway
and the results will be reported in due course.
2.5. Anti-proliferation properties of PhG compounds 1–4 on
human prostate cancer cell lines
Prostate cancer is one of the most commonly occurred non-
cutaneous carcinoma of men.34,35 Typical chemotherapy drugs
for prostatic cancer include paclitaxel, mitroxantrone, etc.
These anti-cancer drugs are highly toxic to normal tissues.36
A
solution to this problem is the co-treatment of the cancer with
a chemotherapeutic agent and anti-proliferation remedies.
Such strategy relieves the toxic effect of the chemotherapeutic
agent. Numerous studies have indicated that natural plant
metabolites such as curcumin and caffeic acid phenethyl ester
(CAPE) that bear a cinnamic acid moiety exhibit anti-prolifer-
ation activities on cancer cell lines.37,38 As the present PhG
compounds 1–4 contain a caffeic acid component, this raises
our interest in studying their anti-proliferation properties. Con-
sidering that the acteoside 2 has been shown to induce the
4 Experimental
4.1. 2,3,4,6-Tetra-O-(2-naphthyl)methyl-D-glucopyranosyl-
β-(1→6)-3-O-allyl-O-(2-naphthyl)methyl-1-thio-
β-D-glucopyranoside (15) (Scheme 3a)
cell-cycle arrest in HL-60 leukemia cell proliferation,39 we evalu- A mixture of glucosyl phosphate 6 (2.1 g, 2.32 mmol), gluco-
ated the anti-proliferation properties of compounds 1–4 on side acceptor 5 (650 mg, 1.55 mmol) and flame dried MS (AW
human prostate cancer cell lines. Fig. 2 depicts the preliminary 300) was suspended in 1 : 2 : 1 CH2Cl2–CH3CN–EtCN (150 mL)
results of this study.
and stirred for 20 min at room temperature under an N2
2930 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
atmosphere. The resulting reaction mixture was cooled at β-anomer of the disaccharide 16, Rf 0.45 (hexane–EtOAc, 7.5/
1
−60 °C and stirred for an additional 20 min, followed by 3.5); [α]2D5 −55.0 (c 0.1, CHCl3); H NMR (600 MHz, CDCl3): δ =
addition of TMSOTf (282 μL, 1.55 mmol). After reaction for 7.81–7.75 (m, 10H, ArH), 7.73–7.71 (m, 3H, ArH), 7.69–7.64 (m,
4 h, the temperature was raised to −50 °C. The progress of the 4H, ArH), 7.60 (d, J = 8.4 Hz, 1H, ArH), 7.55–7.52 (m, 2H, ArH),
glycosylation was monitored by TLC. After 24 h reaction at 7.47–7.41 (m, 10H, ArH), 7.40–7.37 (m, 3H, ArH), 7.36–7.35 (m,
−50 °C, the reaction was quenched by Et3N. The resulting 1H, ArH), 7.14 (d, J = 8.4 Hz, 1H, ArH), 6.64 (d, J = 1.8 Hz, 1H,
mixture was filtered over celite, and concentrated for flash ArH), 6.60 (d, J = 7.8 Hz, 1H, ArH), 6.53 (dd, J = 8.4, 1.8 Hz, 1H,
chromatography purification (hexane–EtOAc, 7/3) over silica ArH), 5.92–5.86 (m, 1H, allyl-H), 5.21 (dd, J = 17.4, 1.2 Hz, 1H),
gel to furnish the desired disaccharide thioglycoside 15 5.16–5.08 (m, 3H), 4.95 (d, J = 10.8 Hz, 2H), 4.92 (d, J = 11.4
(1.20 g, 65%) as a white glassy solid. For disaccharide thiogly- Hz, 1H), 4.84 (d, J = 11.4 Hz, 1H), 4.78 (d, J = 12.6 Hz, 1H),
coside 15, Rf 0.4 (hexane–EtOAc, 1/2); [α]2D5 = −42.1 (c 0.2, 4.68–4.65 (m, 3H), 4.60 (d, J = 7.8 Hz, 1H), 4.42–4.40 (m, 1H),
CHCl3); 1H NMR (600 MHz, CDCl3): δ = 7.83–7.80 (m, 4H, 4.38 (dd, J = 13.2, 6.0 Hz, 1H), 4.25–4.23 (m, 1H), 4.19 (dd, J =
ArH), 7.78–7.75 (m, 7H, ArH), 7.73 (d, J = 7.8 Hz, 1H, ArH), 12.6, 6.0 Hz, 1H), 4.12–4.09 (m, 1H), 3.90 (dd, J = 10.8, 4.8 Hz,
7.71–7.68 (m, 3H, ArH), 7.66 (s, 1H, ArH), 7.61 (d, J = 7.8 Hz, 1H), 3.80 (dd, J = 10.8, 1.8 Hz, 1H), 3.77–3.70 (m, 3H),
1H, ArH), 7.57–7.53 (m, 3H), 7.47–7.45 (m, 6H, ArH), 7.44–7.42 3.65–3.59 (m, 2H), 3.57–3.51 (m, 3H), 3.34–3.33 (m, 2H),
(m, 6H, ArH), 7.41–7.39 (m, 3H, ArH), 7.37 (dd, J = 8.4, 1.2 Hz, 2.75–2.72 (m, 3H), 0.93 (s, 9H), 0.91 (s, 9H), 0.13 (s, 3H), 0.11
1H, ArH), 7.15 (dd, J = 8.4, 1.2 Hz, 1H, ArH), 7.0 (d, J = 8.4 Hz, (s, 3H), 0.07 (s, 3H), 0.06 (s, 3H); 13C NMR (150 MHz, CDCl3):
2H, ArH), 5.91–5.85 (m, 1H, allyl-H), 5.20 (dd, J = 17.4, 1.8 Hz, δ = 146.6, 145.3, 136.1, 136.0, 135.5, 135.2, 133.39, 133.36,
1H), 5.13–5.10 (m, 3H), 4.97 (d, J = 10.8 Hz, 2H), 4.96 (d, J = 133.31, 133.2, 133.1, 133.06, 133.03, 132.99, 132.96, 131.6,
11.2 Hz, 1H), 4.88 (d, J = 11.4 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H), 128.3, 128.2, 128.1, 128.09, 128.03, 128.00, 127.9, 127.78,
4.74 (d, J = 10.8 Hz, 1H), 4.69 (d, J = 7.8 Hz, 1H), 4.67 (d, J = 9.0 127.73, 127.70, 127.68, 127.65, 126.88, 126.85, 126.7, 126.5,
Hz, 1H), 4.65 (d, J = 9.6 Hz, 1H), 4.59 (d, J = 7.8 Hz, 1H), 4.32 126.4, 126.3, 126.1, 126.06, 126.03, 126.01, 125.98, 125.92,
(dd, J = 12.6, 5.4 Hz, 1H), 4.23–4.19 (m, 2H), 3.96 (dd, J = 11.2, 125.89, 125.88, 125.87, 125.82, 121.8, 121.6, 120.9, 117.1 (allyl-
1
1
5.4 Hz, 1H), 3.81 (dd, J = 10.8, 1.8 Hz, 1H), 3.77–3.71 (m, 3H), C), 104.0 (C-1′, JC–H = 161.9 Hz), 103.8 (C-1, JC–H = 161.9 Hz),
3.62–3.58 (m, 2H), 3.55–3.51 (m, 2H), 3.37 (t, J = 9.0 Hz, 1H), 84.7, 83.8, 82.0, 81.6, 77.8, 75.7, 75.1, 74.9, 74.8, 74.7, 74.6,
3.34 (t, J = 9.0 Hz, 1H), 2.75 (s, 1H, OH), 2.19 (s, 3H, CH3); 13C 74.2, 73.7, 71.2, 70.9, 69.4, 68.8, 35.5, 26.01, 26.00, 18.4, −4.00,
NMR (150 MHz, CDCl3): δ = 137.6, 136.1, 136.0, 135.7, 135.58, −4.1; HRMS-EI (m/z): [M + Na]+ calcd for C90H102NaO13Si2 ,
+
135.57, 135.0, 133.38, 133.32, 133.2, 133.14, 133.10, 133.04, 1469.6751; found, 1469.6747.
133.00, 132.9, 132.3, 130.0, 129.7, 128.3, 128.14, 128.12,
128.03, 128.01, 127.9, 127.78, 127.77, 127.71, 127.70, 127.6,
126.88, 126.85, 126.7, 126.5, 126.4, 126.36, 126.32, 126.15,
126.10, 126.0, 125.99, 125.97, 125.93, 125.90, 125.86, 125.85,
125.84, 117.1 (allyl-C), 103.9 (C-1′, JC–H = 161.9 Hz), 88.0 (C-1),
85.9, 84.7, 82.1, 80.5, 78.6, 77.8, 75.8, 75.3, 75.1, 74.95, 74.91,
4.3. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
4-O-[3,4-bis-(O-tert-butyldimethylsilyl)caffeoyl]-
6-O-[2,3,4,6-tetra-O-(2-naphthyl)methyl-β-D-glucopyranosyl)]-
3-O-allyl-2-O-(2-naphthyl)methyl-β-D-glucopyranoside 17
(Scheme 3b)
74.3, 73.7, 71.2, 69.5, 68.8, 31.0, 21.1 (CH3); HRMS-EI (m/z): 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (178 mg,
[M + Na]+ calcd for C77H72NaO10S+, 1211.4738; found, 1211.4742.
