Synthesis and Bioevaluation of Diphyllin Glycosides as Novel Anticancer Agents

Article abstract of DOI:10.1002/ardp.201200035

A series of diphyllin glycosides were synthesized from diphyllin by phase transfer catalysis glycosylation, deprotection, and etherification, and the structures were established by ¹H NMR, ¹³C NMR, and HRMS. These glycosides were evaluated for their in vitro cytotoxicity against HCT-116, A549, and A549T cancer cell lines by MTT assay, and most of them were cytotoxic at submicromolar concentrations. They were also effective against the paclitaxel-resistant cell line A549T. The kDNA decatenation assay indicated that most of these compounds inhibited topoisomerase IIα-mediated kDNA decatenation. In addition, the in vitro tubulin polymerization study showed that compounds 5 and 6 had antimicrotubule activity with a paclitaxel-like mode of action. Taken together, these results suggest that these diphyllin glycosides act on both TopoII and tubulin. Copyright

Full text of DOI:10.1002/ardp.201200035

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
1
Full Paper
Synthesis and Bioevaluation of Diphyllin Glycosides
as Novel Anticancer Agents
Yu Zhao, Chunyan Ni, Yuting Zhang, and Li Zhu
Institute of Nautical Medicine, Nantong University, Nantong, People’s Republic of China
A series of diphyllin glycosides were synthesized from diphyllin by phase transfer catalysis
glycosylation, deprotection, and etherification, and the structures were established by 1H NMR,
13C NMR, and HRMS. These glycosides were evaluated for their in vitro cytotoxicity against HCT-
116, A549, and A549T cancer cell lines by MTT assay, and most of them were cytotoxic at
submicromolar concentrations. They were also effective against the paclitaxel-resistant cell line
A549T. The kDNA decatenation assay indicated that most of these compounds inhibited
topoisomerase IIa-mediated kDNA decatenation. In addition, the in vitro tubulin polymerization
study showed that compounds 5 and 6 had antimicrotubule activity with a paclitaxel-like mode
of action. Taken together, these results suggest that these diphyllin glycosides act on both TopoII and
tubulin.
Keywords: Cytotoxicity / Diphyllin glycosides / Synthesis / TopoII / Tubulin
Received: January 29, 2012; Revised: March 16, 2012; Accepted: March 21, 2012
DOI 10.1002/ardp.201200035
Introduction
moiety endows D11 with an optimal conformation with a
high binding affinity for TopoII via hydrogen bonding to the
entrance of the ATPase pocket [9, 10]. In a docking simulation,
the sugar moiety is hydrogen-bonded to the residue Arg98 at
the entrance of the ATP-binding pocket. In this hydrogen
bond interaction, oxygen atoms of the acetyl groups act as
hydrogen bond donor. Therefore, sugar moieties and acetyl
‘‘protecting groups’’ play very important roles in its potential
inhibitory activity on TopoII, which promoted us to design
and synthesize more diphyllin glycosides possessing various
sugar moieties bearing ester or ether to search for potential
TopoII inhibitors.
DNA-topoisomerase II (TopoII) controls DNA topology by tran-
sient cleavage of the DNA double helix, which has been
established as important molecular target of anticancer
drugs [1, 2]. The DNA TopoII inhibitors doxorubicin and
etoposide (Figure 1) are widely used clinically; they exert
their cytotoxic effects by trapping TopoII in covalent com-
plexes with DNA, thereby stabilizing a normally transient
intermediate in the enzyme’s catalytic cycle [3]. However, due
to development of resistance to these TopoII inhibitors
observed frequently in the clinic, a new type of potent inhibi-
tor of TopoII has been desired [4].
Herein, we report the synthesis, cytotoxicity, TopoII inhibi-
tory activity, and tubulin polymerization stimulation activity
of these novel diphyllin glycosides (1–12, Scheme 1).
Diphyllin (Figure 1), an arylnaphthalene lignan lactone,
isolated from many traditional medicinal plants, has been
reported to possess anticancer and antiviral activities [5, 6].
Previously, we have successfully synthesized several natural Results and discussion
diphyllin glycosides and their analogues [7, 8]. Among these
semisynthetic glycosides, an acetylated D-quinovose glycoside
D11 (Figure 1) showed significant inhibitory effects on TopoII.
Further biological assays disclosed that its peculiar sugar
Synthesis of diphyllin glycosides
The synthetic route used to synthesize the title compounds is
outlined in Scheme 1. The starting material diphyllin was
easily prepared for 2-bromo-4,5-dimethoxybenzaldhyde in
five steps in 42% total yield according to the procedure
reported [11]. The phase transfer catalysis (PTC) glycosylations
of diphyllin with per-acetylated glycosyl bromide donors
were performed in 0.1 mol/L aqueous NaOH in the presence
Correspondence: Dr. Li Zhu, Institute of Nautical Medicine, Nantong
University, Nantong 226001, People’s Republic of China.
E-mail: zhulili65@163.com
Fax: þ86 513 85051796
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
Y. Zhao et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Figure 1. Diphyllin, D11, and etoposide.
of tetrabutylammonium bromide (TBAB, 1.0 equiv) at 408C
to give per-acetyl glycosides 1–4, respectively. Subsequently,
deacetylations with K2CO3 in methanol were carried
out smoothly to produce deprotected glycosides 13–16.
Etherification of 13–16 with methyl iodide in the presence
of aqueous NaOH in DMSO were performed to produce per-
methoxy glycosides 5–8 in good yields. However, ethylation
of 13–16 with ethyl bromide in the same conditions failed
to give per-ethyl glycosides. The 1H NMR spectra indicated
several partially ethylated glycosides were produced under
this condition. By in situ produced ethyl iodine with ethyl
bromide and sodium iodide, the ethylation reaction was
successfully conducted which gave per-ethoxyl glycoside as
single product.
