Design, synthesis and biological evaluation of novel glycosylated diphyllin derivatives as topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.11.011

Recently, a novel glycosylated diphyllin derivative 11 which exhibiting potent anticancer activity by targeting topoisomerase IIα was reported by our group. In order to provide more molecules for structure-activity relationship (SAR) studies, 12 new glyco

Full text of DOI:10.1016/j.ejmech.2011.11.011

European Journal of Medicinal Chemistry 47 (2012) 424e431
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel glycosylated diphyllin
derivatives as topoisomerase II inhibitors
,
b,
Da-Kuo Shi a, Wei Zhang b, Ning Ding b, Ming Li a , Ying-Xia Li
**
*
a School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
b School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, a novel glycosylated diphyllin derivative 11 which exhibiting potent anticancer activity by
targeting topoisomerase II was reported by our group. In order to provide more molecules for structure-
Received 22 September 2011
Received in revised form
3 November 2011
Accepted 4 November 2011
Available online 13 November 2011
a
activity relationship (SAR) studies, 12 new glycosylated diphyllin analogs have been designed, synthe-
sized, and evaluated for their biological activities. The SAR analysis revealed that (i) the sugar moiety on
the diphyllin is essential for the anticancer activity; (ii) equatorial C40eOH on the sugar is superior to the
axial one, and (iii) a proper cyclic lipophilic group at the C40 and C60 of sugar might enhance the anti-
cancer activity.
Keywords:
Diphyllin
Ó 2011 Elsevier Masson SAS. All rights reserved.
Topo II inhibitors
Synthesis
Anticancer
Structureeactivity relationship
1. Introduction
activities. Such sugar appendages add important features to the
shape and the stereoelectronic properties of a molecule and thus
Diphyllin (1, Fig. 1) is a typical 1-arylnaphthalene lignan isolated
from the plant Justicia procumbens L. (Acanthaceae), which was
found to have a wide spectrum of biological activities, such as
cytotoxicity [1,2], antimicrobial [3] and antiviral [4]. Among these,
the cytotoxicity is the most attractive, making arylnaphthalene
a good lead compound for anticancer drug design and development
[5,6]. On the other hand, the role of carbohydrates in biologically
active natural products attracts much more attentions than before
[7]. With deep insights into the understanding of aglycone and
glycoside activities, followed by the interesting stories of morphine-
6-glucuronide [8], rebeccamycin analogs with uncommon sugars [9]
and subtle sugar modification of digitoxin [10] etc, the emphasis on
the key requirement of sugars and in particular the uncommon
sugars is emerging as a new driving force in the development of new
type distinct from the conventional ones.
wield remarkable influences which range from modulating phar-
macology and pharmacokinetic properties to dictating specificity
on a tissue, cellular and/or molecular level [12,13]. This concept
appeals a surge of interests in the development of novel glycosy-
lated compounds for cancer therapy.
In a previous paper, we reported that 11(Fig. 1) [14], a novel
diphyllin glycoside with acetylated
a potent topoisomerase II (Topo II
moiety endows 11 an optimal conformation with a high binding
affinity for Topo II via hydrogen bonding to the entrance of ATPase
D
-quinovose sugar moiety, as
a
a) inhibitior. This peculiar sugar
a
pocket, thereby helping achieve more potent Topo II inhibition
activity compared to the aglycon diphyllin.
With the availability of the synthesized diphyllin in hand [15,16],
and in particularly bearing in mind the critical contribution of sugar
attachments to the outcome of molecular shape and biological
diverse functions, we are encouraged to challenge the glycosylation
manipulation to the diphyllin, with the aim to probe insights into
the SAR-based nature behind the glycosylated diphyllin analogs via
the design, synthesis and biological evaluation.
In recent years, lots of diphyllin glycoside derivatives have been
isolated from the Acanthaceae plants continuously. Sophie Sus-
plugas et. al. obtained three kinds of diphyllin glycosides (2e4,
Fig.1) from Justicia patentiflor Hemsl [11], which have attracted us to
enjoy these research due to their special structure and biological
2. Chemistry
* Corresponding author. Tel./fax: þ86 21 51980127.
The correlation between the structural feature of sugar moiety
and the cytotoxic potency revealed that the glycoside 2 with sugar
** Corresponding author. Tel.: þ86 532 82032150.
E-mail addresses: lmsnouc@ouc.edu.cn (M. Li), liyx417@fudan.edu.cn (Y.-X. Li).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.011

D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
425
which are incorporated by
b-glycosidic bonds. The 1, 2-trans-
glycosidic bonds could be formed effectively by glycosylation of
diphylin with peracetylated 1,2-cis-glycosyl bromides under phase
transfer conditions (TBAB, CHCl3, 0.1 mol Lꢀ1 NaOH in H2O, 40 ꢁC)
[15]. Subsequently, deacetylation was carried out with K2CO3 in
methanol to produce target molecule without destroying the
lactone residue (Scheme 1 and Table 1). This synthetic method
allowed us to obtain analogs 5e14 with
syl(8), 6-OMe- -glucosyl (9),6-OBn- -glucosyl (13),
(5), -arabinosyl (6), 6-OMe- -galactosyl (10), -fucosyl (12) and 6-
OBn- -galactosyl (14) residue respectively.
With glucoside 7 in hand, we further synthesized the cyclic
acetals (15e17), which was smoothly achieved by reaction of
D-glucosyl (7),
D-xylo-
D
D
D
-galactosyl
L
D
D
D
b-D-
diphyllin glucoside with the corresponding aldehyde or ketone
acetal in thepresence of catalytic amountof p-TsOH$H2O (Scheme 2).
3. Results and discussion
3.1. In Vitro cytotoxicity
Fig. 1. Structures of diphyllin and its glycosylated analogs.
Preliminarily, the cytotoxic activity of all glycosylated diphyllin
derivatives wereevaluated towardthe human colon carcinoma HCT-
116, human breast carcinoma MCF-7 and oral epidermoid carcinoma
KB cell lines [17,18]. For comparative purposes, compounds 2, 3, 4
and Etoposide (VP-16) were also included in the assay.
As shown in Table 2, compared with aglycon diphyllin,
compounds in Series A and Series C showed more potent cytotox-
icities, while compounds in Series B were less potent. Their activity
was followed by the order: series C > series A > diphylin > series B.
These results indicated the sugar moiety attached on diphyllin is
a key element for its cytotoxicity. Series A were much more potent
than series B due to the change of 4-equatorial-OH to 4-axial-OH of
sugar, implicating that axial-OH at C4 did not favor the activity.