0.932 mmol) and 4-(dimethyl-amino)pyridine (37 mg,
0.310 mmol), were added to a mixture of compound 9
(380 mg, 0.932 mmol) and 17 (450 mg, 0.310 mmol) in CH2Cl2
(20 mL) at 0 °C. After stirring for 24 h at room temperature,
the reaction mixture was diluted with CH2Cl2 (75 mL) and
washed with H2O (2 × 50 mL). The organic layer were dried
4.2. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
6-O-[2,3,4,6-tetra-(2-naphthyl)methyl-β-D-glucopyranosyl)]-
3-O-allyl-2-O-(2-naphthyl) methyl-β-D-glucopyranoside 16
(Scheme 3a)
A
mixture of thioglucosyl disaccharide 15 (750 mg, over MgSO4, filtered, and concentrated for flash chromato-
0.633 mmol), TBS-protected phenylethyl alcohol 8 (220 mg, graphy purification (hexane–EtOAc, 9/1) over silica gel to
0.575 mmol) and flame dried MS (AW 300) was suspended in furnish the desired disaccharide glucoside 17 (342 mg, 60%,
2 : 1 : 1 CH2Cl2–CH3CN–EtCN (42.0 mL) and stirred for 20 min as a E : Z = >19 : 1) as a colourless gummy solid. For the
at room temperature under an N2 atmosphere. The reaction E-isomer of compound 17, Rf 0.5 (hexane–EtOAc, 1/4); [α]D25
1
mixture was cooled at −60 °C and stirred for an additional −14.3 (c 0.4, CHCl3); H NMR (400 MHz, CDCl3): δ = 7.81–7.75
20 min, followed by the addition of NIS (144 mg, 0.633 mmol) (m, 7H, ArH), 7.72–7.67 (m, 7H, ArH), 7.66–7.64 (m, 2H, ArH),
and TMSOTf (52 μL, 0.287 mmol) and then it was brought to 7.61 (d, J = 6.8 Hz, 1H, ArH), 7.58–7.56 (m, 1H, ArH), 7.52 (d,
−40 °C and stirred for 18 h. The progress of the glycosylation J = 8.4 Hz, 1H), 7.48 (dd, J = 7.2, 1.2 Hz, 1H, ArH), 7.45–7.35
coupling was observed by TLC. Upon the completion of the (m, 14H, ArH), 7.32 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 7.09 (dd, J =
reaction it was quenched by Et3N and Na2S2O3. The resulting 8.0, 0.8 Hz, 1H, ArH), 6.99 (d, J = 2.0 Hz, 1H, ArH), 6.93 (dd,
mixture was filtered, and concentrated for flash chromato- J = 8.4, 2.0 Hz, 1H, ArH), 6.77 (d, J = 8.4 Hz, 1H, ArH), 6.71 [d,
graphy purification (hexane–EtOAc, 4/1) over silica gel to J = 12.4 Hz, 0.04H, ArHCvCH (Z)], 6.60 (d, J = 2.0 Hz, 1H,
obtain the β-anomer of disaccharide 16 (540 mg, 60%, esti- ArH), 6.53 (d, J = 8.2 Hz, 1H, ArH), 6.42 (dd, J = 8.2, 2.0 Hz, 1H,
mated α : β = >19 : 1) as a white amorphous solid. For the ArH), 6.21 [d, J = 16.0 Hz, 1H, ArHCvCH (E)], 5.85–5.75 (m,
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2931

View Article Online
Paper
Organic & Biomolecular Chemistry
1 H, allyl-H), 5.70 [d, J = 12.8 Hz, 0.04 H, ArHCvCH (Z)], 5.28 (s, 127.94, 127.76, 127.74, 127.70, 127.66, 127.61, 127.0, 126.9,
1H), 5.19–5.13 (m, 2H), 5.06 (d, J = 11.2 Hz, 2H), 4.99 (t, J = 9.2 Hz, 126.7, 126.47, 126.44, 126.1, 126.03, 126.00, 125.98, 125.94,
1H), 4.93–4.88 (m, 3H), 4.82 (d, J = 11.2 Hz, 1H), 4.73 (d, 125.89, 125.86, 125.81, 125.7, 122.5, 121.8, 121.6, 121.1, 120.8,
J = 12.8 Hz, 1H), 4.69 (d, J = 11.6 Hz, 1H), 4.63–4.54 (m, 3H), 120.6, 114.5, 104.18 (C-1′), 103.26 (C-1), 84.7, 82.1, 81.1, 77.7,
4.46 (d, J = 8.0 Hz, 1H), 4.27 (dd, J = 12.8, 5.6 Hz, 1H), 75.7, 75.0, 74.9, 74.6, 74.4, 74.1, 73.7, 71.1, 70.8, 69.0, 68.6,
4.12–4.04 (m, 3H), 3.81 (d, J = 8.8 Hz, 2H), 3.74–3.69 (m, 4H), 35.4, 29.7, 26.0, 25.96, 25.93, 18.5, 18.49, 18.44, 18.42, −4.0,
3.61–3.56 (m, 2H), 3.49–3.45 (m, 2H), 2.70–2.57 (m, 2H), 0.978 −4.0, −4.1, −4.1; HRMS-EI (m/z): [M
+
Na]+ calcd for
C
108H132NaO16Si4+, 1819.8485; found, 1819.8495.
(s, 9H), 0.976 (s, 9H), 0.92 (s, 9H), 0.90 (s, 9H), 0.20 (s, 6H),
0.18 (s, 6H), 0.097 (s, 3H), 0.091 (s, 3H), 0.04 (s, 3H), 0.03 (s,
3H); 13C NMR (100 MHz, CDCl3): δ = 166.4 (CvO), 149.9,
147.4, 146.7, 146.1, 145.4, 136.3, 136.2, 136.1, 135.8, 135.7,
135.2, 133.56, 133.51, 133.4, 133.3, 133.2, 133.1, 133.0, 131.8,
128.4, 128.3, 128.29, 128.24, 128.19, 128.16, 128.13, 127.96,
127.92, 127.8, 127.7, 127.2, 127.0, 126.8, 126.7, 126.6, 126.5,
4.5. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
4-O-[3,4-bis-(O-tert-butyldimethylsilyl)-caffeoyl]-
6-O-[2,3,4,6-tetra-(2-naphthyl)methyl-β-D-glucopyranosyl)]-
3-O-[2,3,4-tri-O-(2-naphthyl)methyl-α-L-rhamnopyranosyl]-
2-O-(2-naphthyl)methyl-β-D-glucopyranoside 1a (Scheme 3b)
126.26, 126.24, 126.19, 126.11, 126.05, 126.03, 125.98, 125.93, A mixture of NAP-protected thiorhamnosyl donor 7 (115 mg,
122.6, 122.0, 121.7, 121.4, 121.0, 120.8, 117.1 (allyl-C), 114.9, 0.166 mmol) and flame dried MS (AW 300) was suspended in
104.3 (C-1′), 103.8 (C-1), 84.8, 82.3, 81.9, 81.6, 77.8, 77.4, 75.9, CH2Cl2 (3.3 mL) then stirred for 20 min at room temperature
75.2, 75.0, 74.8, 74.3, 73.8, 71.1, 70.9, 69.3, 68.8, 35.6, 26.2, under an N2 atmosphere. The reaction mixture was cooled at
26.17, 26.14, 26.11, 18.7, 18.6, 18.5, 0.2, −3.80, −3.82, −3.84, −10 °C and stirred for an additional 20 min, followed by the
−3.9; HRMS-EI (m/z): [M + Na]+ calcd for C111H136NaO16Si4 , addition of N-formyl morpholine (45 μL, 0.445), NIS (38 mg,
+
1859.8798; found, 1859.8780.
0.166 mmol) and TMSOTf (30 μL, 0.166 mmol). After the pre-
activation of donor 1.5 h, acceptor 17 (200 mg, 0.111 mmol)
was added to the reaction mixture. The progress of glycosyla-
tion coupling was observed by TLC. Upon the completion of
the reaction after 48 h, it was quenched by Et3N and Na2S2O3.
