Tomensides A-D, new antiproliferative phenylpropanoid sucrose esters from Prunus tomentosa leaves

Article abstract of DOI:10.1016/j.bmcl.2014.04.018

To search for novel cytotoxic constituents against cancer cells as lead structures for drug development, four new 3-phenylpropanoid-triacetyl sucrose esters, named tomensides A-D (1-4), and three known analogs (5-7) were isolated from the leaves of Prunus tomentosa. Their structures were elucidated by spectroscopic analyses (1D, 2D NMR, CD and HRESIMS). The cytotoxic activities of all isolates against four human cancer cell lines (MCF-7, A549, HeLa and HT-29) were assayed, and the results showed that these isolates displayed stronger inhibitory activities compared with positive control 5-fluorouracil. Tomenside A (1) was the most active compound with IC₅₀ values of 0.11-0.62 μM against the four tested cell lines. The structure-activity relationship (SAR) of the isolates was also discussed. The primary screening results indicated that these 3-phenylpropanoid-triacetyl sucrose esters might be valuable source for new potent anticancer drug candidates.

Full text of DOI:10.1016/j.bmcl.2014.04.018

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2459–2462
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tomensides A–D, new antiproliferative phenylpropanoid sucrose
esters from Prunus tomentosa leaves
⇑
Wei Zhao , Xiao-Xiao Huang , Li-Hong Yu, Qing-Bo Liu, Ling-Zhi Li, Qian Sun, Shao-Jiang Song
Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, PR China
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To search for novel cytotoxic constituents against cancer cells as lead structures for drug development,
four new 3-phenylpropanoid-triacetyl sucrose esters, named tomensides A–D (1–4), and three known
analogs (5–7) were isolated from the leaves of Prunus tomentosa. Their structures were elucidated by
spectroscopic analyses (1D, 2D NMR, CD and HRESIMS). The cytotoxic activities of all isolates against four
human cancer cell lines (MCF-7, A549, HeLa and HT-29) were assayed, and the results showed that these
isolates displayed stronger inhibitory activities compared with positive control 5-fluorouracil. Tomenside
Received 18 November 2013
Revised 7 March 2014
Accepted 7 April 2014
Available online 13 April 2014
Keywords:
Prunus tomentosa
3-Phenylpropanoid-triacetyl sucrose esters
Cytotoxicities
Structure–activity relationships (SAR)
A (1) was the most active compound with IC50 values of 0.11–0.62 lM against the four tested cell lines.
The structure–activity relationship (SAR) of the isolates was also discussed. The primary screening results
indicated that these 3-phenylpropanoid-triacetyl sucrose esters might be valuable source for new potent
anticancer drug candidates.
Ó 2014 Elsevier Ltd. All rights reserved.
Natural products based drug discovery has become a major
strategy in modern pharmaceutical research and development,
and roughly half of the currently used drugs are directly or
indirectly derived from natural products.1,2 To search for novel
cytotoxic constituents against cancer cells as lead structures for
drug development, many medicinal plants have been screened
using the in vitro cytotoxic activity assay. Of these plants, the
70% ethanol extract of the leaves of Prunus tomentosa was found
to exhibit a potent cytotoxic effect. The wild Prunus tomentosa,
which is widely distributed in China, Japan and Korea, belongs to
the Rosaceae family.3 Bioassay-directed fractionation of this
extract led to the isolation of four new 3-phenylpropanoid-triace-
tyl sucrose esters, tomensides A–D (1–4), along with three known
analogs (5–7). This study describes the isolation and structural elu-
cidation of these isolates, as well as the evaluation of their inhibi-
tory effects on cancer cells. Moreover, the structure–activity
relationships of some compounds are summarized in the Letter.