Comparing the compounds in series A, it was shown that the C6
functional groups at the sugar strongly affect the bioactivity, the
residue of 4C1 chair conformation was more potent than glycoside 3
and 4 with sugar residue of 1C4 chair conformation, and glycoside 4
with acetylated sugar residue was more potency than 3 [11].
Besides, glycoside 2 showed potent cytotoxicity to drug-resistant
cell strain MCF-7R with IC50 40 nM, while glycoside 3 exhibited
no activity to this cell strain. Starting from the most potent glyco-
side 2 and maintaining the 1, 2-trans glycosidic bond, first to
explore the importance of functional groups of C6 in the sugar,
replacing the methyl group (2) with CH2OH (7), H (8), CH2OMe (9)
and CH2OBn (13) groups respectively lead to the first series (Series
A) of analogs. In order to determining the effect of the C4eOH
orientation of sugar on the bioactivity, the resulting corresponding
C4 epimers of Series A lead to the second series (Series B) of
analogs. Furthermore, to block simultaneously 4,6-dihydroxy
groups of the glucose moiety (6), the third series (Series C) of the
cyclic acetal derivatives (15e17) have also been designed and
prepared (Fig. 2).
analog 13 with 6-OBn-
cytotoxicity, and in the order 11(6-Me-
>> 7 ( -glucosyl) > 9 (6-OMe- -glucosyl). More interestingly, the
D-glucosyl moiety showed the most potent
D
-glucosyl) >8 ( -xylosyl)
D
D
D
Retrosynthetically, the analogs (5e14) can be logically discon-
nected into two fragmentsddiphyllin and the sugar residues,
4, 6-cyclic acetal derivatives of series C (15e17) showed much more
potent cytotoxicity than other analogs by up to 1000-fold,
Fig. 2. Structures of designed diphyllin glycosylated derivatives.

426
D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
Scheme 1. Reagents and conditions: a) CHCl3, H2O, TBAB, NaOH, 40 ꢁC; b) K2CO3, MeOH.
indicating that a small size lipophilic group masked on the 4eOH
and 6eOH favored the bioactivity.
myelogenous leukemia K562 and its adriamycin-selected MDR cell
subline K562/A02 as well as oral epidermoid carcinoma KB and its
vincristine (VCR)-selected MDR KB/VCR subline [19e21] (Table 3).
These compounds retained better inhibition against selected oral
epidermoid carcinoma cellular subclones resistant to VCR (KB/VCR)
compared to selected leukemia cellular subline resistant to adria-
mycin (K562/A02). In particular, the KB/VCR cell line exhibited
a low level of cross-resistance to 2, 8 and 11 (RI (resistance
index) ¼ 2.9e6.2) except 15 (RI ¼ 25.3), whereas higher cross-
resistance to these compounds was observed in the adriamycin
resistant subline K562/A02 (RI ¼ 9.5e19.4). These suggest targeting
Topo II may contribute to the higher resistant index of diphyllin and
its derivates.
3.2. Multidrug resistance analysis
To further investigate their drug resistance profile, diphyllin,
compounds 2, 8, 11 and 15 were selected to test against chronic
Table 1
Results of the glycosylation reaction of donors with diphyllin.
a
Entry Donor
Product
Yield (%)b
71
1
7a
7
3.3. Drug inhibition of kDNA decatenation induced by
topoisomerase II (Topo II)
2
3
8a
9a
8
9
63
79
The relative compound activity in inhibiting Topo II-mediated
kDNA decatenation was studied using catenated kinetoplast DNA
as a substrate for human Topo II and VP-16 as a Topo II-specific
inhibitor [22]. The preliminary experiments indicated that the
compounds containing quinovopyranosyl, glucopyranosyl, xylo-
pyranosyl, 6-O-methyl-pyranosyl residue (2, 4, 11 and 13) and
acetal glycosides (15, 16 and 17) exhibited potent inhibitory activity
(Fig. 3), whereas the remaining compounds did not inhibit Topo II
4
5
13a
5a
13 67
catalytic activity at a concentration of 100
mM. The most potent
cytotoxic compounds 13, 15, 16, and 17 inhibited most strongly
Topo II catalytic activity. These results demonstrate that there is
a good correlation between cytotoxicity in cancer cell cultures and
Topo II inhibitory activity.
5
6
80
59
3.4. Cell cycle arrest and apoptosis
6
7
6a
Previous studies have indicated patentiflorin A(2) caused G0/G1
arrest [11]. On these grounds the action on the cell cycle of these
compounds were studied by flow cytometry. To gain further insight
into the mode of action of these compounds, we examined the
effects of compounds 2, 8, 11, 12 and 15 which were determined cell
cycle accumulation at 24 h by propidium iodide staining and flow
cytometry quantification in HL-60 cells.
10a
10 58
Cell cycle analysis of HL-60 cells showed that cells were arrested
at the G0/G1 and G2/M phase following treatment with 2, 8, 11 and
15 (Table 4), whereas no cell cycle arrest was observed for 12. This
cell cycle arrest was dose- and time-dependent with a relative
potency of the assayed compounds being 15 > 11 > 8 > 12, which
was in agreement with the cytotoxicity results for these
compounds. Higher concentrations and longer incubation times are
required for the less potent analogs in order to get a similar cell
cycle response to that achieved by the most potent compounds.
Similar results were obtained with human colon adenocarcinoma
HCT-116 cells (data not shown).
8
12a
14a
12 54
9
14 70
a
R represents 4-O-diphyllin group.
Two steps for glycosylation and deacetylation.
b

D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
427
Scheme 2. Reagents and conditions: a) R1R2CH(OMe)2, p-TsOH$H2O.
3.5. DNA fragmentation during drug-induced apoptosis
inhibitory activity of these compounds toward Topo II investigated
by Topo II-induced kDNA decatenation assay matched their cyto-
toxicity well, suggesting Topo II is one of the main targets of this
series of compounds. Using the human leukemia HL-60 cell line,
selected compounds were found to induce G0/G1 arrest and DNA
fragmentation characteristic of apoptotic cell death. These results
suggest that the sugar moiety on the C4 of diphyllin is a key
element for its anti-tumor activity.