The resulting mixture was filtered, and concentrated for flash
4.4. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
4-O-[3,4-bis-(O-tert-butyldimethylsilyl)-caffeoyl]-
6-O-[2,3,4,6-tetra-(2-naphthyl)methyl-β-D-glucopyranosyl)]-
2-O-(2-naphthyl)methyl-β-D-glucopyranoside 18 (Scheme 3b)
To a solution of compound 17 (850 mg, 0.462 mmol), in chromatography purification (hexane–Et2O–CH2Cl2, 1/1/6) over
acetone (10 mL), sodium acetate (190 mg, 2.314 mmol) and silica gel to furnish the fully protected echinacoside 1a as a
acetic acid (1.0 mL, 95%) were added. To the above reaction colorless gummy solid (157 mg, 60%, ca. E : Z 10 : 1 to 7 : 1;
mixture palladium dichloride (164 mg, 0.925 mmol) was during chromatography purification, 1a was exposed to UV
added at 0 °C, and stirred for 5 h at room temperature. Upon illustration which increased the proportion of the Z-isomer in
the completion of the reaction as monitored by TLC, the reac- the mixture, the 10 : 1 E : Z ratio was obtained before chromato-
tion mixture was filtered through celite, and concentrated for graphy). For the E-isomer of 1a, Rf 0.25 (hexane–Et2O–CH2Cl2,
1
flash chromatography purification (hexane–EtOAc, 9/1) over 1/2/5); [α]2D5 −25.7 (c 0.3, CHCl3); H NMR (600 MHz, CDCl3):
silica gel to furnish the desired caffeic acid conjugated gluco- δ = 7.82–7.66 (m, 19H, ArH), 7.65–7.56 (m, 9H, ArH), 7.55–7.48
side 18 (655 mg, 78%, E : Z = >19 : 1) as a yellow gummy solid. (m, 6H, ArH), 7.45–7.39 (m, 15H, ArH), 7.37–7.34 (m, 4H, ArH),
For glucoside 18, Rf 0.4 (hexane–EtOAc, 1/4, TLC developed 7.31–7.28 (m, 1H, ArH), 7.24–7.19 (m, 2H, ArH), 7.12–7.09 (m,
twice); [α]D −16.6 (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3): 1H, ArH), 7.02–7.01 (m, 1H, ArH), 6.89 (dd, J = 8.4, 1.8 Hz, 1H,
δ = 7.85–7.81 (m, 6H, ArH), 7.99–7.72 (m, 8H, ArH), 7.70–7.68 ArH), 6.72 [d, J = 13.2 Hz, 0.26 H, ArHCvCH (Z)], 6.70 (d, J =
(m, 2H, ArH), 7.66–7.61 (m, 3H, ArH), 7.58–7.53 (m, 2H, ArH), 8.4 Hz, 1H, ArH), 6.59 [d, J = 13.2 Hz, 0.21H, ArHCvCH (Z)],
7.48–7.44 (m, 9H, ArH), 7.43–7.41 (m, 3H, ArH), 7.39–7.36 (m, 6.54 (d, J = 1.2 Hz, 1H, ArH), 6.44 (d, J = 8.4 Hz, 1H, ArH), 6.38
2H, ArH), 7.15 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 7.03 (d, J = 2.0 Hz, (dd, J = 8.4, 1.8 Hz, 1H), 6.29 [d, J = 16.2 Hz, 1H, ArHCvCH
1H, ArH), 6.95 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 6.80 (d, J = 8.0 Hz, (E)], 5.78 [d, J = 13.2 Hz, 0.14H, ArHCvCH (Z)], 5.31 (d, J =
1H, ArH), 6.67 (d, J = 2.0 Hz, 1H, ArH), 6.61 (d, J = 8.4 Hz, 1H, 6.0 Hz, 1H), 5.21 (d, J = 10.8 Hz, 1H), 5.18 [d, J = 11.4 Hz,
ArH), 6.50 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 6.27 [d, J = 16.0 Hz, 0.29H, (Z)], 5.10–5.04 (m, 2H), 4.99 [d, J = 11.4 Hz, 0.3H, (Z)],
1H, ArHCvCH (E)], 5.23 (d, J = 11.2 Hz, 1H), 5.11 (d, J = 11.6 4.95–4.93 (m, 3H), 4.91–4.88 (m, 2H), 4.77–4.69 (m, 3H),
Hz, 1H), 5.01–4.94 (m, 4H), 4.88 (d, J = 11.8/11.6 Hz, 1H), 4.77 4.66–4.63 (m, 2H), 4.61 (d, J = 7.8 Hz, 1H), 4.57 (d, J = 12.0 Hz,
(d, J = 12.4 Hz, 1H), 4.69–4.59 (m, 5H), 4.52 (d, J = 8.0 Hz, 1H), 1H), 4.49–4.41 (m, 2H), 4.34–4.28 (m, 1H), 4.22 (d, J = 12.0 Hz,
4.18–4.13 (m, 2H), 3.90–3.82 (m, 3H), 3.79–3.75 (m, 4H), 1H), 4.14 (d, J = 12.6 Hz, 1H), 4.09–4.06 (m, 2H), 4.01–3.96 (m,
3.66–3.62 (m, 2H), 3.57–3.54 (m, 1H), 3.39 (dd, J = 8.8, 8.0 Hz, 1H), 3.90 (dd, J = 9.0, 2.4 Hz, 1H), 3.87–3.79 (m, 3H), 3.76 (d,
1H), 2.73–2.64 (m, 2H), 1.02 (s, 9H), 1.01 (s, 9H), 0.98 (s, 9H), J = 2.4 Hz, 2H), 3.73–3.71 (m, 2H), 3.68 (s, 1H), 3.65–3.51 (m,
0.95 (s, 9H), 0.24 (s, 6H), 0.22 (s, 6H), 0.157 (s, 3H), 0.153 (s, 4H), 3.43 (t, J = 8.4 Hz, 1H), 2.59–2.57 (m, 1H), 1.32 [d, J = 6.6
3H), 0.09 (s, 6H); 13C NMR (100 MHz, CDCl3): δ = 166.8 (CvO), Hz, 1.14H, (Z) Rha-CH3], 1.19 (d, J = 6.6 Hz, 3H, Rha-CH3),
149.8, 147.2, 146.5, 146.3, 145.2, 136.17, 136.12, 135.6, 135.5, 0.98 (s, 9H), 0.97 (s, 2.4H), 0.96 (s, 9H), 0.94 (s, 3H), 0.92 (s,
133.38, 133.33, 133.2, 133.1, 133.09, 133.04, 132.95, 132.92, 9H), 0.88 (s, 9H), 0.19 (s, 3H), 0.18 (s, 3H), 0.14 (s, 3H), 0.13 (s,
131.6, 128.3, 128.2, 128.16, 128.10, 128.04, 128.00, 127.97, 3H), 0.079 (s, 6H), 0.014 (s, 3H), 0.011 (s, 3H); 13C NMR
2932 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
(150 MHz, CDCl3): δ = 166.4 (CvO), 149.8, 147.3, 146.5, 146.3, HRMS-EI (m/z): [M + Na]+ calcd for C35H46NaO20+, 809.2475;
145.2, 136.4, 136.3, 136.2, 136.1, 136.0, 135.9, 135.8, 135.6, found, 809.2479.
135.5, 133.36, 133.31, 133.29, 133.25, 133.21, 133.1, 133.0,
132.97, 132.94, 132.91, 132.8, 132.7, 131.4, 128.9, 128.27,
128.23, 128.1, 128.06, 128.03, 128.00, 127.9, 127.8, 127.77,
127.74, 127.71, 127.68, 127.63, 127.5, 127.0, 126.7, 126.5,
4.7. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl-
3-O-allyl-2,6-di-O-(2-naphthyl)methyl-1-β-D-glucopyranoside 19
(Scheme 4a)
126.46, 126.44, 126.3, 126.19, 126.12, 126.06, 126.01, 125.96, A mixture of thioglucoside 13 (1.9 g, 3.14 mmol) and TBS-pro-
125.91, 125.87, 125.83, 125.77, 125.70, 125.66, 125.60, 125.5, tected phenylethyl alcohol 8 (1.0 g, 2.61 mmol) and flame
125.49, 125.43, 125.3, 123.9, 122.7, 121.7, 121.5, 121.1, 120.7, dried MS (AW 300) was suspended in 1 : 2 : 1 CH2Cl2–CH3CN–
120.5, 120.2, 115.3, 114.6, 104.1 (C-1″, JC–H = 161.9 Hz), 103.4 EtCN (176 mL) and stirred for 20 min at room temperature
(C-1′, JC–H = 161.9 Hz), 99.6 (C-1, JC–H = 173.9 Hz), 84.7, 82.5, under an N2 atmosphere. The reaction mixture was cooled at
82.4, 82.1, 80.4, 80.2, 80.1, 80.0, 79.0, 78.0, 77.7, 76.7, 76.4, −60 °C and stirred for an additional 20 min. To the mixture
75.7, 75.0, 74.9, 74.8, 74.7, 74.6, 74.3, 74.2, 73.7, 72.6, 72.4, NIS (891 mg, 3.92 mmol) and TMSOTf (166 μL, 0.916 mmol)
72.4, 70.8, 69.7, 69.5, 69.0, 68.7, 68.6, 35.3, 26.0, 25.99, 25.94, was added. The progress of glycosylation was monitored by
18.6, 18.5, 18.47, 18.42, 18.39, −3.96, −4.03, −4.09, −4.17, TLC. Upon the completion of the reaction (after 3 h), the reac-
+
−4.19. HRMS-EI (m/z): [M + Na]+ calcd for C147H166NaO20Si4 , tion was quenched by the addition of NaHCO3(aq) and
2386.0942; found, 2386.0830.