Cytotoxicity of compounds 1–12 against four cancer
cell lines
The title compounds were evaluated for their cytotoxic activi-
ties against four cancer cell lines including HCT-116, RM-1,
A549, and A549T (paclitaxel-resistant cell line) using the MTT
growth inhibition assay. The IC50 values are summarized in
Table 1 and represent the concentration inducing 50%
decrease of cell growth after 3 days of incubation.
It is significant to note that these derivatives were more
cytotoxic than etoposide in four cancer cell lines. From the
IC50 values, it is clear that most of these derivatives displayed
potent cytotoxic activities against HCT-116, RM-1, and A549
cancer cell lines with IC50 values in the range of mM–nM.
Their in vitro cytotoxicities against A549 cell line were better
Scheme 1. Synthesis of diphyllin glycosides 1–12.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Diphyllin Glycosides as Novel Anticancer Agents
3
Table 1. Cytotoxicity against four cancer cell lines of glycosides
1–12
displayed inhibitory effect on the catalytic activity of TopoII
at a concentration of 500 mM. Compounds 5, 10, and 12
possessed potent inhibitory activities as the same as etopo-
side. Further potency assay for compound 5 revealed that
it inhibits TopoII in a concentration-dependent manner.
However, three cytotoxic compounds 6, 8, and 9 showed weak
TopoII inhibitory activity at the concentration of 500 mM.
These results indicated that there was no good correlation
between their cytotoxicity and TopoII inhibitory ability. They
might exert their primary cytotoxicity through other modes
of action. Therefore, an in vitro tubulin polymerization assay
was performed on compounds 5 and 6 to investigate their
effect on tubulin.
Compounds
Cancer cell (mM)
HCT116
RM-1
A549
A549/T
1
2
3
4
5
6
7
8
9
10
11
12
8.95
5.53
5.58
6.93
0.12
1.09
0.07
0.16
9.31
0.74
3.38
0.71
1.10
13.5
1.68
1.19
1.24
1.09
1.04
1.20
0.11
0.95
1.20
1.10
1.16
1.12
0.11
24
0.62
0.86
0.69
0.72
0.05
0.01
0.21
0.06
0.16
0.76
0.07
0.71
0.10
21.40
13.22
9.08
9.26
8.51
0.69
0.87
0.87
0.89
8.55
8.37
8.97
4.77
108.14
85.85
Stimulation of tubulin polymerization in vitro by
glycosides 5, 6, and paclitaxel
Paclitaxel
Etoposide
Effects of glycosides 5 and 6 on polymerization of porcine
brain tubulin in vitro were assessed and compared with those
of the known tubulin depolymerizing agent colchicine and
tubulin polymerizing agent paclitaxel [12, 13]. The incu-
bation of purified porcine tubulin with 1, 10, and 100 mM
of 5 and 6 induced tubulin polymerization as determined by
spectrophotometric measurements of turbidity increases. In
the concentration of 100 mM, glycosides 5 and 6 caused an
apparent increase in the rate and extent of microtubule
assembly (Fig. 3). Hence, 5 and 6 seemed to stabilize micro-
tubules (like paclitaxel) rather than destabilize them (like
colchicine). Taken together, these results suggest that these
diphyllin glycosides act on both TopoII and tubulin [14].
than those of HCT-116 and RM-1 cell lines. Per-methoxyl
glycosides 5–8 exhibited potent cytotoxic activity with IC50
values in the range of 0.01–0.21 mM in the A549 cell line
while other series showed moderate activities. Furthermore,
glycosides 5–8 were also effective against the A549T cell line
with IC50 values in the range of 0.69–0.89 mM, which were by
far lower than those of paclitaxel and etoposide. These results
disclosed that the methoxyl groups played an important role
for the cytotoxicity of these compounds.
Inhibition of human topoisomerase II kDNA
decatenation by compounds 1–12
Conclusion
To further understand the action mechanisms of antitumor
activity of these compounds, their effect on the catalytic
activity of human TopoII was evaluated using kDNA decate-
nation assay. As shown in Fig. 2, most of the compounds
In summary, novel diphyllin glycosides containing various
sugar moieties were synthesized and evaluated for their
cytotoxic activities against four cancer cell lines. These
Figure 2. Effects of diphyllin glycosides and
etoposide on human TopoII-mediated decate-
nation activity of kDNA.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
Y. Zhao et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Figure 3. Effects of compounds 5 and 6 on tubulin polymerization in vitro. The polymerization of porcine brain tubulin was monitored in the
presence of 5, 6, paclitaxel, and colchicine at concentrations ranging from 1 to 100 mmol/L. Absorbance at 340 nm was recorded at 378C
every minute for 60 min. A for compound 5; B for compound 6.
compounds displayed potent cytotoxic activities against
the four cancer cell lines with IC50 value in the range of
mM–nM. Per-methoxyl glycosides exhibited more potent
cytotoxic activity than other series, and they were also
effective on the A549T cell line. The kDNA decatenation assay
indicated most of compounds displayed inhibitory effect on
the catalytic activity of TopoII. However, their cytotoxicity
did not match well with their TopoII inhibitory ability.
Further in vitro tubulin polymerization assay showed that
compounds 5 and 6 had antimicrotubule activity with a
paclitaxel-like mode of action. These diphyllin glycosides
exerted their cytotoxicity through targeting both TopoII
and tubulin.
