Pharesinosides A-G, acylated glycosidic acid methyl esters derivatized by NH2 silica gel on-column catalyzation from the crude resin glycosides of Pharbitis Semen

Article abstract of DOI:10.1016/j.tet.2017.03.059

Application of NH₂ silica gel column chromatography using CH₂Cl₂-MeOH was found to show a satisfactory resolution for separation of the crude resin glycosides of Pharbitis Semen (the seeds of Pharbitis nil), led to the isolation of seven new acylated glycosidic acid methyl esters, Pharesinosides A-G (1–7), along with four known ones (8–11). These compounds (1–11) were considered to be generated via methyl esterification of the carboxyl group in acylated glycosidic acids. Their structures including stereochemistry were elucidated on the basis of a combination of the spectroscopic and chemical methods. All isolates were evaluated for anti-tumor migration activities with human colon cancer cell line HCT-116, and compound 7 exhibited a potent migration inhibitory activity.

Full text of DOI:10.1016/j.tet.2017.03.059

Accepted Manuscript
Pharesinosides A-G, acylated glycosidic acid methyl esters derivatized by NH silica
2
gel on-column catalyzation from the crude resin glycosides of Pharbitis Semen
Li-Juan Bai, Jian-Guang Luo, Chen Chen, Ling-Yi Kong
PII:
S0040-4020(17)30308-3
10.1016/j.tet.2017.03.059
TET 28565
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 19 January 2017
Revised Date: 18 March 2017
Accepted Date: 21 March 2017
Please cite this article as: Bai L-J, Luo J-G, Chen C, Kong L-Y, Pharesinosides A-G, acylated glycosidic
acid methyl esters derivatized by NH silica gel on-column catalyzation from the crude resin glycosides
2
of Pharbitis Semen, Tetrahedron (2017), doi: 10.1016/j.tet.2017.03.059.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Leave this area blank for abstract info.
Pharesinosides A-G, acylated glycosidic acid methyl
esters derivatized by NH2 silica gel on-column
catalyzation from the crude resin glycosides of
Pharbitis Semen
Li-Juan Bai, Jian-Guang Luo*, Chen Chen and Ling-Yi Kong*
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Pharesinosides A-G, acylated glycosidic acid methyl esters derivatized by NH2 silica
gel on-column catalyzation from the crude resin glycosides of Pharbitis Semen
Li-Juan Bai, Jian-Guang Luo , Chen Chen and Ling-Yi Kong
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China
ARTICLE INFO
ABSTRACT
Application of NH2 silica gel column chromatography using CH2Cl2-MeOH was found to show
a satisfactory resolution for separation of the crude resin glycosides of Pharbitis Semen (the
seeds of Pharbitis nil), led to the isolation of seven new acylated glycosidic acid methyl esters,
Pharesinosides A-G (1-7), along with four known ones (8-11). These compounds (1-11) were
considered to be generated via methyl esterification of the carboxyl group in acylated glycosidic
acids. Their structures including stereochemistry were elucidated on the basis of a combination
of the spectroscopic and chemical methods. All isolates were evaluated for anti-tumor migration
activities with human colon cancer cell line HCT-116, and compound 7 exhibited a potent
migration inhibitory activity.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Pharbitis nil
Convolvulaceae
Resin glycosides
Anti-tumor migration activity
2009 Elsevier Ltd. All rights reserved
.
Corresponding author. Tel/fax: +86-25-8327-1405; luojg@cpu.edu.cn (Jian-Guang Luo)
Corresponding author. Tel/fax: +86-25-8327-1405; cpu_lykong@126.com (Ling-Yi Kong)

2
Tetrahedron
ACCEPTED MANUSCRIPT
1. Introduction
by several methods (column chromatographies on silica gel,
reversed-phase C18 (RP-C18), MCI gel CHP 20P, Sephadex LH-
20 and HPLC (Fig. 1A-i)) was not achieved. Inspired by the
successful example,20 the next fractionation of MeOH-soluble
fraction (Fr. C) via a NH2 silica gel column chromatography
(CC) with CH2Cl2-MeOH resulted in three subfractions (Ca, Cb
and Cc). Subfraction Cb was rich in resin glycosides detected by
1H NMR spectrum, in which a remarkable methoxyl signal was
arisen to suggest the methyl esterification of glycosidic acids (Fig.
1B-ii). Simultaneously, this subfraction was found to show a
satisfactory resolution in the HPLC chromatogram (ELSD
detector) (Fig. 1A-ii). Therefore, the subfraction Cb was further
subjected to RP-C18 and ultimately purified by preparative HPLC
to yield pure compounds 1-11 (Fig. 2).
Resin glycosides, primarily isolated from the morning glory
family (Convolvulaceae), are a class of unusual amphipathic
secondary metabolites composed of hydrophobic (fatty acid
aglycone) and hydrophilic (oligosaccharide) moieties.1 The
chemical structures are so complex and diverse that they
exhibited various pharmacological activities, such as cytotoxic,2, 3
multidrug resistance (MDR) reversal4-6 and antiviral7, 8 activities.
Pharbitis Semen, the seeds of Pharbitis nil, is used as a purgative
drug in Korea, China, and Japan.9 Resin glycosides were reported
to be responsible for its purgative property as traditional Chinese
medicine.9 Previous investigations on its crude resin glycosides
have been confined to characterization of the glycosides acids
and organic acids as alkaline hydrolysis products of the mixture
for separation difficulties.10-12 Recently, intact structures were
disclosed by derivatization using indium (III) chloride in
methanol. Seven oligoglycosides of hydroxyl fatty acid methyl
esters were reported, involving 2-methyl-3-hydroxybutyric acid
(nilic acid, Nla) and 2-methylbutyric acid (Mba) as esterifying
moieties of the oligosaccharide core.13 The existence of a free β-
hydroxyl carboxylic acid moiety maybe resulted in the poor
resolution of this type of acylated glycoside acids.13-15 As part of
our continuing endeavor to study on the novel active resin
glycosides from plants in the Convolvulaceae,16-19 we directed
toward CH2Cl2 emulsion layer of methanol extract of the seeds of
P. nil. A satisfactory resolution of resin glycosides was achieved
on NH2 silica gel using CH2Cl2-MeOH, finally resulting in the
isolation of seven new acylated glycosidic acid methyl esters,
Pharesinosides A-G (1-7), along with four known ones (8-11).
Herein, we described their isolation and structure elucidation, as
well as anti-tumor migration activity.
2.1. Structure elucidation
Compound 1 was obtained as colorless gum. Its molecular
formula was deduced as C55H96O30 by HRESIMS data at m/z
1259.5869 [M + Na]+ (calcd for C55H96NaO30, 1259.5879). The
1H NMR spectrum showed signals assigned to two nilyl units,
one methoxyl group, two nonequivalent methylene protons
adjacent to a carbonyl group and three methyl groups assignable
to 6-deoxyhexosyl units (Table 1). Five anomeric signals [δH 4.88
(1H, d, J = 7.6 Hz), 5.86 (1H, d, J = 6.9 Hz), 5.28 (1H, d, J = 6.8
Hz), 5.35 (1H, s), 6.33 (1H, s); δC 103.1, 102.5, 106.1.102.8,
102.2] in the NMR spectra (Tables 1 and 4) indicated that
compound 1 was a pentasaccharide. The 1H and 13C NMR signals
of the sugar moiety in 1 were assigned by comprehensive
analysis of HSQC, HMBC and TOCSY spectra. Alkaline
hydrolysis of 1 furnished nilic acid (15) and a glycosidic acid.
The glycosidic acid was characterized as pharbitic acid C (12,
1
Fig. 3) by comparison of H NMR and MS spectra with those of
an authentic sample,12 which was obtained from the alkaline
hydrolysis products of the resin glycoside mixture (Fr. Cb). The
connectivities of pharbitic acid C were further confirmed from
the HMBC correlations: H-1 of Glc (δH 4.88) to C-11 (δC 81.5),
H-1 of Qui (δH 5.28) to C-4 of Rha′ (δC 84.5), H-1 of Rha′ (δH
6.33) to C-2 of Glc′ (δC 79.0), H-1 of Glc′ (δH 5.86) to C-2 of Glc
(δC 80.3), H-1 of Rha (δH 5.35) to C-6 of Glc (δC 68.8). The nilic
acid was converted into p-bromophenacyl ester (17, Fig. 3),
which was purified by HPLC and the (-)-α-methoxy-α-
trifluoromethylphenylacetic acid (MTPA) ester of 17 was further
obtained. On the other hand, after basic hydrolysis of Fr. Cb, the
corresponding products (19) using the Mosherʹs method were
gained, which proved to be a pair of (-)-MTPA esters of p-
bromophenacyl nilate (19a:19b, approximate 4:1). Compared
normal-phase HPLC analysis of (-)-MTPA ester of 17 with 19
(19a and 19b), one peak in (-)-MTPA ester of 17 with the same
retention time as 19a confirmed the absolute configuration of 17
and nilic acid in the structure 1 was 2R, 3R (S44, Supplementary
Data). Specification of ester linkage sites was established by the
key HMBC correlations between protons of sugars and acyl
carbons of the fatty acids, i.e. H-3 of Rha (δH 5.79) with C-1 of
fragment A (δC 174.2). Finally, the location of methoxyl group
was determined by the cross peak between the signal of δH 3.66
(OCH3) with δC 173.4 (C-1) (Fig. 4). Thus, the structure of
compound 1 was identified as (3S, 11S)-dihydroxytetradecanoic
acid methyl ester 11-O-β-
mnopyranosyl-(1→2)-O-β-
-O-(3R-hydroxy-2R-methylbutyryl)-hydroxy-2R-methylbutyryl]-
α- -rhamnopyranosyl-(1→6)}-O-β- -glucopyranoside, named
Pharesinoside A.
D
-quinovopyranosyl-(1→4)-O-α-
-glucopyranosyl-(1→2)-O-{[3-O-3R
L-rha-
Fig. 1. HPLC (RP-C18)-ELSD (A) and 1H NMR (pyridine-d5) (B)
analysis of MeOH-soluble fraction (Fr. C) (i) and subfraction Cb
(ii) from the seeds of P. nil.
D
L
D
2. Results and discussion
Compound 2, colorless gum, the molecular formula was
determined as C56H98O32 based on the HRESIMS ion peak at m/z
1305.5922 [M + Na]+ (calcd for C56H98NaO32, 1305.5933). One
Attempted separation of MeOH-soluble fraction of CH2Cl2
emulsion layer from the methanolic extract of the seeds of P. nil

