Multidrug resistance-reversal effects of resin glycosides from Dichondra repens

Article abstract of DOI:10.1016/j.bmcl.2014.12.083

Investigation of hydrophobic extract of Dichondra repens (Convolvulaceae) led to the isolation of three new resin glycosides dichondrins A-C (1-3), and three known resin glycosides cus-1, cus-2, and cuse 3. All the isolated resin glycosides with an acyclic core were evaluated for their multidrug resistance reversal activities, and the combined use of these compounds at a concentration of 25 μM increased the cytotoxicity of vincristine by 1.03-1.78-fold.

Full text of DOI:10.1016/j.bmcl.2014.12.083

Bioorganic & Medicinal Chemistry Letters 25 (2015) 795–798
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multidrug resistance–reversal effects of resin glycosides from
Dichondra repens
⇑
Wei-bin Song , Wen-qiong Wang , Shu-wei Zhang, Li-jiang Xuan
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203,
People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Investigation of hydrophobic extract of Dichondra repens (Convolvulaceae) led to the isolation of three
new resin glycosides dichondrins A–C (1–3), and three known resin glycosides cus-1, cus-2, and cuse
3. All the isolated resin glycosides with an acyclic core were evaluated for their multidrug resistance
Received 24 October 2014
Revised 18 December 2014
Accepted 26 December 2014
Available online 3 January 2015
reversal activities, and the combined use of these compounds at a concentration of 25
the cytotoxicity of vincristine by 1.03–1.78-fold.
lM increased
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
Keywords:
(http://creativecommons.org/licenses/by/4.0/).
Dichondra repens
Resin glycoside acids
Multidrug-resistance (MDR) reversal
activity
Resin glycosides, primarily found in the family Convolvulaceae,
are unusual amphipathic metabolites with structures including
hydrophobic (fatty acid aglycone) and hydrophilic (oligosaccha-
ride) moieties.1 To date, hundreds of resin glycosides have been
isolated from different genera of the family Convolvulaceae, and
these resin glycosides exhibit various pharmacological activities,
such as cytotoxicity,2 multidrug resistance (MDR) reversal,3 iono-
phoretic,4 and phytogrowth-inhibitory activities,5 as well as effects
on the central nervous system.6 It is worthy of note that the resin
glycosides have gained increasing attention as a novel P-glycopro-
tein inhibitor, which is attributed to their intriguing structures and
abundant natural resources. Therefore, the discovery of various
structural resin glycosides from the family Convolvulaceae and
the investigation of their MDR reversal activities arouse our
interest.
Dichondra repens, grown alone or in association with turfgrass
in subtropical and Mediterranean regions, is a perennial plant with
persistent leaves, and has traditionally been used for the treatment
of jaundice and dysentery in China.7 However, as a member of
family Convolvulaceae, the resin glycosides of D. repens are rarely
reported. Therefore, we initiated the isolation and MDR reversal
activities studies of resin glycosides from D. repens.
were isolated from acetone–H2O (4:1, v/v) extract of D. repens.
Their chemical structures were determined by extensive applica-
tion of high resolution 2D NMR techniques, HRESIMS and chemical
methods. Furthermore, all isolated compounds showed MDR
reversal activity at 25 lM when combined with vincristine. The
structures of the three known compounds were elucidated based
on the comparison of their HRESIMS, 1H, and 13C NMR data with
the reported data.
Dichondrin A (1) was obtained as white amorphous powder.
The molecular formula was determined as C50H88O25 based on
the 13C NMR data (Table 1) and the HRESIMS ion at m/z
1111.5531 [M+Na]+ (calcd 1111.5507). The 1H NMR spectrum of
1
showed seven methyl groups, four anomeric protons and
long-chain fatty acid signals. The 13C NMR and DEPT spectra of 1
showed 50 carbons including four anomeric carbons and three
carbonyl carbons.
The NMR data of 1 could be divided into two parts: resonances
in the anomeric region and those representative of the aglycone
moieties. The 1H NMR spectrum of 1 showed two methine groups
(dH 2.80 and 2.76) due to H-2 of 3-hydroxy-2-methybutyryl (Nla)
moieties, two oxygenated methine groups (dH 4.30 and 4.28) due
to H-3 of Nla, two primary methyl groups (dH 1.36 and 1.35) due
to H-4 of Nla, and two methyl groups (dH 1.29 and 1.25) due to
2-Me of Nla in the aglyconic region, which suggested 1 contained
two 3-hydroxy-2-methybutyryl moieties. After alkaline hydrolysis,
In this Letter, three new resin glycosides dichondrins A–C (1–3)
(Fig. 1), and three known compounds cus-1, cus-2,8 and cuse 3,9
the 2R, 3R absolute configuration of Nla was determined by its spe-
ꢀ28).10 In addition, the 11-hydroxyhexadeca-
23
⇑
Corresponding author. Tel./fax: +86 21 20231968.
E-mail address: ljxuan@mail.shcnc.ac.cn (L.-j. Xuan).
These authors contributed equally to this work.
cific rotation ([
a]
D
noyl moiety (Ag) was suggested by the diagnostic signals of the
http://dx.doi.org/10.1016/j.bmcl.2014.12.083
0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