To better characterize the biological activity of these
compounds, we tested the effects of a selected series of derivatives
on programmed cell death (apoptosis), using the human leukemia
cell line HL-60 as the experimental system. We examined whether
these compounds induced DNA fragmentation, which is considered
the end point of the apoptotic pathway [23]. As shown in Fig. 4, all
the compounds (10
m
M, 36 h) induced DNA fragmentation. Mean-
In addition, all acetals of diphyllin glucoside showed much more
potent cytotoxicity and Topo II inhibitory activity compared with
the other derivates. Therefore, these derivatives can provide some
information about glycoside modification to natural products and
should be considered as interesting lead compounds for anticancer
compounds research.
while 2, 11 and 12 displayed similar values in the percentage of
apoptotic cells, and 15 was the most potent compound to induce
apoptosis.
4. Conclusion
In summary, new glycosylated diphyllin derivatives containing
different sugars or chemical modified sugars were prepared and
evaluated against several human tumor cell lines. All the
5. Experimental
5.1. Chemistry
compounds showed IC50 cytotoxicity levels in the mM-nM range. In
general, those compounds containing 40, 60-cyclic acetal glycosy-
lated derivatives (Series C) were more potent than other series. The
results suggest that a moderate size cyclic lipophilic group at the C40
and C60 of sugar is very important to the bioactivity. By comparing
the three series of compounds, glycosides with equatorial C40eOH
in the sugars are more active than those with axial C40eOH, indi-
cating the equatorial C40eOH played a very important role.
Furthermore, some derivatives showed multi-drug resistant
(MDR) activity against two MDR cell lines (K562/A02 and KB/VCR),
which were much lower than those of ADR, VCR, and VP-16. The
Solvents were purified in the usual way. TLC was performed on
precoated Merck silica Gel 60 F254 plates. Flash chromatography
was performed on silica gel (300e400 mesh Qingdao, China).
Optical rotations were determined with a PerkineElmer Model
241 MC polarimeter. 1H NMR and 13C NMR spectra were taken on
a JEOL JNM-ECP 600 MHz spectrometer with tetramethylsilane
(TMS) as an internal standard, and chemical shifts were recorded in
d
values. The high resolution mass spectra were obtained on a Q-
TOF Global Mass (ESIMS) instrument.
5.1.1. General procedure of the diphyllin glycoside compounds
(5e14)
Table 2
Inhibitory effect of compounds 5e17 response for 50% growth inhibition (IC50
,
mM)
Step A: To the solution of diphyllin (1.0 mmol) and TBAB
(1.0 mmol) in CHCl3 (20 mL) was added aqueous 0.1 molꢂLꢀ1 NaOH
(20 mL). After stirring for 10 min at 40 ꢁC, the corresponding
substituted sugar bromide (1.5 mmol) was added, and the two-
phase reaction mixture was stirred for 6 h at 40 ꢁC. Then CHCl3
on human tumor cell lines (HCT-116, MCF-7, KB).
Series
Compound
IC50, mM
HCT-116
MCF-7
KB
VP-16
Diphyllin
0.91
3.54
1.09
0.54
0.85
0.78
2
3
4
7
0.99
>10
0.27
9.67
0.44
1.42
0.11
2.44
0.093
>10
0.14
>10
0.41
Table 3
The effect of Diphyllin, 2, 8, 11 and 15 to the resistant tumor cell lines.
Series A
8
0.82
1.55
Compd
IC50 (mM)
9
>10
0.27
0.036
>10
>10
>10
6.43
0.008
0.043
0.016
>10
0.38
0.028
4.11
>10
>10
1.81
0.004
0.053
0.0002
4.5
KB
KB/VCR
RI
K562
K562/A02
RI
11
13
5
0.44
0.015
4.89
6.88
4.1
3.05
0.003
0.010
Diphyllin
2
8
11
0.78
3.15
0.573
1.19
4.0
6.2
2.9
3.0
25.3
180.3
NT
1.67
1.43
4.402
0.808
0.044
5.16
0.43
NT
23.46
16.14
58.82
7.66
0.853
242.8
56.83
NT
14.1
11.3
13.4
9.5
19.4
47.1
132.2
NT
Series B
Series C
0.093
0.41
0.44
0.003
0.85
NT
6
10
12
15
16
17
1.32
15
0.076
153.26
NT
VP-16
ADR
VCR
0.0006
0.003
1.61
536.7

428
D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
Fig. 3. Drug inhibition of kDNA decatenation induced by topoisomerase II (Topo II).
Table 4
5.1.1.1. 4-O-
79.9%, Rf 0.31 (CHCl3:MeOH, 10:1), [
1H NMR (600 MHz, DMSO-d6) :
b-D-galactopyranosyldiphyllin (5). White solid, yield
25
Effect of 2, 8, 11, 12 and 15 on cell cycle in HL-60 cells.
a]
¼ ꢀ36.0ꢁ (c1.00, MeOH);
D
d
8.20 (d, 1 H, J ¼ 5.0 Hz, ArH), 7.04
Treatment
Compd
% of cells
G0/G1
(d, 1 H, J ¼ 7.8 Hz, ArH), 6.98 (s, 1 H, ArH), 6.93 (d, 1 H, J ¼ 1.4 Hz,
ArH), 6.81e6.78 (dd, 1 H, J ¼ 7.8, 1.4 Hz, ArH), 6.13 (s, 2 H, OCH2O),
5.80 (d, 1 H, J ¼ 5.0 Hz, OH-200), 5.70 (d, 1 H, J ¼ 14.6 Hz, H-9a), 5.49
(dd, 1 H, J ¼ 14.6, 3.2 Hz, H-9b), 5.00 (d, 1 H, J ¼ 5.5 Hz, OH-300), 4.71
(d, 1 H, J ¼ 7.8 Hz, H-100), 4.63 (d, 1 H, J ¼ 4.6 Hz, OH-400), 3.95 (s, 3 H,
OCH3), 3.79e3.77 (m, 1 H, H-200), 3.66 (s, 3 H, OCH3), 3.66e3.65 (m,
1 H, H-400), 3.60e3.58 (m, 2 H, H-600a, 600b), 3.48e3.44 (m, 1 H, H-
500), 3.43e3.41 (m, 1 H, H-300); 13C NMR (150 MHz, DMSO-d6):
Dose (M)
S
G2/M
Control
2
39.0
45.9
54.4
45.6
47.0
46.4
55.0
38.6
40.5
53.6
56.2
38.4
38.0
22.8
35.5
33.8
36.3
19.3
30.0
34.6
23.4
19.2
22.6
16.1
22.8
18.9
19.2
17.4
25.7
31.5
24.9
22.9
24.6
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ5
8
11
12
15
d
169.1 (C]O), 151.4 (C-5), 149.9 (C-4), 146.9 (C-40), 146.8 (C-30),
144.9 (C-7), 134.7 (C-70), 129.7 (C-1), 129.6 (C-8), 128.2 (C-10), 126.7
(C-2), 123.5 (C-60), 118.8 (C-80), 110.8 (C-20), 107.9 (C-3), 105.6 (C-100),
105.3 (C-50), 101.8 (C-6), 101.0 (OCH2O), 75.8 (C-500), 73.0 (C-300),
70.8 (C-200), 68.4 (C-400), 67.3 (C-9), 60.9 (C-600), 55.7 (OCH3), 55.1
(OCH3); HRMS calcd for C27H27O12 543.1503, found 543.1476.