4-O-(20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl)
diphyllin (1)
20
White solid, yield 72.9%; ½aꢁD ꢂ24 (c 0.3, CHCl3); IR nmax
(KBr, cmꢂ1): 707, 808, 929, 1165, 1226, 1263, 1369, 1454,
1507, 1619, 1753, 2920, 2959, 3089; 1H NMR (CDCl3): d 7.50
(s, 1 H, ArH), 7.05 (s, 1 H, ArH), 6.94 (d, J ¼ 7.8 Hz, 1 H, ArH),
6.78 (t, J ¼ 3.9 Hz, 2 H, ArH), 6.06 (d, J ¼ 12.3 Hz, 2 H, CH2), 5.52
(d, J ¼ 8.4 Hz, 1 H, CH), 5.47 (t, J ¼ 3.9 Hz, 1 H, CH), 5.41
(dd, J ¼ 1.8, 14.7 Hz, 1 H, CH), 5.31 (t, J ¼ 9.3 Hz, 1 H, CH),
5.2–5.14 (m, 2 H, CH2), 4.3 (q, J ¼ 6, 12.3 Hz, 1 H, CH),
4.14–4.08 (m, 2 H, CH2), 4.04 (s, 3 H, OCH3), 3.78 (s, 3 H,
OCH3), 2.09 (s, 3 H, CH3), 2.09 (s, 3 H, CH3), 2.04 (s, 3 H, CH3),
2.03 (s, 3 H, CH3), 2.02 (s, 3 H, CH3); 13C NMR (CDCl3): d 170.4,
170.1, 169.4, 169.3, 152.0, 150.5, 147.6, 144.0, 136.4, 130.7, 128.1,
126.9, 126.2, 123.5, 119.2, 110.6, 108.2, 106.2, 101.3, 100.9, 72.6,
71.7, 68.2, 66.8, 61.9, 56.2, 55.9, 21.0, 20.9, 20.6; EIMS
calcd for C35H34O16 (MþNa)þ 733.1747, found 733.1739; mp
129–1338C from ethanol.
Experimental
4-O-(20,30,40,60-Tetra-O-acetyl-b-D-galactopyranosyl)
diphyllin (2)
Syntheses
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 (200–300 mesh, Qingdao, China).
1H NMR and 13C NMR spectra were taken on a Bruker 300 MHz
spectrometer with tetramethylsilane (TMS) as an internal
standard, and chemical shifts were recorded in d values. The
high resolution spectra were obtained on a Q-TOF Global Mass
(ESIMS).
20
White solid, yield 69.4%; ½aꢁD þ43 (c 0.3, CHCl3); IR nmax
(KBr, cmꢂ1): 713, 810, 928, 1174, 1224, 1266, 1369, 1455,
1505, 1626, 1753, 2893, 2937, 3102; 1H NMR (CDCl3): d 7.51
(s, 1 H, ArH), 7.03 (s, 1 H, ArH), 6.94 (d, J ¼ 7.8 Hz, 1 H, ArH),
6.78–6.74 (m, 2 H, ArH), 6.06 (d, J ¼ 11.4 Hz, 2 H, CH2), 5.72
(t, J ¼ 7.8 Hz, 1 H, CH), 5.51 (t, J ¼ 15 Hz, 2 H, CH2), 5.46
(d, J ¼ 18.9 Hz, 1 H, CH), 5.35 (d, J ¼ 15.2 Hz, 1 H, CH),
5.16–5.09 (m, 2 H, CH2), 4.06 (s, 3 H, OCH3), 3.78 (s, 3 H,
OCH3), 2.20 (s, 3 H, CH3), 2.09 (s, 9 H, CH3), 2.06 (s, 3 H, CH3);
13C NMR (CDCl3): d 170.4, 170.1, 169.9, 169.6, 151.9, 150.5, 147.6,
144.3, 136.1, 130.7, 128.2, 125.8, 125.6, 123.5, 119.3, 110.7, 108.2,
106.1, 101.3, 100.6, 71.9, 70.7, 69.1, 67.0, 61.9, 56.3, 55.8, 21.0,
20.7, 20.6, 20.5; EIMS calcd for C35H34O16 (MþNa)þ 733.1747,
found 733.1739; mp 143–1468C from ethanol.
Synthesis of per-acetyl diphyllin glycosides 1–4
Typical procedure: To the solution of diphyllin (0.53 mmol) and
TBAB (0.53 mmol) in CHCl3 (20 mL) aqueous 0.1 mol/L NaOH
(20 mL) was added. After stirring for 10 min at 408C, per-acetyl
glycosyl bromide (302 mg, 0.79 mmol) was added, the two-phase
mixture was stirred for 6 h at 408C. Then CHCl3 (20 mL)
was added and the resulting organic phase was washed with
brine (2 ꢀ 20 mL), dried Na2SO4, and the solvent was evaporated
under reduce pressure. The residue was purified by flash chroma-
tography (EtOAc/petroleum ether, 1:2) to afford 1–4 as white
solid.
4-O-(20,30,40-Tri-O-acetyl-b-D-xylopyranosyl) diphyllin (3)
20
White solid, yield 65.3%; ½aꢁD ꢂ27 (c 0.3, CHCl3); IR nmax
(KBr, cmꢂ1): 713, 811, 931, 1166, 1229, 1261, 1366, 1453,
1503, 1621, 1755, 2925, 2965, 3081; 1H NMR (CDCl3): d 7.49
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Diphyllin Glycosides as Novel Anticancer Agents
5
(s, 1 H, ArH), 7.04 (s, 1 H, ArH), 6.94 (d, J ¼ 7.8 Hz, 1 H, ArH),
6.79–6.73 (m, 2 H, ArH), 6.06 (d, J ¼ 13.2 Hz, 2 H, CH2), 5.46
(dd, J ¼ 1.8, 7.5 Hz, 1 H, CH), 5.41 (d, J ¼ 3.0 Hz, 2 H, CH2),
5.32 (t, J ¼ 8.7, 1 H, CH), 5.12 (d, J ¼ 7.5 Hz, 1 H, CH), 5.09
(s, 1 H, CH), 4.12–4.03 (m, 2 H, CH2), 4.05 (s, 3 H, –OCH3), 3.78
(s, 3 H, OCH3), 2.10 (s, 3 H, CH3), 2.08 (s, 3 H, CH3), 2.05 (s, 3 H, CH3);
13C NMR (CDCl3): d 170.1, 169.8, 169.4, 152.0, 150.4, 147.6, 143.9,
136.4, 130.8, 128.2, 127.8, 126.3, 123.6, 119.3, 110.6, 108.2, 106.3,
101.5, 100.4, 71.7, 71.4, 68.7, 66.8, 62.8, 60.4, 56.3, 55.9, 21.1,
20.9, 20.7; EIMS calcd for C32H340O14 (MþNa)þ 661.1537, found
661.1529; mp 105–1088C from ethanol.