3
ACCEPTED MANUSCRIPT
Fig. 2. Structures of compounds 1-11
nilyl unit, six anomeric protons δH 4.91 (d, J = 7.7Hz), 5.88 (d, J
= 6.3Hz), 5.43 s, 6.31 s, 6.36 s, 5.16 (d, J = 7.8Hz) and their
corresponding carbon signals δC 103.1, 102.4, 102.8, 102.3,
101.6, 105.7 were observed in the 1H and 13C NMR spectra
(Tables 1 and 4), indicating that 2 was a hexasaccharide and
acylated by only one nilic acid. Basic hydrolysis of 2 afforded
nilic acid and a glycosidic acid. The former was converted into
the p-bromophenacyl ester and then purified by HPLC to give p-
bromophenacyl nilate (17′). The comparison between the (-)-
MTPA ester of 17′ and 19 in the normal-phase HPLC analysis
indicated that (-)-MTPA ester of 17′ was same as 19a and the
absolute configuration of 17′ was 2R, 3R. Thus, nilic acid in the
structure 2 was existed as 2R, 3R-form. The latter was identified
)]-O-β-D-glucopyranoside, named Pharesinoside B.
Compound 3, a white, amorphous powder, gave a quasi-
molecular ion at 1505.6970 [M + Na]+ (calcd for C66H114NaO36,
1505.6982) and the molecular formula was determined to be
C66H114O36. The 1H NMR spectrum exhibited six methyl doublets
between δH 1.28 and δH 1.43, three methine signals at δH 2.88-
3.21, confirming the existence of three nilyl units in the structure
(Table 1). The NMR spectra of 3 were similar to those of the
known compound 10 except for the absence of a Mba unit. The
alkaline hydrolysis product nilic acid was treated with p-
bromophenacyl bromide and separated by pre-HPLC to give p-
bromophenacyl nilate (17′′). 17′′ was further converted into (-)-
MTPA ester and the analysis carried out in the normal-phase
HPLC suggested that it was a (-)-MTPA ester mixture of (2R,
3R) and (2S, 3S) p-bromophenacyl nilate at the ratio of
approximately 2:1, indicating that 17′′ was a 2:1 mixture of p-
bromophenacyl-(2R, 3R) nilate and its enantiomer and the
structure 3 contained two 2R, 3R-nilic acid units and a 2S, 3S-
nilic acid unit. These results were consistent with those of 10.
The esterification positions of the oligosaccharide core were
elucidated via HMBC long-range couplings of δH 5.83 (H-4,
1
as pharbitic acid D (13) by comparison of the H NMR and MS
spectra with those of an authentic sample.12 The HMBC
correlation from H-4 of Qui (δH 5.22) to C-1 of 2R, 3R-nilic acid
(δC 175.4) suggested that nilic acid was connected to OH-4 of
Qui. Therefore, the structure of compound 2 was determined as
(3S, 11S)-dihydroxytetradecanoic acid methyl ester 11-O-α-
rhamnopyranosyl-(1→3)-O-[4-O-(3R-hydroxyl-2R-methylbutyr-
yl)]-β- -quinovopyranosyl-(1→4)-O-α- -rhamnopyranosyl-(1→
2)-O-β- -glucopyranosyl-(1→2)-O-[α- -rhamnopyranosyl-(1→6
L-
D
L
D
L
Rha)
with
δC
176.0
(C-1,
Fig. 3. Structures of compounds 12-19