(20 mL) was added and the resulting organic phase was washed
with water (2 ꢃ 20 mL), dried (Na2SO4), and the solvent evaporated
under reduced pressure. The residue was purified by flash chro-
matography (1:1, EtOAc-petroleum ether) to afford a solid.
Step B: The solid above (0.8 mmol) was dissolved in CH3OH
(20 mL), and then K2CO3 (138 mg, 1.0 mmol) was added. After
stirring for 60 min at room temperature the solution was neutral-
ized with 1 molꢂLꢀ1 HCl and concentrated under reduced pressure.
CHCl3 (20 mL) was added and the resulting organic phase was
washed with water (2 ꢃ 20 mL) and dried (Na2SO4), and the solvent
was evaporated under reduced pressure to get a residue, which was
purified by flash chromatography (25:1, CH2Cl2 :MeOH) to afford
a solid.
5.1.1.2. 4-O-
0.27 (CHCl3:MeOH, 20:1), [
(600 MHz, DMSO-d6) :
a-L-arabinosyldiphyllin (6). White solid, yield 58.8%, Rf
25
a
]
D
¼ ꢀ7.5ꢁ (c1.04, CHCl3); 1H NMR
d
8.17 (s, 1 H, ArH), 7.04 (d, 1 H, J ¼ 8.3 Hz,
ArH), 6.98 (s, 1 H, ArH), 6.92 (d, 1 H, J ¼ 1.9 Hz, ArH), 6.80e6.78 (m,
1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.76 (d, 1 H, J ¼ 5.5 Hz, OH-400), 5.53
(dd, 1 H, J ¼ 15.1, 2.3 Hz, H-9a), 5.45 (d, 1 H, J ¼ 15.1 Hz, H-9b), 5.04
(d, 1 H, J ¼ 5.5 Hz, OH-300), 4.71 (d, 1 H, J ¼ 6.8 Hz, H-100), 4.75 (d, 1 H,
J ¼ 3.2 Hz, OH-200), 3.95 (s, 3 H, OCH3), 3.85 (dd, 1 H, J ¼ 13.7, 6.4 Hz,
H-200), 3.78 (d, 1 H, J ¼ 11.4 Hz, H-500a), 3.71 (brs, 1 H, H-400), 3.67 (s,
3 H, OCH3), 3.53e3.51 (m, 1 H, H-300), 3.45 (d, 1 H, J ¼ 11.4 Hz, H-
500b); 13C NMR (150 MHz, DMSO-d6):
d 169.0 (C]O), 151.4 (C-5),
Fig. 4. The diphyllin and its glycoside derivatives caused apoptosis in HL-60 cells by DNA fragmentation electrophoresis.

D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
429
149.9 (C-4), 146.9 (C-40), 146.8 (C-30), 144.5 (C-7), 134.4 (C-70), 129.6
(C-1), 128.9 (C-8), 128.2 (C-10), 126.6 (C-2), 123.5 (C-60), 118.6 (C-80),
110.7 (C-20), 107.9 (C-3), 105.4 (C-100), 104.8 (C-5), 101.8 (C-6), 101.0
(OCH2O), 72.2 (C-300), 70.8 (C-200), 67.2 (C-400), 67.0 (C-9), 65.7 (C-
500), 55.8 (OCH3), 55.1 (OCH3); HRMS calcd for C26H25O11 513.1397,
found 513.1409.
ArH), 7.04 (dd, 1 H, J ¼ 7.7, 1.9 Hz, ArH), 6.98 (d, 1 H, J ¼ 2.6 Hz, ArH),
6.92 (d, 1 H, J ¼ 1.9 Hz, ArH), 6.81e6.78 (m, 1 H, ArH), 6.12 (s, 2 H,
OCH2O), 5.65 (dd, 1 H, J ¼ 14.8, 2.6 Hz, H-9a), 5.46 (dd, 1 H, J ¼ 14.8,
1.9 Hz, H-9b), 4.71 (d, 1 H, J ¼ 8.3 Hz, H-100), 3.95 (s, 3 H, OCH3), 3.78
(dd,1 H, J ¼ 9.3, 8.3 Hz, H-200), 3.66 (s, 3 H, OCH3), 3.66e3.65 (m,1 H,
H-500), 3.63 (d, 1 H, J ¼ 2.6 Hz, H-400), 3.56e3.49 (m, 2 H, H-6a, 6b),
3.43 (dd, 1 H, J ¼ 9.3, 3.2 Hz, H-300), 3.23 (s, 3 H, OCH3); 13C NMR
5.1.1.3. 4-O-
71.3%, Rf 0.30 (CHCl3:MeOH,10:1), [
NMR (600 MHz, DMSO-d6) :
b
-
D
-glucopyranosyldiphyllin (7). White solid, yield
(150 MHz, DMSO-d6): d 169.1 (C]O), 151.4 (C-5), 149.9 (C-4), 146.8
25
a
]
D
¼ ꢀ30.9ꢁ (c1.00, MeOH); 1H
(C-40), 146.7 (C-30), 144.9 (C-7), 134.8 (C-70), 129.8 (C-1), 129.6 (C-8),
128.2 (C-10), 126.8 (C-2), 123.5 (C-60), 118.7 (C-80), 110.8 (C-20), 107.9
(C-3), 105.6 (C-100), 105.3 (C-5), 101.8 (C-6), 101.1 (OCH2O), 73.3 (C-
500), 72.9 (C-300), 72.0 (C-600), 70.6 (C-200), 68.6 (C-400), 67.2 (C-9), 58.2
(OCH3), 55.7 (OCH3), 55.1 (OCH3); HRMS calcd for C28H29O12
557.1659, found 557.1663.