3.66–3.65 (m, 1 H, CH), 3.60–3.58 (m, 2 H, CH2), 3.48–3.44 (m, 1 H,
CH), HRMS, calcd for C27H27O12 543.1503, found 543.1476.
4-O-b-D-Xylopyranosyl) diphyllin (15)
White solid, yield 80.5%, 1H NMR (DMSO): 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 (dd, 1 H, J ¼ 14.6, 1.4 Hz, ArH), 6.80–
6.78 (m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.76 (t, 1 H,
J ¼ 5.5 Hz, OH), 5.51 (dd, 1 H, J ¼ 15.1, 2.3 Hz, CH2), 5.45 (d, 1
H, J ¼ 15.1 Hz, CH2), 5.26 (d, 1 H, J ¼ 5.0 Hz, OH), 5.10 (dd, 1 H,
J ¼ 5.0, 1.4 Hz, OH), 4.76 (d, 1 H, J ¼ 7.8 Hz, CH), 3.94 (s, 3 H,
OCH3), 3.81 (dd, 1 H, J ¼ 11.5, 4.6 Hz, CH), 3.67 (s, 3 H, OCH3),
3.49–3.43 (m, 2 H, CH2), 3.27–3.24 (m, 1 H, CH), 3.15–3.11 (m, 1 H,
CH); HRMS, calcd for C27H27O12 513.1397, found 513.1396.
4-O-(20,30,40-Tri-O-acetyl-a-L-arabinopyranosyl) diphyllin (4)
20
White solid, yield 69.2%; ½aꢁD ꢂ13 (c 0.3, CHCl3); IR nmax (KBr,
cmꢂ1): 715, 808, 932, 1164, 1225, 1263, 1379, 1457, 1506, 1590,
1749, 2936, 3083; 1H NMR (CDCl3): d 7.50 (s, 1 H, ArH), 7.04 (s, 1 H,
ArH), 6.94 (d, J ¼ 7.8 Hz, 1 H, ArH), 6.78–6.74 (m, 2 H, ArH), 6.06
(d, J ¼ 13.5 Hz, 2 H, –CH2–), 5.67 (dd, J ¼ 7.2, 9.6 Hz, 1 H, CH),
5.43 (d, J ¼ 6 Hz, 2 H, –CH2–), 5.35 (s, 1 H, CH), 5.16 (dd, J ¼ 3.3,
9.3 Hz, 1 H, CH), 5.08 (d, J ¼ 7.2 Hz, 1 H, CH), 4.06 (s, 3 H, OCH3),
3.78 (s, 3 H, OCH3), 4.13–3.67 (m, 2 H, CH2), 2.20 (s, 3 H, CH3), 2.09
(s, 3 H, CH3), 2.06 (s, 3 H, CH3); 13C NMR (CDCl3): d 170.2, 170.1,
169.5, 169.4, 151.9, 150.4, 147.5, 144.2, 136.2, 130.7, 128.2, 127.2,
126.2, 123.5, 119.3, 110.6, 108.2, 106.2, 101.4, 100.5, 70.2, 69.4,
67.3, 66.9, 63.9, 56.3, 55.9, 20.9, 20.7; EIMS calcd for C32H340O14
(MþNa)þ 661.1537, found 661.1529; mp 143–1468C from
ethanol.
4-O-a-L-Arabinopyranosyl diphyllin (16)
White solid, yield 83.1%, 1H NMR (DMSO): d 8.17 (d, 1 H,
J ¼ 2.3 Hz, ArH), 7.04 (d, 1 H, J ¼ 8.3 Hz, ArH), 6.93–6.92 (m, 1
H, ArH), 6.80–6.78 (m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.76 (t, 1 H,
J ¼ 5.5 Hz, OH), 5.53 (dd, 1 H, J ¼ 15.1, 2.3 Hz, CH2), 5.45 (d, 1 H,
J ¼ 15.1 Hz, CH2), 5.04 (d, 1 H, J ¼ 5.5 Hz, OH), 4.75 (d, 1 H,
J ¼ 3.2 Hz, OH), 4.71 (d, 1 H, J ¼ 6.8 Hz, CH), 3.95 (s, 3 H,
OCH3), 3.85 (dd, 1 H, J ¼ 13.7, 6.4 Hz, CH), 3.78 (d, 1 H,
J ¼ 11.4 Hz, CH), 3.71 (s, 1 H, CH), 3.67 (s, 3 H, OCH3), 3.53–
3.51 (m, 1 H, CH), 3.45 (d, 1 H, J ¼ 11.4 Hz, CH); HRMS, calcd
for C27H27O12 513.1397, found 513.1409.
Synthesis of per-methoxy diphyllin glycosides 5–8
Synthesis of intermediates 13–16
Typical procedure: To the solution of glycosides 13–16
(0.5 mmol) in DMSO (20 mL), 50% NaOH (10 mmol) was added.
Then, MeI (10 mmol) was added slowly, and the mixture was
stirred overnight at room temperature. After the reaction was
completed, the mixture was filtered and evaporated under
reduced pressure. The residue was purified by flash chromatog-
raphy (1:3, EtOAc/petroleum ether) to afford white solid.
Typical procedure: The per-acetyl glycosides 1–4 (0.32 mmol)
were dissolved in CH3OH (20 mL), and then K2CO3 (0.72 mmol)
was added. After stirring for 30 min at room temperature, the
solution was neutralized with 1 mol/L HCl, then CHCl3 (20 mL)
was added and the resulting organic phase was washed with
brine (2 ꢀ 20 mL) and dried Na2SO4, then the solvent was evap-
orated under reduced pressure. The residue was purified by flash
chromatography (CHCl3/MeOH, 10:1) to afford 13–16 as white
solid.
