Synthesis and identification of cytotoxic diphyllin glycosides as vacuolar H+-ATPase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.06.002

The concise syntheses of two natural diphyllin glycosides Cleistanthin-A (CA), Cleistanthoside-A (CleA) and its derivative, Cleistanthoside-A tetraacetate (CleT), have been achieved. They were evaluated for their in vitro anti-proliferative activities against MCF-7, HeLa, HepG2, HCT-116, U251 cancer cell lines by MTT assay. Both of CA and CleT were anti-proliferative to these cancer cells at nanomolar concentrations. They have been shown to inhibit the activity of vacuolar H⁺-ATPase (V-ATPase) in HepG2 cells and neutralize the pH of lysosomes at nanomolar concentrations.

Full text of DOI:10.1016/j.ejmech.2014.06.002

Accepted Manuscript
+
Synthesis and Identification of Cytotoxic Diphyllin Glycosides as Vacuolar H -ATPase
Inhibitors
Zhitao Zhang , Jinlong Ma , Li Zhu , Zhao Yu
PII:
S0223-5234(14)00518-2
10.1016/j.ejmech.2014.06.002
EJMECH 7044
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2014
Revised Date: 26 May 2014
Accepted Date: 1 June 2014
Please cite this article as: Z. Zhang, J. Ma, L. Zhu, Z. Yu, Synthesis and Identification of Cytotoxic
+
Diphyllin Glycosides as Vacuolar H -ATPase Inhibitors, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.06.002.
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ACCEPTED MANUSCRIPT
Synthesis and Identification of Cytotoxic Diphyllin Glycosides as
Vacuolar H+-ATPase Inhibitors
Zhitao Zhang, Jinlong Ma, Li Zhu, Yu Zhao*
Institute of Nautical Medicine, Nantong University, Nantong 226001, China
Corresponding author. Tel.: +86 513 85051797; fax: +86 513 85051796.
E-mail addresses: zhaoyoo@163.com
Three diphyllin glycosides CA, CleA and CleT were synthesized. CA and CleT
showed were identified as novel vacuolar H+ ATPase inhibitors.

ACCEPTED MANUSCRIPT
Synthesis and Identification of Cytotoxic Diphyllin Glycosides as Vacuolar
H+-ATPase Inhibitors
Zhitao Zhang, Jinlong Ma, Li Zhu, Yu Zhao*
Institute of Nautical Medicine, Nantong University, Nantong 226001, China
Corresponding author. Tel.: +86 513 85051797; fax: +86 513 85051796.
E-mail addresses: zhaoyoo@163.com
Abstract
The concise syntheses of two natural diphyllin glycosides Cleistanthin-A (CA),
Cleistanthoside-A (CleA) and its derivative, Cleistanthoside-A tetraacetate (CleT), have been
achieved. They were evaluated for their in vitro anti-proliferative activities against MCF-7,
HeLa, HepG2, HCT-116, U251 cancer cell lines by MTT assay. Both of CA and CleT were
anti-proliferative to these cancer cells at nanomolar concentrations. They have been shown to
inhibit the activity of vacuolar H+-ATPase (V-ATPase) in HepG2 cells and neutralize the pH
of lysosomes at nanomolar concentrations.
Keywords Cleistanthin-A; Cleistanthoside-A; synthesis; vacuolar H+-ATPase; inhibitor;
1. Introduction
Scientific evidence suggests that the acidic tumor microenvironment is critical to
managing cancer development, progression and metastasis[1]. In particular, V-ATPases play
a major role in tumor invasion and metastasis development, which are responsible for
microenvironment acidification [2-3]. These results propose V-ATPases as a key target for
new strategies in cancer treatment [4-8]. Thus, inhibitors of V-ATPases could be promising
novel therapeutics for cancer treatment. Most of the known V-ATPases inhibitors are natural
compounds of microbial origin such as salicylihalamides and bafilomycin [9].
Fig.1
Diphyllin, an arylnaphthalene lignan lactone, isolated from many traditional medicinal
plants, has been reported to possess anticancer and antiviral activities [10]. Recently, diphyllin
has been noted to potently inhibit V-ATPase and thereby lysosomal acidification in osteoclasts
[11]. Previously, we found that some diphyllin glycosides possessed inhibitory effects on
cancer cell proliferation and DNA-topoisomerase II [12,13]. However, their TopoII inhibitory
abilities did not match well with their antiproliferative activities, which prompted us to
elucidate their mode of action. Some diphyllin glycosides, such as Cleistanthin-A (CA) and
1