4
Tetrahedron
ACCEPTED MANUSCRIPT
Table 1. 1H NMR Data of Compounds 1-3 (500 MHz, in pyridine-d5)a
Positionb
Positionb
1
2
3
1
2
3
Glc-1
4.88 d (7.6)
4.30 m*
4.91 d (7.7)
4.33 m*
4.50 m*
3.98 m*
4.01 m*
4.10 m*
4.56 m*
5.88 d (6.3)
4.23 m*
4.23 m*
4.10 dd (8.5)
3.88 m*
4.30 m*
4.45 m*
5.43 s
4.92 d (7.6)
4.32 m*
Qui-1
5.28 d (6.8)
4.02 m*
5.16 d (7.8)
4.04 dd (8.5)
4.44 m*
5.14*
2
2
3.95 m*
4.25 m*
5.14 dd (9.5)
3
4.52 dd (8.7)
3.92 dd (8.8)
4.01 m*
4.50 m*
3
4.11 m*
4
3.95 m*
4
3.70 dd (8.3)
3.66 m
5.22 dd (9.5)
5
3.97 m*
5
3.55 dq (12.3, 6.1) 3.51 dq (12.5, 6.1)
6a
4.08 m*
4.10 m*
6
1.57 d (6.0)
1.35 d (7.1)
1.28 d (7.1)
6b
4.47 m*
4.49 m*
Ag-1
Glc′-1
5.86 d (6.9)
4.26 m*
5.87 d (6.9)
4.24 m*
2
2.73 m*
4.45 m*
3.95 m*
0.98 t (7.0)
3.66 s
2.74 m*
4.45 m*
3.95 m*
0.99 t (7.0)
3.64 s
2.74 m*
4.50 m*
3.95 m*
1.00 t (7.1)
3.64 s
2
3
3
4.27 m*
4.24 m*
11
4
4.10 m*
4.10 m*
14
5
3.88 m*
3.86 m*
OCH3
6a
4.29 m*
4.28 m*
A-1
6b
4.45 m*
4.22 m*
2
3.01 dq (7.0)
5.62 dq (6.5)
1.35 d (6.5)
1.21 d (6.6)
3.21 dq (7.2)
5.59 dq (6.5)
1.42 d (6.0)
1.38 d (7.5)
Rha-1
5.35 s
5.42 s
3
2
4.72 br s
5.79 m
4.51 m*
4.51 m*
4.26 m*
4.35 m*
1.68 d (6.1)
6.31 s
4.46 m*
4
3
4.55 dd (9.8, 3.2)
5.83 dd (9.7)
4.35 m*
5
4
4.41 m*
A-1'
5
4.39 m*
2'
2.76*
2.96 dq (6.9)
4.42 m*
6
1.63 d (5.0)
6.33 s
1.50 d (6.3)
6.29 s
3'
4.31 m*
Rha′-1
4'
1.35 d (6.5)
1.20 d (7.0)
1.39 d (6.0)
1.28 d (7.1)
2
4.72 br s
4.86 dd (9.4, 2.6)
4.45 m*
4.70 br s
4.76 dd (9.3, 3.3)
4.39 m*
5.00 (12.5, 6.1)
1.86 d (6.3)
6.36 s
4.70 br s
4.76 dd (9.3, 3.5)
4.37 m*
5'
3
2R,3R-Nla-1
4
2
2.86 dq (7.5)
4.37 m*
5
5.05 dq (12.5, 6.2)
1.92 d (6.1)
5.04 m*
3
6
1.84 d (6.3)
6.13 s
4
1.36 d (6.9)
1.28 d (7.1)
Rha′′-1
5
2
3
4
5
4.61 m*
4.60 m*
4.19 m*
4.35 m*
4.73 br s
4.46 m*
2S,3S-Nla-1
2
3
4
2.88 dq (7.0)
4.33 m*
4.28 m*
4.35 m*
1.43 d (6.5)
6
1.75 d (6.3)
1.73 d (6.1)
5
1.33 d (7.1)
a Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). Chemical shifts marked with asterisk (*) indicate overlapped
signals.
b Abbreviations: Glc = glucose; Rha = rhamnose; Mba = 2S-methylbutanoyl; Nla = 3-hydroxy-2-methylbutyryl; Ag = aglycone.
2S, 3S-nilic acid), δH 5.14 (H-4, Qui) with δC 173.1 (C-1,
fragment A). Thus, the structure of 3 was elucidated as (3S, 11S)-
between protons of sugars and acyl carbons of the fatty acids.
The key HMBC correlations: H-2 of Rha (δH 5.76) to C-1 of
fragment A (δC 174.0), H-2 of Rha′ (δH 5.93) to C-1 of Mba (δC
176.6), respectively, indicated the ester sites were at OH-2 Rha
and OH-2 of Rha′. The absolute configuration of nilic acid was
determined as 2R, 3R according to the aforementioned method.
2-methylbutyric acid was confirmed as S by comparison of
optical rotation value of its p-bromophenacyl ester with p-
bromophenacyl 2S-methylbutyrate (18), obtained from the resin
glycoside mixture (Fr. Cb) and determined by 1H NMR spectrum
and optical rotation value comparison with the literature.21
Consequently, the structure of compound 4 was established as
(3S, 11S)-dihydroxytetradecanioc acid methyl ester 11-O-β-D-
quinovopyranosyl-(1→4)-O-[2-O-(2S-methylbutyryl)]-α-L-rham-
dihydroxytetradecanoic acid methyl ester 11-O-α-
nosyl-(1→3)-O-[4-O-3R-O-(3R-hydroxy-2R-methylbutyryl)-hyd-
roxy-2R-methylbutyryl]-β- -quinovopyranosyl-(1→4)-O-α- -rha
mnopyranosyl-(1→2)-O-β- -glucopyranosyl-(1→2)-O-[4-O-(3S-
hydroxy-2S-methylbutyryl)-α- -rhamnopyranosyl-(1→6)]-O-β-
-glucopyranoside, as Pharesinoside C.
L-rhamnopyra-
D
L
D
L
D
Compound 4 was isolated as colorless gum and gave the
molecular formula C60H104O31, as determined by HRESIMS m/z
1343.6440 [M + Na]+ (calcd for C60H104NaO31, 1343.6454). On
the basis of alkaline hydrolysis, 4 yielded 2-methylbutyric acid
(Mba, 16), nilic acid and pharbitic acid C (12),12 which were
similar to compound 1 apart from the appearance of 2-methyl
butyric acid. The difference also laid in the ester linkage sites
nopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-O-[2-O-3R-O-
(3R-hydroxy-2R-methylbutyryl)-hydroxy-2R-methylbutyryl]-α-

5
ACCEPTED MANUSCRIPT
Table 2. 1H NMR Data of Compounds 4-6 (500MHz, in pyridine-d5)a
Positionb
4
5
6
Positionb
4
5
6
Glc-1
4.90 d (7.6)
4.29 m*
4.88 d (7.7)
4.18 m*
4.90 d (7.0)
4.27 m*
4
3.70 m*
3.71 m*
1.61 d (5.5)
5.16 dd (9.5)
3.61 m
5.18 dd (9.5)
3.63 m
2
5
3
4.53 dd (8.7)
3.94 m*
4.58 m*
4.48 m*
6
1.30 d (7.0)
1.35 d (6.5)
4
3.94 m*
3.94 m*
Ag-1
5
3.94 m*
3.93 m*
3.99 m*
2
2.74 m
2.74 m
2.74*
6a
4.04 m*
4.14 m*
4.11 m*
3
4.43 m*
3.91 m*
1.03 t (7.1)
3.65 s
4.43 m*
3.96 m*
1.03 t (7.0)
3.64 s
3.36 m*
3.95 m*
1.01 t (7.1)
3.65 s
6b
4.44 m*
4.42 m*
4.45 m*
11
14
OCH3
A-1
2
Glc′-1
5.91 d (6.7)
4.22 m*
5.71 d (7.1)
4.25 m*
5.82 d (6.9)
4.21 m*
2
3
4.22 m*
4.22 m*
4.21 m*
4
4.07 m*
4.06 dd (8.8)
3.85 m*
4.08 dd (8.5)
3.81 m*
3.04 dq (6.9)
5.57 dq (6.5)
1.37 d (6.3)
1.28 d (7.1)
3.22 dq (7.3)
5.56 dq (6.5)
1.42 d (6.0)
1.38 d (6.5)
3.22 dq (7.0)
5.59 dq (6.5)
1.43 d (6.5)
1.39 d (6.5)
5
3.85 m*
3
6a
4.29 m*
4.28 m*
4.28 m*
4
6b
4.40 m*
4.45 m*
4.41 m*
5
Rha-1
5.24 s
5.45 s
5.39 s
A-1'
2'
2
5.76 br s
4.36 m*
4.62 br s
5.76 dd (9.1, 3.1)
4.39 m*
2.81 dq (7.0)
4.34 m*
2.94 dq (6.8)
4.45 m*
2.95 dq (7.0)
4.42 m*
3
4.64 dd (9.5, 3.6)
4.14 dd (9.4)
4.37 dd (9.5, 6.2)
1.69 d (6.3)
6.24 s
4.50 m*
3'
4
5.76 dd (9.8)
4.36 m*
4'
1.35 d (6.3)
1.25 d (7.1)
1.40 d (6.0)
1.30 d (7.0)
1.38 d (6.0)
1.28 d (6.9)
5
4.41 m*
5'
6
1.52 d (6.3)
6.33 s
1.68 d (5.8)
6.20 s
A'-1
2
Rha′-1
3.02 dq (7.1)
5.53 dq (6.5)
1.36 d (6.3)
1.30 (7.0)
3.02 dq (7.0)
5.60 dq (6.0)
1.34 d (6.5)
1.24 d (7.0)
2
5.93 br s
4.72 br s
6.23 dd (9.6, 2.9)
4.56 dd (9.5)
5.18 dd (9.5)
1.84 d (6.0)
6.02 s
5.93 br s
4.99 dd (9.5, 3.6)
4.28 m*
3
3
5.00 dd (9.4, 3.6)
4.32 m*
4
4
5
5
5.12 m
5.07 m
A'-1'
2'
6
1.96 d (6.1)
1.90 d (6.1)
6.06 s
2.78 dq (7.1)
4.39 m*
2.78*
Rha′′-1
3'
4.31 m*
2
4.74 br s
4.48 m*
4.71 br s
4.45 m*
4'
1.35 d (6.3)
1.27 d (6.9)
1.33 d (6.5)
1.23 d (7.0)
3
5'
4
4.21 m*
4.23 m*
Mba-1
2
5
4.34 m*
4.29 m*
2.51 ddq (7.0)
1.50 m*
2.54 ddq (7.1)
1.45 *
2.52 ddq (7.0)
1.53 m*
6
1.71 d (6.1)
4.94 m*
1.70 d (6.1)
5.30 d (7.9)
3.99 m*
3a
3b
4
Qui-1
5.34c
1.78 m*
1.87 *
1.81 m*
2
4.05 m*
3.88 m*
0.91 dd (7.3)
0.88 dd (7.4)
0.93 dd (7.4)
3
4.07 m*
4.18 m*
4.23 m*
5
1.15 d (6.9)
1.22 d (7.7)
1.17 d (6.9)
a Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). Chemical shifts marked with asterisk (*) indicate overlapped
signals.
b Abbreviations: Glc = glucose; Rha = rhamnose; Mba = 2S-methylbutanoyl; Nla = 3-hydroxy-2-methylbutyryl; Ag = aglycone.
c Signal is deformed by virtual coupling
L
-rhamnopyranosyl-(1→6)]-O-β-
D
-
glucopyranoside,
named
anomeric proton signals were placed at H-4 of Rha, H-3 of Rha′
in 5, while at H-3 of Rha and H-2 of Rha′ in 6. A further analysis
of HMBC led to assignments of the exact locations of the acyl
groups on the oligosaccharide skeletons: from H-4 of Rha (δH
5.76) to C-1 of fragment A′ (δC 174.0), from H-3 of Rha′ (δH
6.23) to C-1 of Mba (δC 177.2) and from H-4 of Qui (δH 5.16) to
C-1 of fragment A (δC 173.2) in 5 suggested that the ester
linkages were located at OH-4 of Rha, OH-3 of Rha′ and OH-4
of Qui, while at OH-3 of Rha, OH-2 of Rha′ and OH-4 of Qui in
compound 6. The absolute configurations of all nilic and 2-
Pharesinoside D.
Compounds 5 and 6 shared the same molecular formula
C76H130O39 based on the [M + Na]+ HRESIMS ions at m/z
1689.8066 and 1689.8073, respectively. The same alkaline
hydrolysis products, nilic acid, 2-methylbutyric acid and
pharbitic acid D (13)12 suggested that they were the positional
isomers. Comparison of the NMR data (Tables 2 and 4) of
compounds 5 and 6 showed the existence of four nilyl units, one
Mba unit and the major difference was that two downshift non-