d
8.18 (d, 1 H, J ¼ 5.0 Hz, ArH), 7.04 (d,
1 H, J ¼ 7.8 Hz, ArH), 6.98 (s, 1 H, ArH), 6.93 (d, 1 H, J ¼ 1.9 Hz, ArH),
6.82e6.78 (dd, 1 H, J ¼ 7.8, 1.9 Hz, ArH), 6.13 (s, 2 H, OCH2O), 5.98 (d,
1 H, J ¼ 4.1 Hz, OH-200), 5.77 (d, 1 H, J ¼ 15.1 Hz, H-9a), 5.48 (dd, 1 H,
J ¼ 15.1, 3.7 Hz, H-9b), 5.25 (d, 1 H, J ¼ 4.6 Hz, OH-300), 5.09 (d, 1 H,
J ¼ 5.5 Hz, OH-400), 4.75 (d, 1 H, J ¼ 7.8 Hz, H-100), 4.71e4.68 (m, 1 H,
OH-600), 3.95 (s, 3 H, OCH3), 3.79e3.77 (m, 1 H, H-600a), 3.67 (s, 3 H,
OCH3), 3.50e3.44 (m, 2 H, H-600b, H-200), 3.31e3.28 (m, 1 H, H-5”),
3.24e3.22 (m, 1 H, H-300), 3.17e3.16 (m, 1 H, H-400); 13C NMR
5.1.1.7. 4-O-
53.8%, Rf 0.40 (CHCl3:MeOH, 10:1), [
1H NMR (600 MHz, DMSO-d6) :
b-D-fucopyranosyldiphyllin (12). White solid, yield
25
a
]
D
¼ ꢀ57.3ꢁ (c1.00, CH2Cl2);
d
8.18 (d, 1 H, J ¼ 2.8 Hz, ArH), 7.03
(150 MHz, DMSO-d6):
d
169.2 (C]O), 151.4 (C-5), 149.9 (C-4), 146.9
(dd, 1 H, J ¼ 7.8, 1.9 Hz, ArH), 6.98 (d, 1 H, J ¼ 3.2 Hz, ArH), 6.93 (d,
1 H, J ¼ 1.9 Hz, ArH), 6.81e6.78 (m, 1 H, ArH), 6.12 (s, 2 H, OCH2O),
5.77 (brs, 1 H, OH-200), 5.52 (dd, 1 H, J ¼ 14.8, 3.7 Hz, H-9a), 5.47 (d,
1 H, J ¼ 14.8 Hz, H-9b), 4.96 (brs, 1 H, OH-300), 4.71 (d, 1 H, J ¼ 7.7 Hz,
H-100), 4.68 (brs, 1 H, OH-400), 3.94 (s, 3 H, OCH3), 3.76e3.72 (m, 1 H,
H-200), 3.66 (s, 3 H, OCH3), 3.63e3.60 (m, 1 H, H-500), 3.47e3.46 (m,
1 H, H-400), 3.43e3.42 (m, 1 H, H-300), 1.18 (dd, 3 H, J ¼ 6.4, 1.2 Hz,
(C-30), 146.8 (C-40), 144.8 (C-7), 134.9 (C-70), 129.9 (C-1) 129.6 (C-8),
128.2 (C-10), 126.7 (C-2), 123.5 (C-60), 118.8 (C-80), 110.9 (C-20), 107.9
(C-3), 105.4 (C-100), 105.0 (C-50), 101.7 (C-6), 101.1 (OCH2O), 77.2 (C-
300), 76.3 (C-500), 73.7 (C-200), 70.0 (C-400), 67.3 (C-9), 61.2 (C-600), 55.8
(OCH3), 55.2 (OCH3); HRMS calcd for C27H27O12 543.1503, found
543.1481.
CH3); 13C NMR (150 MHz, DMSO-d6):
d 169.1 (C]O), 151.3 (C-5),
5.1.1.4. 4-O-
(CHCl3:MeOH, 20:1), [
(600 MHz, DMSO-d6) :
b
-
D
-xylosyldiphyllin (8). White solid, yield 63.3%, Rf 0.28
149.9 (C-4), 146.9 (C-40), 146.8 (C-30), 145.0 (C-7), 134.8 (C-70), 129.9
(C-1), 129.7 (C-8), 128.2 (C-10), 126.9 (C-2), 123.5 (C-60), 118.6 (C-80),
110.8 (C-20), 107.9 (C-3), 105.7 (C-100), 105.3 (C-5), 101.8 (C-6), 101.1
(OCH2O), 73.2 (C-300), 70.8 (C-400), 70.5 (C-200), 70.4 (C-500), 67.1 (C-
9), 55.7 (OCH3), 55.1 (OCH3), 16.5 (CH3); HRMS calcd for C27H27O11
527.1553, found 527.1576.