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Cleistanthoside-A tetraacetate (CleT) (Figure 1), showed potent anti-proliferative activities at
nanomolar concentration[14, 15]. To the best of our knowledge, there is still no report on the
total synthesis of Cleistanthoside-A (CleA) and its tetraacetate (CleT).
Herein, we report the total syntheses, anti-proliferative activities and V-ATPase
inhibitory effects of these glycosides. CA and CleT exhibited anti-proliferative activities at
nanomolar concentrations against five cancer cell lines and showed potent V-ATPase
inhibitory activities. Our results firstly disclose that diphyllin glycosides exert their
anti-proliferative activities mainly through inhibiting V-ATPase.
2. Results and discussion
Retrosynthetically, CleT can be traditionally divided into two fragments, diphyllin and
the disaccharide residue, which were linked by a β-glycosidic bond. Syntheses of diphyllin
have been reported by several groups[16,17]. Thus, CleT could be achieved by the
glycosylation of diphyllin with disaccharide donor. However, the particularity of phenolic
hydroxyl makes our numerous attempts failed. Finally, we turned our attention to the
traditional linear synthesis. In the process of synthesis, phase transfer catalysis (PTC) and
Schmidt glycosylation were sequentially employed to afford two β-glycosyl bonds in
satisfactory yields. The stereochemistry of CleT was confirmed by conversion to CleA, which
was identical to the literature data [18].
To assemble CA in reasonable manner, a xylopyranosyl bromide 7 was prepared
(Scheme1). A six-step reaction was successfully applied to obtain donor 7 from commercial
available compound 1[19]. The bromide 1 was converted to 2 through acetalization reaction
[20] in 97% yield. Deacetylation of 2 with NaOMe afforded the desired 3 in 85% yield.
Methylation of 3 in the presence of 50% NaOH and MeI afforded 4 in 89% yield. Compound
4 was subjected to cleavage of the acetal group to give diol 5 in 80% yield. Nextly,
acetylation of the C-3 and C-4 hydroxyl groups proceeded under Ac2O and pyridine
conditions. Finally, Compound 6 was treated with 35% HBr-HOAc to provide 7 in 72% yield.
Scheme 1
Diphyllin was easily prepared from 2-bromo-4, 5-dimethoxybenzaldehyde in five steps and
42.5% yield according to the procedure reported by Charlton. Glycosylation of diphyllin with
donor 7 was achieved in 0.1 M NaOH/CHCl3 in the presence of tetra-butylammonium
bromide (TBAB) at 40 °C, affording diphyllin monoglycoside 8 in 76% good yield [21-23].
2

