Synthesis of C3-Neoglycosides of digoxigenin and their anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2017.12.086

Cardiac glycosides exhibit significant anticancer effects and the glycosyl substitution at C₃ position of digoxigenin is pivotal for their biological activity. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and explore more potent anticancer agents, a series of C₃-O-neoglycosides and C₃-MeON-neoglycosides of digoxigenin were synthesized by the Koenigs-Knorr and neoglycosylation method, respectively. In addition, digoxigenin bisdigitoxoside and monodigitoxoside were prepared from digoxin by sodium periodate (NaIO₄) oxidation and 6-aminocaproic acid hydrolysis. The SAR analysis revealed that C₃-O-neoglycosides of digoxigenin exhibited stronger cytotoxicity and induction of Nur77 expression of tumor cells than C₃-MeON-neoglycosides. Also, 3β-O-glycosides exhibited stronger anticancer effects than 3α-O-glycosides. Among them, 3β-O-(β-L-fucopyranosyl)-digoxigenin (3i) showed the highest activity on induction of Nur77 expression and translocation from the nucleus to cytoplasm, leading to cancer cell apoptosis.

Full text of DOI:10.1016/j.ejmech.2017.12.086

Accepted Manuscript
Synthesis of C -Neoglycosides of digoxigenin and their anticancer activities
3
Xiao-san Li, Yi-chang Ren, Yu-zhou Bao, Jie Liu, Xiao-kun Zhang, You-wei Zhang,
Xue-Long Sun, Xin-sheng Yao, Jin-Shan Tang
PII:
S0223-5234(17)31117-0
10.1016/j.ejmech.2017.12.086
EJMECH 10064
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 October 2017
Revised Date: 4 December 2017
Accepted Date: 27 December 2017
Please cite this article as: X.-s. Li, Y.-c. Ren, Y.-z. Bao, J. Liu, X.-k. Zhang, Y.-w. Zhang, X.-L. Sun, X.-s.
Yao, J.-S. Tang, Synthesis of C -Neoglycosides of digoxigenin and their anticancer activities, European
3
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.086.
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ACCEPTED MANUSCRIPT
A series of C -O-neoglycosides and C -MeON-neoglycosides of digoxigenin were synthesized.
3
3
The SAR analysis revealed that C -O-neoglycosides of digoxigenin exhibited stronger
3
cytotoxicity and induction of Nur77 expression of tumor cells than C -MeON-neoglycosides.
3
Among them, 3β-O-(β-L-fucopyranosyl)-digoxigenin (3i) showed the highest activity on
induction of Nur77 expression and translocation from the nucleus to cytoplasm, leading to cancer
cell apoptosis.

ACCEPTED MANUSCRIPT
Synthesis of C -Neoglycosides of Digoxigenin and Their Anticancer Activities
3
a
a
b
b
b
Xiao-san Li , Yi-chang Ren , Yu-zhou Bao , Jie Liu , Xiao-kun Zhang , You-wei
c
d
a*
a*
Zhang , Xue-Long Sun , Xin-sheng Yao , Jin-Shan Tang
a
Institute of Traditional Chinese Medicine and Natural Products, College of
Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China.
b
School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, People’s
Republic of China.
c
Department of Pharmacology, Case Comprehensive Cancer Center, Case Western
Reserve University School of Medicine, Cleveland, OH 44106, USA
d
Department of Chemistry, Chemical and Biomedical Engineering and Center for
Gene Regulation in Health and Disease (GRHD), Cleveland State University, 2121
Euclid Avenue, Cleveland, OH 44115, USA
Corresponding author at: Institute of Traditional Chinese Medicine and Natural
Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s
Republic of China.
E-mail address: tyaoxs@jnu.edu.cn (X.-S. Yao); gztangjinshan@126.com (J.-S. Tang)

ACCEPTED MANUSCRIPT
ABSTRACT:
Cardiac glycosides exhibit significant anticancer effects and the glycosyl
substitution at C position of digoxigenin is pivotal for their biological activity. In
3
order to study the structure-activity relationship (SAR) of cardiac glycosides toward
cancers and explore more potent anticancer agents, a series of C -O-neoglycosides
3
and C -MeON-neoglycosides of digoxigenin were synthesized by the Koenigs-Knorr
3
and neoglycosylation method, respectively. In addition, digoxigenin bisdigitoxoside
and monodigitoxoside were prepared from digoxin by sodium periodate (NaIO4)
oxidation and 6-aminocaproic acid hydrolysis. The SAR analysis revealed that
C -O-neoglycosides of digoxigenin exhibited stronger cytotoxicity and induction of
3
Nur77 expression of tumor cells than C -MeON-neoglycosides. Also,
3
3β
-O-glycosides exhibited stronger anticancer effects than 3
α-O-glycosides. Among
them, 3 -O-( -fucopyranosyl)-digoxigenin (3i) showed the highest activity on
β
β-L
induction of Nur77 expression and translocation from the nucleus to cytoplasm,
leading to cancer cell apoptosis.
Key words: Digoxigenin, O-neoglycosylation, MeON-noeglycosylation, cytotoxicity,
Nur77 nuclear receptor, anticancer, apoptosis.

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1
. Introduction
Cardiac glycosides have been used for the treatment of congestive heart failure
CHF) for centuries due to their strong inhibitory activity toward the ubiquitous cell
(
+
+
surface Na /K -ATPase [1-3]. Recent studies demonstrate that cardiac glycosides
exhibit potent antiproliferative and apoptotic effects on cancer cells through complex
signal transduction mechanisms [4-6]. Our previous studies showed that cardiac
glycosides exerted apoptotic effects through the Nur77 dependent apoptotic pathway,
a novel signaling pathway for them against cancer cells, which has great potential for
novel anticancer agent discovery [7-9]. However, the cardiotoxicity and neurotoxity
of cardiac glycosides limit their extensive application in clinical practice [10]. In
order to get more potent leading compounds with fewer side effects, it is requisite to
study the structural–activity relationship (SAR) of cardiac glycosides against cancers.
Preliminary SAR analysis showed that the sugar substitution at the C of digoxigenin
3
(3β,12β,14-Trihydroxy-5β,20(22)-cardenolide) is pivotal for their anticancer effects
[
11-14]. Recent studies showed that subtle variation of sugars for cardiac glycosides
dramatically affected their antiproliferative effects, even the mechanism of action
15-17]. Herein, we report the synthesis of a series of C -O-neoglycosides and
[
3
C -MeON-neoglycosides of digoxigenin by the Koenigs-Knorr [17] and
3
neoglycosylation methods [15]. Meanwhile, digoxigenin bisdigitoxoside and
monodigitoxoside were obtained from digoxin by sodium periodate (NaIO ) oxidation
4
and 6-aminocaproic acid hydrolysis. Also, the anticancer effects of these new cardiac
glycosides were evaluated and discussed.
2
. Results and discussion
2
.1.Chemistry
2
.1.1 Synthesis of C -O-neoglycosides of digoxigenin by the Koenigs-Knorr method
3
The synthesis of C -O-neoglycosides of digoxigenin was started from the synthesis of
3
1
-bromo glycosyl donors from commercially available reducing sugars. The sugars
were firstly converted to their corresponding peracetylated derivatives under the
condition of Ac O and anhydrous pyridine at 90°C. The peracetylated derivatives of
2

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sugars were then reacted with HBr (33%, w/w) in AcOH at room temperature to yield
1
-bromo glycosyl donors (1a-1o). On the other hand, the synthesis of digoxigenin
aglycones was initiated from commercially available digoxin. Peracetylation of
digoxin (Dig) with Ac O in pyridine and subsequent regioselective acid-catalyzed
2
hydrolysis of the sugar chain at C led to digoxigenin aglycones Dig-1a and Dig-1b
3
[
18]. In order to study the effects of C configuration on their anticancer bioactivity,
3
we prepared 3α-OH-digoxigenin (Dig-3a) as well from intermediate Dig-1a. Briefly,
intermediate Dig-1a was oxidized to yield ketone Dig-2a with Dess-Martain
periodinane in high yield, which was subsequently reduced to yield
3
α
-OH-digoxigenin (Dig-3a) and 3β-OH-digoxigenin (Dig-1b) with NaBH (Scheme
4
1
).
Scheme 1. Synthesis of 3
α- and 3β-OH-digoxigenin. Reagents and conditions: a.
Ac O, DMAP, CH Cl /pyr, r.t., 5 h; b. 1M H SO , MeOH, 90°C, 3 h; c. Dess-Martin
2
2
2
2
4
Periodinane, CH Cl , r.t., 13 h, 84%; d. [i] NaBH , MeOH, 0°C, 2h , [ii] H O, r.t.
2
2
4
2
With the glycosyl donors (1a-1o) and acceptors (Dig-1a, Dig-1b, and Dig-3a) in
hands, the C -O-neoglycosides of digoxigenin (3a-3o and 5a-5e) were synthesized by
3
the Koenigs-Knorr reaction method and subsequent deacetylation of the acetyl groups
of C and sugars under the condition of CH ONa/CH OH (Scheme 2). We found that
1
2
3
3
when the aglycone Dig-1b was used as glycosyl acceptor, only C -O-neoglycosides
3
(
3m-3o) were obtained, indicating the regioselective glycosylation of C -OH.
3
Therefore, the aglycone Dig-3a was directly subjected to glycosylation with 1-bromo
glycosyl donors to get C -O-neoglycosides (5a-5e). Overall, a series of
3
C -O-neoglycosides of pyranosides including ribose, xylose, lyxose, arabose were
3
obtained in high yield [19-21]. The configuration of glycosidic bond was identified by
the J value of JH1′-H2′ and ROESY or NOESY spectrum (Supporting Information, Table
S1).

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Scheme 2. Synthesis of C -O-neoglycosides of digoxigenin by the Koenigs-Knorr
3
method.
2
.1.2 Synthesis of C -MeON-neoglycosides of digoxigenin by neoglycosylation
3
The synthesis of C -MeON-neoglycosides was initiated by preparation of
3
MeON-digoxigenin as glycosyl acceptor. Briefly, the ketone Dig-2a was reacted with
methoxyamine hydrochloride salt to produce corresponding methoxyimines at C-3
(
Dig-3, Z/E), which was subsequently reduced by NaCNBH and then deacetylation
3
of the C -OAc with K CO to give a separable 1:1 mixture of MeON-digoxigenin
1
2
2
3
Dig-4/5 (β/α, 1/1). Then, glycosyl acceptors Dig-4/5 were reacted with reducing sugar
in DMF/AcOH (3:1) at 50 °C for 48 h to obtain C -MeON-neoglycosides 6a-6d and
3
7
a-7d (Scheme 3) [15]. It is interesting that furanosides were obtained for ribose and
xylose [19-21]. The glycosidic bond configuration of all the C -MeON-neoglycosides
3
1
was characterized according to the H NMR spectral analysis (see Support
Information Table 1). In addition, the digoxigenin bisdigitoxoside (8a) and
monodigitoxoside (9b) were obtained via sodium periodate (NaIO ) oxidation and
4
6
-aminocaproic acid hydrolysis from digoxin (Scheme 4) [22].