796
W.-b. Song et al. / Bioorg. Med. Chem. Lett. 25 (2015) 795–798
HO
HO
Rha
O
O
R
2O
Glc
O
O
O
Glc
OH
HO
HO
HO
O
HO
O
O
O
HO
OH
O
O
O
HO
OH
Rha'
O
Rha
Glc'
O
O
O
R1
OH
HO
O
HO
O
OH
O
O
OH
Rha'
OH
R
HO
O
OH
HOOC
3
R
H5 R2=H
R2=H
1=C2
R1=H
R1=H
Cuse 3
Cus-1
Cus-2
1
2
R=H
R=OH
R
2=OAc
H5 R
2=OAc
R
1=C2
Figure 1. Chemical structures of compounds 1–3.
Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1–3 (in ppm)
Position
1a
2a
3b
a-Anomer
b-Anomer
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
Glc-1
2
3
4
4.90 d (7.7)
4.11 m
4.27 m
3.90 dd (8.5, 8.5)
3.97 m
3.99 m, 4.46 m
5.24 d (7.8)
4.08 m
4.14 d (3.8)
4.40 m
3.72 m
4.12 m, 4.30 m
5.32 br s
5.84 dd (3.4, 1.4)
4.64 m
4.54 dd (9.2, 9.2)
4.34 m
1.50 d (6.2)
5.82 br s
4.66 m
5.66 t (9.8)
4.32 m
102.1
83.6
78.1
71.6
76.3
68.8
105.9
77.2
76.4
78.5
77.6
61.7
99.3
73.3
72.7
72.9
68.5
18.3
102.9
72.7
75.2
74.1
70.5
18.7
176.8
35.2
5.24 d (7.9)
4.12 m
4.29 m
3.92 m
4.00 m
4.01 m, 4.55 m
5.24 d (7.9)
4.04 m
4.16 m
4.44 dd (8.7,8.7)
3.73 m
4.32 m, 4.16 m
5.33 br s
5.86 m
4.66 m
4.57 m
102.1
84.1
78.2
71.7
76.4
68.9
106.3
77.5
76.5
78.5
77.9
68.9
99.5
73.4
73.1
70.7
67.5
18.5
103.0
72.9
75.4
74.3
70.7
18.9
177.0
44.4
68.7
80.3
14.7
175.7
49.5
69.8
21.3
13.9
175.0
49.4
69.7
21.1
13.7
5.19 d (3.6)
3.39 dd (9.7,3.5)
3.79 m
3.75 m
3.81 m
93.2
82.1
73.7
72.2
73.5
62.7
4.57 d (7.7)
3.28 m
3.81 m
3.75 m
3.27 m
96.8
80.5
78.9
72.2
77.9
62.8
5
6
3.68 m, 3.81 m
3.60 m, 3.77 m
Glc0-1
2
3
4
5
6
Rha-1
2
3
4
4.94 d (1.4)
5.23 m
4.20 dd (9.9, 3.3)
5.03 dd (9.9, 9.9)
4.06 dd (9.9, 6.3)
1.16 d (6.3)
4.83 d (1.3)
3.70 m
3.51 dd (9.4, 3.5)
3.32 dd (9.4, 9.4)
3.57 dd (9.4, 6.3)
1.18 d (6.3)
100.8
73.0
76.5
74.0
68.3
17.9
104.0
72.1
72.3
72.8
70.6
17.9
174.5
35.2
5.24 br s
5.26 m
4.11 dd (9.9, 3.3)
5.03 dd (9.9, 9.9)
4.32 dd (9.9, 6.3)
1.16 d (6.3)
4.79 (1.3)
3.70 m
3.51 dd (9.4, 3.5)
3.32 dd (9.4, 9.4)
3.57 dd (9.4, 6.3)
1.18 d (6.3)
99.0
73.2
76.1
74.0
67.9
17.8
104.0
72.1
72.3
72.8
70.6
17.8
174.5
35.2
5
6
4.35 m
1.52 d (6.2)
5.86 br s
4.68 m
5.68 d (9.8)
4.34 br s
4.95 m
Rha0-1
2
3
4
5
6
Ag-1
2
3
11
16
Nla-1
2
4.92 m
1.66 d (6.2)
1.69 d (6.0)
2.47 m
2.51 m, 2.87 m
4.53 m
3.91 m
2.40 m
2.40 m
3.92 m
0.90 t (7.0)
80.3
14.6
175.6
49.3
69.6
21.1
13.8
174.9
49.1
69.6
20.9
13.7
3.68 m
0.91 t (7.2)
71.9
14.2
175.2
49.2
70.3
20.8
14.1
3.68 m
0.91 t (7.2)
71.9
14.2
175.2
49.2
70.3
20.8
14.1
0.92 t (6.8)
2.80 m
4.34 m
1.34 d (6.2)
1.22d (7.0)
2.80
4.27
1.34 d (6.3)
1.23 d (6.9)
2.45 m
3.86 m
1.19 d (6.2)
1.12 d (7.2)
2.45 m
3.86 m
1.19 d (6.2)
1.12 d (7.2)
3
4
5
Nla0-1
2
3
4
5
2.76 m
4.33 m
1.32 d (6.3)
1.19 d (7.0)
2.77
4.27
1.33 d (6.3)
1.20 d (7.0)
a
NMR data recorded in pyridine.
NMR data recorded in MeOH.
b
methyl triplet (dH 0.90), methylene group (dH 2.47), and oxygen-
ated methine (dH 3.92). After acid hydrolysis, the resulting 11-
hydroxyhexadecanoic acid showed a fragment ion at m/z 201
[MꢀCH3(CH2)4]+ by the EIMS data, suggesting the 11-OH group
of Ag. The S configuration of 11-hydroxyhexadecanoic acid was
determined by Mosher’s method as we reported before.11
In the anomeric region, the 1H–1H COSY showed four spin sys-
tems, which were attributed to two hexose and two 6-deoxyhexose