Na2S2O3, and stirred for 15 min at room temperature. Then
the resulting mixture was filtered to remove MS and concen-
trated for flash chromatography purification (elution: hexane–
EtOAc, 4/1) to furnish the desired phenylethyl-β-glucoside 19
4.6. Echinacoside 1 (Scheme 3b)
A mixture of 1a (146 mg, 0.061 mmol), DDQ (224 mg, (1.40 g, 63%, estimated α : β = >19 : 1) as a pale yellow gummy
0.94 mmol) in CH2Cl2–MeOH (3 : 1), (6 mL) was stirred at liquid. For the glucoside 19, Rf = 0.4 (hexane–EtOAc, 2/1);
room temperature for 8 h. Upon the completion of the reac- [α]2D5 = −3.5 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3): δ =
tion, the reaction mixture was subjected to chromatography 7.80–7.76 (m, 7H, ArH), 7.70 (s, 1H, ArH), 7.46–7.41 (m, 6H,
purification (CH2Cl2–MeOH, 9/1) over silica gel to furnish the ArH), 6.72–6.65 (m, 3H, ArH), 5.98–5.89 (m, 1H, allyl-H), 5.25
deprotected product (46 mg, 61%). Rf 0.3 (CH2Cl2–MeOH, 9/1); (d, J = 17.2 Hz, 1H), 5.15 (d, J = 10.0 Hz, 1H), 4.93 (d, J = 11.2
the further naphthyl deprotected compound (46 mg, Hz, 1H), 4.77–4.71 (m, 3H), 4.45–4.40 (m, 2H), 4.24 (dd, J =
0.037 mmol) was dissolved in pyridine (2.0 mL) and Et3N 12.4, 6.0 Hz, 1H), 4.17–4.12 (m, 1H), 3.82 (dd, J = 10.4, 3.6 Hz,
(10 μL, 0.074 mmol) and HF·Et3N (142 µL 1.56 M in pyridine) 1H), 3.77–3.69 (m, 2H), 3.58 (t, J = 8.8 Hz, 1H), 3.50–3.47 (m,
was added at room temperature. After stirring for 3 h, TLC 1H), 3.42–3.34 (m, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.74 (bs, 1H,
showed the disappearance of the starting material. The reac- OH), 0.96 (s, 18H), 0.15 (s, 6H), 0.13 (s, 6H); 13C NMR
tion mixture was directly loaded to the silica gel column for (100 MHz, CDCl3): δ = 146.6, 145.3, 135.9, 135.4, 135.1, 133.36,
flash chromatography purification (elution: MeOH–H2O, 10/0.5) 133.30, 133.08, 133.04, 131.6, 128.3, 128.0, 127.9, 127.7, 127.6,
and followed by (ii) size exclusion chromatography (Superdex 126.8, 126.6, 126.3, 126.1, 125.99, 125.96, 125.8, 125.7, 121.86,
30 prep grade gel) (elution: MeOH) to obtain the fully depro- 121.80, 120.9, 117.2, 103.8 (C-1), 83.7, 81.6, 74.7, 74.3, 74.1,
tected echinacoside 1 (20 mg, 68%, E : Z = 10 : 1), as a pale 73.8, 71.6, 71.0, 70.3, 35.7, 26.0, 18.4, −3.9, −4.0; HRMS-EI
+
yellow glassy solid. For E-isomer of 1, [α]D25 = −71.1 (c 0.4, (m/z): [M + Na]+ calcd for C51H69O8NaSi2 , 865.4525; found,
1
CH3OH); H NMR (600 MHz, CD3OD): δ = 7.60 [d, J = 16.2 Hz, 865.4513.
1H, ArHCvCH (E)], 7.05 (d, J = 1.8 Hz, 1H, ArH), 6.96 (dd, J =
8.4, 1.8 Hz, 1H), 6.88 [d, J = 12.8 Hz, 0.1H, ArHCvCH (Z)], 6.77
(d, J = 8.4 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 6.68 (d, J = 7.8 Hz,
1H), 6.57 (dd, J = 7.8, 1.8 Hz, 1H), 6.27 [d, J = 15.6 Hz, 1H,
4.8. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
4-O-[3,4-bis-(O-tert-butyldimethylsilyl)-caffeoyl]-3-O-allyl-
2,6-di-O-(2-naphthyl)methyl-1-β-D-glucopyranoside 20
(Scheme 4a)
ArHCvCH (E)], 5.77 [d, J = 12.8 Hz, 0.1H, ArHCvCH (Z)], 5.18
(d, J = 1.2 Hz, 1H), 5.01 (t, J = 9.6 Hz, 1H), 4.39 (d, J = 7.8 Hz, 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (530 mg,
1H), 4.29(d, J = 7.8 Hz, 1H), 4.04 (ddd, J = 9.5, 8.0, 6.7 Hz, 1H), 2.78 mmol) and 4-(dimethyl-amino)-pyridine (112 mg,
3.94 (dd, J = 12.0, 2.4 Hz, 1H), 3.91 (dd, J = 3.0, 1.2 Hz, 1H), 0.925 mmol) were added TBS-protected caffeic acid 9 (1.13 g,
3.83 (dd, J = 12.0, 2.4 Hz, 1H), 3.81 (t, J = 9.6 Hz, 1H), 3.78–3.75 2.77 mmol) and phenylethyl glucoside 19 (800 mg,
(m, 1H), 3.74–3.71 (m, 1H), 3.65 (dd, J = 5.4, 1.2 Hz, 1H), 3.63 0.925 mmol) in a CH2Cl2 solution (40 mL) at 0 °C. After stir-
(dd, J = 5.4, 0.6 Hz, 1H), 3.58–3.54 (m, 2H), 3.39 (dd, J = 9.0, ring for 24 h from 0 °C to RT, the reaction mixture was diluted
8.4 Hz, 1H), 3.35 (s, 1H), 3.33 (d, J = 9.0 Hz, 1H), 3.28 (dd, J = with CH2Cl2 (150 mL) and washed with H2O (2 × 100 mL). The
9.6, 1.8 Hz, 1H), 3.23–3.18 (m, 2H), 2.82–2.77 (m, 2H), 1.08 (d, organic layer were dried over MgSO4, filtered, and concentrated
J = 6.0 Hz, 3H, CH3); 13C NMR (150 MHz, CD3OD): δ = 168.4 for flash chromatography purification (hexane–CH2Cl2–Et2O,
(CvO), 149.9, 148.2, 146.8, 146.1, 144.6, 131.4, 127.6, 123.2, 12/2/0.8) over silica gel to furnish the caffeic acid conjugated
121.3, 117.1, 116.5, 116.3, 115.2, 114.6, 104.6 (C-1″), 104.2 glucoside 20 (690 mg, 60% estimated E : Z = >19 : 1) as a pale
(C-1′), 103.0 (C-1), 81.6, 77.9, 77.8, 76.1, 75.1, 74.7, 73.7, 72.4, yellow sticky solid. For glucoside 20, Rf = 0.55 (hexane–EtOAc,
72.3, 72.0, 71.4, 70.5, 70.4, 69.4, 62.6, 36.5, 18.4 (CH3); 1/4); [α]D = −0.80 (c 0.3, CHCl3); 1H NMR (500 MHz, CDCl3):
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2933

View Article Online
Paper
Organic & Biomolecular Chemistry
δ = 7.82–7.78 (m, 3H, ArH), 7.72–7.70 (m, 5H, ArH), 7.57 [d, J = 4.10. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl-ethyl-
16.0 Hz, 1H, ArHCvCH (E)], 7.44–7.37 (m, 6H, ArH), 6.99–6.96 4-O-[3,4-bis-(O-tert-butyldimethylsilyl)-caffeoyl]-2,6-di-
(m, 2H, ArH), 6.81 (d, J = 8.0 Hz, 1H, ArH), 6.76 (d, J = 1.5 Hz, O-(2-naphthylmethyl)-3-O-(α-L-rhamnopyranosyl)-
1H, ArH), 6.72 (d, J = 8.5 Hz, 1H, ArH), 6.69 (dd, J = 8.5, 2.0 Hz, β-D-glucopyranoside 22 (Scheme 4b)
1H, ArH), 6.18 [d, J = 15.5 Hz, 1H, ArHCvCH (E)], 5.85–5.79
(m, 1H, allylic-H), 5.17 (dd, J = 17.0, 1.5 Hz, 1H), 5.11–5.06 (m,
2H), 4.94 (d, J = 11.0 Hz, 1H), 4.80 (d, J = 11.0 Hz, 1H), 4.68 (s,
2H), 4.51 (d, J = 8.0 Hz, 1H), 4.30 (dd, J = 12.5, 5.5 Hz, 1H),
A mixture of NAP-protected thiorhamnosyl donor 7 (68 mg,
0.098 mmol) and flame dried MS (AW 300) was suspended in
CH2Cl2 (2 mL) then stirred for 20 min at room temperature
under an N2 atmosphere. The reaction mixture was cooled at
4.22–4.17 (m, 1H), 4.13 (dd, J = 12.5, 6.5 Hz, 1H), 3.77 (dd, J =
−10 °C and stirred for an additional 20 min, which was fol-
17.0, 8.0 Hz, 1H), 3.69–3.59 (m, 4H), 3.52 (dd, J = 9.0, 8.0 Hz,
lowed by the addition of N-formyl morpholine (33 μL,
1H), 2.92–2.89 (m, 2H), 1.00 (s, 9H), 0.99 (s, 9H), 0.97 (s, 18H),
0.329 mmol), NIS (22.4 mg, 0.098 mmol) and TMSOTf
(18.0 μL, 0.098 mmol). After preactivation of the thioglycosyl
donor 7, the thioglucoside acceptor 21 (100 mg, 0.082 mmol)
0.23 (s, 6H), 0.21 (s, 6H), 0.167 (s, 3H), 0.165 (s, 3H), 0.14 (s,
6H); 13C NMR (125 MHz, CDCl3): δ = 166.0 (CvO), 149.7,
147.2, 146.6, 145.6, 145.3, 135.9, 135.4, 135.1, 133.3, 133.2,
was added to the reaction mixture. The progress of glycosyla-
133.0, 132.9, 131.6, 128.1, 128.06, 128.04, 127.9, 127.8, 127.6,
tion coupling was observed by TLC. Upon completion of reac-
126.8, 126.5, 126.3, 125.98, 125.95, 125.82, 125.81, 125.7,
tion after 48 h, the reaction was quenched by Et3N and
122.4, 121.87, 121.81, 121.2, 120.9, 120.6, 116.9, 114.9, 103.6
Na2S2O3 addition. The resulting mixture, filtered to remove MS
(C-1), 81.8, 81.5, 74.9, 74.1, 73.8, 71.15, 71.01, 69.9, 35.7, 26.0,
and the filtrate was concentrated for flash chromatography
25.9, 18.5, 18.49, 18.46, −3.96, −3.99, −4.02, −4.06; HRMS-EI
purification (hexane–Et2O–CH2Cl2, 9/1/1) over silica gel to
furnish fully protected acteoside 22 (87 mg, 60%, E : Z =
(m/z): [M + H]+ calcd for C72H103O11Si4 , 1255.6572; found,
+
1255.6538.