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Scheme 3. Synthesis of C -MeON-neoglycosides of digoxigenin by the
3
neoglycosylation method. Reagents and conditions: a. MeONH ⋅HCl, MeOH/pyr, r.t.,
2
1
5
3 h; b. NaCNBH , MeOH, r.t.; c. K CO , MeOH, r.t., 4h; d. DMF/AcOH (v/v 3:1),
3 2 3
0°C, 48 h.
Scheme 4. Synthesis of digoxigenin bisdigitoxoside (8a) and monodigitoxoside (9b)
from Digoxin (Dig). Reagents and conditions: a.[i] NaIO , MeOH, r.t., 3h; [ii]
4
6
-Aminocaproic acid, MeOH, r.t., 4h.
2
.2.Biology
Our previous study suggested that some cardiac glycosides showed strong
inhibitory effects on the proliferation of cancer cells [8,9]. Further study demonstrated
that induction of Nur77 expression and its subsequent translocation from the nucleus

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to the cytoplasm are critical events in apoptosis induction by cardiac glycosides in
cancer cells, which underlay the new mechanism of action for cardiac glycosides
toward cancer cell [8,9,12]. In this study, the cytotoxicity of all new glycosides
toward NIH-H460 cancer cells was assessed by MTT assay. Digoxin was used as
positive control. As a result, glycosides 3a, 3g, 3h and 3i showed significant
inhibitory activities on the proliferation of NIH-H460 cancer cells at a concentration
of 50 nM (Fig. 1). Also, SAR analysis revealed that neoglycosides with O-linkage
exhibited stronger cytotoxic activities than MeON-neoglycosides, especially for the
3β-O-neoglycosides.
1
1
1
.6
.4
.2
1
0
0
0
0
.8
.6
.4
.2
0
Fig. 1. Antiproliferative effects of neoglycosides on NIH-H460 cancer cells.
NIH-H460 cells were incubated with neoglycosides (50 nM) for 48h and cell viability
was assayed by the MTT method.
Next, the induction of Nur77 expression for the newly synthesized neoglycosides
was evaluated by western blot. As a result, most of C -O-neoglycosides, especially for
3
3
c, 3g-3k, 5b and 8a, Dig-3a exhibited significant induction of Nur77 expression at a
concentration of 20 nM compared to digoxin (Fig. 2).

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Fig. 2. Induction of Nur77 expression by neoglycosides. NIH-H460 cancer cells were
incubated with neoglycosides (20 nM) for 3 h, and Nur77 expression was determined
by Western blotting using anti-Nur77 antibody.
Previous study suggested that subcellular localization of induced Nur77 determines
its biological function. We chose 3β-O-neoglycosides 3g, 3h, and 3i that showed
significant inhibitory effects on the proliferation of NIH-H460 cancer cells to evaluate
their subcellular localization of the induced Nur77. We found that these compounds
could significantly induce the translocation of the induced Nur77 from the nucleus to
cytoplasm after 9 h treatment. Meanwhile, they could induce the Nur77 expression of
cancer cells in a time dependent manner (Supporting Information Fig. S3). Among
them, 3β-O-neoglycoside 3h could significantly induce the translocation of the
induced Nur77 from the nucleus to cytoplasm after 6 h (Fig. 3). We then tested the
apoptotic level of NIH-H460 cells after treating with 3g, 3h and 3i by Flow
Cytometry after staining with PI and Annixin V. Results showed apoptotic ratio of 3g
was about 14.79%, 3h was about 14.85% and 3i was 34.03% (Fig. 4). These results
demonstrate 3g, 3h and 3i was good cancer cell apoptosis inducer.

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Fig. 3. Effects of 3
β
-O-neoglycosides 3h on translocation of the induced Nur77
protein from the nucleus to the cytoplasm. NIH-H460 cancer cells were treated with
-O-neoglycosides 3h (20 nM) for 3, 6, and 9 h. Endogenous Nur77 expression was
3β
immunostained with anti-Nur77 antibody. Cells were costained with DAPI to
visualize the nuclei.

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Fig. 4. Pro-apoptotic effects of 3β-O-neoglycosides 3g, 3h, and 3i toward NIH-H460
cancer cells. NIH-H460 cancer cells were treated with 3β-O-neoglycosides 3g, 3h,
and 3i (20 nM) for 12 h.
3
. Conclusions
In conclusion, we have successfully synthesized a series of C -O-glycosides of
3
digoxigenin by the Koenigs-Knorr reaction and a series of C -MeON-neoglycosides
3
of digoxigenin by neoglycosylation. Their anticancer activities were investigated by
examine the induction of Nur77 expression and its translocation from the nucleus to
cytoplasm together with cytotoxicity toward NIH-H460 cancer cells. Overall, we
found that 3β-O-neoglycosides showed the strongest cytotoxicity. Preliminary
structure-activity relationship (SAR) analysis revealed that cardiac glycosides with
O-linkage exhibited stronger cytotoxic activities and induction of Nur77 expression
than MeON-neoglycosides. Especially, 3
cytotoxic activity; which is consistent with the literature [23]. Meanwhile,
-O-neoglycosides 3g, 3h, and 3i could induce the Nur77 expression and its
translocation from the nucleus to cytoplasm, leading the apoptosis of cancer cells.
β-O-neoglycosides exhibited the strongest
3β

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Their potential anticancer effects deserve future study in vivo.
4
4
4
. Experimental
.1. Chemistry
.1.1 General materials
All reagents were obtained from commercial suppliers as follows. All reducing sugars,
N,N-dimethylformamide (DMF), pyridine (Pyr), 4-dimethylaminopyridine (DMAP),
Dess-Martin periodinane, methoxylamine hydrochloride (CH ONH ⋅HCl), Sodium
3
2
cyanoborohydride (NaCNBH ), Sodium borohydride (NaBH ), 6-aminocaproic acid,
3
4
Sodiumperiodate (NaIO ), Silver oxide (Ag O), Silver carbonate (Ag CO ),
4
2
2
3
hydrogen bromide (33% in acetic acid) and sodium methoxide (CH ONa) were
3
purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China).
Acetic anhydrides (Ac O), sulfuric acid (H SO ), hydrochloric acid (HCl), acetic acid
2
2
4
(
AcOH), were purchased from Tianjin Tie Xiang Commerce Development Co., LTD
Tianjin, China). Digoxin was purchased from Shanghai Raise Chemical Technology
(
Co., LTD (Shanghai, China).
4
.1.2 General methods.
TLC analysis was performed on precoated silica gel GF254 plates (Qingdao
Haiyang Chemical Group Corp., Qingdao). Reactions were monitored by UV and/or
by treatment with 5% Vanillin-sulfuric acid and subsequent heating. Silica gel
(100-200, 300-400 mesh) for column chromatography was from Qingdao Haiyang
Chemical Group Corp., Qingdao, China. Optical rotations were measured on a P-1020
digital polarimeter (JASCO Corporation Tokyo, Japan). UV spectra were measured on
a Jasco V-550 UV/vis spectrophotometer. NMR spectra were measured on a Bruker
AV 300, 400, and 600 instruments. The chemical shifts are expressed in δ (ppm)
relative to the chemical shifts of solvent resonances using CDCl , CD OD, and
3
3
pyridine-d with 0.05% v/v TMS (Cambridge Isotope Laboratories, MA, USA), and
5
coupling constants (J) are given in hertz. All NMR spectra were recorded at ambient
temperature. HRESIMS data was obtained on a Micromass Q-TOF mass spectrometer.
Analytical HPLC was performed on a Shimadzu LC-20AB liquid chromatography

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system equipped with an SPD-M20A diode array detector using a Luna C18column
(
4.6 × 250 mm, 5 µm, Phenomenex, Torrance, California, USA) by following methods
at a wavelength of 220 nm, flow rate = 1 ml/min. Semi-preparative reverse-phase
HPLC was conducted on a Gemini C18 column (5 m, 10 × 250 mm, Phenomenex,
µ
Torrance, California, USA) using the following methods at a wavelength of 220 nm,
flow rate =3 ml/min.
4
.1.3. General procedure for preparation of 1-bromo-peracetylated glycosyl donors
To a solution of reducing sugars (4 mmol) in anhydrous pyridine (10mL) was
added Ac O (20 mL) and AcOH (3 mL) at room temperature. After stirring at 90°C
2
for 6h, the reaction was quenched with MeOH and the resulting mixture was
concentrated under reduced pressure. The residue was diluted with EtOAc and was
washed with 1M HCl, saturated NaHCO and H O successively, dried over anhydrous
3
2
Na SO and then concentrated under reduced pressure. The peracetylated sugars were
2
4
obtained and used without further purification.
To a solution of peracetylated sugars (1.28 mmol) in CH Cl (7 mL) was slowly
2
2
added a 33% (w/w) solution of HBr in AcOH and stirred for 3 h at room temperature.
The reaction mixture was diluted with CH Cl and washed with ice water, saturated
2
2
NaHCO and water successively, dried over anhydrous Na SO and then concentrated
3
2
4
under reduced pressure. The crude 1-bromo-peracetylated glycosyl donors (1a-1o)
was obtained and used without further purification.
4
.1.4. General procedure for preparation of C -O-neoglycoside library
3
Glycosylation of digoxigenin aglycones with 1-bromo glycosyl donors was
accomplished following the Koenigs-Knorr reaction method. To a solution of
digoxigenin aglycones (Dig-1a, Dig-1b, and Dig-3a, 0.25 mmol) in CH Cl was
2
2
added a mixture solution of 1-bromo-peracetylated sugars (0.80 mmol) and
Ag O/Ag CO (1:1 w/w) (1.2 mmol) in anhydrous CH Cl (8 mL). The reaction
2
2
3
2
2
mixture was added molecular sieves (4Å) and stirred under N atmosphere for 72 h at
2
room temperature. The crude product was obtained by filtered and concentrated under
reduced pressure. The residue was purified by semipreparative RP-HPLC (65%
MeOH-H O) to obtain glycosylation products (2a-2o and 4a-4e).
2