6
Tetrahedron
ACCEPTED MANUSCRIPT
Table 3. 1H NMR Data of Compound 7 (500MHz, in pyridine-
d5)a
methylbutyric acids in 5 and 6 were determined to be 2R, 3R and
2S, respectively, using the foregoing mentioned method.
Therefore, the structures of compounds 5 and 6 were established
as depicted.
Positionb
7
Positionb
7
Glc-1
4.94 d (7.6)
4.32 m*
4.51 m*
3.94 m*
3.99 m*
4.12 m*
4.55 m*
5.87 d (7.1)
4.23 m*
4.22 m*
4.09 dd (8.5)
3.84 m*
4.29 m*
4.44 m*
5.46 s
5
4.35 m*
Compound 7, obtained as white amorphous powders, gave the
HRESIMS quasimolecular ion of [M + Na]+ at m/z 1517.7340
and the molecular formula was assigned as C68H118O35. The NMR
spectra of 7 showed close similarity with the known compound
PM-3,13 except for the esterification position of Mba unit at the
oligosaccharide core. The same basic hydrolysis products, nilic
acid, 2-methylbutyric acid and pharbitic acid B (14)12 suggested
that they were positional isomers like 5 and 6. A long-range
correlation between H-2 of Rha′ (δH 5.91) and C-1 of Mba unit
(δC 176.6) indicated that the exact position of Mba unit was at
OH-2 of Rha′ (Fig. 4). Thus the structure of 7 was defined as (3S,
2
6
1.70 d (6.1)
5.30 d (7.9)
4.00 m*
3
Qui-1
4
2
5
3
4.24 m*
6a
4
5.15 dd (9.5)
3.61 m
6b
5
Glc′-1
6
1.34 d (6.2)
2
Ag-1
2
11S)-dihydroxyhexadecanoic acid methyl ester 11-O-α-
opyranosyl-(1→3)-O-[4-O-3R-O-(3R-hydroxy-2R-methylbutyry-
l)-hydroxy-2R-methylbutyryl]-β- -quinovopyranosyl-(1→4)-O-
[2-O-(2S-methylbutyryl)]-α- -rhamnopyranosyl-(1→2)-O-β-
-rhamnopyranosyl-(1→6)]-O-β-
L-rhamn-
3
2.73 m
4
3
4.43 m*
3.96 m*
0.89 t (7.5)
3.65 s
D
5
11
16
OCH3
A-1
2
L
D-
6a
glucopyranosyl-(1→2)-O-[α-
L
D-
glucopyranoside.
6b
Four known compounds 8-11 were also isolated from the
crude resin glycosides of the seeds of P. nil. Their structures
Rha-1
1
were identified by comparison of H, 13C NMR and HRESIMS
2
4.49 m*
4.49 m*
4.23 m*
4.34 m*
1.67 d (6.1)
6.23 s
3.20 dq (7.0)
5.58 dq (6.5)
1.41 d (6.3)
1.37 d (6.2)
data with those of the literature.13
3
3
4
4
Although the isolation of a pure form could not be achieved,
we further illustrated the detailed structures of the crude resin
glycosides of Pharbitis Semen. These resin glycosides of
Pharbitis Semen are naturally existed to be a mixture of
monomers composed of free carboxylic acid forms, which
resulted in poor resolution of these compounds.13 Such acylated
glycosidic acids possess a polar free β-hydroxyl carboxylic acid
headgroup linked to a hydrophobic alkyl tail (alkyl chain and
acylated glycosidic core). As amphiphilic molecules, they are
capable of demonstrating liquid crystalline phase behavior and
forming micelle systems.22 Since the intermolecular hydrogen
bonds between headgroups (free β-hydroxyl carboxylic acid)
make the micelle be stable, the acylated glycosidic acids are
difficult to be separated in the aqueous solution.23 Therefore, for
HPLC analysis (RP-C18, MeOH-H2O or CH3CN-H2O), the
acylated glycosidic acid mixture exhibited an inseparable broad
peak with larger retention time (Fig. 1A-i), while the acylated
glycosidic acid methyl ester mixture (the polar free carboxylic
acid headgroups were destroyed) showed a satisfactory resolution
and smaller retention time (Fig. 1A-ii). Additionally, NH2 silica
gel may serve as a potential alkaline catalyst leading to methyl
5
5
6
A-1’
2’
Rha′-1
2.97 dq (6.6)
4.45 m*
2
5.91 br s
4.99 dd (9.2, 3.0)
4.30 m*
5.09 m
3’
3
4’
1.39 d (6.3 )
1.28 d (7.0)
4
5’
5
Mba-1
2
6
1.90 d (6.1)
6.05 s
2.52 ddq (7.0)
1.53 m*
Rha′′-1
3a
3b
4
2
3
4
4.73 br s
4.48 m*
4.22 m*
1.81 m*
0.91 dd (7.5)
5
1.16 d (7.0)
a Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is
given in parentheses (Hz). Chemical shifts marked with asterisk (*) indicate
overlapped signals.
b Abbreviations: Glc = glucose; Rha = rhamnose; Mba = 2S-methylbutanoyl;
Nla = 3-hydroxy-2-methylbutyryl; Ag = aglycone.
Fig. 4. Key HMBC correlations of compounds 1 and 7
Table 4. 13C NMR Data of Compounds 1-7 (125 MHz, in pyridine-d5)a