Phenylpropanoid sucrose esters (PSEs) belong to the phenyl-
propanoid glycoside (glycoconjugate) class of compounds. As the
name indicates, PSEs have a sucrose core connected to one or more
Ph–CH@CH–CO– moieties through an ester linkage.4 Over the past
three decades, nearly 150 PSEs have been isolated from the fami-
lies Rosaceae, Sparganiaceae, Polygonaceae, Rutaceae, Liliaceae, Arec-
aceae and Smilacaceae.4–14 The PSEs have broad range of biological
activities such as antitumor,9,12–16 antioxidant10,11 and anti-
inflammatory.10
Repeated column chromatography of the 70% ethanol extract of
the leaves of Prunus tomentosa resulted in the isolation of four new
3-phenylpropanoid-triacetyl sucrose esters (1–4) and three known
analogs (5–7)17 (Fig. 1). The chemical structures of the known
compounds were identified as mumeose C (5),18 3-p-O-couma-
royl-1,30,60-O-triacetyl sucrose (6)19 and 3-p-O-coumaroyl-1,20,
60-O-triacetyl sucrose (7)20 by comparing the spectroscopic data
with those reported literatures.
Tomenside A (1)21 was obtained as yellow amorphous powder.
The molecular formula of 1 was established as C27H34O16 on the
basis of a quasi-molecular ion at m/z 637.1745 [M+Na]+ (calcd
for C27H34O16Na, 637.1739) in HRESIMS. The 1H NMR spectrum
(Table 1) showed characteristic signals at dH 6.81 (d, J = 8.6 Hz,
2H), 7.52 (d, J = 8.6 Hz, 2H) of p-hydroxyphenyl group and a set
of trans-double-bond signals [dH 6.42, (d, J = 15.8 Hz), 7.72 (d,
J = 15.8 Hz)], together with the carbon signals (Table 2) at dC
168.4, 114.3, 147.9 suggesting the presence of p-coumaroyl moi-
ety. The characteristic anomeric proton signal was appeared at dH
⇑
Corresponding author at present address: School of Traditional Chinese Materia
5.39 with
a small coupling constant (J = 3.6 Hz), as well as
Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang Liaon-
ing 110016, PR China. Tel.: +86 24 23986088; fax: +86 24 23986510.
E-mail address: songsj99@163.com (S.-J. Song).
twelve-oxygenated carbon signals including those for two anomer-
ic carbons (dC 103.3, 93.0) in 13C NMR spectrum, together with the
correlations between dH 5.39 (H-10) and dC 103.3 (C-2) in HMBC
These authors made equal contribution to the Letter.
http://dx.doi.org/10.1016/j.bmcl.2014.04.018
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2460
W. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2459–2462
O
6''
OR7
5'
7''
1''
5''
4''
OH
X
OR1
O
6'
3'
9''
1
2
4'
8''
8''
O
R6O
R5O
2''
7''
1''
1'
OR3
5
3''
O
OH
2'
OR4
O
6
X'
5''
3
4
9''
R2O
OH
OCH3
3''
R1
COCH3
COCH3
H
COCH3
H
COCH3
COCH3
R2
R3
R4
H
H
H
R5
H
R6
H
R7
1
2
3
4
5
6
7
X
X
X
X'
X
X
X
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
COCH3
H
H
H
H
H
H
H
COCH3
COCH3
H
COCH3
COCH3
H
COCH3
COCH3
COCH3
H
H
H
H
H
COCH3
Figure 1. Structures of compounds 1–7.
spectrum confirmed that 1 possessed a disaccharide moiety. More-
over, the result of alkaline hydrolysis22,23 and detailed analysis of
1H NMR, 13C NMR, HMBC and HSQC showed that the two sugars
indicated that 2 had the structure of 3-p-O-coumaroyl-1,40,60-O-tri-
acetyl sucrose.
The molecular formula of tomenside C (3)25 was assigned
were comprised of b-
connected through a 2 ? 1 linkage (a sucrose moiety).
D
-fructose and
a
-
D
-glucose moieties
C27H34O16 according to the quasi-molecular ion at m/z 637.1744
[M+Na]+ (calcd for C27H34O16Na, 637.1739) in HRESIMS. The IR,
UV and NMR spectra showed 3 possessed a p-coumaroyl moiety,
two sugars moieties and three acetyl groups, in a manner similar
to 1, except for the positions of the three acetyl groups. The two
sugars moieties were identified as sucrose moiety by alkaline
hydrolysis.22,23 The correlation between H-3 (dH 5.40) and C-900
(dC 168.1) in HMBC spectrum indicated the p-coumaroyl was
located at the same position with 1. The structure of 3 was identi-
fied due to the correlations observed between H-30 (dH 4.88) and dC
172.5, H-40 (dH 5.23) and dC 172.1, H-60 (dH 4.16) and dC 171.5. Thus,
the structure of 3 was characterized as 3-p-O-coumaroyl-30,40,
60-O-triacetyl sucrose.