>19 : 1) as a gummy solid. For the E-isomer of acteoside 22, Rf
= 0.4 (hexane–Et2O–CH2Cl2, 8/1.5/1); [α]2D5 = −22.91 (c = 0.3,
1
CHCl3); H NMR (500 MHz, CDCl3): δ = 7.80 (J = 8.0 Hz, 1H,
4.9. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]-ethyl
ArH), 7.73–7.67 (m, 10H, ArH), 7.64–7.63 (m, 3H, ArH),
4-O-[3,4-bis-(O-tert-butyldimethylsilyl)-caffeoyl]-2,6-di-
7.60–7.54 (m, 4H, ArH), 7.48–7.32 (m, 14H, ArH), 7.26 (dd, J =
O-(2-naphthyl)methyl-1-β-D-glucopyranoside 21 (Scheme 4a)
8.0, 1.5 Hz, 1H, ArH), 7.21–7.19 (m, 2H, ArH), 7.01 (dd, J = 8.5,
To a solution of caffeic acid conjugated glucoside 20 (2.0 g,
1.0 Hz, 1H, ArH), 6.97 (d, J = 2.0 Hz, 1H, ArH), 6.88 (dd, J = 8.0,
1.593 mmol) in acetone (20 mL), sodium acetate (653 mg,
2.0 Hz, 1H, ArH), 6.72 (dd, J = 8.5, 3.0 Hz, 1H, ArH), 6.67 (d, J =
2.0 Hz, 1H, ArH), 6.62–6.59 (m, 2H, ArH), 6.20 (dd, J = 16.0, 3.5
Hz, 1H, ArHCvCH (E)] 5.30 (s, 1H), 5.12 (td, J = 9.5, 3.5 Hz,
1H), 4.98 (d, J = 12.0 Hz, 1H), 4.92 (dd, 11.5, 2.5 Hz, 1H), 4.76
(dd, J = 12.0, 2.4 Hz, 1H), 4.70 (s, 3H), 4.67–4.65 (m, 2H),
4.53–4.48 (m, 2H), 4.24 (dd, J = 12.5, 2.0 Hz, 1H), 4.16–4.14 (m,
2H), 3.98 (td, J = 9.5, 3.5 Hz, 1H), 3.89 (dt, J = 9.5, 2.5 Hz, 1H),
3.77–3.63 (m, 7H), 3.59 (td, J = 9.5, 3.5 Hz, 1H), 3.47–3.44 (m,
7.97 mmol) and acetic acid (2.0 mL, 95%) were added. To the
above reaction mixture palladium dichloride (564 mg,
3.187 mmol) was added and stirred from 0 °C to room temp-
erature for 5 h until the completion of the reaction. The reac-
tion mixture was then filtered over a celite pad to remove the
sodium salts and the filtrate was concentrated for flash chrom-
atography purification (hexane–CH2Cl2–Et2O, 8/2/0.8) over
silica gel to furnish the desired thioglucosyl acceptor 21 (1.3 g,
1H), 2.84–2.79 (m, 2H), 1.16 (d, J = 6.0 Hz, 3H), 0.99 (s, 9H),
70%, E : Z = >19 : 1). For thioglucosyl acceptor 21, Rf = 0.4
0.96 (s, 9H), 0.95 (s, 9H), 0.94 (s, 2.97, 9H), 0.21 (s, 3H), 0.20 (s,
(hexane–EtOAc, 1/4); [α]2D5 −3.4 (c 0.3, CHCl3); 1H NMR
3H), 0.14 (s, 6H), 0.13 (s, 6H), 0.09 (s, 6H); 13C NMR (125 MHz,
(400 MHz, CDCl3): δ = 7.81–7.69 (m, 8H, ArH), 7.55 [d, J = 15.6
Hz, 1H, ArHCvCH (E) ], 7.44–7.36 (m, 6H, ArH), 6.97 (s, 1H,
ArH), 6.92 (d, J = 8.4 Hz, 1H, ArH), 6.79–6.68 (m, 4H, ArH), 6.15
[d, J = 16.0 Hz, 1H, ArHCvCH (E) ], 5.06 (t, J = 9.2 Hz, 1H),
4.95 (d, J = 11.6 Hz, 1H), 4.77 (d, J = 11.6 Hz, 1H), 4.72 (d, J =
12.0 Hz, 1H), 4.66 (d, J = 12.0 Hz, 1H), 4.50 (d, J = 7.6 Hz, 1H),
CDCl3): δ = 166.2 (CvO), 149.6, 147.1, 146.5, 146.0, 145.2,
136.3, 136.2, 135.9, 135.8, 135.3, 133.27, 133.22, 133.1, 133. 0,
132.9, 132.8, 132.7, 132.6, 131.3, 128.1, 127.9, 127.88, 127.84,
127.76, 127.72, 127.65, 127.59 127.58, 127.53, 127.4, 126.5,
126.2, 126.1, 125.95, 125.93, 125.81, 125.80, 125.74, 125.72,
125.59, 125.4, 125.3, 122.6, 121.6, 121.0, 120.8, 120.3, 114.7,
4.23–4.17 (m, 1H), 3.83–3.75 (m, 2H), 3.70–3.63 (m, 3H),
103.4 (C-1′, JC–H = 159.3 Hz), 99.4 (C-1, JC–H = 171.3 Hz), 82.5,
3.44–3.40 (m, 1H), 2.90 (t, J = 6.0 Hz, 2H), 2.66 (s, 1H), 0.99 (s,
80.0, 79.9, 78.7, 74.4, 74.2, 73.8, 73.7, 72.5, 72.3, 71.0, 69.8,
18H), 0.97 (s, 18H), 0.22 (s, 6H), 0.20 (s, 6H), 0.17 (s, 6H), 0.14
68.6, 35.5, 25.9, 25.8, 18.4, 18.3, −4.0, −4.09, −4.18; HRMS-EI
(s, 6H); 13C NMR (100 MHz, CDCl3): δ = 166.7 (CvO), 149.7,
147.1, 146.6, 146.0, 145.3, 135.6, 135.3, 133.3, 133.2, 133.0,
132.9, 131.5, 128.3, 128.2, 128.0, 127.88, 127.80, 127.67,
127.62, 127.0, 126.5, 126.06, 126.04, 125.98, 125.91, 125.7,
122.5, 121.84, 121.83, 121.1, 120.9, 120.5, 114.7, 103.6 (C-1),
+
(m/z): [M + H]+ calcd for C108H133O15Si4 , 1781.8716; found,
1781.8731.