7
ACCEPTED MANUSCRIPT
Positionb
1
2
3
4
5
6
7
Positionb
1
2
3
4
5
6
7
Glc-1
103.1 103.1 103.1 103.2 103.2 103.1 103.2
CH2
35.4
31.1
30.7
30.7
26.7
25.7
19.6
35.5
31.0
30.7
30.6
26.7
25.8
19.5
35.5
31.0
30.7
30.6
26.7
25.8
19.5
35.5
31.0
30.7
30.6
26.7
25.7
19.5
35.4
31.1
30.7
30.6
26.7
25.4
19.1
35.4
31.1
30.7
30.6
26.7
25.7
19.5
35.3
32.8
31.0
30.7
30.6
26.7
25.9
25.8
23.4
173.2
44.8
71.4
17.3
13.2
175.2
48.6
69.2
20.5
12.7
2
80.3
79.6
72.6
76.7
68.8
80.0
79.8
72.3
76.7
68.5
80.2
79.7
72.2
76.6
68.7
80.0
79.7
72.0
76.3
68.7
80.4
79.1
71.8
76.5
68.6
80.1
79.7
72.3
76.7
68.8
80.2
79.7
72.3
76.9
68.6
CH2
3
CH2
4
CH2
5
CH2
6
CH2
Glc′-1
102.5 102.4 102.4 102.1 102.9 102.3 102.2
CH2
2
79.0
79.7
73.0
78.2
63.7
79.4
79.7
73.0
78.2
63.6
79.6
79.6
73.0
78.1
63.6
78.5
80.0
73.0
78.3
63.6
99.3
74.5
70.8
74.7
70.1
19.0
98.9
73.7
70.5
84.7
68.1
19.3
78.0
79.8
73.1
77.9
63.9
79.1
79.3
73.0
78.1
63.6
78.8
79.4
73.0
78.2
63.6
CH2
3
CH2
4
A-1
174.2
45.3
71.9
16.9
12.9
175.2
49.1
69.5
21.3
13.8
173.1 174.0 173.2 173.1
5
2
44.8
71.3
17.3
13.2
45.3
71.9
17.3
13.2
44.8
71.4
17.2
13.4
44.7
71.4
17.2
13.1
6
3
Rha-1
102.8 102.8 102.8
102.8 102.9 102.9
4
2
70.1
76.8
71.4
70.4
19.1
72.8
73.2
74.6
70.2
19.1
72.4
71.0
76.0
67.5
18.7
72.8
70.9
76.0
67.5
18.8
101.4
71.4
75.2
78.4
68.4
19.2
70.0
76.8
71.2
70.3
19.3
99.0
73.8
70.4
83.8
68.1
19.1
72.7
73.1
74.5
70.1
19.2
98.9
73.8
70.5
83.6
68.0
19.2
5
3
A-1′
175.2 175.2 175.2 175.3
4
2′
48.6
69.2
20.7
12.8
49.0
69.4
21.2
13.5
48.5
69.2
20.5
12.6
48.6
69.2
20.6
12.8
5
3′
6
4′
Rha′-1
102.2 102.3 102.4
5′
2
72.5
72.9
84.5
68.4
19.3
72.5
72.8
84.6
68.4
19.2
72.8
72.9
84.5
68.3
19.2
2R,3R-Nla-1
175.4
49.6
69.3
21.5
14.9
3
2
3
4
5
4
5
6
Rha′′-1
101.6 102.7
102.9 102.8 102.9 2S, 3S-Nla-1
176.0
49.0
70.2
70.2
13.8
2
72.6
72.5
74.9
69.9
19.2
72.5
72.8
74.4
70.5
19.3
73.0
72.8
74.5
70.4
19.4
72.8
72.8
74.4
70.4
19.2
72.9
72.7
74.4
70.4
19.3
2
3
3
4
4
5
6
5
A′-1
174.0 174.0
Qui-1
2
106.1 105.7 105.5 106.1 104.3 105.2 105.1
2
45.5
71.7
17.4
18.8
45.2
71.8
16.9
12.7
76.7
78.6
77.2
73.5
19.0
77.7
77.2
74.7
71.2
18.6
77.1
80.0
75.2
70.9
18.7
76.7
78.5
77.1
73.6
19.2
76.1
80.4
75.4
70.7
18.6
76.7
80.0
75.3
70.9
18.7
77.0
80.3
75.3
70.9
18.7
3
3
4
4
5
5
A′-1′
175.1 175.2
6
2′
49.0
69.3
21.2
13.6
49.0
69.5
21.3
13.8
Ag-1
2
173.4 173.4 173.4 173.4 173.4 173.4 173.4
3′
43.9
68.8
81.5
15.1
43.9
68.8
81.5
15.1
43.9
68.7
81.3
15.1
43.9
69.4
81.6
15.1
43.9
68.7
81.2
15.1
43.9
68.1
81.5
15.1
43.9
68.7
81.8
4′
3
5′
11
14
16
OCH3
CH2
Mba-1
176.6 177.2 176.6
176.6
41.8
27.5
12.1
17.2
2
3
4
5
41.8
27.5
12.1
17.2
42.2
27.1
12.3
17.1
41.8
27.5
12.1
17.3
14.8
51.7
38.6
35.9
51.8
38.6
38.2
51.8
38.6
38.2
51.7
38.6
38.2
51.7
38.6
38.1
51.7
38.6
38.1
51.7
38.6
38.1
CH2
a Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz).Chemical shifts marked with asterisk (*) indicate overlapped
signals.
b Abbreviations: Glc = glucose; Rha = rhamnose; Mba = 2S-methylbutanoyl; Nla = 3-hydroxy-2-methylbutyryl; Ag = aglycone

8
Tetrahedron
esterification of carboxylic acid groups by using CH2Cl2-
ACCEPTE
D
MANUSCRIPT
MeOH,24-27 which facilitated the isolation and structure
illustration of acylated glycosidic acids from Pharbitis Semen.
Interestingly, solid-supported NH2 materials have been used for a
environmentally friendly and reusable solid-base catalyst for
some condensation reactions in the anhydrous conditions due to
its high surface area and its functionalized pore channels of large
diameter.24,28-30 The present NH2 silica gel-catalyzed methyl
esterification of carboxylic acids is considered to be useful for
the isolation of carboxylic acid containing compounds from
complex mixtures.
The isolated compounds were evaluated for cytotoxic
activities against MDA-MB-231 human breast cancer cell lines
and HCT-116 human colon cancer cell lines. The results showed
that compounds were inactive against two cell lines (IC50>100
µM), except for 5, 7 with weak cytotoxic activities towards HCT-
116 cells (IC50 = 35.17 ± 1.56 and 27.53 ± 1.05 µM, respectively).
Oxaliplatin was used as the positive control with IC50 values of
8.65 ± 1.62µM and 3.95 ± 0.96 µM towards MDA-MB-231 and
HCT-116 cell lines.
Fig. 5. Effect of individual compounds on HCT-116 cells
migration.
Fig. 6. Wound healing and transwell assay on HCT-116 cells with compound 7 in different concentrations.
The individual compounds were further screened for their
inhibitory effects on migration of cancer cells through wound
healing assay with HCT-116 cells. Among the tested compounds,
and analytical applications (for chiral recognition), or as
biomaterials.31
Experimental Section
compound
7
exhibited preferential activity (Fig. 5),
demonstrating that the C16 aglycone in 7 is important for the
observed activity. Detailed assays by transwell and wound
healing indicated that compound 7 suppressed cell migration
effectively in a dose dependent manner (Fig. 6). These data
suggest that 7 represents a potential tumor migration inhibitory
agent.
3.1. General experimental procedures
Optical rotations were measured with a JASCO P-1020
polarimeter. IR spectra were recorded in KBr-disc on a Bruker
Tensor 27 spectrometer. 1D and 2D NMR spectra were recorded
on a Bruker AVIII-500 NMR instrument in pyridine-d5 at 500
MHz (1H) and 125 MHz (13C), and chemical shifts were
recorded as δ values. Mass experiments were performed on an
Agilent 1100 Series LC/MSD ion-trap mass spectrometer
(ESIMS) and Agilent 6520B Q-TOF spectrometer (HRESIMS),
respectively. Column chromatography (CC) was carried out
using silica gel (Qingdao Haiyang Chemical Co., Ltd.), MCI gel
CHP 20P (Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex
LH-20 (75-150 ꢀm, Pharmacia, Sweden), NH2 silica gel (40-75
ꢀm, Fuji Silysia Chemical Ltd, Japan) and RP-C18 (40-63 ꢀm,
Fuji, Japan). Thin-layer chromatography was performed on pre-
coated silica gel GF254 plates (Qingdao Marine Chemical Co.,
Ltd., China) and detected by spraying with 10% H2SO4-EtOH.
Analytical reversed-phase and normal-phase HPLC were
determined on an Agilent 1100 series system with an Agilent
Eclipse XDB-C18 column (150 × 4.6 mm, 5 ꢀm) and Shimadzu
2.2. Conclusion
In summary, 11 compounds including 7 new and 4 known
resin glycoside methyl esters, which were considered to be
generated via methyl esterification by NH2 silica gel CC with
CH2Cl2 -MeOH, were isolated and identified from the seeds of P.
nil. Although the separation of pure compounds from the crude
resin glycosides of P. nil was not succeeded, it provided a
convenient and useful sample processing method to solve the
separation problems. Moreover, the anti-tumor migration
bioactivity of these compounds was reported for the first time,
which makes for the enrichment of pharmacological activity of
resin glycosides. Notably, the structure characteristics in the
compounds make further investigations on the mechanisms of
action of these compounds be of value for biological, chemical