4-O-(20,30,40,60-Tetra-O-methoxy-b-D-glucopyranosyl)
diphyllin (5)
20
White solid, yield 55.7%; ½aꢁD þ17 (c 0.3, CHCl3); IR nmax
4-O-b-D-Glucopyranosyl diphyllin (13)
(KBr, cmꢂ1): 714, 812, 930, 1214, 1264, 1390, 1433, 1511,
1624, 1764, 2833, 2933, 3070; 1H NMR (CDCl3): d 7.85 (s, 1 H,
ArH), 7.06 (s, 1 H, ArH), 6.95 (dd, J ¼ 2.7, 8.7 Hz, 1 H, ArH),
6.85–6.76 (m, 2 H, ArH), 6.07 (d, J ¼ 14.1 Hz, 2 H, CH2), 5.56–
5.41 (m, 2 H, CH2), 4.72 (d, J ¼ 7.2 Hz, 1 H, CH), 4.06 (s, 3 H, OCH3),
3.80 (s, 1 H, CH), 3.82 (s, 3 H, OCH3), 3.79 (s, 3 H, OCH3), 3.68 (s, 3 H,
OCH3), 3.54 (s, 6 H, –OCH3), 3.42–3.38 (m, 1 H, CH), 3.32–3.25
(t, J ¼ 8.7 Hz, 1 H, CH), 3.25–3.22 (m, 2 H, CH2), 3.19 (s, 1 H, CH);
13C NMR (CDCl3): d 169.9, 151.9, 150.1, 147.5, 144.5, 136.5, 131.4,
130.8, 128.4, 127.3, 123.6, 119.3, 110.7, 108.2, 106.2, 104.8, 101.3,
100.7, 86.8, 83.9, 79.4, 77.4, 71.2, 67.6, 61.3, 61.0, 60.6, 59.3, 56.1,
55.9; EIMS calcd for C31H34O12 (MþNa)þ 621.1950, found
621.1943; mp 143–1468C from ethanol.
White solid, yield 89.1%, 1H NMR (DMSO): d 8.18 (d, 1 H,
J ¼ 5.0 Hz, ArH), 7.04 (dd, 1 H, J ¼ 7.8, 2.3 Hz, ArH), 6.98 (d, 1
H, J ¼ 4.1 Hz, ArH), 6.93 (dd, 1 H, J ¼ 24.3, 1.9 Hz, ArH), 6.82–6.78
(m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.98 (t, 1 H, J ¼ 4.1 Hz, OH),
5.77 (d, 1 H, J ¼ 15.1 Hz, CH2), 5.48 (dd, 1 H, J ¼ 15.1, 3.7 Hz,
CH2), 5.25 (d, 1 H, J ¼ 4.6 Hz, OH), 5.19 (dd, 1 H, J ¼ 5.5, 1.8 Hz,
OH), 4.75 (d, 1 H, J ¼ 7.8 Hz, CH), 4.71–4.68 (m, 1 H, OH), 3.95 (s, 3
H, OCH3), 3.79–3.77 (m, 1 H, CH), 3.67 (s, 3 H, OCH3), 3.50–3.44
(m, 2 H), 3.31–3.28 (m, 1 H, CH), 3.24–3.22 (m, 1 H, CH), 3.17–3.16
(m, 1 H, CH), HRMS, calcd for C27H27O12 543.1503, found
543.1481.
4-O-b-D-Galactopyranosyl diphyllin (14)
White solid, yield 81.4%, 1H NMR (DMSO): d 8.20 (d, 1 H,
J ¼ 5.0 Hz, ArH), 7.04 (d, 1 H, J ¼ 7.8 Hz, ArH), 6.98 (d, 1 H,
J ¼ 3.2 Hz, ArH), 6.93 (dd, 1 H, J ¼ 21.1, 1.4 Hz, ArH), 6.81–
6.78 (m, 1 H, ArH), 6.13 (s, 2 H, OCH2O), 5.80 (t, 1 H,
J ¼ 5.0 Hz, OH), 5.70 (d, 1 H, J ¼ 14.6 Hz, CH2), 5.49 (dd, 1 H,
J ¼ 14.6, 3.2 Hz, CH2), 5.00 (d, 1 H, J ¼ 5.5 Hz, OH), 4.71 (d, 1 H,
J ¼ 7.8 Hz, CH), 4.63 (d, 1 H, J ¼ 4.6 Hz, OH), 3.95 (s, 3 H, OCH3),
3.79–3.77 (m, 1 H, CH), 3.67 (s, 3 H, OCH3), 3.66 (s, 3 H, OCH3),
4-O-(20,30,40,60-Tetra-O-methoxy-b-D-galactopyranosyl)
diphyllin (6)
20
White solid, yield 47.6%; ½aꢁD þ14 (c 0.3, CHCl3); IR nmax (KBr,
cmꢂ1): 719, 807, 930, 1210, 1265, 1386, 1457, 1506, 1590, 1624,
1762, 2835, 2928, 3088; 1H NMR (CDCl3): d 7.90 (s, 1 H, ArH), 7.06
(s, 1 H, ArH), 6.95 (dd, J ¼ 1.5, 7.5 Hz, 1 H, ArH), 6.84–6.76 (m, 2 H,
ArH), 6.07 (d, J ¼ 13.5 Hz, 2H, CH2), 5.56–5.41 (m, 2 H, CH2), 4.72
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
Y. Zhao et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
(d, J ¼ 5.7 Hz, 1 H, CH), 4.05 (s, 3 H, OCH3), 3.82 (s, 3 H, OCH3), 3.79
(s, 3 H, OCH3), 3.75 (dd, J ¼ 1.8, 9.6 Hz, 1 H, CH), 3.69–3.63 (m, 2 H,
CH2), 3.62 (s, 3 H, OCH3), 3.56 (s, 3 H, OCH3), 3.33 (s, 3 H, OCH3),
3.53–3.47 (m, 1 H, CH), 3.45–3.41 (m, 1 H, CH), 3.28–3.21 (m, 1 H,
CH); 13C NMR (CDCl3): d 169.9, 151.8, 150.1, 147.5, 144.6, 136.4,
131.3, 130.8, 128.5, 127.3, 123.6, 119.3, 110.7, 108.2, 106.2, 105.2,
101.2, 100.9, 84.4, 80.7, 74.7, 73.6, 70.6, 67.8, 61.6, 59.2, 58.3,
56.2, 55.8; EIMS calcd for C31H34O12 (MþNa)þ 621.1950, found
621.1943; mp 229–2318C from ethanol.