On further inspection of the HMBC spectrum, the structure of 1
could be assigned unambiguously due to the correlations observed
between H-1 (dH 4.14, 4.12) and dC 172.9, H-4 (dH 4.43) and dC
172.6, H-60 (dH 4.55, 4.10) and dC 172.1, together with the correla-
tion between H-3 (dH 5.47) and the carbonyl carbon of p-couma-
royl (dC 168.4) group (Fig. 2). Thus, the structure of 1 was
elucidated as 3-p-O-coumaroyl-1,4,60-O-triacetyl sucrose.
Tomenside B (2)24 was obtained as yellow amorphous powder
with the molecular formula C27H34O16, based on quasi-molecular
ion m/z 637.1747 [M+Na]+ (calcd for C27H34O16Na, 637.1739). The
IR, UV and NMR spectra indicated that 2 possessed a p-coumaroyl
moiety, two sugars moieties and three acetyl groups. The result of
alkaline hydrolysis22,23 revealed the existence of a sucrose moiety.
In HMBC spectrum, there are the correlations between H and
carbonyl carbons of acetyl, H-1 (dH 4.22, 4.17) and dC 172.7, H-40
(dH 4.76) and dC 172.2, H-60 (dH 4.09, 4.20) and dC 172.0, together
with correlation between H-3 (dH 5.43) and C-900 (dC 168.1),
The molecular formula of tomenside D (4)26 was determined to
be C28H36O17 by HRESIMS at m/z 667.1846 [M+Na]+ (calcd for C28-
H36O17Na, 667.1845). The IR and UV spectra together with 1H and
13C NMR suggested a similar structure to 1, except for the presence
of a methoxyl group at C-300 and the positions of three acetyl
groups. This evidence suggested that 4 possessed one feruloyl
Table 1
1H NMR data of compounds 1–4 (dH values, measured in CD3OD, at 600 MHz)a
H
1
2
3
4
1
3
4
5
4.14 d (11.6) 4.12 d (11.6)
5.47 d (8.6)
4.43 m
4.22 d (11.8) 4.17 d (11.8)
5.43 d (8.1)
4.34 t (8.2)
3.96 m
3.68 d (12.3) 3.60 d (12.3)
5.40 d (7.0)
4.15 d (11.5) 4.06 d (11.5)
5.42 d (8.4)
4.33 dd (8.4)
3.93 m
3.81 mb
4.08 m
3.99 m
6
4.38 dd (12.0, 2.8) 4.44 mb
2.11 s
3.82 mb 2H
2.11 s
4.33 mb 2H
3.86 mb 2H
1-OAc
2.11 s
4-OAc
2.08 s
5.39 d (3.6)
3.39 dd (9.8, 3.6)
3.57 dd (9.8, 9.4)
3.25 t (9.4)
10
5.49 d (3.6)
3.49 dd (9.9, 3.6)
3.70 t (9.9)
4.76 dd (9.9, 9.5)
4.26 m
4.09 dd (12.0, 2.1) 4.20 dd (12.0, 5.8)
5.54 d (3.6)
3.72 dd (9.9, 3.6)
4.88 dd (9.9, 9.7)
5.23 t (9.7)
5.57 d (3.6)
20
4.62 dd (10.1, 3.6)
30
3.84 mb
40
3.37 t (9.4)
4.10 m
4.51 dd (11.8, 1.7) 4.16 dd (11.8, 6.2)
2.10 s
50
4.12 m
3.81 m
60
4.55 br d (10.5) 4.10 mb
4.16 mb 2H
20-OAc
30-OAc
40-OAc
60-OAc
200
1.79 s
1.99 s
2.07 s
7.54 d (8.6)
6.81 d (8.6)
6.81 d (8.6)
7.54 d (8.6)
7.74 d (15.9)
6.42 d (15.9)
1.95 s
2.08 s
2.11 s
2.09 s
7.28 d (1.8)
7.52 d (8.6)
6.81 d (8.6)
6.81 d (8.6)
7.52 d (8.6)
7.72 d (15.8)
6.42 d (15.8)
7.52 d (8.2)
6.82 d (8.2)
6.82 d (8.2)
7.52 d (8.2)
7.72 d (15.9)
6.40 d (15.9)
300
500
6.82 d (8.2)
7.14 dd (8.2, 1.8)
7.71 d (15.8)
6.47 d (15.8)
3.90 s
600
700
800
300-OCH3
a
Assignments are based on 1H,13C NMR, HMBC, and HSQC experiments.