9
LC-2010CHT series system with a Welch Ultimate SiO2 column
1317 [M + Cl]- and 1300 [M + NH4]+; HRESIMS m/z
ACCEPTED MANUSCRIPT
(250 × 4.6mm, 5 ꢀm), respectively. Preparative HPLC was
carried out on a Shimadzu LC-6AD Series instrument with a
shim-pack RP-C18 column (20 × 200 mm). An Alltech 3300
evaporative light scattering detector (ELSD, Grace, Columbia,
MD, USA) was combined with HPLC for detection of
compounds. All solvents were of analytical grade (Jiangsu
Hanbon Science and Technology. Co., Ltd.).
1305.5922 [M + Na]+ (calcd for C56H98NaO32, 1305.5933).
3.4.3. Pharesinoside C (3). White amorphous powder; [α]25
-
D
51.3 (c 1.3, MeOH); IR (KBr) νmax 3428, 2929, 2854, 1732,
1643, 1455, 1384, 1073 cm-1; 1H and 13C NMR data, see Tables 1
and 4; ESIMS m/z 1505 [M + Na]+ and 1517 [M + Cl]-;
HRESIMS m/z 1505.6970 [M + Na]+ (calcd for C66H114NaO36,
1505. 6982).
3.2. Plant material
3.4.4. Pharesinoside D (4). Colorless gum; [α]25 -17.1 (c 1.1,
D
The dried seeds of P. nil were purchased from Hebei province
of China in April 2014, and were authenticated by Professor
Min-Jian Qin, Department of Medicinal Plants, China
Pharmaceutical University. A voucher specimen (No. 2014-PBC)
is deposited in the Department of Natural Medicinal Chemistry,
China Pharmaceutical University.
MeOH); IR (KBr) νmax 3436, 2931, 1729, 1632, 1454, 1383,
1073 cm-1; 1H and 13C NMR data, see Tables 2 and 4; ESIMS m/z
1343[M + Na]+ and 1355[M + Cl]-; HRESIMS m/z 1343.6440 [M
+ Na]+ (calcd for C60H104NaO31, 1343.6454).
3.4.5. Pharesinoside E (5). Colorless gum; [α]25 -28.8 (c 1.5,
D
MeOH); IR (KBr) νmax 3448, 2927, 1728, 1639, 1384, 1075 cm-1;
1H and 13C NMR data, see Tables 2 and 4; ESIMS m/z 1701 [M +
Cl]- and 1689 [M + NH4]+; HRESIMS m/z 1689.8066 [M + Na]+
HRESIMS m/z 1689.8066 [M + Na]+ (calcd for C76H130NaO39,
1689.8081).
3.3. Extraction and isolation
The dried and powdered seeds of P. nil (500 g) were extracted
with ultrasonication in MeOH (2 l × 4). After removal of solvent,
the residue (47.1 g) was suspended in H2O and successively
extracted with petroleum ether (Fr. A), CH2Cl2 (Fr. B), EtOAc
(Fr. D) and n-BuOH (Fr. E). Serious emulsification appeared
when extracted with CH2Cl2 and no effective methods to break it.
Emulsion layer was dissolved in MeOH (45 ml) with sonication
for 10 min, then stand overnight to afford MeOH-soluble (Fr. C)
3.4.6. Pharesinoside F (6). Colorless gum; [α]25 -30.6 (c 1.0,
D
MeOH); IR (KBr) νmax 3438, 2930, 1730, 1632, 1454, 1384,
1076 cm-1; 1H and 13C NMR data, see Tables 2 and 4; ESIMS m/z
1701 [M + Cl]- and 1689 [M + NH4]+; HRESIMS m/z 1689.8073
[M + Na]+ (calcd for C76H130NaO39, 1689. 8081).l
and -insoluble (Fr. C´) fractions. Fr.
C (14 g) was
3.4.7. Pharesinoside G (7). White amorphous powder; [α]25
-
D
chromategraphed over a NH2 silica gel CC and eluted with a step
gradient CH2Cl2-MeOH (5:1-0:1, v/v) to get fractions Ca, Cb and
Cc, monitored by HPLC (ELSD) and TLC. The elution condition
of HPLC-ELSD analysis of MeOH-soluble fraction (Fr. C) and
subfraction Cb was: 0 min, 70% MeOH; 15 min, 80% MeOH; 25
min, 85% MeOH; 35 min, 90% MeOH; 40 min, 100% MeOH;
50min, 100% MeOH. Fr. Cb was rich in resin glycosides and run
on an open RP-C18 CC with a continuous gradient of MeOH-H2O
(6:4 to 10:0) to afford eight fractions (Fr. Cb1-8). Fr. Cb2 was
further subjected to a RP-C18 CC with a gradient elution (MeOH-
H2O, 5:5 to 10:0, v/v) followed by preparative HPLC using
CH3CN-H2O (33:67, v/v) with 0.1% formic acid to give 1 (19.9
mg) and 2 (10.3 mg). Fr. Cb4 was submitted to a RP-C18 CC
using a step gradient of CH3CN-H2O to give seven subfractions
(Fr. Cb4a-g). Fr. Cb4b and Fr. Cb4e were purified by preparative
HPLC with MeOH-H2O (74:26, v/v) to yield compounds 3 (15.5
mg), 8 (8.7 mg) and 9 (14.2 mg). Separation of Fr. Cb4d was
achieved by preparative HPLC with CH3CN-H2O (41:59, v/v)
adding 0.1% formic acid to afford compound 4 (12.1 mg). Fr.
Cb6 was directly separated by preparative HPLC using MeOH-
H2O (80:20, v/v) as mobile phase to furnish compound 11
(41.7mg). Fr. Cb7 was further performed on a RP-C18 CC and 5
(11.1 mg) was obtained from subfraction Cb7b through the
preparative HPLC (MeOH-H2O, 80:20, v/v); Compounds 6 (14.5
mg), 7 (11.7 mg) and 10 (10.3 mg) were obtained successively
from subfractions Cb7f, Cb7h, Cb7g through the preparative
HPLC with 50% CH3CN in water.
45.0 (c 1.0, MeOH); IR (KBr) νmax 3416, 2932, 1735, 1637, 1457,
1
1384, 1071 cm-1; H and 13C NMR data, see Tables 3 and 4;
ESIMS m/z 1529 [M + Cl]- and 1517 [M + Na]+; HRESIMS m/z
1517.7340 [M + Na]+ (calcd for C68H118NaO35, 1517.7346).
3.5. Alkaline hydrolysis of resin glycoside mixture and
preparation of (-)-MTPA ester as authentic samples
Resin glycoside mixture Fr. Cb (2.6 g) was dissolved in 3%
K2CO3 (40ml) and heated at 95°C for 2h. After cooling, the
reaction mixture was acidified to pH 4.0 with 1 N HCl and
extracted successively with CH2Cl2 (40 ml × 3) and Et2O (40 ml
× 4). Two extracts were washed with water and dried over
Na2SO4 to give organic acid fractions. The CH2Cl2 layer organic
acid fraction (150 mg) was treated with triethylamine (20 drops)
and p-bromophenacyl bromide (306 mg) in dry acetone (6 ml) for
1h at room temperature. The mixture was filtered and the filtrate
was fractioned between H2O (15 ml) and ether (15 ml × 4). The
organic phase was further separated by semi-preparative HPLC
with MeOH-H2O (7:3, v/v) to yield p-bromophenacyl 2-
methylbutyrate (18, 62 mg).21 The same treatment of ether layer
organic acid fraction provided p-bromophenacyl nilate (10 mg).21
Two p-bromophenacyl esters existed proved the presence of 2-
methylbutyric acid and nilic acid units in the original structures.13
The absolute configuration of 2-methylbutyric acid was
identified as S by comparison of the 1H NMR spectrum and
specific rotation value ([α]25 +13.6) of p-bromophenacyl 2-
D
methylbutyrate with those of the literature data.21
3.4. Characteristics of compounds
3.4.1. Pharesinoside A (1). Colorless gum; [α]25 -21.8 (c 1.0,
p-Bromophenacyl 2S-methylbutyrate (18): Colorless needles;
1
[α]25 +13.6 (c 1.5, CHCl3); ESIMS m/z 300 [M + H]+; H NMR
D
D
MeOH); IR (KBr) νmax 3439, 2931, 1727, 1633, 1454, 1384,
1073 cm-1; 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z
1271 [M + Cl]- and 1254 [M + NH4]+; HRESIMS m/z 1259.5869
[M + Na]+ (calcd for C55H96NaO30, 1259. 5879).
(500 MHz, CDCl3): δH: 0.98 (3H, t, J = 7.4 Hz, H3-4), 1.24 (3H,
d, J = 7.0 Hz, H3-5), 1.56, 1.79 (each 1H, m, H-3), 2.56 (1H, m,
H-2), 5.28 (2H, s, OCH2CO), 7.63 (2H, d, J = 8.5 Hz, Ar-H),
7.78 (2H, d, J = 8.5 Hz, Ar-H).
3.4.2. Pharesinoside B (2). Colorless gum; [α]25 -59.6 (c 1.3,
p-Bromophenacyl nilate: Colorless needles; [α]25 -9.8 (c 1.4,
D
D
CHCl3); ESIMS m/z 314 [M-H]-; 1H NMR (500 MHz, CDCl3):
δH: 1.25 (3H, d, J = 7.1 Hz, H3-5), 1.30 (3H, d, J = 6.3 Hz, H3-
4), 2.62 (1H, dq, J = 7.0, 7.0 Hz, H-2), 3.97 (1H, dq, J= 7.0, 6.0
MeOH); IR (KBr) νmax 3419, 2932, 1729, 1631, 1452, 1384,
1070 cm-1; 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z