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The NaOMe solution was prepared by dissolving 10 mg of NaOMe (0.18mmol) in
0 mL of MeOH until the pH value was 9. The glycosylation product (2a-2o and
a-4e) was dissolved in the preparative NaOMe solution (2mL) and the reaction
1
4
mixture was stirred for 5 h at room temperature. The reaction solution was neutralized
with AcOH (pH = 7) and then concentrated under reduced pressure. The residue was
purified by semipreparative RP-HPLC (35%-40% MeOH-H O) to obtain
2
3β
-O-neoglycosides 3a-3o and 3α-O-neoglycosides 5a-5e. All compounds were
1
13
characterized by analyses of H and C NMR spectra as well as ESI-MS.
4
.1.4.1. 3β-O-(β-D-glucopyranosyl)-digoxigenin (3a). Obtained as a white solid (12.8
mg, 61%) from
3
β
-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-12-O-acetyl
2
D
9
1
-digoxigenin (2a). [α]
+19.6 (c 0.70, MeOH); H NMR (400 MHz, Pyridine-d ): δ
5
H
6
.25 (1H, s, H-22) , 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8
Hz, H-21b), 4.95 (1H, d, J = 8.0 Hz, H-1'), 4.57 (1H, d, J = 10.0 Hz), 4.40 (1H, br s,
H-3), 4.26 (2H, d, J = 6.1 Hz), 4.01 (2H, m) 3.74 (1H, m, H-17), 3.72 (1H, m, H-12),
13
2
.11-1.76 (15H, m), 1.76-1.36 (4H, m), 1.23 (3H, s, H-18), 0.83 (3H, s, H-19); C
NMR (100 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23), 117.7 (C-22), 85.7
5
(C-14), 74.9 (C-12), 74.5 (C-3), 74.4 (C-21), 57.2 (C-13), 46.9 (C-17), 42.0 (C-8),
33.3 (C-9), 35.7 (C-10), 33.8 (C-15), 37.1 (C-5), 27.3 (C-6),31.2 (C-11), 30.8 (C-4),
28.2 (C-16), 27.5 (C-2), 31.2 (C-1), 24.1 (C-19), 22.8 (C-7), 10.5 (C-18), 103.3 (C-1'),
75.7 (C-2'), 78.8 (C-3'), 72.2 (C-4'), 78.8 (C-5'), 63.3 (C-6'); ESI-MS m/z 575.5 [M +
＋
＋
Na] ; 1127.5 [2M + Na] .
.1.4.2. 3 -O-( -mannopyranosyl)-digoxigenin (3b). Obtained as a white solid (4.8
mg, 74%)
acetyl-digoxigenin (2b). [α]
4
β
α-D
from
3β
-O-(2,3,4,6-tetra-O-acetyl-
α
-D-mannopyranosyl)-12-O-
2
D
9
1
+13.2 (c 0.40, MeOH); H NMR (600 MHz,
Pyridine-d ): δ 6.26 (1H, s, H-22) , 5.49 (1H, d, J = 1.2 Hz, H-1'), 5.28 (1H, dd, J =
5
H
1
8.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.66-4.60 (3H, m), 4.57
1H, m), 4.44-4.39 (2H, m), 4.27 (1H, br s, H-3), 3.76 (1H, m, H-17), 3.72 (1H, m,
H-12), 2.15-1.75 (11H, m), 1.68 (2H, m), 1.56 (1H, m), 1.46 (2H, m), 1.32 (3H, m),
(
1
3
1
.25 (3H, s, H-18), 0.85 (3H, s, H-19); C NMR (150 MHz, Pyridine-d ): δc177.0
5
(C-20), 175.2 (C-23), 117.8 (C-22), 85.7 (C-14), 74.9 (C-12), 74.4 (C-21), 72.1 (C-3),

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5
2
2
6
4
7.1 (C-13), 47.0 (C-17), 42.0 (C-8), 33.2 (C-9), 35.8 (C-10), 33.9 (C-15), 37.8 (C-5),
7.4 (C-6), 31.1 (C-11), 32.7 (C-4), 28.3 (C-16), 24.6 (C-2), 31.2 (C-1), 24.3 (C-19),
2.6 (C-7), 10.5 (C-18), 103.8 (C-1'), 73.1 (C-2'), 73.6 (C-3'), 69.8 (C-4'), 76.0 (C-5'),
＋
＋
3.7 (C-6'); ESI-MS m/z 553.3 [M + H] ;1127.4 [2M + Na] .
.1.4.3. 3 -O-( -galactopyranosyl)-digoxigenin (3c). Obtained as a white solid
22.5 mg, 75%) from
-galactopyranosyl)-12-O-acetyl-digoxigenin (2c). [α]
): δ 6.25 (1H, s, H-22) ,
β
β-D
(
3β
-O-(2,3,4,6-tetra-O-acetyl-β-D
29
1
D
+5.2 (c 0.80, MeOH); H NMR (400 MHz, Pyridine-d
5
H
5
.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.86
(
1H, d, J = 7.8 Hz, H-1'), 4.60 (1H, d, J = 3.0 Hz), 4.50-4.45 (3H, m), 4.40 (1H, br s,
H-3), 4.18 (1H, dd, J = 9.8, 3.6 Hz), 4.09 (1H, t, J = 5.8 Hz), 3.75(1H, m, H-17),
.71(1H, m, H-12), 2.18-1.68 (15H, m), 1.64-1.28 (4H, m), 1.23 (3H, s, H-18), 0.81
3
1
3
(
3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23),
5
117.7 (C-22), 85.7 (C-14), 74.9 (C-12), 74.6 (C-3), 74.4 (C-21), 57.1 (C-13), 46.9
(C-17), 41.9 (C-8), 33.2 (C-9), 35.7 (C-10), 33.8 (C-15), 37.0 (C-5), 27.3 (C-6), 31.1
(C-11), 30.7 (C-4), 28.3 (C-16), 27.5 (C-2), 31.1 (C-1), 24.0 (C-19), 22.8 (C-7), 10.6
(C-18), 104.1 (C-1'), 73.0 (C-2'), 75.8 (C-3'), 70.6 (C-4'), 77.3 (C-5'), 63.3 (C-6');
＋
＋
ESI-MS m/z 575.7 [M + Na] ; 1127.8 [2M + Na] .
.1.4.4. 3 -O-( -arabinopyranosyl)-digoxigenin (3d). Obtained as a white solid
28.3 mg, 76%) from
-arabinopyranosyl)-12-O-acetyl-digoxigenin (2d). [α]
4
β
α-D
(
2
D
9
3β
-O-(2,3,4-tri-O-acetyl-
α
-
D
1
+
24.5 (c 0.80, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) , 5.28
5
H
(
1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.73 (1H, d,
J = 7.2 Hz, H-1'), 4.34 (1H, br s, H-3), 4.32 (1H, m), 4.29 (1H, m), 4.16 (1H, m), 3.76
1H, m, H-17), 3.72 (1H, m, H-12), 2.16-1.68 (15H, m), 1.61-1.31 (4H, m), 1.23 (3H,
(
1
3
s, H-18), 0.80 (3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.1 (C-20),
5
1
75.1 (C-23), 117.7 (C-22), 85.8 (C-14), 74.8 (C-12), 74.3 (C-3), 74.4 (C-21), 57.0
(C-13), 46.8 (C-17), 41.9 (C-8), 33.1 (C-9), 35.7 (C-10), 33.8 (C-15), 37.3 (C-5), 27.4
(C-6), 31.1 (C-11), 32.8 (C-4), 28.1 (C-16), 25.0 (C-2), 31.1 (C-1), 24.0 (C-19), 22.5
(C-7), 10.5 (C-18), 103.8 (C-1'), 75.0(C-2'), 72.8 (C-3'), 69.9 (C-4'), 67.3 (C-5') ;

ACCEPTED MANUSCRIPT
＋
＋
ESI-MS m/z 523.6 [M + H] ; 1067.6 [2M + Na] .
.1.4.5. 3 -O-( -xylopyranosyl)-digoxigenin (3e). Obtained as a white solid (18.8
mg, 67%) from 3 -O-(2,3,4-tri-O-acetyl- -xylopyranosyl)-12-O-acetyl-digoxigenin
+20.3 (c 0.70, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.25 (1H, s,
4
β
β-D
β
β-D
2
D
9
1
(2e). [α]
5
H
H-22) , 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b),
4
4
1
.82 (1H, d, J = 7.4 Hz, H-1'), 4.34 (1H, br s, H-3), 4.33 (1H, m), 4.29-4.14 (2H, m),
.03 (1H, t, J = 6.4 Hz), 3.74 (1H, m, H-17), 3.72 (1H, m, H-12), 2.16-1.68 (15H, m),
1
3
.57-1.34 (4H, m), 1.24 (3H, s, H-18), 0.85 (3H, s, H-19); C NMR (100 MHz,
Pyridine-d ): δc 177.1 (C-20), 175.2 (C-23), 117.7 (C-22), 85.7 (C-14), 75.0 (C-12),
5
7
3
3
7
4
4.9 (C-3), 74.4 (C-21), 57.1 (C-13), 46.9 (C-17), 42.0 (C-8), 33.3 (C-9), 35.7 (C-10),
3.8 (C-15), 37.1 (C-5), 27.3 (C-6), 31.1 (C-11), 31.0 (C-4), 28.2 (C-16), 27.5 (C-2),
1.0 (C-1), 24.1 (C-19), 22.6 (C-7), 10.5 (C-18), 100.2 (C-1'), 75.5 (C-2'), 78.9 (C-3'),
＋
＋
1.6 (C-4'), 67.5 (C-5'); ESI-MS m/z 523.4 [M + H] ; 1067.6 [2M + Na] .
.1.4.6. 3 -O-( -lyxopyranosyl)-digoxigenin (3f). Obtained as a white solid (10.7
-O-(2,3,4-tri-O-acetyl- -lyxopyranosyl)-12-O-acetyl-digoxigenin
+17.8 (c 0.55, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s,
β
α-D
mg, 56%) from 3
β
α-D
2
D
9
1
(
2f). [α]
H-22) , 5.42 (1H, d, J = 1.2 Hz, H-1'), 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13
1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.67 (1H, s), 4.56 (1H, br s, H-3), 4.25 (2H, m),
.14 (1H, m), 3.75 (1H, m, H-17), 3.73 (1H, m, H-12), 2.18-1.69 (13H, m), 1.64-1.35
5
H
(
4
1
3
(
6H, m), 1.26 (3H, s, H-18), 0.86 (3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc
5
1
77.0 (C-20), 175.1 (C-23), 117.8 (C-22), 85.7 (C-14), 74.9 (C-12), 72.8 (C-3), 74.4
(C-21), 57.1 (C-13), 47.0 (C-17), 42.0 (C-8), 33.2 (C-9), 35.8 (C-10), 33.9 (C-15),
3
2
6
4
7.7 (C-5), 27.3 (C-6), 31.2 (C-11), 32.9 (C-4), 28.3 (C-16), 24.7 (C-2), 31.0 (C-1),
4.3 (C-19), 22.7 (C-7), 10.5 (C-18), 100.5 (C-1'), 72.5 (C-2'), 73.8 (C-3'), 69.3 (C-4'),
＋
＋
5.4 (C-5'); ESI-MS m/z 523.4 [M + H] ; 1067.7 [2M + Na] .
.1.4.7. 3 -O-( -ribopyranosyl)-digoxigenin (3g). Obtained as a white solid (8.6
β
β-D
mg, 57%) from 3
β
-O-(2,3,4-tri-O-acetyl-β-D-ribopyranosyl)-12-O-acetyl-digoxigenin
2
D
9
1
(2g). [α]
+7.8 (c 0.60, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s,
5
H
H-22), 5.39 (1H, d, J = 4.2, H-1'), 5.27 (1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.13 (1H,
dd, J = 18.0, 1.8 Hz, H-21b), 4.47 (1H, m), 4.28 (1H, m), 4.21 (1H, m), 4.21 (1H, br s,