The signals were overlapped.
b

W. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2459–2462
2461
Table 2
13C NMR data of compounds 1–4 (dC values, measured in CD3OD, at 125 MHz)a
Table 3
Cytotoxic activities of compounds 1–7 in vitro (IC50
,
l
M)
M)
C
1
2
3
4
Compounds
IC50
(
l
1
65.9
64.3
65.4
66.2
MCF-7a
A549a
HeLaa
HT-29a
2
3
4
5
103.3
78.6
73.8
81.0
66.0
172.1
20.7
172.6
20.9
93.0
72.7
74.7
72.0
72.2
65.7
103.5
79.6
73.6
84.4
63.5
172.2
20.9
105.6
79.9
74.7
85.4
63.5
103.4
79.3
73.6
84.2
63.7
172.1
21.0
1
2
3
4
5
6
7
0.62
1.22
2.93
0.77
0.98
1.44
3.44
11.50
0.29
0.49
1.47
0.45
1.12
2.56
3.20
44.05
0.11
0.97
3.80
1.09
4.32
1.33
2.49
7.34
0.18
1.48
2.78
0.46
2.45
2.10
3.96
17.20
6
1-OAc
4-OAc
5-Fub
10
93.1
72.4
72.6
72.6
70.1
66.6
92.8
69.7
74.4
70.3
70.9
63.9
90.8
72.1
74.2
72.1
71.7
65.0
172.6
20.6
a
Key to cell lines used: A549 (human lung cancer cells), MCF-7 (human breast
cancer cells), HeLa (Henrietta Lacks strain of cancer cells) and HT-29 (colon cancer
cell line).
20
30
40
b
5-Fu (5-fluorouracil) was used as positive control.
50
60
20-OAc
were evaluated in vitro for their inhibitory abilities against the
growth of MCF-7, A549, HeLa and HT-29 cancer cells using the
MTT method.27–30 The IC50 values of the PSEs along with the
positive control 5-fluorouracil are summarized in Table 3. The
results showed that all PSEs displayed stronger inhibitory activities
compared with positive control. Among all tested compounds,
Tomenside A (1) showed the most potent inhibitory activities with
30-OAc
40-OAc
60-OAc
171.5
20.5
172.1
20.7
172.5
20.8
127.3
131.5
116.8
161.4
116.8
131.5
147.7
114.6
168.1
172.0
20.7
172.7
20.8
127.1
131.4
116.9
161.7
116.9
131.4
147.7
114.3
168.1
172.9
20.7
172.8
20.8
100
127.1
131.5
116.8
161.5
116.8
131.5
147.9
114.3
168.4
127.6
112.0
149.4
150.9
116.5
124.5
148.2
114.5
168.3
56.5
200
IC50 values of 0.11–0.62 lM against the four tested cell lines.
300
The structural requirements of the active constituents were
examined. Panda et al. reported that the substitution on phenyl
ring did not influence the cytotoxic activity against Hela cancer
cell.15,16 Whereas, the stronger cytotoxic activities of 4 compared
to 7 imply the importance of the substitution on phenyl ring in
mediating the cytotoxic activities of the PSEs. Kuo et al.23 and
Panda et al.15,16 independently suggested that acetyl moieties on
the sucrose core in PSEs might be responsible for mediating the
observed activities. In our research, we also found that the activity
data seems to vary with the position of acetyl moieties on the
sucrose core. Compound 1 was the most active compound against
the four cancer cell lines in the isolated PSEs. In consideration of
the structures of 1–3, 5–7, it was suggested that the acetyl moiety
at the position of C-4 maight enhance the cytotoxicities of the PSEs.