CH), 1.31 (t, J ¼ 7.2 Hz, 3 H, CH3), 1.25 (t, J ¼ 6.6 Hz, 3 H, CH3),
1.20 (t, J ¼ 8.1 Hz, 3 H, CH3), 1.14 (t, J ¼ 8.7 Hz, 3 H, CH3);
13C NMR (CDCl3): d 169.9, 151.8, 150.1, 147.5, 144.5, 136.2,
131.0, 130.8, 128.5, 127.2, 119.4, 107.1, 101.2, 75.6, 74.3, 70.1,
69.0, 68.9, 67.6, 16.4, 15.8, 15.3, 14.9; EIMS calcd for C35H42O12
(MþNa)þ 677.2576, found 677.2569; mp 127–1308C from
ethanol.
4-O-(20,30,40,60-Tetra-O-ethoxy-b-D-galactopyranosyl)
diphyllin (10)
4-O-(20,30,40-Tri-O-methoxy-b-D-xylopyranosyl) diphyllin (7)
20
White solid, yield 52.6%; ½aꢁD ꢂ8 (c 0.3, CHCl3); IR nmax
20
(KBr, cmꢂ1): 715, 808, 932, 1216, 1262, 1386, 1453, 1506,
1626, 1761, 2871, 2924, 3079; 1H NMR (CDCl3): d 7.87 (s, 1 H,
ArH), 7.05 (s, 1 H, ArH), 6.95 (dd, J ¼ 2.7, 8.1 Hz, 1 H, ArH),
6.83–6.76 (m, 2 H, ArH), 6.07 (d, J ¼ 14.1 Hz, 2 H, CH2), 5.50
(s, 2 H, CH2), 4.77 (d, J ¼ 8.1 Hz, 1 H, CH), 4.10–4.08 (m, 1 H,
CH), 4.04 (s, 3 H, OCH3), 4.01 (s, 1 H, CH), 3.78 (s, 3 H, OCH3), 3.70
(t, J ¼ 8.1 Hz, 2 H, CH2), 3.66 (d, J ¼ 4.5 Hz, 1 H, CH), 3.65–3.41
(m, 8 H, OCH2), 3.30 (dd, J ¼ 3.3, 9.9 Hz, 1 H, CH), 1.27 (s, 3 H,
CH3), 1.25 (s, 3 H, CH3), 1.24 (s, 3 H, CH3), 1.23 (s, 3 H, CH3);
13C NMR (CDCl3): 169.9, 151.7, 150.1, 147.4, 144.8, 135.9, 130.7,
130.2, 128.6, 127.1, 123.6, 119.3, 110.7, 108.2, 106.1, 104.6, 101.2,
100.7, 82.8, 78.7, 74.0, 73.8, 68.9, 68.8, 67.8, 66.9, 66.3, 56.3, 55.8,
15.9, 15.7, 15.6, 15.1; EIMS calcd for C35H42O12 (MþNa)þ
677.2576, found 677.2569; mp 79–818C from ethanol.
White solid, yield 48.6%; ½aꢁD þ11 (c 0.3, CHCl3); IR nmax (KBr,
cmꢂ1): 713, 806, 925, 1258, 1388, 1453, 1510, 1622, 1759, 2828,
2929, 3079; 1H NMR (CDCl3): d 7.85 (s, 1 H, ArH), 7.09 (s, 1 H, ArH),
6.97 (d, 1H, J ¼ 7.5 Hz, ArH), 6.85–6.78 (m, 2 H, ArH), 6.09 (d, 2 H,
J ¼ 12.9 Hz, CH2), 5.54–5.39 (m, 2 H, CH2), 4.79 (d, J ¼ 7.8 Hz,
CH), 4.08 (s, 3 H, OCH3), 4.04–3.99 (m, J ¼ 7.2 Hz, CH), 3.83 (s, 3 H,
OCH3), 3.81 (s, 3 H, OCH3), 3.68 (s, 3 H, OCH3), 3.49 (s, 3 H, OCH3),
3.41–3.86 (m, 2 H, CH2), 3.26–3.20 (t, J ¼ 8.7 Hz, CH), 3.10–3.03
(t, J ¼ 10.8 Hz, CH); 13C NMR (CDCl3): 169.8, 151.9, 150.2, 147.5,
144.3, 136.4, 131.0, 130.9, 128.3, 127.1, 123.6, 119.3, 110.7, 108.2,
106.3, 105.2, 101.2, 100.7, 85.5, 83.5, 79.4, 67.3, 63.5, 61.3, 60.7,
58.8, 56.1, 55.9; EIMS calcd for C29H30O11 (MþNa)þ 577.1688,
found 577.1680; mp 94–978C from ethanol.