ACCEPTED MANUSCRIPT
H-3, overlap), 4.16 (2H, s), 3.76 (1H, m, H-17), 3.74 (1H, m, H-12), 2.16-2.09 (3H,
m), 2.02 (1H, m), 1.94 (2H, m), 1.89-1.76 (6H, m), 1.66 (2H, m), 1.59 (3H, m), 1.35
1
3
(
2H, m), 1.25 (3H, s, H-18), 1.18 (1H, m), 0.89 (3H, s, H-19); C NMR (100 MHz,
Pyridine-d ): δc 177.1 (C-20), 175.2 (C-23), 117.7 (C-22), 85.7 (C-14), 74.9 (C-12),
5
7
3
3
7
4
3.5 (C-3), 74.4 (C-21), 57.2 (C-13), 46.9 (C-17), 42.0 (C-8), 33.3 (C-9), 35.8 (C-10),
3.9 (C-15), 37.4 (C-5), 27.3 (C-6), 31.2 (C-11), 30.6 (C-4), 28.3 (C-16), 27.5 (C-2),
1.4 (C-1), 24.3 (C-19), 22.7 (C-7), 10.5 (C-18), 100.9 (C-1'), 73.3 (C-2'), 69.0 (C-3'),
＋
＋
0.9 (C-4'), 65.6 (C-5'); ESI-MS m/z 523.5 [M + Na] ;1067.4 [2M + Na] .
.1.4.8. 3 -O-( -rhamnopyranosyl)-digoxigenin (3h). Obtained as a white solid
20.4 mg, 60%) from
-rhamnopyranosyl)-12-O-acetyl-digoxigenin (2h). [α]
β
α-L
(
2
D
9
3β
-O-(2,3,4-tri-O-acetyl-
α
-L
1
+
11.6 (c 0.70, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22) , 5.41
5
H
(1H, br s, H-1'), 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz,
H-21b), 4.54 (1H, br s, H-3), 4.53 (1H, m), 4.28 (2H, m), 4.16 (1H, s), 3.76 (1H, m,
H-17), 3.73 (1H, m, H-12), 2.16-1.73 (13H, m), 1.67 (3H, d, J = 5.2 Hz), 1.58 (4H, m),
1
3
1
.36 (1H, m), 1.25 (3H, s, H-18), 1.18 (1H, m), 0.85 (3H, s, H-19); C NMR (100
MHz, Pyridine-d ): δc 177.1 (C-20), 175.2 (C-23), 117.7 (C-22), 85.7 (C-14), 74.8
5
(
(
(
(
C-12), 73.3 (C-3), 74.4 (C-21), 57.1 (C-13), 46.9 (C-17), 41.9 (C-8), 33.2 (C-9), 35.7
C-10), 33.8 (C-15), 37.4 (C-5), 27.1 (C-6), 31.2 (C-11), 30.5 (C-4), 28.2 (C-16), 27.5
C-2), 31.4 (C-1), 24.3 (C-19), 22.6 (C-7), 10.5 (C-18), 100.2 (C-1'), 72.5 (C-2'), 73.3
＋
C-3'), 74.4 (C-4'), 70.4 (C-5'), 19.0 (C-6'); ESI-MS m/z 537.3 [M + H] ; 1095.7 [2M
＋
+
Na] .
.1.4.9. 3
mg, 65%) from 3
4
β-O-(β-L-fucopyranosyl)-digoxigenin (3i). Obtained as a white solid (26.6
β
-O-(2,3,4-tri-O-acetyl-β-L-fucopyranosyl)-12-O-acetyl-digoxigenin
2
D
9
1
(2i). [α]
+16.2 (c 0.70, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s,
5
H
H-22) , 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b),
.72 (1H, d, J = 7.8, H-1'), 4.36 (2H, m,), 4.11 (1H, m), 4.08 (1H, br s, H-3), 3.81 (1H,
m, H-17), 3.74 (1H, m, H-12), 2.16-1.71 (15H, m), 1.56 (1H, m), 1.55 (3H, d, J = 6.4
4
1
3
Hz), 1.43 (3H, m), 1.28 (1H, m), 1.23 (3H, s, H-18), 0.80 (3H, s, H-19); C NMR
(
100 MHz, Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23), 117.7 (C-22), 85.7 (C-14),
5

ACCEPTED MANUSCRIPT
7
3
2
7
1
4
5
3
4.9 (C-12), 74.4 (C-3), 74.4 (C-21), 57.1 (C-13), 46.9 (C-17), 42.0 (C-8), 33.2 (C-9),
5.7 (C-10), 33.8 (C-15), 37.2 (C-5), 27.4 (C-6), 31.1 (C-11), 32.9 (C-4), 28.2 (C-16),
5.2 (C-2), 30.9 (C-1), 24.0 (C-19), 22.6 (C-7), 10.5 (C-18), 103.7 (C-1'), 73.1 (C-2'),
＋
5.9 (C-3'), 72.8 (C-4'), 71.6 (C-5'), 17.8 (C-6'); ESI-MS m/z 537.5 [M + Na] ;
＋
095.4 [2M + Na] .
.1.4.10. 3
β
-O-(β
-D-cellobiosyl)-digoxigenin (3j). Obtained as a white solid (8.0 mg,
1%)
from
β
-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-
β
-D-cellobiosyl)-12-O-acetyl-digoxigenin (2j).
2
9
1
[
α]
.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 5.43
1H, br s), 5.22 (1H, d, J = 7.8, H-1'), 4.90 (1H, d, J = 7.8, H-1''), 4.53 (3H, m),
.40-4.20 (5H, m), 4.15-3.99 (3H, m), 3.93 (1H, m), 3.76 (1H, m, H-17), 3.72 (1H, m,
H-12), 2.16-1.65 (13H, m), 1.55 (2H, m), 1.45-1.27 (2H, m), 1.24 (3H, s, H-18), 0.84
D
+7.2 (c 0.50, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) ,
5
H
5
(
4
1
3
(
3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23),
5
1
17.8 (C-22), 85.7 (C-14), 75.0 (C-12), 74.8 (C-3), 74.4 (C-21), 57.2 (C-13), 46.9
(C-17), 42.0 (C-8), 33.3 (C-9), 35.8 (C-10), 33.9 (C-15), 37.1 (C-5), 27.3 (C-6), 31.2
(C-11), 30.7 (C-4), 28.2 (C-16), 27.5 (C-2), 31.2 (C-1), 24.1 (C-19), 22.7 (C-7), 10.6
(C-18), 105.3 (C-1'),75.2 (C-2'), 76.8 (C-3'), 81.8 (C-4'), 77.3 (C-5'), 62.3 (C-6'),
1
03.2 (C-1''), 75.2 (C-2''), 78.6 (C-3''), 71.9 (C-4''), 78.8 (C-5''), 62.8 (C-6''); ESI-MS
＋
＋
m/z 737.5[M + Na] ;1451.5 [2M + Na] .
4
2
3
.1.4.11. 3
β
-O-(
β
-
D
-lactobiosyl)-digoxigenin (3k). Obtained as a white solid (3.5 mg,
7%)
from
β
-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-
β
-D-lactobiosyl)-12-O-acetyl-digoxigenin (2k).
2
9
1
[
α]
.44 (1H, s), 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz,
H-21b), 5.13 (1H, d, J = 8.2, H-1'), 4.89 (1H, d, J = 8.0, H-1''), 4.58-4.45 (5H, m),
D
+5.2 (c 0.50, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) ,
5
H
5
4
3
.42-4.29 (4H, m), 4.15 (2H, m), 4.07 (1H, m), 3.90 (1H, m), 3.76 (1H, m, H-17),
.72 (1H, m, H-12), 2.14-1.51 (15H, m), 1.45-1.26 (3H, m), 1.24 (3H, s, H-18), 0.84
1
3
(
3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.1 (C-20), 175.2 (C-23),
5
117.8 (C-22), 85.7 (C-14), 75.0 (C-12), 74.8 (C-3), 74.4 (C-21), 57.2 (C-13), 47.0