Saeed et al.31 also concluded that increased lipophilicity of
molecules seemed to be responsible for enhanced cytotoxicity. In
addition, the lipophilicity can be influenced by the number of
the acetyl units on the sucrose core. It is notable that the
3-phenylpropanoid-triacetyl sucrose esters display higher
cytotoxic activities compared to the PSEs with one or two acetyl
400
500
600
700
800
900
300-OCH3
Assignments are based on 1H,13C NMR, HMBC, and HSQC experiments.
a
moiety, two sugars moieties and three acetyl groups. The result of
alkaline hydrolysis22,23 also revealed the existence of a sucrose
moiety. Correlations between H-1 (dH 4.15, 4.06) and dC 172.8,
H-20 (dH 4.62) and dC 172.6, H-60 (dH 4.51, 4.16) and dC 172.1,
together with correlation between H-3 (dH 5.42) and C-900
(dC 168.3) in HMBC spectrum, suggested that 4 had the structure
of 3-O-feruloyl-1,20,60-O-triacetyl sucrose.
Some PSEs were found to possess anti-tumour activity, and
their structure–activity relationships were also reported in
previous researches.9,12–16 In our study, the isolated PSEs (1–7)
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
O
O
OH
HO
OH
HO
O
O
OH
OH
O
O
OH
OH
O
O
1
2
O
O
O
O
O
O
OH
O
O
O
O
O
O
O
HO
HO
HO
HO
OH
OH
O
O
O
OH
O
O
O
O
OH
OH
O
O
OCH3
3
4
Figure 2. Observed key HMBC correlations of compounds 1–4.

2462
W. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2459–2462
units from Fagopyrum tataricum (L.) Gaertn.13 The results suggest
that the number of acetyl moieties also plays a useful role in the
cytotoxic activities of the PSEs.
In summary, four new and three known PSEs were isolated from
the leaves of Prunus tomentosa. The cytotoxic activities of all
isolates against four were assayed and the SAR of the PSEs was also
discussed. This study not only helps to provide sufficient materials
for the SAR studies, but also indicates that PSEs may be a poten-
tially valuable source for new potent anticancer drug candidates.
solvent system to obtain 19 fractions (D1–D19). Fractions D9 (0.5 g), D10
(0.7 g) and D12(0.5 g) were cut into 8 fractions (E1–E8), 9 fractions (F1–F8) and
11 fractions (G1–G11) respectively by preparative HPLC eluted with MeOH/
H2O (40:60). 1 (12 mg, tR 11.1 min) was obtained from F8 (39 mg) by semi-
preparative HPLC eluted with CH3CN/H2O (23:77). 2 (22 mg, tR 9.8 min) and 3
(12 mg, tR 10.7 min) were obtained from F6 (91 mg) with CH3CN/H2O (21:79).
4 (4.5 mg, tR 14.4 min) was obtained from E8 (48 mg) with CH3CN/H2O (20:80).
5 (9 mg, tR 8.8 min) was obtained from F5 (31 mg) with CH3CN/H2O (20:80). 6
(14 mg, tR 10.3 min) was obtained from E6 (37 mg) with CH3CN/H2O (20:80). 7
(32 mg, tR 14.5 min) was obtained from F7 (56 mg) with CH3CN/H2O (23:77).
18. Nakamura, Seikou; Fujimoto, Katsuyoshi; Yoshikawa, Masayuki Phytochemistry
2013, 92, 128.
19. Yoshinari, K.; Sashida, Y.; Mimaki, Y.; Shimomura, H. Chem. Pharm. Bull. 1990,
38, 415.
Acknowledgment
20. Shimazaki, N.; Mimaki, Y.; Sashida, Y. Phytochemistry 1991, 30, 1475.
20
21. Tomenside A (1) yellow amorphous powder; [
(CH3OH) kmax (log
vmax: 3447, 2922, 2851, 1733, 1634, 1517, 1454, 1384, 1268, 1169, 1108, 1052,
a
]
+41.6 (c 0.35, CH3OH); UV
D
The authors are grateful to Ms. Wen Li and Mr. Yi Sha of the
Department of Instrumental Analysis, Shenyang Pharmaceutical
University, for measuring the NMR.
e
): 313 (4.11) nm, 228 (3.89) nm, 219 (3.85) nm; IR (KBr)
838, 619 cmꢀ1 1H and 13C NMR data: see Tables 1 and 2; HRESIMS m/z:
;
637.1745 [M+Na]+ (calcd for C27H34O16Na, 637.1739).