25
a]
D
¼ ꢀ24.4ꢁ (c1.00, MeOH); 1H NMR
d
8.12 (d, 1 H, J ¼ 2.3 Hz, ArH), 7.04 (d, 1 H,
J ¼ 7.8 Hz, ArH), 6.98 (d, 1 H, J ¼ 5.5 Hz, ArH), 6.92 (d, 1 H, J ¼ 1.4 Hz,
ArH), 6.80e6.78 (m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.76 (dd, 1 H,
J ¼ 5.5, 4.6 Hz, OH-400), 5.51 (dd,1 H, J ¼ 15.1, 2.3 Hz, H-9a), 5.45 (d,1 H,
J ¼ 15.1 Hz, H-9b), 5.26 (d, 1 H, J ¼ 5.0 Hz, OH-300), 5.10 (d, 1 H,
J ¼ 5.0 Hz, OH-200), 4.76 (d, 1 H, J ¼ 7.8 Hz, H-100), 3.94 (s, 3 H, OCH3),
3.81(dd,1 H, J¼ 11.5, 4.6 Hz, H-500a), 3.67 (s, 3 H, OCH3), 3.49e3.43(m,
2 H, H-200, H-400), 3.27e3.24 (m,1 H, H-300), 3.15e3.11 (m,1 H, H-500b);
5.1.1.8. 60-O-Benzyl-4-O-
solid, yield 66.6%, Rf 0.25 (CHCl3:MeOH,10:1), [
CHCl3); 1H NMR (600 MHz, CDCl3) :
b
-D
-glucopyranosyldiphyllin
(13). White
25
a]
¼ ꢀ36.5ꢁ (c1.00,
D
d
7.91 (s, 1 H, ArH), 7.34e7.32
13C NMR (150MHz, DMSO-d6):
d
169.0 (C]O),151.4(C-5),149.9 (C-4),
(m, 2 H, ArH), 7.29e7.27 (m, 2 H, ArH), 7.26 (s, 1 H, ArH), 7.05 (d, 1 H,
J ¼ 2.2 Hz, ArH), 6.93 (dd, 1 H, J ¼ 7.7, 5.0 Hz, ArH), 6.81e6.74 (m,
2 H, ArH), 6.07 (s, 1 H, OCH2O), 6.04 (d, 1 H, J ¼ 3.8 Hz, OCH2O), 5.54
(d,1 H, J ¼ 14.8 Hz, H-9a), 5.40 (d,1 H, J ¼ 14.8, 2.2 Hz, H-9b), 4.81 (d,
1 H, J ¼ 7.7 Hz, H-100), 4.54 (dd, 2 H, J ¼ 20.3, 12.1 Hz, PhCH2), 4.00 (s,
3 H, OCH3), 3.86 (t, 1 H, J ¼ 8.8 Hz, H-500), 3.78 (s, 3 H, OCH3), 3.71 (d,
2 H, J ¼ 3.3 Hz, H-200, H-400), 3.67e3.66 (m, 1 H, H-600a), 3.60 (t, 1 H,
146.9(C-40),146.8(C-30),144.6(C-7),134.9(C-70),129.7(C-1),129.6(C-
8),128.1 (C-10),126.7 (C-2),123.5 (C-60) 118.6 (C-80),110.8 (C-20),107.9
(C-3) 105.7 (C-100),105.4 (C-50),101.6(C-6),101.1 (OCH2O), 76.3 (C-300),
73.5 (C-200), 69.3 (C-400), 67.0 (C-9), 65.9 (C-500), 55.8 (OCH3), 55.2
(OCH3); HRMS calcd for C26H25O11 513.1397, found 513.1396.
5.1.1.5. 60-O-Methyl-4-O-
solid, yield 78.6%, Rf 0.26 (CHCl3:MeOH,10:1), [
CH2Cl2); 1H NMR (600 MHz, DMSO-d6) :
ArH), 7.04 (d, 1 H, J ¼ 7.8 Hz, ArH), 6.98 (s, 1 H, ArH), 6.92 (d, 1 H,
J ¼ 1.8 Hz, ArH), 6.82e6.78 (m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 6.01
(d, 1 H, J ¼ 5.5 Hz, OH-200), 5.67 (dd, 1 H, J ¼ 15.1, 2.8 Hz, H-9a), 5.46
(dd, 1 H, J ¼ 15.1, 2.3 Hz, H-9b), 5.30 (d, 1 H, J ¼ 5.0 Hz, OH-400), 5.22
(d, 1 H, J ¼ 5.5 Hz, OH-300), 4.76 (d, 1 H, J ¼ 7.8 Hz H-100), 3.95 (s, 3 H,
OCH3), 3.67 (s, 3 H, OCH3), 3.64e3.62 (m,1 H, H-600a), 3.49e3.43 (m,
2 H, H-200, H-600b), 3.41e3.39 (m, 1 H, H-500), 3.31e3.28 (m, 1 H, H-
300), 3.26 (s, 3 H, OCH3), 3.18e3.14 (m, 1 H, H-400); 13C NMR
b
-D
-glucopyranosyldiphyllin
(9). White
J ¼ 8.8 Hz, H-600b); 13C NMR (150 MHz, CDCl3):
d 170.1 (C]O), 152.0
25
a]
¼ ꢀ75.5ꢁ (c1.00,
(C-5), 150.1 (C-4), 147.5 (C-30), 144.2 (C-40), 137.0 (C-7), 136.7 (C-70),
130.7 (C-1), 130.4 (C-8), 128.6 (C-10), 128.5 (C-Bn), 128.4 (C-Bn),
128.2 (C-Bn), 128.0 (C-Bn), 127.9 (C-Bn), 127.9 (C-2), 127.1 (C-Bn),
123.5 (C-60), 119.0 (C-80), 110.6 (C-20), 108.1 (C-50), 106.1 (C-3), 104.5
(C-100), 101.2 (C-6), 100.8 (OCH2O), 77.2 (C-PhCH2), 73.8 (C-300), 73.6
(C-500), 71.7 (C-200), 70.1 (C-400), 67.5 (C-9), 56.2 (OCH3-5), 55.8
(OCH3-4); HRMS (ESI) calcd for C34H33O12 (M þ H)þ m/z 633.1972
found 633.1968.
D
d
8.16 (d, 1 H, J ¼ 3.7 Hz,
5.1.1.9. 60-O-Benzyl-4-O-
solid, yield 70.2%, Rf 0.60 (CHCl3:MeOH,10:1), [
CHCl3), 1H NMR (600 MHz, CDCl3) :
d 7.94 (s, 1 H, ArH), 7.33e7.31
b-D-galactopyranosyldiphyllin (14). White
25
(150 MHz, DMSO-d6):
d
169.1 (C]O), 151.4 (C-5), 149.9 (C-4), 146.8
a]
¼ ꢀ25.4ꢁ (c1.00,
D
(C-40), 146.7 (C-30), 144.8 (C-7), 134.9 (C-70), 129.8 (C-1), 129.6 (C-8),
128.2 (C-10), 126.8 (C-2), 123.5 (C-60), 118.7 (C-80), 110.8 (C-20), 107.9
(C-3), 105.3 (C-100), 105.0 (C-5), 101.7 (C-6), 101.1 (OCH2O), 76.2 (C-
300), 74.9 (C-500), 73.6 (C-200), 72.2 (C-600), 69.9 (C-400), 67.2 (C-9), 58.3
(OCH3), 55.7 (OCH3), 55.2 (OCH3); HRMS calcd for C28H29O12
557.1659, found 557.1651.
(m, 2 H, ArH), 7.26 (s, 2 H, ArH), 7.02 (d, 1 H, J ¼ 6.4 Hz, ArH), 6.93 (t,
1 H, J ¼ 6.9 Hz, ArH), 6.80e6.72 (m, 3 H, ArH), 6.07 (s, 1 H, OCH2O),
6.04 (d, 1 H, J ¼ 4.1 Hz, OCH2O), 5.58 (d, 1 H, J ¼ 15.1 Hz, H-9a), 5.45
(dd, 1 H, J ¼ 15.1, 2.3 Hz, H-9b), 4.77 (d, 1 H, J ¼ 7.8 Hz, H-100), 4.52
(dd, 2 H, J ¼ 11.5, 1.4 Hz, OCH2), 4.16e4.13 (m, 1 H, H-200), 4.05 (brs,
1 H, H-400), 3.99 (s, 3 H, OCH3), 3.78 (s, 3 H, OCH3), 3.75 (m, 1 H, H-
300), 3.63e3.62 (m, 2 H, H-6a, H-6b), 3.58 (t, 1 H, J ¼ 5.0 Hz, H-500);
HRMS (ESI) calcd for C34H33O12 (M þ H)þ m/z 633.1972 found
633.1975.