ACCEPTED MANUSCRIPT
(C-17), 42.0 (C-8), 33.3 (C-9), 35.8 (C-10), 33.9 (C-15), 37.1 (C-5), 27.3 (C-6), 31.2
(C-11), 30.7 (C-4), 28.3 (C-16), 27.5 (C-2), 31.2 (C-1), 24.1 (C-19), 22.7 (C-7), 10.8
(C-18), 106.2 (C-1'), 75.2 (C-2'), 76.8 (C-3'), 82.7 (C-4'), 77.2 (C-5'), 62.4 (C-6'),
1
03.2 (C-1''), 72.9 (C-2''), 75.6 (C-3''), 70.5 (C-4''), 77.8 (C-5''), 62.8 (C-6''); ESI-MS
＋
m/z 737.7 [M + Na] .
.1.4.12. 3 -O-(2,3-dideoxy-
5.2 mg,
4
β
β-D-glucal)-digoxigenin (3l). Obtained as a white solid
(
53%)
from
2
D
9
3β
-O-(4,6-di-O-acetyl-2,3-dideoxy-
β
-D-glucal)-12-O-acetyl-digoxigenin (2l). [α]
1
+
32.8 (c 0.65, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22) , 5.28
5
H
(1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.71 (1H, d,
J = 7.8, H-1'), 6.33 (1H, d, J = 9.2, H-3'), 5.97 (1H, d, J = 9.2, H-2'), 5.47 (1H, s),
5
3
.38 (1H, s), 4.73 (1H, m), 4.40 (3H, m), 4.27 (1H, br s, H-3), 3.78 (1H, m, H-17),
.76 (1H, m, H-12), 2.15-1.72 (13H, m), 1.57 (3H, m), 1.42-1.30 (3H, m), 1.26 (3H, s,
13
H-18), 0.92 (3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.1 (C-20), 175.2
5
(C-23), 117.7 (C-22), 85.7 (C-14), 75.0 (C-12), 74.6 (C-3), 74.4 (C-21), 57.2 (C-13),
47.0 (C-17), 42.0 (C-8), 37.8 (C-9), 35.8 (C-10), 33.2 (C-15), 33.9 (C-5), 27.5 (C-6),
31.2 (C-11), 32.9 (C-4), 28.2 (C-16), 25.7 (C-2), 31.2 (C-1), 24.3 (C-19), 22.6 (C-7),
10.6 (C-18), 94.0 (C-1'), 127.5 (C-2'), 135.5 (C-3'), 73.6 (C-4'), 64.4 (C-5'), 63.1
＋
＋
(
C-6') ; ESI-MS m/z 519.5 [M + H] ; 1059.7 [2M + Na] .
.1.4.13. 3 -O-( -glucopyranosyl)-digoxigenin (3m). Obtained as a white solid
12.6 mg, 63%) from 3
4
β
β-L
(
(
β
-O-(2,3,4,6-tetra-O-acetyl-β-L-glucopyranosyl)-digoxigenin
2
D
9
1
2m). [α]
+7.4 (c 0.60, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s,
5
H
H-22) , 5.43 (1H, s), 5.27 (1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0,
.8 Hz, H-21b), 4.93 (1H, d, J = 7.8 Hz, H-1'), 4.55 (1H, m), 4.41(1H, m), 4.39 (1H,
br s, H-3), 4.28 (2H, m), 4.06 (1H, m) 3.95 (1H, m), 3.74 (2H, m), 2.16-1.96 (9H, m),
1
1
3
1
.84 (2H, m), 1.76 (2H, m), 1.55 (3H, m), 1.24 (3H, s, H-18), 0.83 (3H, s, H-19); C
NMR (150 MHz, Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23), 117.8 (C-22), 85.7
5
(C-14), 75.0 (C-12), 74.4 (C-3), 74.3 (C-21), 57.1 (C-13), 47.0 (C-17), 42.0 (C-8),
3
3.3 (C-9), 35.8 (C-10), 33.9 (C-15), 37.3 (C-5), 27.5 (C-6),31.2 (C-11), 32.9 (C-4),
8.3 (C-16), 25.1 (C-2), 30.9 (C-1), 24.1 (C-19), 22.6 (C-7), 10.6 (C-18), 103.3 (C-1'),
2

ACCEPTED MANUSCRIPT
7
5.7 (C-2'), 79.1 (C-3'), 72.2 (C-4'), 78.8 (C-5'), 63.3 (C-6'); ESI-MS m/z 575.5 [M +
＋
＋
Na] ; 1127.5 [2M + Na] .
.1.4.14. 3 -O-( -arabinopyranosyl)-digoxigenin (3n). Obtained as a white solid
7.0 mg, 56%) from 3
4
β
α-L
(
[
β
-O-(2,3,4-tri-O-acetyl-
α
-
L
-arabinopyranosyl)-digoxigenin (2n).
2
D
9
1
α]
+16.2 (c 0.60, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) ,
5
H
5
.40 (1H, s), 5.27 (1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz,
H-21b), 4.76 (1H, d, J = 7.0 Hz, H-1'), 4.44 (1H, t, d = 7.6 Hz), 4.34 (1H, br s, H-3),
.32 (2H, m), 4.17 (1H, m), 3.75 (1H, m, H-17), 3.73 (1H, m, H-12), 2.16-1.68 (13H,
4
1
3
m), 1.56 (2H, m), 1.40 (1H, m), 1.24 (3H, s, H-18), 0.84 (3H, s, H-19); C NMR (150
MHz, Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23), 117.8 (C-22), 85.8 (C-14), 75.1
5
(
C-12), 75.0 (C-3), 74.3 (C-21), 57.2 (C-13), 47.0 (C-17), 42.1 (C-8), 33.3 (C-9), 35.8
C-10), 33.9 (C-15), 37.2 (C-5), 27.6 (C-6), 31.1 (C-11), 31.3 (C-4), 28.3 (C-16), 27.4
C-2), 31.0 (C-1), 24.2 (C-19), 22.7 (C-7), 10.6 (C-18), 104.4 (C-1'), 73.0 (C-2'),
(
(
＋
＋
7
4
5.0(C-3'), 70.0 (C-4'), 67.4 (C-5'); ESI-MS m/z 523.6 [M + H] ; 1067.6 [2M + Na] .
.1.4.15. 3
β
-O-(β
-L
-xylopyranosyl)-digoxigenin (3o). Obtained as a white solid (6.0
2
D
9
mg, 72%) from 3
β
-O-(2,3,4-tri-O-acetyl-
β
-L
-xylopyranosyl)-digoxigenin (2o). [α]
1
+
9.2 (c 0.74, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) , 5.43
5
H
(
1H, s), 5.27 (1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b),
.82 (1H, d, J = 7.5 Hz, H-1'), 4.37 (1H, br s, H-3), 4.36 (1H, m), 4.27 (1H, m), 4.17
1H, t, d = 8.5 Hz), 4.04 (1H, t, J = 8.2 Hz), 3.75 (1H, m, H-17), 3.73 (1H, m, H-12),
.18-1.77 (15H, m), 1.62-1.53 (3H, m), 1.41 (1H, m), 1.24 (3H, s, H-18), 0.85 (3H, s,
4
(
2
1
3
H-19); C NMR (150 MHz, Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23), 117.8
5
(C-22), 85.8 (C-14), 75.0 (C-12), 74.5 (C-3), 74.4 (C-21), 57.2 (C-13), 47.0 (C-17),
42.1 (C-8), 33.3 (C-9), 35.8 (C-10), 33.9 (C-15), 37.4 (C-5), 27.5 (C-6), 31.2 (C-11),
33.0 (C-4), 28.3 (C-16), 25.2 (C-2), 31.0 (C-1), 24.2 (C-19), 22.7 (C-7), 10.6 (C-18),
104.0 (C-1'), 75.5(C-2'), 79.0 (C-3'), 71.7 (C-4'), 67.7 (C-5'); ESI-MS m/z 523.4 [M +
＋
＋
H] ; 1067.6 [2M + Na] .
.1.4.16. 3 -O-( -glucopyranosyl)-digoxigenin (5a). Obtained as a white solid
14.0
4
α
β-D
(
mg,
-O-(2,3,4,6-tetra-O-acetyl-
70.2%)
from
2
D
9
3
α
β
-D
-glucopyranosyl)-digoxigenin (4a). [α]
+19.5 (c

ACCEPTED MANUSCRIPT
1
0
.70, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22) , 5.26 (1H, dd,
5 H
J = 18.0, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 5.05 (1H, d, J = 7.6
Hz, H-1'), 4.60 (1H, d, J = 10.0 Hz), 4.43 (1H, m), 4.30 (2H, m), 4.07 (1H, m), 4.02
(1H, qd, H-3), 3.72 (1H, m, H-17), 3.59 (1H, m, H-12), 1.22 (3H, s, H-18), 0.83 (3H,
1
3
s, H-19); C NMR (150 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23), 117.8
5
(C-22), 85.6 (C-14), 74.9 (C-12), 78.3 (C-3), 74.4 (C-21), 57.1 (C-13), 46.9 (C-17),
4
3
1
5
4
2.1 (C-8), 33.7 (C-9), 35.5 (C-10), 33.9 (C-15), 42.4 (C-5), 27.9 (C-6), 30.9 (C-11),
5.1 (C-4), 28.2 (C-16), 35.6 (C-2), 27.9 (C-1), 23.8 (C-19), 22.8 (C-7), 10.5 (C-18),
02.7 (C-1'), 75.8 (C-2'), 79.1 (C-3'), 72.2 (C-4'), 78.9 (C-5'), 63.3 (C-6'); ESI-MS m/z
＋
＋
53.3 [M + H] ; 1127.3 [2M + Na] .
.1.4.17. 3a-O-( -galactopyranosyl)-digoxigenin (5b). Obtained as a white solid
35.3
β-D
(
mg,
70.1%)
from
2
D
9
3
0
a-O-(2,3,4,6-tetra-O-acetyl-
β
-D
-galactopyranosyl)-digoxigenin (4b). [α]
+30.2 (c
1
.85, MeOH); H NMR (300 MHz, Pyridine-d ): δ 6.24 (1H, s, H-22) , 5.25 (1H, dd,
5
H
J = 18.0, 1.8 Hz, H-21a), 5.11 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.94 (1H, d, J = 7.5
Hz, H-1'), 4.58 (1H, d, J = 10.0 Hz), 4.46 (2H, m), 4.22 (1H, m), 4.11 (1H, m), 3.98
(1H, qd, H-3), 3.70 (1H, m, H-17), 3.54 (1H, m, H-12), 1.20 (3H, s, H-18), 0.80 (3H,
1
3
s, H-19); C NMR (75 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23), 117.6
5
(C-22), 85.5 (C-14), 74.7 (C-12), 78.2 (C-3), 74.3 (C-21), 56.9 (C-13), 46.7 (C-17),
4
3
1
5
4
1.9 (C-8), 33.6 (C-9), 35.3 (C-10), 33.7 (C-15), 42.2 (C-5), 27.7 (C-6), 30.8 (C-11),
5.0 (C-4), 28.0 (C-16), 35.5 (C-2), 27.7 (C-1), 23.6 (C-19), 22.6 (C-7), 10.4 (C-18),
03.2 (C-1'), 73.0 (C-2'), 75.7 (C-3'), 70.5 (C-4'), 77.2 (C-5'), 62.8 (C-6'); ESI-MS m/z
＋
＋
75.7 [M + Na] ; 1127.8 [2M + Na] .
.1.4.18. 3 -O-( -arabinopyranosyl)-digoxigenin (5c). Obtained as a white solid
11.5 mg, 62.7%) from 3
α
α-D
(
(
α
-O-(2,3,4-tri-O-acetyl-α-D-arabinopyranosyl)-digoxigenin
2
D
9
1
4c). [α]
+15.2 (c 0.70, MeOH); H NMR (300 MHz, Pyridine-d ): δ 6.25 (1H, s,
5
H
H-22), 5.28 (1H, dd, J = 18.1, 1.8 Hz, H-21a), 5.13 (1H, dd, J = 18.0, 1.8 Hz, H-21b),
.85 (1H, d, J = 7.4 Hz, H-1'), 4.46 (1H, m), 4.35 (2H, m), 4.23 (1H, m), 3.95 (1H, qd,
H-3), 3.87 (1H, m), 3.74 (1H, m, H-17), 3.65 (1H, m, H-12), 2.16-1.96 (4H, m),
.94-1.64 (10H, m), 1.53 (1H, m), 1.29 (1H, m), 1.23 (3H, s, H-18), 0.98 (1H, m),
4
1