4-O-(20,30,40-Tri-O-methyl-a-L-arabinopyranosyl) diphyllin (8)
20
White solid, yield 62.7%; ½aꢁD þ41 (c 0.4, CHCl3); IR nmax (KBr,
4-O-(20,30,40-Tri-O-ethoxy-b-D-xylopyranosyl) diphyllin (11)
cmꢂ1): 713, 811, 932, 1214, 1263, 1389, 1456, 1508, 1619, 1756,
2829, 2928, 3079; 1H NMR (CDCl3): d 7.89 (s, 1 H, ArH), 7.05 (s, 1 H,
ArH), 6.93 (d, J ¼ 7.5 Hz, 1 H, ArH), 6.81–6.76 (m, 2 H, ArH), 6.06
(dd, J ¼ 1.2, 13.5 Hz, 2 H, CH2), 5.56–5.41 (m, 2 H, CH2), 4.76 (d,
J ¼ 7.2 Hz, 1 H, CH), 4.14 (dd, J ¼ 2.4, 12.9 Hz, 1 H, CH), 4.05 (s, 3
H, OCH3), 3.79 (s, 3 H, OCH3), 3.78 (s, 3 H, OCH3), 3.75–3.72 (m, 2 H,
CH2), 3.54 (s, 3 H, OCH3), 3.50 (s, 3 H, OCH3), 3.34 (dd, J ¼ 3.3,
9.3 Hz, 1 H, CH), 3.21 (d, J ¼ 12.6 Hz, 1 H, CH); 13C NMR (CDCl3): d
169.9, 151.9, 150.1, 147.5, 144.5, 136.2, 130.8, 130.7, 128.5, 127.1,
119.3, 107.1, 106.1, 105.6, 101.7, 100.7, 82.7, 81.8, 75.2, 74.5, 73.5,
67.6, 62.2, 61.9, 58.8, 56.9, 55.5; EIMS calcd for C29H30O11
(MþNa)þ 577.1688, found 577.1680; mp 130–1328C from
ethanol.
20
White solid, yield 41.8%; ½aꢁD þ6 (c 0.3, CHCl3); IR nmax (KBr,
cmꢂ1): 709, 808, 930, 1214, 1259, 1384, 1452, 1506, 1622, 1761,
2893, 2922, 3070; 1H NMR (CDCl3): d 7.53 (s, 1 H, ArH), 7.07 (d,
J ¼ 8.1 Hz, 1H, ArH), 6.95 (d, J ¼ 7.5 Hz, 1H, ArH), 6.81 (m, 2 H,
ArH), 6.07 (d, J ¼ 14.1 Hz, 2 H, CH2), 5.45 (m, 2 H, CH2), 4.32 (q,
1H, CH), 4.11 (d, J ¼ 7.2 Hz, 1 H, CH), 4.05 (s, 3 H, OCH3), 3.99 (q, 1
H, CH), 3.94 (d, J ¼ 7.5 Hz, 1 H, CH), 3.90–3.84 (m, 2 H, CH2), 3.78
(s, 3 H, CH3), 3.67–2.97 (m, 6 H, CH2), 1.25–1.18 (m, 9 H, OCH2);
13C NMR (CDCl3): d 169.9, 151.8, 151.5, 150.2, 147.5, 146.9, 144.4,
136.2, 134.4, 130.8, 128.5, 127.0, 126.6, 125.6, 123.6, 119.3, 110.8,
108.2, 106.2, 104.9, 101.2, 100.7, 84.2, 81.7, 69.1, 68.3, 66.8, 64.3,
56.3, 55.8, 15.9, 15.7; EIMS calcd for C32H36O11 (MþNa)þ
619.2158, found 619.2150; mp 106–1088C from ethanol.
Synthesis of per-ethoxy diphyllin glycosides 9–12
Typical procedure: To the solution of glycosides 13–16
(0.5 mmol) in DMSO (20 mL), 50% NaOH (10 mmol) was added.
Then, EtBr (10 mmol) and NaI (10 mmol) were added slowly, and
the mixture was stirred overnight at room temperature. After
the reaction was completed, the mixture was filtered and evap-
orated under reduced pressure. The residue was purified by flash
chromatography (1:3, EtOAc/petroleum ether) to afford white
solid 9–12.
4-O-(20,30,40-Tri-O-ethyl-a-L-arabinopyranosyl) diphyllin (12)
20
White solid, yield 49.8%; ½aꢁD þ35 (c 0.3, CHCl3); IR nmax (KBr,
cmꢂ1): 768, 808, 922, 1211, 1228, 1353, 1452, 1506, 1622, 1753,
2848, 2922, 3447; 1H NMR (CDCl3): d 7.53 (s, 1 H, ArH), 7.04 (s, 1 H,
ArH), 6.94 (d, J ¼ 7.8 Hz, 1 H, ArH), 6.79 (m, 2 H, ArH), 6.07
(d, J ¼ 13.8 Hz, 2 H, CH2), 5.45 (s, 2 H, CH2), 4.02 (s, 3 H,
OCH3), 4.00 (s, 2 H, CH), 3.96–3.83 (m, 2 H, CH), 3.78 (s, 3 H,
OCH3), 3.74 (s, 2 H, CH2), 3.63–3.30 (m, 6 H, OCH2), 1.23 (s, 9 H,
CH3); 13C NMR (CDCl3): d 169.7, 151.6, 150.3, 149.9, 147.5, 147.0,
134.5, 130.7, 128.6, 126.6, 125.7, 123.7, 119.3, 110.8, 108.2, 106.2,
105.5, 103.1, 100.7, 81.2, 79.4, 73.4, 68.9, 68.3, 66.7, 65.8, 64.7,
56.0, 55.8, 15.9; EIMS calcd for C32H36O11 (MþNa)þ 619.2158,
found 619.2150; mp 175–1788C from ethanol.
4-O-(20,30,40,60-Tetra-O-ethoxy-b-D-glucopyranosyl)
diphyllin (9)
20
White solid, yield 57.1%; ½aꢁD þ6 (c 0.2, CHCl3); IR nmax (KBr,
cmꢂ1): 715, 808, 936, 1211, 1259, 1381, 1458, 1506, 1622, 1761,
2893, 2927, 3070; 1H NMR (CDCl3): d 7.82 (s, 1 H, ArH), 7.06 (s, 1 H,
ArH), 6.95 (dd, J ¼ 2.7, 7.8 Hz, 1H, ArH), 6.84–6.76 (m, 2 H, ArH),
6.07 (d, J ¼ 13.5 Hz, 2 H, CH2), 5.60–5.42 (m, 2 H, CH2), 4.77 (d,
J ¼ 7.8 Hz, 1H, CH), 4.1–4.06 (m, 1 H, CH), 4.04 (s, 3 H, OCH3), 3.99
(t, J ¼ 7.5 Hz, 1 H, CH), 3.89–3.82 (m, 2 H, CH2), 3.78 (s, 3 H, OCH3),
3.68 (t, J ¼ 9 Hz, 1 H, CH), 3.61–3.26 (m, 8 H, OCH2), 3.21 (s, 1 H,
Cell culture
Four cancer cell lines, HCT-116 (human colon carcinoma), RM-1
(murine prostate carcinoma), A549 (human lung carcinoma),
and A549T (lung, paclitaxel resistant), were cultured on RPMI-
1640 medium supplemented with fetal bovine serum (10%),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Diphyllin Glycosides as Novel Anticancer Agents
7
penicillin (100 U/mL), and streptomycin (100 mg/mL) in 25 cm2
The studies in the laboratories were supported by the Natural Science
Foundation of Jiangsu Province (BK2011390) and Priority Academic
Program Development of Jiangsu Higher Education Instititutions (PAPD).
culture flasks at 378C in
5% CO2.
a humidified atmosphere with
The authors have declared no conflict of interest.