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Subsequently, deacetylation with K2CO3 in methanol was performed smoothly to produce CA
in 81% yield without destruction of the lactone residue (Scheme2).
Scheme 2
The key to synthesis of CleT and CleA turned out to be the glycosylation of CA. We
started with donor 12 to explore the glycosylation reaction. TMSOTf and NBS were used
respectively to promote the coupling reactions, but we found they led to glycosidic bond
cleavage of CA. Several other glycosylation conditions were also tried, e.g. AgOTf , TBAB,
failing to give desired product[24-27] (Table 1). Fortunately, the weak lewis acid BF3·Et2O
catalyzed Schmidt glycosylation could afford the desired disaccharide glycoside in satisfied
yield without cleavage of glycosidic bond. The glycosylation of 9 with donor 13 was
performed under the catalysis of BF3·Et2O (1.5 equiv) in the presence of 4 Ǻ MS in CH2Cl2 at
-20 °C to give 10 in 64% yield. Freshly activated 4 Ǻ MS molecular sieves were used in this
condition to remove water from the solvent. Finally, deacetylation with K2CO3 in methanol
was performed smoothly to produce 11 in 83% yield. All of the analytical data of the synthetic
9 and 11 were virtually identical to those recorded for the natural Cleistanthin-A and
Cleistanthoside A.
Table1
Five cancer cell lines from various solid tumors, MCF-7, HeLa, HepG2, HCT-116 and
U251, were used to determine the effects of synthesized glycosides on cell proliferation
employing a MTT assay. The average inhibitory concentrations of 50% were shown in Table 1.
The results indicated that CA and CleT possessed significant anti-proliferative activities in
vitro at nanomolar concentrations, while CleA was not active which suggested a lipothilic
glycosyl moiety was critical to keep their activity. CA and CleT showed the best inhibitory
effect on HepG2 cells with the IC50 values 36 nM and 39 nM, which were much lower than
that of Paclitaxel .
Table 2
Based on the above results, the human liver hepatocellular carcinoma cell line (HepG2) was
used for V-ATPase activity assay (Figure2). HepG2 was treated with CA and CleT in a range
of concentrations (0, 25, 50, 100 nM, respectively) and then the activity of V-ATPase was
detected by V-ATPase activity assay kit.
3

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A dose-dependent decrease in enzymatic activity was observed in Figure 2. The
significant decreasing characteristics of the histogram reflected the high affinity of the
inhibitor for V-ATPase.
Fig.2
It is well known that the V-ATPase inhibition results in a modification of intracellular
pH. Consequently, the lysosome acidity was examined in the human liver hepatocellular
carcinoma (HepG2) cells at various concentrations of CA and CleT (Figure3). HepG2 cells
were stained with a pH-sensitive fluorescence dye (LysoTracker Red) and sequential loaded
with CA, CleT. As shown in Figure 3, the amount of red fluorescence decreased in cells
treated with CA and CleT compared to untreated cells and V-ATPase was completely
suppressed at the concentrations of 100 nM. The results also implied that the modulation of
lysosomes acidity by CA and CleT was consistent with their inhibitory effect on V-ATPase
activity.
Several diphyllin glycosides could inhibit cancer cell proliferation at nanomolar
concentration, but their molecular targets were unknown. Previously, we found that some
diphyllin glycosides possessed inhibitory effects on DNA-topoisomerase II and microtube
polymerization in high concentration. However, their TopoII inhibitory abilities did not match
well with their antiproliferative activities, which prompted us to elucidate their mode of action.
Diphyllin possess antiproliferation activity only in micromolar concentration, it has been
noted to inhibit V-ATPase recently[28]. We deduced that diphyllin glycosides might have
similar effects on V-ATPase. To our delight, our results demonstrated diphyllin glycosides
showed inhibitory activity on V-ATPase at nanomolar concentration. All evidence indicated
that diphyllin glycosides exert their potent anti-proliferative activities mainly through
inhibiting V-ATPase.
Fig.3
3. Conclusion
In summary, we have accomplished the syntheses of three diphyllin glycosides
Cleistanthin-A (CA), Cleistanthoside-A (CleA) and Cleistanthoside-A tetraacetate (CleT).
Both of CA and CleT exhibited strong anti-proliferative activity in vitro against five cancer
cell lines. They showed potent inhibitory activites against V-ATPase with dose-dependent
4