ACCEPTED MANUSCRIPT
1
3
0
.85 (3H, s, H-19); C NMR (75 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23),
5
117.8 (C-22), 85.6 (C-14), 74.9 (C-12), 78.4 (C-3), 74.4 (C-21), 57.1 (C-13), 47.0
(
C-17), 42.1 (C-8), 33.7 (C-9), 35.5 (C-10), 33.8 (C-15), 42.3 (C-5), 27.9 (C-6), 31.0
C-11), 33.5 (C-4), 28.2 (C-16), 36.0 (C-2), 29.7 (C-1), 23.8 (C-19), 22.8 (C-7), 10.6
C-18), 103.6 (C-1'), 73.0 (C-2'), 75.1(C-3'), 70.0 (C-4'), 67.3 (C-5'); ESI-MS m/z
(
(
＋
＋
5
23.6 [M + H] ; 1067.6 [2M + Na] .
4
.1.4.19. 3 -O-( -xylopyranosyl)-digoxigenin (5d). Obtained as a white solid (11.0
α
β-D
2
D
9
mg, 68.3%) from 3
α
-O-(2,3,4-tri-O-acetyl-
β
-D-xylopyranosyl)-digoxigenin (4d). [α]
1
+
20.2 (c 0.80, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s, H-22), 5.27
5
H
(1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.12 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.91 (1H, d,
J = 7.4 Hz, H-1'), 4.42 (1H, dd, J = 11.1, 5.2 Hz), 4.28 (1H, m), 4.19 (1H, t, J = 8.5
Hz), 4.03 (1H, t, J = 8.2 Hz), 3.96 (1H, qd, H-3), 3.78 (1H, t, J = 11.1 Hz, H-17), 3.73
(1H, m, H-12), 3.59 (1H, m), 2.13-1.98 (5H, m), 1.87-1.73 (8H, m), 1.60 (1H,m), 1.52
1
3
(
1H, m), 1.39-1.29 (3H, m), 1.23 (3H, s, H-18), 0.96 (1H,m), 0.86 (3H, s, H-19); C
NMR (175 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23), 117.8 (C-22), 85.6
5
(C-14), 74.9 (C-12), 78.9 (C-3), 74.4 (C-21), 57.1 (C-13), 47.0 (C-17), 42.1 (C-8),
3
2
7
3.8 (C-9), 35.5 (C-10), 33.9 (C-15), 42.5 (C-5), 28.0 (C-6), 31.0 (C-11), 35.4 (C-4),
8.2 (C-16), 35.7 (C-2), 27.9 (C-1), 23.8 (C-19), 22.8 (C-7), 10.6 (C-18), 103.8 (C-1'),
＋
5.6 (C-2'), 79.0 (C-3'), 71.6 (C-4'), 67.7 (C-5'); ESI-MS m/z 523.4 [M + H] ; 1067.6
＋
[
2M + Na] .
.1.4.20. 3
10.4 mg, 51.2%) from 3
4
α-O-(α-L-rhamnopyranosyl)-digoxigenin (5e). Obtained as a white solid
(
(
α
-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-digoxigenin
29
1
4e). [α]
D
+8.2 (c 0.60, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.25 (1H, s,
5
H
H-22), 5.53 (1H, br s, H-1'), 5.28 (1H, dd, J = 18.0, 1.8 Hz, H-21a), 5.13 (1H, dd, J =
8.0, 1.8 Hz, H-21b), 4.57 (2H, m), 4.35 (2H, m), 3.87 (1H, qd, H-3), 3.75 (1H, m,
H-17), 3.70 (1H, m, H-12), 2.09 (3H, m), 1.95-1.76 (6H, m), 1.60-1.27 (6H, m), 1.25
1
1
3
(
3H, s, H-18), 0.91 (1H, m), 0.86 (3H, s, H-19); C NMR (150 MHz, Pyridine-d ): δc
5
1
77.1 (C-20), 175.1 (C-23), 117.8 (C-22), 85.6 (C-14), 74.8 (C-12), 76.1 (C-3), 74.4
C-21), 57.1 (C-13), 47.0 (C-17), 42.1 (C-8), 33.7 (C-9), 35.5 (C-10), 33.8 (C-15),
2.5 (C-5), 27.9 (C-6), 31.0 (C-11), 34.9 (C-4), 28.3 (C-16), 35.6 (C-2), 27.2 (C-1),
(
4

ACCEPTED MANUSCRIPT
2
7
4
3.8 (C-19), 22.8 (C-7), 10.6 (C-18), 99.5 (C-1'), 73.2 (C-2'), 73.3 (C-3'), 74.6 (C-4'),
＋
＋
0.3 (C-5'), 19.1 (C-6'); ESI-MS m/z 537.3 [M + H] ; 1095.7 [2M + Na] .
.1.5. General procedure for preparation of C -MeON-neoglycosideslibrary
3
3
β
-MeON-digoxigenin (Dig-4/
mmol) and free reducing sugar (2.0-2.5 eq) were dissolved in DMF/AcOH (3: 1,
00–1500 µL) and stirred at 50 °C for 48 h, and then was neutralized with AcOH until
β) or 3α-MeON-digoxigenin (Dig-5/α) (0.07-0.14
6
pH = 7. The crude product was obtained by filtered and concentrated under reduced
pressure. The residue was purified by semipreparative RP-HPLC (45% MeOH-H O)
2
to obtain C -MeON-neoglycosides of digoxigenin (6a-6d and 7a-7d). All compounds
3
1
13
were characterized by analyses of H and C NMR spectra as well as ESI-MS.
.1.5.1. 3 -N-( -glucopyranosyl)-N-(O-methyl)-digoxigenin (6a). Obtained as a
4
β
β-D
2
D
9
white solid (6.2 mg, 17.9%) from 3
β
-N-(O-methyl)-digoxigenin (Dig-4). [α]
+8.2 (c
1
0
.70, MeOH); H NMR (300 MHz, CD OD): δ 5.91 (1H, s, H-22) , 4.99 (1H, dd, J =
3
H
1
8.0, 1.8 Hz, H-21a), 4.91 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.10 (1H, d, J = 8.6 Hz,
H-1'), 3.80 (1H, m), 3.73 (3H, s), 3.70-3.57 (3H, m), 3.16 (1H, m), 2.17-1.52 (15H,
1
3
m), 1.29 (6H, m) 1.00 (3H, s, H-18), 0.79 (3H, s, H-19); C NMR (75 MHz, CD OD)
3
δc 178.6 (C-20), 177.4 (C-23), 117.7 (C-22), 86.8 (C-14), 75.5 (C-12), 71.7 (C-21),
6
3
2
7
4
2.7 (OMe), 58.5 (C-13), 57.3 (C-3), 47.0 (C-17), 42.3 (C-8), 37.8 (C-5), 36.7 (C-10),
4.0 (C-15), 33.5 (C-9), 31.7 (C-11), 30.9 (C-1), 30.7 (C-6), 28.4 (C-16), 28.2 (C-4),
4.5 (C-19), 24.1 (C-2), 22.5 (C-7), 9.9 (C-18), 91.0 (C-1'),75.7 (C-2'), 79.6 (C-3'),
＋
＋
1.1 (C-4'), 79.8 (C-5'), 64.5 (C-6'); ESI-MS m/z 582.6 [M + H] ; 1185.7 [2M + Na] .
.1.5.2. 3 -N-( -allopyranosyl)-N-(O-methyl)-digoxigenin (6b). Obtained as a
β
β-D
2
D
9
white solid (8.0 mg, 23.1%) from 3
β
-N-(O-methyl)-digoxigenin (Dig-4). [α]
+12.2
1
(
c 0.73, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22), 5.30 (1H,
5
H
dd, J = 18.0, 1.8 Hz, H-21a), 5.15 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 5.15 (1H, d, J =
8
2
.5 Hz, H-1′), 4.82 (2H, m), 4.44 (2H, m), 3.92 (1H, br s, H-3), 3.87 (1H, br s, H-3),
1
3
.17-1.77 (10H, m), 1.68-1.53 (5H, m) 1.25 (3H, s, H-18), 0.79 (3H, s, H-19); C
NMR (100 MHz, Pyridine-d ): δ 177.1 (C-20), 175.2 (C-23), 117.8 (C-22), 86.7
5
(C-14), 75.0 (C-12), 74.4 (C-21), 63.8 (OMe), 57.6 (C-3), 57.2 (C-13), 47.0 (C-17),
4
2.2 (C-8), 37.3 (C-5), 36.3 (C-10), 34.0 (C-15), 33.6 (C-9), 31.7 (C-11), 31.3 (C-1),