W.-b. Song et al. / Bioorg. Med. Chem. Lett. 25 (2015) 795–798
797
Rha
HO
HO
HO
HO
Rha
O
O
O
Glc
O
Glc
O
O
Nla
HO
HO
Nla
O
HO
HO
O
O
O
O
HO
O
O
HO
O
O
O
OH
O
OH
O
HO
OH
HO
OH
Glc'
Glc'
O
O
Rha'
Rha'
OH
HO
HO
O
O
OH
Nla'
HO
O
O
Nla'
1
2
OH
OH
HOOC
HO
HOOC
Glc
O
HO
HO
OH
O
O
Rha
O
O
O
OH
HO
O
HO
O
O
OH
Rha'
OH
3
Figure 2. Key 1H–1H COSY (bold) and HMBC (arrow) of compounds 1–3.
units (Fig. 2). The sugars obtained from the acid hydrolysates were
further identified as -rhamnose and -glucose by the HPLC analysis
and their corresponding optical rotations. The b-configuration of
-glucose was suggested by the coupling constants of the anomeric
protons at dH 4.90 (d, J = 7.7 Hz, H-1 of b-Glc) and dH 5.24 (d,
J = 7.8 Hz, H-1 of b-Glc0) in the 1H NMR spectrum. The
-configura-
tion of -rhamnose was suggested by the chemical shift of C-5 of
hydroxy-2-methybutyryl)-
glucopyranose.
a-L-rhamnopyranosyl-(1?6)]-O-b-D-
L
D
Dichondrin C (3) was obtained as white amorphous powder,
and its molecular formula was determined as C39H70O18 based on
the HRESIMS peak at m/z 849.4472 ([M+Na]+ (calcd 849.4454)
and 13C NMR data (Table 1). The comparison of the 1H and 13C
NMR data of 3 and cus-2, indicated that 3 lacked an acetyl at
OH-6 of Glc. Furthermore, the anomeric protons at dH 5.19 (d,
D
a
L
rhamnose (dC 68.5, 70.5) in the 13C NMR spectrum.12 Moreover,
the long-range correlations from dH 4.90 (H-1 of b-Glc) to dC 80.3
(C-11 of Ag) indicated that b-Glc was the first hexose unit in
the sugar moiety. Besides, the sequence of the sugar moiety was
determined to be rhamnosyl-(1?4)-glucosyl-(1?2)-[rhamnosyl-
(1?6)]-glucosyl by their long-range HMBC correlations: H-1 of
J = 3.5 Hz, H-1 of
1H NMR spectrum suggested that 3 existed as an equilibrium
mixture of -and b-anomers, and the ratio ( :b) was identified as
8:5 by the integration of anomeric protons. Thus, the structure of
was defined as -rhamnopyranosyl-(1?3)-[1-O-(11S)-11-
hydroxyhexadecanoyl]-[4-O-(2R,3R)-3-hydroxy-2-methybutyryl]-
-rhamnopyranosyl-(1?2)-O- -glucopyranose.
a-Glc), 4.57 (d, J = 7.7 Hz, H-1 of b-Glc) in the
a
a
3
a-L
b-Glc0 (dH 5.24) with C-2 of b-Glc (dC 83.6), H-1 of
with C-4 of b-Glc0 (dC 78.5), H-1 of
-Rha (dH 5.32) with C-6 of
b-Glc (dC 68.8). In addition, the linkages between Nla and sugar moi-
ety, that is, Nla located at OH-2 of
-Rha, Nla0 to H-3 of -Rha0, were
indicated by the HMBC correlations: dH 5.84 (H-2 of -Rha) with dC
175.6 (C-1 of Nla), dH 5.66 (H-3 of
-Rha0) with dC 174.9 (C-1 of Nla0).
a
-Rha0 (dH 5.82)
a
a
-
L
D
We then started the research on the MDR reversal activity of the
a
a
isolated resin glycosides in KB/VCR cells by the SRB method,14 and
the results were presented in Table 2. All the isolated compounds
a
a
Thus, the structure of compound 1 was defined as (11S)-dihydroxy-
Table 2
hexadecanoic acid 11-O-(3-O-(2R,3R)-3-hydroxy-2-methybutyryl)-