5.1.1.6. 60-O-Methyl-4-O-
solid, yield 58.2%, Rf 0.29 (CHCl3:MeOH,10:1), [
CH2Cl2); 1H NMR (600 MHz, DMSO-d6) :
b-D-galactopyranosyldiphyllin (10). White
25
a]
¼ ꢀ65.7ꢁ (c1.00,
D
d
8.19 (d, 1 H, J ¼ 3.2 Hz,

430
D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
5.1.2. General procedure of the diphyllin glycoside compounds
(15e17)
5.2. Biology evaluation
To the solution of 7 (108 mg, 0.2 mmol) in dried CH3CN (10 mL)
was added TsOH (2 mg, 0.01 mmol) and corresponding acetals
(0.3 mmol). The mixture stirred at room temperature for 5 h,
quenched by Et3N (2 drops), The solvent was removed under
vacuum, and the resdue was chromatographed on silica gel (EtOAc :
petroleum ether, 2:1) to get the corresponding derivatives.
5.2.1. Cell growth inhibition assays and multidrug resistance
analysis
Sulforhodamine B (SRB) assay [18]: cells in the logarithmic
phase were seeded to 6 well plate and cultured over night, followed
treating with the corresponding drug for 72 h (Setting 5 above
concentration gradient and
3 double-pored holes for each
concentration). Culture solution was dumped after the drug treat-
ment, and the cells were then treated with 10% precooled TCA in
each well. After that, cells were fixed for 1 h at 4 ꢁC, then washed 5
times with distilled water and dried in the air. 100 mL of 4 mg/ml
SRB (Sigma) in 1% acetic acid was added to each well for staining
over 15 min. Then plate was washed 5 times with 1% acetic acid and
5.1.2.1. 40, 60-ethydenide-4-O-
b-D-glucopyranosyldiphyllin (15).
White solid, yield 73.2%, Rf 0.26 (EtOAc:petroleum ether, 2:1),
25
[
a]
¼ ꢀ67.7ꢁ (c1.00, CH2Cl2), 1H NMR (600 MHz, CDCl3) :
d
7.92 (s,
D
1 H, ArH), 7.02 (d, 1 H, J ¼ 2.8 Hz, ArH), 6.91 (d, 1 H, J ¼ 7.8 Hz, ArH),
6.79e6.75 (m, 1 H, ArH), 6.67e6.66 (m, 1 H, ArH), 6.08 (d, 1 H,
J ¼ 1.4 Hz, OCH2O), 6.03 (d, 1 H, J ¼ 1.4 Hz, OCH2O), 5.36 (s, 2 H, H-
9a, 9b), 4.88 (d, 1 H, J ¼ 7.3 Hz, H-100), 4.76e4.74 (m, 1 H, CH),
4.11e4.08 (m, 1 H, H-600a), 4.01 (s, 3 H, OCH3), 3.91e3.89 (m, 1 H, H-
200), 3.81e3.80 (m, 1 H, H-300), 3.78 (s, 3 H, OCH3), 3.61e3.57 (m, 1 H,
H-600b), 3.46e3.43 (m, 1 H, H-400), 3.24e3.20 (m, 1 H, H-500), 1.36 (d,
dried. SRB in the cells was dissolved in 150 mL of 10 mM TriseHCl
and was measured at 520 nm using a multiwell spectrophotometer.
The inhibition rate on cell proliferation was calculated as (OD520
control - OD520 treated)/OD520 control ꢃ 100%. IC50 value was ob-
tained by Logit method and was determined at least 3 independent
tests. The resistance factor (RF) to each drug was calculated as the
ratio of the IC50 value of resistant cells to that of parental cells.
Methyl Thiazolyl Tetrazolium (MTT) Assay [17]: cells in the
3 H, J ¼ 5.0 Hz, CH3); 13C NMR (150 MHz, CDCl3):
d 169.9 (C]O),
151.9 (C-5), 150.1 (C-4), 147.4 (C-30), 147.3 (C-40), 144.8 (C-7), 136.7
(C-70), 130.9 (C-1) 130.6 (C-8), 128.0 (C-10), 127.0 (C-2), 123.5 (C-60),
118.8 (C-80), 110.6 (C-20), 108.1 (C-50), 106.0 (C-3), 104.9 (C-100), 101.2
(C-6), 100.8 (OCH2O), 99.7 (CeCH), 79.4 (C-400), 74.7 (C-300), 73.7
(C-200), 67.8 (C-9), 67.2 (C-500), 66.4 (C-600), 56.2 (OCH3), 55.7
(OCH3), 20.1 (CeCH3); HRMS calcd for C29H29O12 569.1654, found
569.1661.
logarithmic phase were transferred into 96 well plates in 100
medium and incubated for 24 h. Followed by treating the corre-
sponding drug similar as above. After 72 h of drug exposure, 20
of MTT reagent (5 mg/mL) was added to each well and incubated for
4 h at 37 ꢁC. 100
L of Tris buffer (10% SDS-5% isobutanol-0.01 mol/L
mL of
mL
m
HCl) was added to each microtiter well and incubated for 12e20 h
at the same temperature, then measured at 570 nm using a multi-
well spectrophotometer. The IC50 value and resistance factor (RF)
calculation was similar as the above.
5.1.2.2. 40,
60-Acetonide-4-O-
b-D
-glucopyranosyldiphyllin
(16).