4.11. Acteoside 2 (Scheme 4b)10d,e
81.3, 74.54, 74.52, 73.7, 71.3, 71.0, 69.4, 35.6, 25.99, 25.94, A mixture of 22 (200 mg, 0.112 mmol) and DDQ (255 mg,
25.93, 18.5, 18.4, −4.00, −4.03, −4.07. The glucoside inter- 1.123 mmol) in a 3 : 1 CH2Cl2–MeOH mixture (8.0 mL) was
mediate 21 was taken to the next reaction step without mass stirred at room temperature for 7 h. Upon the completion of
characterization.
the reaction, it was subjected to chromatography purification
2934 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
over silica gel to furnish the denaphthalyted product (100 mg, (d, J = 8.0 Hz, 1H), 4.05–4.01 (m, 1H), 3.74–3.68 (m, 1H),
82%) (hexane–EtOAc, 3/2). The deprotected intermediate from 3.64–3.59 (m, 2H), 3.55–3.48 (m, 2H), 2.81–3.77 (m, 2H); 13C
the previous step (100 mg, 0.107 mmol) was dissolved in pyri- NMR (125 MHz, CD3OD): δ = 168.6 (CvO), 149.6, 147.6, 146.7,
dine (2 mL) to which Et3N (30 μL, 0.214 mmol) and 3HF·Et3N 146.0, 144.6, 131.4, 127.6, 123.0, 121.2, 117.1, 116.5, 116.2,
(411 µL 1.56 M) in pyridine was added. After stirring for 5 h, 115.1, 114.7, 104.3 (C-1), 76.0, 75.7, 75.2, 72.4, 72.1, 62.3, 36.5;
TLC showed the disappearance of the starting material. The HRMS-EI (m/z): [M + Na]+ calcd for C23H26NaO11+, 501.1367;
resulting mixture was purified with (i) flash chromatography found, 501.1366.
purification (elution: CH2Cl2–MeOH, 9/1 gradient to 8/2) and
followed by (ii) size exclusion chromatography (Superdex 30
prep grade gel) (elution: MeOH) to obtain fully deprotected
acteoside 2 (48 mg, 83%, E : Z = 10 : 1) as a white amorphous
4.13. p-Tolyl 6-O-[(E)-3,4-bis-(O-tert-butyldimethylsilyl)-
caffeoyl]-3-O-allyl-2,4-di-O-(2-naphthyl)methyl-1-thio-
β-D-glucopyranoside (23) (Scheme 5b)
solid. For the E-isomer of acteoside 2, Rf = 0.2 (CH2Cl2–MeOH– 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (787 mg,
H2O, 8/2/0.1); [α]D25 = −82.33 (c 0.3, CH3OH); 1H NMR 4.13 mmol) and 4-(dimethylamino)pyridine (DMAP) (200 mg,
(400 MHz, CD3OD): δ = 7.58 [d, J = 16.0 Hz, 1H, ArHCvCH (E)], 1.650 mmol), were added to a mixture of TBS-protected caffeic
7.04 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 8.4, 2.0 Hz, 1H, ArH), acid 9 (1.34 g, 3.300 mmol) and thioglucoside 14 (1.0 g,
6. 85 [d, J = 12.8 Hz, 0.18 H, ArHCvCH (Z)], 6.76 (d, J = 8.4 Hz, 1.650 mmol) in CH2Cl2 (20 mL) at 0 °C. After stirring for 4 h at
1H, ArH), 6.68–6.64 (m, 2H, ArH), 6.55 (dd, J = 8.0, 2.0 Hz, 1H, room temperature, the reaction mixture was diluted with
ArH), 6.25 [d, J = 16.0 Hz, 1H, ArHCvCH (E)], 5.7 [d, J = 12.8 CH2Cl2 (150 mL) and washed with H2O (2 × 100 mL). The
Hz, 0.14 H, ArHCvCH (Z)], 5.18 (d, J = 1.6 Hz, 1H), 4.91 (t, J = organic layer were dried over MgSO4, filtered, and concentrated
9.6 Hz, 1H), 4.37 (d, J = 7.6 Hz, 1H), 4.05–4.03 (m, 1H), 3.90 for flash chromatography purification over silica gel (hexane–
(dd, J = 3.2, 1.6 Hz, 1H), 3.80 (t, J = 9.2 Hz, 1H), 3.75–3.68 (m, EtOAc 9 : 1) to furnish the desired thioglucoside 23 (1.2 g,
1H), 3.62–3.50 (m, 5H), 3.38 (dd, J = 9.2, 8.0 Hz, 1H), 3.27 (d, J 75%, E : Z = >19 : 1). For thioglucoside 23, Rf = 0.4 (hexane–
1
= 9.6 Hz, 1H), 2.80–2.76 (m, 2H), 1.16 (d, J = 6.4 Hz, 0.4 H, EtOAc 1 : 4 v/v); [α]2D5 = +9.7 (c 0.5, CHCl3); H NMR (400 MHz,
Rha-CH3), 1.08 (d, J = 6.4 Hz, 3H, Rha-CH3). 13C NMR CDCl3): δ = 7.90–7.80 (m, 8H, ArH), 7.64 (dd, J = 8.4, 1.6 Hz,
(150 MHz, CD3OD): δ = 168.3 (CvO), 149.7, 148.0, 146.7, 1H), 7.58 (d, J = 15.9 Hz, 1H, HCvCH–O (E)), 7.53–7.47 (m,
146.0, 144.6, 131.4, 127.6, 123.2, 121.2, 117.1, 116.5, 116.3, 5H), 7.46–7.44 (m, 2H, ArH), 7.07–7.05 (m, 3H, ArH), 7.02 (dd,
115.2, 114.6, 104.1 (C-1′), 102.9 (C-1), 81.6, 76.1, 75.9, 73.7, J = 8.3, 2.1 Hz, 1H, ArH), 6.88 (d, J = 8.2 Hz, 1H, ArH), 6.21 (d, J
72.3, 72.2, 72.0, 70.5, 70.3, 62.3, 36.5, 18.4; HRMS-EI (m/z): = 15.9 Hz, 1H, HCvCH–O), 6.09–5.99 (m, 1H), 5.35 (dq, J =
[M + H]+ calcd for C29H37O+15, 625.2127; found, 625.2130.
10.5, 1.7 Hz, 1H), 5.23 (dq, J = 10.5, 1.6 Hz, 1H), 5.12 (d, J =
10.5 Hz, 1H), 5.06 (d, J = 11.0 Hz, 1H), 4.95 (d, J = 10.5 Hz, 1H),
4.84 (d, J = 11.0 Hz, 1H), 4.66 (d, J = 9.7 Hz, 1H), 4.57 (d, J =
4.12. The synthesis of calceolarioside-A (3) (Scheme 5a)
A mixture of 21 (300 mg, 0.247 mmol) and DDQ (224 mg, 11.5 Hz, 1H), 4.49 (dd, J = 12.3, 5.6 Hz, 1H), 4.44–4.39 (m, 2H),
0.988 mmol) in 3 : 1 CH2Cl2–MeOH (9.0 mL) was stirred at 3.71–3.63 (m, 3H), 3.55 (dd, J = 9.5, 8.5 Hz, 1H), 2.25 (s, 3H),
room temperature for 5 h. Upon the completion of the reaction 1.05 (s, 9H), 1.04 (s, 9H), 0.28 (s, 6H), 0.27 (s, 6H); 13C NMR
as monitored by TLC, the reaction mixture was concentrated (100 MHz, CDCl3): δ = 166.8, 149.6, 147.2, 145.0, 137.8, 135.6,
under reduced pressure. The solution was diluted with EtOAc 135.1, 134.9, 133.3, 133.2, 133.15, 133.13, 132.9, 129.5, 128.2,
(50 mL) then washed with NaHCO3(aq), (3 × 50 mL) and H2O (2 128.0, 127.9, 127.75, 127.73, 127.1, 126.9, 126.3, 126.17,
× 50 mL). The collected organic phase was dried over MgSO4, 126.10, 126.07, 125.9, 122.53, 121.2, 120.5, 117.1, 115.3, 87.7,
filtered and concentrated for flash chromatography purifi- 86.5, 80.5, 77.3, 77.0, 75.5, 75.1, 74.67, 63.14, 25.99, 25.97,
cation over silica gel to furnish the partial deprotected inter- 21.0, 18.59, 18.51, −3.98; HRMS (m/z): [M + H]+ calcd for
+
mediate (184 mg, 80%). The intermediate from the previous C59H73O8SSi2 , 997.4559; found, 997.4535.