Results of modulating MDR activities in KB/VCR cells of compounds 1–3, cus-1, cus-2
a
-
L
-rhamnopyranosyl-(1?4)-O-b-
3R)-3-hydroxy-2-methybutyryl)-
b- -glucopyranose.
Dichondrin B (2) possessed the molecular formula C50H88O26
D
-glucopyranose-(1?2)-[(2-O-(2R,
and cuse 3a
a
-L
-rhamnopyranosyl-(1?6)]-O-
Sample
Inhibition ratio% (25
lM)
Vincristine+sampleb
D
IC50 value (lM)
RFc value
based on the 13C NMR data (Table 1) and the HRESIMS ion at m/z
1127.5464 [M+Na]+ (calcd 1127.5460), which showed one more
oxygen atoms than 1. Furthermore, 2 contained the same complex
sugar moiety and Nla groups as 1 according to their 1H and 13C
spectra. However, the H2/H3 correlation in the 1H–1H COSY
revealed long chain fatty acids in 2 had an additional 3-OH. The
3S, 11S configuration of 3,11-hydroxyhexadecanoic acid was
1
2
3
2.52
0.83
1.35
1.64
0.85
ꢀ0.47
0.45
—
0.48
0.43
0.32
0.54
0.42
0.36
0.02
0.57
1.19
1.32
1.78
1.03
1.36
1.58
28.5
—
Cus-1
Cus-2
Cuse 3
Verapamild
Vincristine
determined by the chemical shift difference (
ꢀ0.01,
d2Ha = +0.02) according to the Mosher’s method.13 Thus,
the structure of 2 was defined as (3S,11S)-dihydroxyhexadecanoic
acid 11-O-(3-O-(2R,3R)-3-hydroxy-2-methybutyryl)- -rhamno-
pyranosyl-(1?4)-O-b- -glucopyranose-(1?2)-[(2-O-(2R,3R)-3-
D
d = dS ꢀ dR,
Dd16H =
a
MDR: multidrug resistance.
D
b
Serial dilutions ranging from 0.125 to 1
M sample.
RF: IC50 of VCR alone/IC50 of VCR in the presence of sample.
lM of vincristine in the presence or
absence of 25
l
c
a-L
d
The concentration of the positive control verapamil is 10 lM.
D

798
W.-b. Song et al. / Bioorg. Med. Chem. Lett. 25 (2015) 795–798
enhanced the cytotoxicity of vincristine by 1.03–1.78-fold when
incorporated at the concentration of 25 M, while the cytotoxicity
assay showed the compounds 1, 2, 3, cus-1, cus-2, and cuse 3 were
not toxic at 25 M as their inhibition ratios were less than 50%. In
data for isolates, and procedures of MDR-reversal bioassay) associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2014.12.083.
l
l
addition, the deacylated resin glycosides (cuse 3 and 3) were more
active than the acylated resin glycosides (cus-1 and cus-2), which
demonstrated that the minor variations in the acylation pattern of
oligosaccharide core could affect their MDR reversal activities.
In conclusion, we have made a phytochemical investigation on
the crude extract of Dichondra repens resulting in six resin glyco-
sides with an acyclic core, the three new resin glycosides dichond-
rins A–C (1–3), and three known resin glycosides cus-1, cus-2, and
cuse 3. Moreover, the MDR reversal studies of these compounds
showed that they could increase the cytotoxicity of vincristine by
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Supplementary data
Supplementary data (general experimental procedures, plant
material, extraction and isolation, physical properties and NMR