22. Alkaline hydrolysis: compounds 1 (20 mg), 2 (1.2 mg), 3 (1.5 mg) and 4 (1.0 mg)
were dissolved in 3% KOH in methanol (4 mL) separately, and the solution was
stirred at room temperature for 6 h. The reaction mixture was neutralized with
1 N HCl and then extracted with chloroform. From the water layer, sucrose was
identified by its TLC behavior [Si gel, developed with chloroform/methanol/
water (6:4:1, v/v/v)].
References and notes
1. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
2. Popsavin, V.; Francuz, J.; Zelenovic´, B. S.; Benedekovic´, G.; Popsavin, M.; Kojic´,
´
V.; Bogdanovic, G. Bioorg. Med. Chem. Lett. 2013, 23, 5507.
23. Kuo, Y. H.; Hsu, Y. W.; Liaw, C. C.; Lee, J. K.; Huang, H. C.; Kuo, L. M. Y. J. Nat.
3. Gao, H. S.; Shi, P. B.; Zhang, J. C. Food Sci. 2006, 27, 378.
Prod. 2005, 68, 1475.
4. Panda, P.; Appalashetti, M.; Judeh, Z. M. A. Curr. Med. Chem. 2011, 18, 3234.
5. Shirota, O.; Sekita, S.; Satake, M.; Yan, N.; Hua, W. Y. J. Nat. Prod. 1996, 59, 242.
6. Sun, X. Z.; Zimmermann, M. L.; Campagne, J. M.; Sneden, A. T. J. Nat. Prod. 2000,
63, 1094.
7. Brown, L. L.; Larson, S. R.; Sneden, A. T. J. Nat. Prod. 1998, 61, 762.
8. Wang, J. S.; Di, Y. T.; Yang, X. W.; Li, S. L.; Wang, Y. H.; Hao, X. J. Phytochemistry
2006, 67, 486.
9. Yan, L. L.; Gao, W. Y.; Zhang, Y. J.; Wang, Y. Fitoterapia 2008, 79, 306.
10. Chang, C. L.; Zhang, L. J.; Chen, R. Y.; Kuo, L. M. Y.; Huang, J. P.; Huang, H. C.; Lee,
K. H.; Wu, Y. C.; Kuo, Y. H. J. Nat. Prod. 2010, 73, 1482.
11. Zhang, L. J.; Liao, C. C.; Huang, H. C.; Shen, Y. C.; Yang, L. M.; Kuo, Y. H.
Phytochemistry 2008, 69, 1398.
12. Takasaki, M.; Kuroki, S.; Kozuka, M.; Konoshima, T. J. Nat. Prod. 2001, 64, 1305.
13. Zheng, C. J.; Hu, C. L.; Ma, X. Q.; Peng, C.; Zhang, H.; Qin, L. P. Food Chem. 2012,
132, 433.
14. Takasaki, M.; Konoshima, T.; Kuroki, S.; Tokuda, H.; Nishino, H. Cancer Lett.
2001, 173, 133.
15. Panda, P.; Appalashetti, M.; Natarajan, M.; Mary, C. P.; Venkatraman, S. S.;
Judeh, Z. M. A. Eur. J. Med. Chem. 2012, 58, 418.
16. Panda, P.; Appalashetti, M.; Natarajan, M.; Chan-Park, M. B.; Venkatraman, S.
S.; Judeh, Z. M. A. Eur. J. Med. Chem. 2012, 53, 1.