Cell viability
Cell viability was assessed by the MTT assay. Cells were harvested
from the culture during the exponential growth phase, and
seeded into multiwell culture plates at 5 ꢀ 104–1 ꢀ 105 cells/
mL in fresh medium. After overnight growth, cells were treated
with compounds (predissolved in DMSO) at selected concen-
trations for a period of 3 days. The medium was then discarded
and replaced with MTT dye. Plates were incubated at 378C for 4 h.
The resulting formazan crystals were solubilized in lysis buffer
(SDS 10 g, DMF 25 mL, H2O 25 mL, acetic acid 1 mL, pH 4.7), and
the optical density was read at 570 nm with a microplate reader
(Biotek synergy 2).
References
[1] J. J. Champoux, Annu. Rev. Biochem. 2001, 70, 369–413.
[2] Y. Pommier, E. Leo, H. L. Zhang, C. Marchand, Chem. Biol.
2010, 17, 421–433.
[3] J. T. Bau, E. U. Kurz, Biochem. Pharmacol. 2011, 81, 345–354.
[4] T. Onda, E. Toyoda, O. Miyazaki, C. Seno, S. Kagaya, K.
Okamoto, K. Nishikawa, Cancer Lett. 2008, 259, 99–110.
[5] S. A. Beers, Y. Imakura, H. J. Dai, D. H. Li, Y. C. Cheng, K. H.
Lee, J. Nat. Prod. 1988, 51, 901–905.
Topoisomerase II-mediated kDNA decatenation assay
Gel assay was carried out as previously described [15] to
determine if compounds inhibited the catalytic decatenation
activitiy of TopoII. kDNA consists of highly catenated
networks of circular DNA, which can be decatenated by TopoII
in an ATP-dependent reaction.
[6] S. H. Day, Y. C. Lin, M. L. Tsai, J. Nat. Prod. 2002, 65, 379–381.
[7] Y. Zhao, Y. P. Lu, L. Zhu, J. Carbohydr. Chem. 2008, 27, 113–119.
[8] Y. Zhao, Y. X. Li, Chin. J. Chem. 2007, 25, 679–682.
[9] M. Gui, D. K. Shi, M. Huang, Y. Zhao, Q. M. Sun, J. Zhang,
Q. Chen, J. M. Feng, C. H. Liu, M. Li, Y. X. Li, M. Y. Geng,
J. Ding, Invest. New Drugs 2011, 29, 800–810.
Tubulin polymerization assay
[10] D. K. Shi, W. Zhang, N. Ding, M. Li, Y. X. Li, Eur. J. Med. Chem.
2012, 47, 424–431.
The tubulin polymerization assay kit was purchased from
Cytoskeleton which was used for the detection of polymerization
of tubulin [16]. According to kit manufacture, first 10 mL of
General Tubulin Buffer (80 mM PIPES pH 6.9, 2 mM MgCl2,
0.5 mM EGTA) was pipetted only into one control well (the
96-well half-area plate is pre-warmed to 378C for 30 min). Then,
10 mL of 10ꢀ strength of the compound of interest in General
Tubulin Buffer was pipetted into each well. Tubulin solution
(100 mL/well) was then added to the wells. Then, the plates were
immediately put into the plate reader and read at 340 nm once
per minute for 60 min at 378C using a kinetic protocol. Raw data
were processed using Microsoft Excel software to get the curves of
different treatments to the polymerization of tubulin.
[11] J. L. Charlton, C. J. Oleschuk, G. L. Chee, J. Org. Chem. 1996, 61,
3452–3457.
[12] P. K. Li, B. Pandit, D. L. Sackett, Z. G. Hu, J. Zink, J. D. Zhi,
D. Freeman, R. W. Robey, K. Werbovetz, A. Lewis, C. L. Li, Mol.
Cancer Ther. 2006, 5, 450–456.
[13] S. Arora, X. I. Wang, S. M. Keenan, C. Andaya, Q. Zhang,
Y. Y. Peng, W. J. Welsh, Cancer Res. 2009, 69, 1910–1915.
[14] J. Y. Chang, H. P. Hsieh, W. Y. Pan, J. P. Liou, S. J. Bey,
L. T. Chen, J. F. Liu, J. S. Song, Biochem. Pharmacol. 2003, 65,
2009–2019.
[15] B. B. Hasinoff, H. Kazlowska, A. M. Creighton, W. P. Allan, P.
Thampatty, J. C. Yalowich, Mol. Pharmacol. 1997, 52, 839–845.
Statistical analysis
[16] G. Kuznetsov, K. TenDyke, M. J. Towle, H. S. Cheng, J. K. Liu,
J. P. Marsh, S. E. R. Schiller, M. R. Spyvee, H. Yang, B. M.
Seletsky, C. J. Shaffer, V. Marceau, Y. Yao, E. M. Suh, S.
Campagna, F. G. Fang, J. J. Kowalczyk, B. A. Littefield, Mol.
Cancer Ther. 2009, 8, 2852–2860.
Data were presented as means ꢃ SE and analyzed by SPSS
software. Pictures were processed with Photoshop software.
Mean values were obtained from at least three independent
experiments.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com