ACCEPTED MANUSCRIPT
3
8
5
4
0.5 (C-6), 28.3 (C-16), 27.8 (C-4), 24.4 (C-19), 24.4 (C-2), 22.4 (C-7), 10.6 (C-18),
8.1 (C-1'),74.0 (C-2'), 69.8 (C-3'), 69.7 (C-4'), 77.4 (C-5'), 64.0 (C-6'); ESI-MS m/z
＋
＋
82.5 [M + H] ; 1185.5 [2M + Na] .
.1.5.3. 3 -N-( -xylofuranosyl)-N-(O-methyl)-digoxigenin (6c). Obtained as a white
β
β-D
2
D
9
solid (6.1 mg, 18.6%) from 3
β
-N-(O-methyl)-digoxigenin (Dig-4). [α]
+15.6 (c 0.48,
1
MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.27 (1H, s, H-22) , 5.30 (1H, dd, J =
5
H
1
8.0, 1.8 Hz, H-21a), 5.15 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.62 (1H, d, J = 8.5 Hz,
H-1'), 4.46 (2H, m), 4.28 (1H, m), 4.20 (1H, m), 3.98 (3H, s), 3.87 (1H, br s, H-3),
.78-3.73 (2H, m), 2.17-1.94 (10H, m), 1.86 (2H, m), 1.73-1.36 (6H, m) 1.27 (3H, s,
3
1
3
H-18), 1.14 (1H, m), 0.87 (3H, s, H-19); C NMR (100 MHz, Pyridine-d ): δc 177.1
5
(
(
(
(
(
C-20), 175.2 (C-23), 117.8 (C-22), 85.7 (C-14), 75.0 (C-12), 74.4 (C-21), 64.1
OMe), 57.9 (C-3), 57.2 (C-13), 47.0 (C-17), 42.2 (C-8), 37.7 (C-5), 36.3 (C-10), 33.9
C-15), 33.7 (C-9), 31.6 (C-11), 31.3 (C-1), 30.6 (C-6), 28.3 (C-16), 27.9 (C-4), 24.5
C-19), 24.4 (C-2), 22.5 (C-7), 10.6 (C-18), 92.5 (C-1'),71.5 (C-2'), 72.2 (C-3'), 81.0
＋
C-4'), 69.6 (C-5'); ESI-MS m/z 574.5 [M + Na] .
4
.1.5.4. 3β-N-(D-ribofuranosyl)-N-(O-methyl)-digoxigenin (α/β, 1:1) (6d). Obtained
2
D
9
as a white solid (7.3 mg, 22.2%) from 3
β
-N-(O-methyl)-digoxigenin (Dig-4). [α]
1
+
6.2 (c 0.52, MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.26 (2H, s, H-22) , 5.28
5
H
(
2H, dd, J = 18.0, 1.8 Hz, H-21a), 5.14 (2H, dd, J = 18.0, 1.8 Hz, H-21b), 5.42 (1H, d,
J = 4.0 Hz, H-1'), 5.39 (1H, s, H-1'), 4.46 (2H, m), 4.88 (2H, m), 4.79 (2H, m), 4.71
1H, m), 4.68 (1H, m), 4.03 (3H, s), 3.86 (1H, br s, H-3), 3.74 (3H, s), 1.26 (6H, s,
(
1
3
H-18), 0.90 (3H, s, H-19), 0.80 (3H, s, H-19); C NMR (150 MHz, Pyridine-d ): δ
5
1
8
77.1 (C-20), 177.1 (C-20), 175.2 (C-23), 175.1 (C-23), 117.8 (C-22), 117.8 (C-22),
5.7 (C-14), 85.7 (C-14), 74.9 (C-12), 74.4 (C-21), 64.1 (OMe), 64.0 (OMe), 58.6
(C-3), 57.9 (C-3), 57.2 (C-13), 57.1 (C-13), 47.0 (C-17), 47.0 (C-17), 42.2 (C-8), 42.1
(C-8), 37.7 (C-5), 37.4 (C-5), 36.3 (C-10), 36.3 (C-10), 33.9 (C-15), 33.9 (C-15), 33.7
(C-9), 33.6 (C-9), 31.7 (C-11), 31.6 (C-11), 31.3 (C-1), 31.3 (C-1), 30.6 (C-6), 30.5
(C-6), 28.3 (C-16), 28.3 (C-16), 27.9 (C-4), 27.7 (C-4), 24.5 (C-19), 24.4 (C-19), 24.4
(C-2), 24.4 (C-2), 22.4 (C-7), 22.4 (C-7), 10.6 (C-18), 10.5 (C-18), 97.4 (C-1'), 88.1
(C-1'), 69.4 (C-2'), 69.0 (C-2'), 73.6 (C-3'), 73.1 (C-3'), 85.8 (C-4'), 85.0 (C-4'), 66.4

ACCEPTED MANUSCRIPT
＋
＋
(C-5'), 64.6 (C-5'); ESI-MS m/z 574.5 [M + Na] ; 1125.5 [2M + Na] .
4
.1.5.5. 3 -N-( -glucopyranosyl)-N-(O-methyl)-digoxigenin (7a). Obtained as a
α
β-D
2
D
9
white solid (12.0 mg, 28.8%) from 3
α
-N-(O-methyl)-digoxigenin (Dig-5). [α]
+18.5
1
(
c 0.60, MeOH); H NMR (300 MHz, CD OD): δ 5.91 (1H, s, H-22) , 5.00 (1H, dd,
3
H
J = 18.0, 1.8 Hz, H-21a), 4.91 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.17 (1H, d, J = 8.6
Hz, H-1'), 3.81 (1H, m), 3.70 (3H, s), 3.66 (1H, m), 3.57 (1H, m), 3.36 (3H, m), 3.18
(2H, m), 2.17-1.52 (15H, m), 1.44-1.24 (6H, m), 1.10 (2H, m), 0.96 (3H, s, H-18),
1
3
0
.79 (3H, s, H-19); C NMR (75 MHz, CD OD): δ 178.5 (C-20), 177.3 (C-23), 117.7
3
(C-22), 86.8 (C-14), 75.5 (C-12), 71.3 (C-21), 63.2 (C-3), 62.7 (OMe), 57.3 (C-13),
4
3
9
7.1 (C-17), 43.1 (C-8), 36.7 (C-5), 35.9 (C-10), 35.9 (C-15), 34.0 (C-9), 33.5 (C-11),
2.6 (C-1), 30.6 (C-6), 28.4 (C-16), 28.3 (C-4), 25.5 (C-19), 23.8 (C-2), 23.0 (C-7),
.9 (C-18), 91.0 (C-1'),75.7 (C-2'), 79.7 (C-3'), 71.1 (C-4'), 79.8 (C-5'), 64.6 (C-6');
＋
＋
ESI-MS m/z 582.6 [M + H] ; 1185.7 [2M + Na] .
4
.1.5.6. 3 -N-( -allopyranosyl)-N-(O-methyl)-digoxigenin (7b). Obtained as a
α
β-D
2
D
9
white solid (19.5 mg, 28.1%) from 3
α
-N-(O-methyl)-digoxigenin (Dig-5). [α]
+17.2
1
(
c 0.75, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22), 5.28 (1H,
5
H
dd, J = 18.0, 1.8 Hz, H-21a), 5.14 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 5.24 (1H, d, J =
.5 Hz, H-1′), 4.83 (1H, br s), 4.53-4.40 (3H, m), 4.04 (1H, br s, H-3), 3.80-3.69 (3H,
8
1
3
m), 2.15-1.70 (15H, m) 1.25 (3H, s, H-18), 0.83 (3H, s, H-19); C NMR (100 MHz,
Pyridine-d ): δ 177.1 (C-20), 175.1 (C-23), 117.8 (C-22), 85.7 (C-14), 75.0 (C-12),
5
7
3
2
6
4.4 (C-21), 62.7 (OMe), 63.5 (C-3), 57.2 (C-13), 47.0 (C-17), 42.4 (C-8), 42.2 (C-5),
6.4 (C-10), 35.6 (C-15), 34.0 (C-9), 33.8 (C-11), 32.7 (C-1), 30.9 (C-6), 30.4 (C-16),
8.3 (C-4), 25.6 (C-19), 24.0 (C-2), 23.0 (C-7), 10.6 (C-18), 88.3 (C-1'), 73.9 (C-2'),
＋
9.5 (C-3'), 69.3 (C-4'), 77.4 (C-5'), 64.4 (C-6'); ESI-MS m/z 604.7 [2M + Na] ;
＋
1185.7 [2M + Na] .
4
.1.5.7. 3 -N-( -xylofuranosyl)-N-(O-methyl)-digoxigenin (7c). Obtained as a
α
β-D
2
D
9
white solid (12.7 mg, 38.6%) from 3
α
-N-(O-methyl)-digoxigenin (Dig-5). [α]
+16.1
1
(
c 0.65, MeOH); H NMR (400 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22) , 5.28 (1H,
5
H
dd, J = 18.0, 1.8 Hz, H-21a), 5.14 (1H, dd, J = 18.0, 1.8 Hz, H-21b), 4.68 (1H, d, J =
.8 Hz, H-1'), 4.45 (1H, m), 4.39 (1H, m), 4.24 (1H, m), 4.18 (1H, m), 3.96 (3H, s),
8

ACCEPTED MANUSCRIPT
3
.80-3.56 (5H, m), 2.16-2.05 (4H, m), 1.98-1.73 (8H, m), 1.62-1.51 (3H, m), 1.39 (2H,
1
3
m), 1.26 (3H, s, H-18), 1.04 (1H, m), 0.89 (3H, s, H-19); C NMR (100 MHz,
Pyridine-d ): δc 177.1 (C-20), 175.1 (C-23), 117.8 (C-22), 86.6 (C-14), 74.9 (C-12),
5
7
3
2
7
4
4.4 (C-21), 62.8 (OMe), 64.4 (C-3), 57.2 (C-13), 47.0 (C-17), 42.6 (C-8), 42.2 (C-5),
6.5 (C-10), 35.6 (C-15), 34.0 (C-9), 33.9 (C-11), 32.8 (C-1), 31.0 (C-6), 28.3 (C-16),
8.3 (C-4), 25.6 (C-19), 24.1 (C-2), 23.0 (C-7), 10.6 (C-18), 92.6 (C-1'), 71.4 (C-2'),
＋
＋
1.7 (C-3'), 81.0 (C-4'), 69.5 (C-5'); ESI-MS m/z 552.3 [M + H] ; 1125.5 [2M + Na] .
.1.5.8. 3 -N-( -lactosyl)-N-(O-methyl)-digoxigenin (7d). Obtained as a white solid
α
β-D
2
D
9
(3.0 mg, 6.8%) from 3
α
-N-(O-methyl)-digoxigenin (Dig-5). [α]
+28.2 (c 0.40,
1
MeOH); H NMR (600 MHz, Pyridine-d ): δ 6.26 (1H, s, H-22) , 5.28 (1H, dd, J =
5
H
1
8.0, 1.8 Hz, H-21a), 5.14 (2H, dd, J = 18.0, 1.8 Hz, H-21b), 5.12 (1H, d, J = 6.9 Hz,
H-1''), 4.73 (1H, d, J = 8.3 Hz, H-1'), 4.57-4.46 (6H, m), 4.42 (1H, m), 4.31 (2H, m),
.17 (2H, m), 3.97 (3H, s), 3.83 (1H, m), 3.76 (2H, m), 3.51 (1H, m), 2.18-2.08 (4H,
m), 2.01-1.75 (8H, m), 1.67 (1H, m), 1.56-1.48 (3H, m), 1.41-1.30 (3H, m), 1.26 (3H,
4
1
3
s, H-18), 0.93 (1H, m), 0.85 (3H, s, H-19); C NMR (150 MHz, Pyridine-d ): δc
5
1
77.1 (C-20), 175.2 (C-23), 117.8 (C-22), 85.7 (C-14), 74.9 (C-12), 74.4 (C-21), 62.7
(
(
(
(
OMe), 64.3 (C-3), 57.2 (C-13), 47.0 (C-17), 42.6 (C-8), 42.2 (C-5), 36.5 (C-10), 35.6
C-15), 34.0 (C-9), 33.8 (C-11), 32.6 (C-1), 31.0 (C-6), 28.3 (C-16), 28.3 (C-4), 25.6
C-19), 24.0 (C-2), 23.0 (C-7), 10.6 (C-18), 91.6 (C-1'), 71.5 (C-2'), 78.7 (C-3'), 82.9
C-4'), 78.9 (C-5'), 62.5 (C-6'), 106.3 (C-1''), 72.9 (C-2''), 75.6 (C-3''), 70.5 (C-4''),
＋
7
7.7 (C-5''), 62.9 (C-6''); ESI-MS m/z 766.7 [2M + Na] .
4
.1.6. Synthesis of digoxigenin bisdigitoxoside (8a) and digoxigenin monodigitoxoside
(9b)
To a solution of digoxin (2.0g, 2.56 mmol) in MeOH (360 mL) was added a solution
of NaIO (2.0g, 9.39 mmol) in H O (20 mL). The reaction mixture was stirred at room
4
2
temperature for 3 h and was filtered to remove NaIO precipitation. The filtrate was
3
concentrated under reduced pressure and was diluted with EtOAc followed by
washing with H O. The resulting solution was dried over anhydrous Na SO and then
2
2
4
concentrated to give dialdehyde intermediate as a white amorphous powder (1.95 g,
7.5%), which was subjected to next reaction without further purification. The
9