step (184 mg, 0.196 mmol) was dissolved in pyridine (2.0 mL);
to which, Et3N (54 μL, 0.393 mmol) and 3HF·Et3N (753 µL,
1.56 M in pyridine) were added. After stirring at room tempera-
ture for 3 h, the resulting mixture was subjected for purifi-
4.14. 2-[3,4-Bis(tert-butyldimethylsilyloxy)phenyl]ethyl-
6-O-[(E)-3,4-bis-(O-tert-butyldimethylsilyl)caffeoyl]-3-O-allyl-
2,4-di-O-(2-naphthyl)methyl-β-D-glucopyranose (24)
(Scheme 5b)
cation with (i) flash chromatography over silica gel (CH2Cl2–
MeOH, 9 : 1) and (ii) followed by size exclusion chromato- A mixture of thioglucoside donor 23 (430 mg, 0.431 mmol),
graphy (Superdex 30 prep grade gel) (elution: MeOH) to TBS-protected phenylethyl alcohol 8 (150 mg, 0.392 mmol)
furnish the deprotected calceolarioside-A 3 (39 mg, 85%, E : Z and flame dried MS (AW 300) was suspended in CH2Cl2–
= >19 : 1). For E-isomer of calceolarioside-A 3, Rf = 0.25 CH3CN–EtCN (1 : 2 : 1), (28 mL) and stirred for 20 min at room
(CH2Cl2–MeOH, 9/1); [α]2D5 = −20.0 (c 0.3, CH3OH); 1H NMR temperature under an N2 atmosphere. The resulting reaction
(400 MHz, CD3OD): δ = 7.58 [d, J = 16.0 Hz, 1H, HCvCH–O], mixture was cooled at −60 °C and stirred for an additional
7.04 (d, J = 2.0 Hz, 1H, ArH), 6.94 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 10 min, followed by the addition of NIS (133 mg, 0.589 mmol)
6.77 (d, J = 8.0 Hz, 1H, ArH), 6.69 (d, J = 2.0 Hz, 1H, ArH), 6.66 and TMSOTf (35 μL, 0.196 mmol). The progress of the glycosy-
(d, J = 8.0 Hz, 1H, ArH), 6.55 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 6.28 lation was observed by TLC. After 30 h of reaction, the mixture
(d, J = 16.0 Hz, 1H, HCvCH–O), 4.82 (d, J = 9.6 Hz, 2H), 4.35 was neutralized by NaHCO3(aq) and Na2S2O3. The resulting
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2935

View Article Online
Paper
Organic & Biomolecular Chemistry
1
mixture was filtered, and concentrated for flash chromato- MeOH); H NMR (600 MHz, CD3OD): δ = 7.55 [d, J = 15.9 Hz,
graphy purification over silica gel (elution: hexane–EtOAc, 9 : 1) 1H, HCvCH–O], 7.02 (d, J = 1.6 Hz, 1H), 6.87 (dd, J = 8.2, 1.7
to furnish the protected calceolarioside-B 24 (295 mg, 60%, Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.66 (d, J = 1.43 Hz, 1H), 6.62
E : Z = >19 : 1). For 24, Rf = 0.55 (hexane–EtOAc, 4/1 v/v); [α]D25
=
(d, J = 8.0 Hz, 1H), 6.52 (dd, J = 8.1, 1.6 Hz, 1H), 6.27 [d, J =
1
+22.5 (c 0.5 CHCl3); H NMR (600 MHz, CDCl3): δ = 7.86–7.72 15.9 Hz, 1H, HCvCH–O], 4.50 (bs, 1H), 4.49 (dd, J = 11.8, 1.6
(m, 9H, ArH), 7.51–7.37 (m, 7H, ArH), 6.96 (d, J = 1.8 Hz, 1H, Hz, 1H), 4.43–4.31 (m, 2H), 3.94 (dd, J = 16.7, 8.0 Hz, 1H), 3.70
ArH), 6.94 (dd, J = 8.3, 1.8 Hz, 1H, ArH), 6.80 (d, J = 8.2 Hz, 1H, (dd, J = 16.9, 8.1 Hz, 1H), 3.51 (ddd, J = 8.1, 6.2, 1.5 Hz, 1H),
ArH), 6.70–6.66 (m, 3H, ArH), 6.13 [d, J = 15.9 Hz, 1H, 3.39–3.33 (m, 2H), 3.20 (t, J = 8.2 Hz, 1H), 2.79–2.76 (m, 2H);
HCvCH–O], 6.04–5.97 (m, 1H), 5.30 (d, J = 17.1 Hz, 1H), 5.18 13C NMR (150 MHz, CD3OD): δ = 169.1 (CvO), 149.5, 147.2,
(d, J = 10.2 Hz, 1H), 5.01 (d, J = 11.0 Hz, 1H), 4.90 (d, J = 11.3 146.7, 146.0, 144.6, 131.3, 127.6, 123.1, 121.2, 117.0, 116.5,
Hz, 1H), 4.77 (t, J = 11.3 Hz, 1H), 4.46 (dd, J = 12.2, 5.5 Hz, 116.3, 115.0, 104.5, 77.9, 75.4, 75.0, 72.3, 71.7, 64.6, 36.6;
1H), 4.45–4.42 (m, 3H), 4.34 (dd, J = 12.2, 5.7 Hz, 1H), 4.14 (dd, HRMS-EI (m/z): [M + Na]+ calcd for C23H26NaO11+, 501.1367;
J = 16.4, 7.4 Hz, 1H), 3.73 (dd, J = 17.0, 7.7 Hz, 1H), 3.65–3.54 found, 501.1368.
(m, 4H), 3.47 (t, J = 8.3 Hz, 1H), 2.88–2.84 (m, 2H), 1.00 (s, 9H),
4.16. Anti-proliferation assay
0.99 (s, 9H), 0.956 (s, 9H), 0.950 (s, 9H), 0.22 (s, 6H), 0.21 (s,
6H), 0.15 (s, 3H), 0.14 (s, 3H), 0.12 (s, 6H); 13C NMR (150 MHz,
CDCl3): δ = 167.1 (CvO), 149.5 147.2, 146.7, 145.4, 145.1,
135.9, 135.3, 135.1, 133.4, 133.3, 133.1, 133.0, 131.5, 129.7,
128.4, 128.13, 128.12, 128.03, 128.00, 127.74, 127.71, 127.2,
126.9, 126.4, 126.2, 126.1, 126.04, 126.01, 125.8, 122.4, 121.84,
121.80, 121.1, 120.9, 120.6, 117.0, 115.2, 103.9 (C-1), 84.4, 82.0,
77.1, 75.1, 74.9, 74.7, 73.1, 71.23, 63.1, 35.7, 26.0, 25.9, 18.6,
18.5, 18.4, −3.93, −3.95, −3.98, −4.06; HRMS-EI (m/z): [M + H]+
Human hormone-refractory prostate cancer PC-3 cells were
seeded in 96-well plates in medium with 5% FBS. After 24 h,
the cells were fixed with 10% trichloroacetic acid (TCA) to rep-
resent a cell population at the time of the compound addition
(t0) (baseline). After the additional incubation of 0.1%
dimethylsulfoxide (DMSO) or the PhG compound (1, 2, 3 or 4)
for 48 h, the cells were fixed with 10% TCA and Sulforhoda-
mine B (SRB) at 0.4% (w/v) in 1% acetic acid was added to
stain the cells. Unbound SRB was washed out by 1% acetic
acid and SRB bound cells were solubilized with 10 mM Trizma
base. The absorbance was read at a wavelength of 515 nm.
Using the following absorbance measurements, such as time
zero (t0), control growth (C), and cell growth in the presence of
the compound (tx), the percentage growth was calculated at
each of the compound concentrations levels. The percentage
growth inhibition was calculated as: [1 − (tx − t0)/(C − t0)] ×
100%.
+
calcd for C72H103O11Si4 , 1255.6572; found, 1255.6539.
4.15. The synthesis of calceolarioside-B (4) (Scheme 5b)
To a solution of protected calceolarioside-B 23 (136 mg,
0.108 mmol) in acetone (2 mL), sodium acetate (47 mg,
0.541 mmol) and acetic acid (0.2 mL, 95%) were added. To the
above reaction mixture palladium dichloride (38 mg,
0.216 mmol) was added at 0 °C, and stirred for 5 h at room
temperature. Upon the completion of the reaction as moni-
tored by TLC, the reaction mixture was filtered over a celite
bed to remove the catalyst, and the filtrate was concentrated
for flash chromatography purification over silica gel (elution:
hexane–EtOAc, 4/1) to furnish the partially deprotected
product (105 mg, 80%). The intermediate from the preceding
step (105 mg, 0.086 mmol) and DDQ (39 mg, 0.172 mmol) in
3 : 1 CH2Cl2–MeOH (5 mL) was stirred at room temperature for
5 h. Upon the completion of the NAP deprotection as moni-
tored by TLC, the reaction mixture was concentrated under
reduced pressure and the residue was diluted with EtOAc
(50 mL) then washed with NaHCO3(aq), (3 × 50 mL) and H2O
(2 × 50 mL). The EtOAc solution was dried over MgSO4, filtered
and concentrated for flash chromatography purification over
silica gel (elution: hexane–EtOAc, 1/1) and the product was
treated with Dowex 50 W × 4 (in Na+) to furnish the partially
deprotected intermediate (68 mg, 86%). The intermediate
from the preceding NAP deprotection step (68 mg,
0.072 mmol) was dissolved in pyridine (2 mL); to which, Et3N
(20 μL, 0.145 mmol) and 539 µL of 3HF·Et3N (1.56 M in pyri-
dine) were added. After stirring for 3 h at RT, the resulting
mixture was subjected for flash chromatography purification
over silica gel (elution: CH2Cl2–MeOH = 9 : 1) to furnish the
calceolarioside-B 4 (30 mg, 88%, E : Z = >19 : 1). For calceolario-
side-B 4: Rf = 0.35 (CH2Cl2–MeOH. 9 : 1); [α]2D5 = −29.0 (c 0.3,
Acknowledgements
Thanks are given to the National Science Council (NSC) of
Taiwan for financial support (NSC 102–2113-M-009-009) and
Prof. J.-H. Guh of the School of Pharmacy in National Taiwan
University (NTU) for performing the anti-proliferation assay on
PC-3 human prostate cancer cells.
Notes and references
1 J. Pan, C. Yuan, C. Lin, Z. Jia and R. Zeng, Pharmazie, 2003,
58, 767–775.
2 G. Fu, H. Pang and Y.-H. Wong, Curr. Med. Chem., 2008, 15,
2592–2613.