17. Isolation: The leaves of Prunus tomentosa obtained from Liaoning Province,
China. Dried leaves (9.3 kg) of Prunus tomentosa were extracted with 70% (v/v)
EtOH. The combined extract (1200 g) was suspended in 3 L water and
partitioned with PE, EtOAc, and water-saturated n-butanol, successively, and
the EtOAc extract showed stronger cytotoxic activities than other extracts. The
EtOAc extract (120 g) was subjected to silica gel column chromatography with
a CH2Cl2/MeOH gradient solvent system to obtain four fractions (A1–A4),
which were combined according to TLC analysis. Fraction A2 (75 g) was
subjected to a CHP20 CC to remove chlorophyll by collecting fractions (B1,
69 g) of MeOH/H2O gradient solvent system. The fraction B1 was
chromatographed on Polyamide CC (100 mesh) eluted with a gradient of
EtOH/H2O to obtain 3 fractions, C1 (18 g), C2 (32 g), C3 (19 g). C1 was subjected
20
24. Tomenside B (2) yellow amorphous powder; [
(CH3OH) kmax (log
vmax: 3423, 2920, 2850, 1727, 1605, 1515, 1444, 1384, 1251, 1167, 1048, 836,
a
]
+27.4 (c 0.20, CH3OH); UV
D
e): 314 (4.18) nm, 228 (3.90) nm, 219 (3.86) nm; IR (KBr)
591 cmꢀ1 1H and 13C NMR data: see Tables 1 and 2; HRESIMS m/z: 637.1747
;
[M+Na]+ (calcd for C27H34O16 Na, 637.1739).
20
25. Tomenside C (3) yellow amorphous powder; [
a
]
D
+23.9 (c 0.25, CH3OH); UV
(CH3OH) kmax (log
e): 313 (4.09) nm, 228 (3.88) nm, 218 (3.84) nm; IR (KBr)
vmax: 3447, 2922, 2851, 1742, 1606, 1516, 1450, 1384, 1243, 1170, 1053.6,
836,619, 591 cmꢀ1 1H and 13C NMR data: see Tables 1 and 2; HRESIMS m/z:
;
637.1744 [M+Na]+ (calcd. for C27H34O16 Na, 637.1739).
20
26. Tomenside D (4) yellow, amorphous powder; [
(CH3OH) kmax (log
a
]
D
+34.2 (c 0.22, CH3OH); UV
e
): 331 (4.17) nm, 236(3.93) nm, 228(3.90) nm; IR (KBr)
m
max: 3442, 2920, 2851, 1729, 1632, 1517, 1453, 1384, 1250, 1129, 1050,
619 cmꢀ1
;
1H and 13C NMR data: see Tables 1 and 2; HRESIMS m/z: 667.1846
[M+Na]+ (calcd for C28H36O17 Na, 667.1845).
27. Cytotoxicity assay: MTT agent, against MCF-7, A-549, Hela and HT-29, was
based on the lit.28–30 procedure. In brief, all cells were cultured in RPMI-1640
medium. Test samples were prepared at four concentrations. After the cell
lines were seeded in a 96-well microplate for 4 h, 20
in each well and incubated at 37 °C for 2 days, then 20
mL) was added. After further 4 h of incubation, 100 L of 10% sodium dodecyl
l
l
L of sample was placed
L MTT solution (5 mg/
l
sulfate in 0.01 M HCl solution was added to each well and the formazan
crystals in each well were dissolved by stirring with a pipette, and their
absorbances were measured on a microtiter plate reader, at a wavelength of
550 nm. 5-Fluorouracil was used as a positive control.
28. Wang, Y.; Wang, W. J.; Su, C.; Zhang, D. M.; Xu, L. P.; He, R. R.; Wang, L.; Zhang,
J.; Zhang, X. Q.; Ye, W. C. Bioorg. Med. Chem. Lett. 2013, 23, 654.
29. Li, X.; Tian, Y.; Yang, S. X.; Zhang, Y. M.; Qin, J. C. Bioorg. Med. Chem. Lett. 2013,
23, 2945.
30. Liu, F. Z.; Ren, J. W.; Tang, J. S.; Liu, X. Z.; Che, Y. S.; Yao, X. S. Fitoterapia 2013,
87, 78.
31. Saeed, S.; Rashid, N.; Jones, P. G.; Ali, M.; Hussain, R. Eur. J. Med. Chem. 2010, 45,
1323.
to silica gel column chromatography (CC) with
a CH2Cl2/MeOH gradient