10
Tetrahedron
ACCEPTED MANUSCRIPT
Hz, H-3), 5.33, 5.43 (each 1H, d, J = 16.5 Hz, OCH2CO), 7.65
MTT assay. Cells were plated in 96-well microplates (6,000 cells
(2H, ddd-like, J = 8.5, 2.0, 2.0 Hz, Ar-H), 7.76 (2H, ddd-like, J =
8.5, 2.0, 2.0 Hz, Ar-H).
/well, 200 ꢀl) and routinely cultured in a humidified incubator for
24h. The medium was aspirated off and exchanged for medium
containing each compound at various concentrations for
additional 24h. Then, 20 ꢀl MTT (5 mg/ml stock solution) was
added to each well and incubated for 4h. After the medium was
removed, 150 ꢀl DMSO was added to dissolve the formazan
crystals by constant shaking for 10 min. The optical density was
measured at a test wavelength of 570 nm and a reference
wavelength of 630 nm using a Universal Microplate Reader
(Spectramax Plus 384, Molecular Devices, Sunnyvale, CA,
USA). Cell viability was calculated by the following formula:
Cell viability % = (A treatment group/A control group) ×100%.
These differences are expressed in percentage, and cytotoxic
activity was indicated as an IC50 value. The data were presented
as the means ± SEM for at least three independent experiments.
p-Bromophenacyl nilate (2.9 mg) was treated with DMAP
(2.8mg),
(R)-(-)-α-methoxy-α-(trifluoromethyl)-phenylacetyl
chloride (7.4 ꢀl, 10 mg) in dry pyridine (150 ꢀl) and the reaction
mixture was left to stand at room temperature until the reaction
was complete as evidenced by no more formation of crystalline
pyridine hydrochloride.32 The solvent was removed under a N2
stream and gave a (-)-MTPA ester after purified by pre-HPLC.
The 1H NMR spectrum with two serial of signals indicated that it
was a (-)-MTPA ester mixture of p-bromphenacyl-(2R, 3R)-nilate
and p-bromphenacyl-(2S, 3S)-nilate in the ratio of approximately
4:1 based on the signal intensities due to H3-5 (δH 1.24, 1.31) of
the (2R, 3R)- and (2S, 3S)-form.21, HPLC analysis of it on
33
Ultimate SiO2 column [250 × 4.6 mm, 5 ꢀm; solvent, n-hexane-
isopropanol (4:1, v/v)] also have a satisfactory resolution and two
peaks (tR = 12.1 and 10.5 min) with area ratio of 4:1 (S44,
Supplementary Data) were consistent with the results of NMR
spectrum, thus they were identified respectively as 2R, 3R- and
2S, 3S-forms. The (-)-MTPA ester mixture obtained was used as
an authentic sample for absolute configuration determination of
individual compounds.
3.8. Antimigration activity
The compounds were further evaluated for their suppression
effects on tumor migration with HCT-116 cell line by wound
healing and transwell assay.
Wound healing assay. Cells (1 × 105/ml) were seeded into a
6-well plate. After incubated for 24h (80-90% confluence), cells
were switched to serum-free medium and cultured for another
12h. Then, the cells were gently scratched in the middle of each
well with a 10 ꢀl plastic pipette tip to create a mechanical wound
and incubated with different test substances. Images were taken
at 0 and 24h after scraping by a phase-contrast microscope.
The aqueous phase was extracted with n-butanol (40ml × 3)
and solvent was removed in vacuo. Residue was separated by
preparative HPLC with a shim-pack RP-C18 column (20 × 200
mm) using CH3CN:H2O with 0.1% formic acid (25:75, v/v) to
afford pharbitic acid C (12) and pharbitic acid D (13).
Transwell assay. The cell migration was determined using a
transwell chamber (Corning, USA) with a pore size of 8 ꢀm. 5 ×
104 cells suspended in FBS-free medium were placed in the upper
chamber, whereas complete medium was added to the lower
chamber. After incubation for 24h at 37 °C, the cells in the upper
chamber were carefully removed with a cotton swab, and the
cells that had traversed to the reverse face of the membrane were
fixed with paraformaldehyde, and stained with crystal violet.
3.6. Alkaline hydrolysis and preparation of (-)-MTPA esters
of individual compounds for absolute configuration
determination
Compounds 1-7 (4 mg each) were dissolved in 3% K2CO3
(0.8-1 ml) and heated at 80°C for 2h. 1N HCl was added to adjust
pH to 4 and then extracted with ether (3 × 1ml), respectively. The
upper layer was washed with H2O and dried over anhydrous
Na2SO4. After removal of reagent, the organic acid residue in dry
acetone (0.1-0.2 ml) was neutralized with triethylamine (1-2
drops) and 1.5 mg p-bromophenacyl bromide was added to stay
at room temperature for 1h. The reaction mixture was
concentrated and fractioned with ether to get a residue, which
was subjected to HPLC to give p-bromophenacyl nilate from 1-7
and p-Bromophenacyl 2S-methylbutyrate from 4-7. Each
individual solution of p-bromophenacyl nilate, DMAP (1.2-
1.5mg) and (-)-MTPACl (2 ꢀl) in dry pyridine stayed overnight
to furnish (-)-MTPA ester, respectively. On the basis of the
comparison between each (-)-MTPA ester of p-bromophenacyl
nilate and the authentic sample mentioned above in the normal-
phase HPLC with same analysis conditions [Ultimate SiO2
column, 250 × 4.6 mm, 5 ꢀm; solvent, n-hexane-isopropyl
alcohol (4:1, v/v)], the absolute configurations of nilic acid in the
structures were determined as shown (S44, Supplementary Data).
Migrated cells were counted by using
microscopy.
a phase-contrast
Acknowledgments
This work was supported financially by the National Natural
Science Foundation of China (No. 81573570), the project funded
by the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD), and the Program for
Changjiang Scholars and Innovative Research Team in
University (IRT_15R63).
Supplementary Data
Supplementary data associated with this article can be found
in the online version.
The aqueous phase of 1-7 was separately extracted with n-
butanol (1 ml × 3) and the solvent was removed in vacuo.
Residue was subjected to preparative HPLC with a shim-pack
RP-C18 column (20 × 200 mm) using CH3CN-H2O with 0.1%
formic acid (25:75, v/v) to give pharbitic acid B (14) from
compound 7, pharbitic acid C (12) from compounds 1, 4, and
pharbitic acid D (13) from 2-3, 5-6.
References and notes
1. Kinghorn, A. D.; Falk, H.; Kobayashi, J. In Progress in the
Chemistry of Organic Natural Products; Budzikiewicz, H.;
Pereda-Miranda, R.; Rosas-Ramírez, D.; Castañeda-Gómez, J.;
Eds.; Springerwien: New York, 2010; Vol. 92, pp 77-153.
2. Cao, S. -G.; Guza, R. C.; Wisse, J. H.; Miller, J. S.; Evans, R.;
Kingston, D. G. I. J. Nat. Prod. 2005, 68, 487-492.
3. Yin, Y. -Q.; L, Y.; Kong, L. -Y. J. Agric. Food Chem. 2008, 56,
2363-2368.
4. Song, W. -B.; Wang, W. -Q.; Zhang, S. -W.; Xuan, L. -J. Bioorg.
Med. Chem. Lett. 2015, 25, 795-798.
3.7. Cytotoxicity assay.
The isolated compounds were evaluated for their
cytotoxicities against MDA-MB-231, HCT-116 cell lines by
5. Bautista, E.; Fragoso-Serrano, M.; Pereda-Miranda, R. J. Nat.