White solid, yield 71.4%, Rf 0.21 (EtOAc:petroleum ether, 2:1),
25
[
a]
¼ 23.1ꢁ (c1.00, CH2Cl2), 1H NMR (600 MHz, DMSO-d6) :
d 8.14
D
(d, 1 H, J ¼ 4.1 Hz, ArH), 7.04 (dd, 1 H, J ¼ 7.8, 2.8 Hz, ArH), 6.98 (d,
1 H, J ¼ 3.2 Hz, ArH), 6.93 (d, 1 H, J ¼ 1.4 Hz, ArH), 6.81e6.78 (m, 1 H,
ArH), 6.21 (d,1 H, J ¼ 5.2 Hz, OH-200), 6.13 (s, 2 H, OCH2O), 5.48e5.42
(m, 3 H, H-9a, H-9b, OH-300), 4.91 (d, 1 H, J ¼ 7.8 Hz, H-100), 3.94 (s,
3 H, OCH3), 3.86e3.83 (m, 1 H, H-600a), 3.78e3.74 (m, 1 H, H-500),
3.66 (s, 3 H, OCH3), 3.57e3.52 (m, 2 H, H-600b, H-200), 3.45e3.41 (m,
1 H, H-400), 3.32e3.27 (m, 1 H, H-300), 1.48 (s, 3 H, CH3), 1.31 (s, 3 H,
5.2.2. Drug inhibition of kDNA decatenation induced by
topoisomerase II
Topo II activity was measured by the ATP-dependent decatena-
tion of kDNA [22]. The standard reaction mixture was a total volume
of 15
5 mM MgCl2, 1 mM ATP, 0.5 mM dithiothreitol (DTT), 0.5 mM EDTA,
50 mg/mL of BSA, 20 g/mL of kDNA, 1 unit of Topo II and the cor-
responding compound. After incubation at 37 ꢁC for 15 min, the
reaction was terminated by addition of 1 L of 10% SDS. Electro-
mL which containing with 50 mM TriseHCl (pH 7.7), 50 mM KCl,
CH3); 13C NMR (150 MHz, DMSO-d6):
d
169.9 (C]O), 151.9 (C-5),
m
150.1 (C-4), 147.4 (C-30), 147.3 (C-40), 144.1 (C-7), 136.7 (C-70), 131.0
(C-1) 130.7 (C-8), 128.0 (C-10), 127.0 (C-2), 123.5 (C-60), 118.9 (C-80),
110.6 (C-20), 108.1 (C-50), 106.1 (C-3), 104.8 (C-100), 101.2 (C-6), 100.8
(OCH2O),100.1 (CeCH), 79.5 (C-400), 74.6 (C-300), 73.9 (C-200), 67.8 (C-
9), 67.2 (C-500), 66.7 (C-600), 56.2 (OCH3), 55.8 (OCH3), 32.1 (CeCH3),
30.6 (CeCH3), 24.9 (CeCH3); HRMS calcd for C30H31O12 583.1816,
found 583.1810.
m
phoresis was carried out in a 1% agarose gel in 1 ꢃ TAE (40 mM Tris
base, 40 mM acetate acid and 1 mM EDTA) at 4 V/cm for 1 h. DNA
bands were stained with 0.5 mg/mL of ethidium bromide (E.B.)
solution and photographed through a Gel Document System.
5.2.3. Cell cycle analysis of HL-60 cells [24]
5.1.2.3. 40, 60-Isobutydenide-4-O-
b-
D
-glucopyranosyldiphyllin (17).
HL-60 cells in the logarithmic phase were seeded to 6 well plate
and treated with the corresponding drug for 24 h. Trypsin was
digested and collected into 1.5 mL centrifuge tubes, Cells were
harvested, followed by washing once with cold phosphate buffer
White solid, yield 60.3%, Rf 0.30 (EtOAc: petroleum ether, 2:1),
25
[
a]
¼ ꢀ20.2ꢁ (c1.00, CH2Cl2), 1H NMR (600 MHz, CDCl3) :
d 7.95 (s,
D
1 H, ArH), 7.08 (brs, 1 H, ArH), 6.96e6.93 (m, 1 H, ArH), 6.84e6.82
(m, 1 H, ArH), 6.81e6.77 (m, 1 H, ArH), 6.09 (s, 1 H, OCH2O), 6.05 (d,
1 H, J ¼ 3.7 Hz, OCH2O), 5.40 (s, 2 H, H-9a, 9b), 4.93 (d, 1 H,
J ¼ 7.4 Hz, H-100), 4.32 (d, 1 H, J ¼ 4.6 Hz, CH), 4.13e4.11 (m, 1 H, H-
600a), 4.04 (s, 3 H, OCH3), 3.95e3.92 (m, 1 H, H-200), 3.82e3.81 (m,
1 H, H-300), 3.80 (s, 3 H, OCH3), 3.59e3.56 (t, 1 H, J ¼ 10.1 Hz, H-600b),
3.23e3.21 (m, 2 H, H-400, H-500), 1.88e1.82 (m, 1 H, CH), 0.94 (t, 6 H,
saline (PBS). Adding 300
and fixed overnight in 70% ethanol at 4 ꢁC. The mixture was spun
for 5 min at 300 g, resuspended with 500 L PBS containing 10 g/
mL RNase for 15 min at 37 ꢁC, and then added 2
g/mL PI. Staining
mL PBS and the cells were resuspended,
m
m
m
at room temperature in dark for 30 min. Filtered and then analyzed
using an FACS-Calibur cytometer. At least 10,000 events were
counted for each sample.
J ¼ 7.3 Hz, 2 ꢃ CH3); 13C NMR (150 MHz, CDCl3):
d 169.9 (C]O),
151.9 (C-5), 150.1 (C-4), 147.4 (C-30), 147.3 (C-40), 144.1 (C-7), 136.7
(C-70), 131.00 (C-1) 130.7 (C-8), 128.0 (C-10), 127.0 (C-2), 123.5 (C-60),
118.9 (C-8 ), 110.6 (C-20), 108.1 (C-50), 106.1 (C-3), 106.0 (CeCH),
104.8 (C-100), 101.2 (C-6), 100.8 (OCH2O), 79.5 (C-400), 74.6 (C-300),
73.9 (C-200), 67.8 (C-9), 67.2 (C-500), 66.7 (C-600), 56.2 (OCH3), 55.8
(OCH3), 32.1 (CeCH3), 17.2 (CeCH3), 16.8 (CeCH3); HRMS calcd for
C31H33O12 597.1972, found 597.1964.
5.2.4. DNA fragmentation electrophoresis
The HL-60 cells were seeded into a six well plate. The corre-
sponding drugs was added at the 10 mM concentrations for 36 h.
DNA fragmentation was extracted using the method as described
[23,24]. Concisely, harvested cells were lysed by the addition of an
equal volume of 1.2% SDS. By adding 350 mL precipitation solution

D.-K. Shi et al. / European Journal of Medicinal Chemistry 47 (2012) 424e431
431
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