3 (a) S. De Marino, C. Festa, F. Zollo, F. Incollingo, G. Raimo,
G. Evangelista and M. Iorizzi, Food Chem., 2012, 133,
21–28; (b) A. López-Munguía, Y. Hernández-Romero,
J. Pedraza-Chaverri, A. Miranda-Molina, I. Regla and
C. Edmundo, PLoS One, 2011, 6, e20115; (c) Y.-H. Liu,
Y.-L. Lu, C.-H. Han and W.-C. Hou, Botanical Studies, 2013,
54, 6.
2936 | Org. Biomol. Chem., 2014, 12, 2926–2937
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
4 (a) Q. Xiong, K. Hase, Y. Tezuka, T. Tani, T. Namba and
S. Kadota, Planta Med., 1998, 64, 120–125; (b) C. Phakeovilay,
W. Disadee, et al., J. Nat. Med., 2013, 67, 228–233.
5 (a) R. Radev, J. Clin. Med., 2010, 3, 20–23; (b) M. S. Butler,
J. Nat. Prod., 2004, 67, 2141–2153; (c) I. Ali, P. Sharma,
K. A. Suri, N. K. Satti, P. Dutt, F. Afrin and I. A. Khan,
(b) Arun B. Ingle, C.-S. Chao, W.-C. Hung and K.-K. T. Mong,
Org.Lett., 2013, 15, 5290–5293.
18 (a) Z. B. Szabó, A. Borbás, I. Bajza and A. Lipták, Tetra-
hedron: Asymmetry, 2005, 16, 83–95; (b) J. Xia, S. A. Abbas,
R. D. Locke, C. F. Piskorz, J. L. Aderfer and K. L. Matta,
Tetrahedron Lett., 2000, 41, 169–173.
J. Med. Microbiol., 2011, 60, 1326–1336; (d) G. Grynkiewicz, 19 E. Attolino, I. Cumpstey and A. J. Fairbanks, Carbohydr.
W. Sseja and J. Boryski, Acta Pol. Pharm., 2008, 65, 655–676. Res., 2006, 341, 1609–1618.
6 C. Jiménez and R. Riguerub, Nat. Prod. Rep., 1994, 11, 20 M. P. DeNinno, J. B. Etienne and K. C. Duplantier, Tetra-
591–606. hedron Lett., 1995, 36, 669–672.
7 (a) T. Wang, X. Zhang and W. Xie, Am. J. Chin. Med., 2012, 21 (a) A. Borbás, Z. B. Szabó, L. Szilágyi, A. Bényei and
40, 1123–1141; (b) L. Han, L. Ji, M. B. Yiadom, W. Li,
X. Song and X. Gao, Molecules, 2012, 17, 8276–8284;
(c) C. Chen, X.-H. Zhao, H.-L. Yue, Y.-L. Li and T. Chen,
J. Chromatogr. Sci., 2013, DOI: 10.1093/chromsci/bmt048.
8 Q. Zhang, C. Jia, H. Xu, Y. Wang, M. Zhang, C. Huo, Q. Shi
and S. Yu, Mini-Rev. Org. Chem., 2012, 9, 303–318.
A. Lipták, Tetrahedron, 2002, 58, 5723–5732; (b) C.-R. Shie,
Z.-H. Tzeng, S. S. Kulkarni, B.-J. Uang and C.-Y. Hsu,
Angew. Chem., Int. Ed., 2005, 44, 1665–1668.
22 R. R. France, R. G. Compton, B. G. Davis, A. J. Fairbanks,
N. V. Rees and J. D. Wadhawan, Org. Biomol. Chem., 2004,
2, 2195–2202.
9 V. Kren and L. Martinkova, Curr. Med. Chem., 2001, 8, 23 T. Oshitar, M. Shibasaki, T. Yoshizawa, M. Tomita,
1303–1338.
K.-I. Takao and S. Kobayashi, Tetrahedron, 1997, 53, 10993–
10 (a) S.-Q. Zang, Z. J. Li, A. B. Wang, M.-S. Cai and R. Feng,
11006.
Carbohydr. Res., 1997, 299, 281–285; (b) Q. Li, S.-C. Li, 24 (a) S. Hashimoto, T. Honda and S. J. Ikegami, J. Chem. Soc.,
H. Li, M.-S. Cai and Z.-J. Li, Carbohydr. Res., 2005, 340,
1601–1604; (c) S. K. Das, K. A. Reddy and K. Mukkanti,
Carbohydr. Res., 2007, 342, 2309–2315; (d) T. Kawada,
R. Asano, K. Makino and T. Sakuno, Eur. J. Org. Chem.,
Chem. Commun., 1989, 685–687; (b) T. Tsuda, S. Nakamura
and S. Hashimoto, Tetrahedron, 2004, 60, 10711–10737;
(c) O. J. Plante, E. R. Palmacci, R. B. Andrade and
P. H. Seeberger, J. Am. Chem. Soc., 2001, 123, 9545–9554.
2000, 2723–2727; (e) T. Kawada, R. Asano, S. Hayashida 25 B. Mukhopadhyay and R. A. Field, Carbohydr. Res., 2006,
and T. Sakuno, J. Org. Chem., 1999, 64, 9268–9271; 341, 1697–1701.
(f) H. I. Duynstee, M. C. de Koning, H. Ovaa, G. A. van der 26 D. E. Bogucki and J. L. Charlton, Can. J. Chem., 1997, 75,
Marel and J. H. van Boom, Eur. J. Org. Chem., 1999, 2623– 1783–1794.
2632; (g) F.-Y. Zhou, J. She and Y.-G. Wang, Carbohydr. Res., 27 K. Bock and C. Pedersen, J. Chem. Soc., Perkin Trans. 2,
2006, 341, 2469–2477; (h) J. Li and Y.-G. Wang, Synth. 1974, 293–297.
Commun., 2004, 34, 515–522; (i) T. Kawada, R. Asano, 28 (a) A. Hassner and V. Alexanian, Tetrahedron Lett., 1978, 19,
K. Makino and T. Sakuno, J. Wood Sci., 2002, 48, 512–515;
( j) G. Gu, Y. Zhao and Z. Guo, Carbohydr. Res., 2013, 380,
174–180.
4475–4478; (b) B. Neises and W. Steglich, Angew. Chem., Int.
Ed. Engl., 1978, 17, 522–524.
29 J. Rothenburger and E. Haslinger, Liebigs Ann. Chem., 1994,
1994, 1113–1115.
11 (a) H. L. Frush and H. S. Isbell, J. Res. Natl. Bur. Stand.,
1941, 27, 413–428; (b) K. Igarashi, T. Honma and J. Irisawa, 30 In our study, we note that some extent of E to Z isomeriza-
Carbohydr. Res., 1970, 15, 329–337; (c) H. Paulsen and
C.-P. Herold, Chem. Ber., 1970, 103, 2450–2462; (d) B. Capon
tion occurs in prolonged exposure of the intermediates
(having the caffeoyl group) to daylights or UV irradiation.
and S. P. McManus, Neighbouring Group Participation, 31 A. B. Smith, III, R. A. Rivero, K. J. Hale and H. A. Vaccaro,
Plenum Press, New York, 1976. J. Am. Chem. Soc., 1991, 113, 2092–2112.
12 A. Stoll, J. Renz and A. Brack, Helv. Chim. Acta, 1950, 33, 32 H. Kobayashi, H. Karasawa, T. Miyase and S. Fukushima,
1877–1893. Chem. Pharm. Bull., 1984, 32, 3009–3014.
13 F. Pardo, F. L. Perich, et al., J. Ethnopharmacol., 1993, 39, 33 Y.-J. Chen, H.-G. Zang and X. Li, Chem. Nat. Compd., 2009,
221–222. 45, 330–332.
14 M. Nicoletti, C. Galeffi, et al., Gazz. Chim. Ital., 1986, 776, 431. 34 Z. Culig, et al., Cancer Res., 1994, 54, 5474–5478.
15 M. E. Kaighn, K. S. Narayan, Y. Ohnuki, J. F. Lechner and 35 A. Shen and M. M. Shen, Trends Genet., 2002, 18, S1–S5.
L. W. Jones, Invest. Urol., 1979, 17, 16–23.
16 (a) C.-S. Chao, C.-W. Li, M.-C. Chen, S.-S. Chang and 37 S. Yogosawa, Y. Yamada, S. Yasuda, Q. Sun, K. Takizawa
K.-K. T. Mong, Chem. - Eur. J., 2009, 15, 10972–10982; and T. Sakai, J. Nat. Prod., 2012, 75, 2088–2093.
(b) C.-S. Chao, C.-Y. Lin, S.-K. Mulani, W.-C. Hung and 38 H. P. Lin, S.-S. Jiang and C.-P. Chuu, PLoS One, 2012, 7,
K.-K. T. Mong, Chem. - Eur. J., 2011, 17, 12193–12202. e31286.
17 (a) S.-R. Lu, Y.-H. Lai, J.-H. Chen, C.-Y. Liu and 39 K.-W. Lee, H.-J. Kim, Y.-S. Lee, H.-J. Park, J.-W. Choi, J. Ha
36 A. van Bokhoven, et al., Prostate, 2003, 57, 205–225.
K.-K. T. Mong, Angew. Chem., Int. Ed., 2011, 50, 7315–7320;
and K.-T. Lee, Carcinogenesis, 2007, 28, 1928–1936.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2926–2937 | 2937