11
Prod. 2015, 78, 168-172.
6. Figueroa-Gonzalez, G.; Jacobo-Herrera, N.; Zentella-Dehesa, A.;
Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93-97.
7. Ono, M.; Takigawa, A.; Kanemaru, Y.; Kawakami, G.; Kabata,
K.; Okawa, M.; Kinjo, J.; Yokomizo, K.; Yoshimitsu, H.; Nohara,
T. Chem. Pharm. Bull. 2014, 62, 97-105.
8. Ono, M.; Kawakami, G.; Takigawa, A.; Kabata, K.; Okawa, M.;
Kinjo, J.; Yokomizo, K.; Yoshimitsu, H.; Nohara, T. Chem.
Pharm. Bull. 2014, 62, 839-844.
20. Wang, W. -Q.; Song, W. -B.; Lan, X. -J.; Huang, M.; Xuan, L.
ACCEPTED MANUSCRIPT
-J. J. Nat. Prod. 2014, 77, 2234-2240.
21.
O
NO, M.; TAKAGI-TAKI, Y.; HONDA-YAMADA, F.; NODA, N.;
M
IYAHARA, K. Chem. Pharm. Bull. 2010, 58, 666-672.
22. Chong, T. T.; Hashim, R.; Bryce, R. A. J. Phys. Chem. B. 2006,
110, 4978-4984.
23. Bogusz, S.; Venable, R. M.; Pastor, R. W. J. Phys. Chem. B. 2000,
104, 5462-5470.
24. Xie, W. -L.; Zhao, L. -L. Fuel. 2013, 103, 1106-1110.
25. Liu, Y.; Zhao, G. -M.; Zhu, W. -C.; Wang, J.; Liu, G.; Zhang, W. -
X.; Jia, M. -J. J. Braz. Chem. Soc, 2010, 21, 2254-2261.
26. Werkhoven, P. R.; Elwakiel, M.; Meuleman, T. J.; Quarles van
Ufford, H. C.; Kruijtzer, J. A. W.; Liskamp, R. M. J. Org. Biomol.
Chem. 2016, 14, 701–710.
9. Bensky, D.; Gamble, A. In Chinese Herbal Medicine: Materia
Medica; Revised, Ed.; Eastland: Seattle, 1993; p 121.
10. Kawasaki, T.; Okabe, H.; Nakatsuka, I. Chem. Pharm. Bull. 1971,
19, 1144-1149.
11. Okabe, H.; Koshito, N.; Tanaka, K.; Kawasaki, T. Chem. Pharm.
Bull. 1971, 19, 2394-2408.
12.
O
NO, M.; NODA, N.; KAWASAKI, T.; MIYAHARA, K. Chem.
27. Fischer, G. M.; Jüngst, C.; Isomäki-Krondahl, M.; Gauss, D.;
Möller, H. M.; Daltrozzo, E.; Zumbusch, A. Chem. Commun.
2010, 46, 5289–5291.
Pharm. Bull. 1990, 38, 1892-1897.
13. Ono, M.; Takigawa, A.; Mineno, T.; Yoshimitsu, H.; Nohara, T.;
Ikeda, T.; Fukuda-Teramachi, E.; Noda, N.; Miyahara. K. J. Nat.
Prod. 2010, 73, 1846-1852.
28. Wang, X. -G.; Lin, K. S. K.; Chan, J. C. C.; Cheng, S. J. Phys.
Chem. B. 2005, 109, 1763-1769.
14. Ono, M.; Akiyama, K.; Yamamoto, K.; Mineno, T.; Okawa, M.;
Kinjo, J.; Miyashita, H.; Yoshimitsu, H.; Nohara, T. Chem.
Pharm. Bull. 2014, 62, 830–835.
29. Wang, X. -G.; Tseng, Y. -H.; Chan, J. C. C.; Cheng, S. J. Catal.
2005, 233, 266-275.
30. Kubota, Y.; Goto, K.; Miyata, S.; Goto, Y.; Fukushima, Y.; Sugi,
Y.; Chem. Lett. 2003, 32, 234-235.
15. Akiyama, K.; Yamamoto, K.; Mineno, T.; Okawa, M.; Kinjo, J.;
Yoshimitsu, H.; Nohara, T.; Ono, M. Chem. Pharm. Bull. 2014, 62,
125–133.
31. Xie, J.; Bogliotti, N. Chem. Rev. 2014, 114, 7678−7739.
32. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512-519.
33. Pereda-Miranda, R.; Hernández-Carlos, B. Tetrahedron, 2002, 58,
3145-3154.
16. Fan, B. -Y.; Luo, J. -G.; Gu, Y. -C.; Kong, L. -Y. Tetrahedron.
2014, 70, 2003-2014.
17. Fan, B. -Y.; Gu, Y. -C.; He, Y.; Li, Z. -R.; Luo, J. -G.; Kong, L. -
Y. J. Nat. Prod. 2014, 77, 2264−2272.
18. Yu, B. -W.; Luo, J. -G.; Wang, J. -S.; Zhang, D. -M.; Yu, S. -S.;
Kong, L. -Y. J. Nat. Prod. 2011, 74, 620-628.
Click here to remove instruction text...
19. Yu, B. -W.; Luo, J. -G.; Wang, J. -S.; Zhang, D. -M.; Yu, S. -S.;
Kong, L. -Y. Phytochemistry. 2013, 95, 421-427.

12
Tetrahedron
ACCEPTED MANUSCRIPT
Legends
Fig. 1 HPLC (RP-C18)-ELSD (A) and 1H NMR (pyridine-d5) (B) analysis of MeOH-soluble fraction (Fr. C) (i) and
subfraction Cb (ii) from the seeds of P. nil.
Fig. 2. Structures of compounds 1-11
Fig. 3. Structures of compounds 12-19
Fig. 4. Key HMBC correlations of compounds 1 and 7
Fig. 5. Effect of individual compounds on HCT-116 cells migration. HCT-116 cells were treated with or without
compounds (12.5µM) for 24h and then subjected to a wound healing assay.
Fig. 6. Wound healing and transwell assay on HCT-116 cells with compound 7 in different concentrations. A.
Wound healing assay on HCT-116 cells with compound 7 in different concentrations for 24h and the migration
ability of the cells was monitored with an inverted microscope. The wound closure area was calculated by measuring
the diminution of the wound bed surface upon time; B. The effect of compound 7 on the migration of HCT-116 was
detected by transwell assay. Cells were treated with different concentrations of compound 7 for 24h and detected
through crystal violet staining. The cell number from the transwell assay was counted. (*p < 0.05, compared with the
control).