ACCEPTED MANUSCRIPT
dialdehyde intermediate (1.95 g, 2.51 mmol) was dissolved in MeOH (60mL) and was
added aminocaproic acid (420 mg, 3.21 mmol) in small portions. The reaction
mixture was stirred for 4 h and then quenched with NaHCO . The mixture solution
3
was concentrated at 40 °C under reduced pressure and was extracted with EtOAc
followed by washing with H O. The resulting solution was dried over anhydrous
2
Na SO and was evaporated to dryness under reduced pressure. The residue was
2
4
purified by silica gel column chromatography (Petroleum ether/EtOAc, 1:3) to give
3β
-O-(2,6-dideoxy-
β
-D
-ribopyranosyl-(1→4)-O-2,6-dideoxy-
β
-D-ribopyranosyl)-digo
1
xigenin (digoxigenin bisdigitoxoside, 8a) as white solids (1.20 g, 74%). H NMR
(
400 MHz, Pyridine-d ): δ 6.24 (1H, s, H-22), 5.27 (1H, dd, J = 17.8, 1.8 Hz, H-21a),
5 H
5
.12 (1H, dd, J = 17.8, 1.8 Hz, H-21b), 4.41 (1H, dd, J = 7.1, 2.0 Hz, H-1'), 4.69 (1H,
dd, J = 7.1, 2.0 Hz, H-1''), 4.26 (1H, br s), 4.27 (2H, m), 3.75 (1H, m, H-17), 3.72 (1H,
m, H-12), 3.54 (2H, m), 2.40 (2H, m), 2.11 (4H, m), 1.97-1.51 (15H, m), 1.49 (3H, d,
1
3
J = 6.1 Hz), 1.43 (3H, d, J = 6.1 Hz), 1.23 (3H, s, H-18), 0.89 (3H, s, H-19); C
NMR (100 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1 (C-23), 117.8 (C-22), 85.8
5
(C-14), 74.9 (C-12), 74.4 (C-3), 74.3 (C-21), 57.1 (C-13), 46.9 (C-17), 42.0 (C-8),
3
2
3.3 (C-9), 35.8 (C-10), 33.9 (C-15), 37.4 (C-5), 27.5 (C-6), 31.2 (C-11), 31.2 (C-4),
8.2 (C-16), 27.3 (C-2), 30.8 (C-1), 24.3 (C-19), 22.7 (C-7), 10.6 (C-18), 96.9
(C-1'),39.5 (C-2'), 67.9 (C-3'), 83.8 (C-4'), 69.0 (C-5'), 19.1 (C-6'), 100.3 (C-1''), 39.8
(C-2''), 68.9 (C-3''), 73.6 (C-4''), 70.8 (C-5''), 19.3 (C-6''); ESI-MS m/z 673.7 [M + Na]
＋
＋
;
1323.5 [2M + Na] .
Following procedure
the
for
preparation
of
compound
8a,
3β
-O-(2,6-dideoxy-β-D-ribopyranosyl)-digoxigenin (digoxigenin monodigitoxoside,
9
b) was obtained as white solid (690 mg, 71.9 %) by silica gel column
1
chromatography eluted with Petroleum ether/EtOAc (1:3). H NMR (300 MHz,
Pyridine-d ): δ 6.24 (1H, s, H-22), 5.27 (1H, dd, J = 17.8, 1.8 Hz, H-21a), 5.12 (1H,
5
H
dd, J = 17.8, 1.8 Hz, H-21b), 4.45 (1H, dd, J = 7.1, 2.0 Hz, H-1'), 4.30 (1H, br s, H-3),
4
2
.31 (1H, m), 3.73 (1H, m, H-17), 3.70 (1H, m, H-12), 3.65 (1H, m), 2.45 (1H, m),
.13 (4H, m), 1.98-1.66 (10H, m), 1.59 (3H, d, J = 6.1 Hz), 1.39 (2H, m), 1.23 (3H, s,
13
H-18), 0.90 (3H, s, H-19); C NMR (75 MHz, Pyridine-d ): δc 177.0 (C-20), 175.1
5

ACCEPTED MANUSCRIPT
(C-23), 117.7 (C-22), 85.7 (C-14), 74.9 (C-12), 74.6 (C-3), 74.4 (C-21), 57.1 (C-13),
46.9 (C-17), 42.0 (C-8), 33.3 (C-9), 35.8 (C-10), 33.8 (C-15), 37.4 (C-5), 27.5 (C-6),
31.2 (C-11), 31.2 (C-4), 28.2 (C-16), 27.4 (C-2), 30.8 (C-1), 24.3 (C-19), 22.7 (C-7),
10.6 (C-18), 97.0 (C-1'),40.4 (C-2'), 69.1 (C-3'), 73.4 (C-4'), 70.7 (C-5'), 19.5 (C-6');
＋
＋
ESI-MS m/z 521.4 [M + H] ; 1063.5 [2M + Na] .
4
4
.2. Biology assay
.2.1 Cell Culture.
The NIH-H460 cell line was purchased from the American Type Culture Collection
and maintained in RPMI 1640 medium containing 10% FBS and 1%
units/mL penicillin, and 100 mg/mL streptomycin.
L-glutamine, 100
4
.2.2 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
MTT assay was performed according to the method reported previously [24]. The
3
cells were seeded in 96-well plates at 3×10 /well in the complete medium and then
substituted by the medium containing 50 nM glycosides for 48 h incubation. Next, 10
µL of 5 µg/mL MTT was directly added to each well while the cells continue to
incubate at 37 °C for 4 hr. At the end of experiments, 100 µL of DMSO was used to
dissolve formazan and measured at 560 nm on Victor 3V which is a multiwall plate
reader from Perkin Elmer.
4
.2.3. Western blotting analysis for Nur77 expression
Equal amounts of the lysates were electrophoresed on an 8% SDS-PAGE gel and
transferred onto polyvinylidene difluoride (PVDF) membranes, which were then
blocked with5% nonfat milk in TBST [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L
NaCl, and 0.1% Tween 20] for 1 h, incubated with various primary antibodies
overnight at 4 °C, and incubated with secondary antibodies for 1 h. Immunoreactive
products were detected by using chemiluminescence with an enhanced
chemiluminescence system (ECL, Amersham Biosciences). The dilution of the
primary antibody was anti-Nur77 (Cell Signal, 3960) in 1:1000. The blots were
reprobed with anti-β-actin antibody (Sigma, A3854) in 1:10000 for loading control.
4
.2.4. Immunofluorescence microscopy
Cells mounted on glass slides were permeabilized with PBS containing 0.1% Triton

ACCEPTED MANUSCRIPT
X-100 for 15 min, and blocked with 1% bovine serum albumin (BSA) in PBS for 30
min at room temperature, followed with incubation with Nur77 antibodies at 37°C for
1
h and detected by Cy3-labeled anti-rabbit IgG (Chemicon International) at room
temperature for 30 min. Cells were co-stained with 4',6'-diamidino-2-phenylindole
DAPI) (Sigma) to visualize nuclei. The images were taken under an LSM-510
confocal laser scanning microscope system (Carl Zeiss, Oberkochen, Germany).
(
4
.2.5 Flow cytometry assay
Apoptosis was determined by dual staining using Annexin V: FITC and propidium
5
iodide (Invitrogen). Briefly, cells were seeded into 24-well cell culture plates (1×10
cells/well) and treated with glycosides. Cells were dissociated from wells with 0.25%
trypsin, spun at 1,500 rpm for 5 min, resuspended in Annexin V binding buffer, and
stained with Annexin V: FITC for 15 min and propidium iodide for 5 min. Cells were
analyzed using the FACS Calibur System (BD Biosciences, San Jose, CA, USA). The
relative proportion of Annexin V-positive cells, representing apoptotic cells, was
determined using FlowJo software (FlowJo LLC, Ashland, OR, USA).
Acknowledgement
We are grateful to Ms. L. Wang, Ms. X.Y. Shen, and Dr. Y. Yu for the HR ESI-MS and
NMR measurements. This work was supported by National Natural Science
Foundation of China (No 81673320, 81728022, 81502406), and the National 111
Project of China (No. B13038).
References.
1
. T. Hashimoto, H. Rathore, D. Satoh, G.Hang, J. F. Griffin, A. H. L. From,K.
Ahmed,D. S. Fullerton, Cardiac glycosides. 6. Ditoxigenin C16 acetates,
+
+
formates, methoxycarbonates and digitoxosides. Synthesis and Na ,K -ATPase
inhibitory activities, J. Med. Chem.29 (1986) 997–1003.
2
. Cornelius F, Kanai R, et al. F. Cornelius, R. Kanai, C. Toyoshima, A structural
view on the functional importance of the sugar moiety and steroid hydroxyls of
cardiotonic steroids in binding to Na,K-ATPase, J. Biol. Chem. 288 (2013)
6602–6616.