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characteristics. Lysosomal pH analysis illuminated that they inhibited the HepG2 lysosomal
acidification at nanomolar concentrations. All evidence indicated that diphyllin glycosides
exert their potent anti-proliferative activities mainly through inhibiting V-ATPase. The
synthesis of their derivatives has become a feasible task that would facilitate in-depth studies
of their antitumor activities and molecular targets.
4. Experimental
4.1 Materials and General Methods Commercial grade reagents and solvents were used
without further purification, except where noted. Flash chromatography was performed on
silica gel. The purity of all compounds was judged by TLC analysis (single-spot/two-solvent
1
systems) using a UV lamp. H NMR and 13C NMR spectra were taken on 400 MHz
spectrometer with tetramethylsilane (TMS) as an internal standard, and chemical shifts were
recorded in ppm values.
4.1.1 3, 4-Di-O-acetyl-1, 2-O-[(R, S)-ethylidenyl]-D-xylopyranose (2)
To a stirring solution of bromide 1 (2 g, 5.9 mmol) in dry MeCN was added TBAI (1.39
g, 3.76 mmol) and NaBH4 (1 g, 6.4 mmol) at 0 °C. The reaction mixture was stirred for the
next 24 h at room temperature. The precipitate filtered and the isolated solid washed with
CH2Cl2, followed by washing with cold 10% HCl (2×10 mL), brine (2×40 mL). Organic layer
was isolated and dried on NaSO4. Under reduced pressure, solvent was evaporated to obtain
crude product 2, which was purified by column chromatorraphy with EtOAc/petroleum ether
1
(1:5 v/v) to give 1.27 g of compound 2 (83%). H NMR (400 Hz, CDCl3) (R/S: 2.8/1): R: δ
5.45 (d, J=4.4 Hz, 1H), 5.25 (t, J=2.8 Hz, 1H), 5.15 (q, J=5.2,10 Hz, 1H), 4. 90 (m, 1H),
3.96-3.86 (m, 3H), 2.12, 2.10 (2s, 6H), 1. 52 (d, J=4.8 Hz, 3H); S: 5.61 (q, J=4.8, 10 Hz, 1H),
5.43 (d, J=4 Hz, 1H), 5.32 (t, J=5.6 Hz, 1H), 4.82 (q, J=6, 10.8 Hz, 1H), 4.10 (t, J=4.4 Hz,
1H), 3.80-3.72 (m, 2H), 2.12, 2.08 (2s, 6H), 1. 39 (d, J=4.8 Hz, 3H); LRMS (ESI-TOF) (m/z)
[M+Na]+ calcd for C11H16O7Na 283.1, found 283.0, Spectral data for 2 matched those
previously reported [19] .
4.1.2 3, 4-Di-O-methyl-1, 2-O-[(S)-ethylidenyl]-D-xylopyranose (4)
To a stirring solution of acetal 2 (4 g, 15.38 mmol) in MeOH was added NaOMe (18.2
mg, 0.34 mmol) at 0 °C. The reaction mixture was stirred for 2 h at room temperature. The
mixture was evaporated under reduced pressure to obtain crude product 3, which was purified
5

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by column chromatorraphy with EtOAc/ petroleum ether (1:1 v/v) to give 2.3 g of compound
1
3 in 85% yield. H NMR (400 Hz, CDCl3) (R/S: 2.1/1): R: δ 5.34 (d, J=2.8 Hz, 1H), 5.23 (q,
J=4.8, 9.6, 1H), 4.16-3.62 (m, 5H) ,1.52 (d, J=4.8 Hz, 3H); S: δ 5.58 (q, J=4.8, 9.6 Hz, 1H),
5.39 (d, J=3.2, 1H), 4.16-3.62 (m, 5H) ,1.39 (d, J=5.2 Hz, 3H).
A solution of diol 3 (3 g, 17 mmol) and 50% NaOH (50 mL) in DMSO was stirred
vigorously, and then MeI (6.4 g, 45 mmol) was dropwise added. The reaction mixture was
stirred for 2 h at room temperature. The reaction mixture was diluted with CH2Cl2 (3×100
mL), washed with water (1×100 mL), brine (2×50 mL) and the organic layer was dried on
NaSO4. Purification by column chromatography on silica eluting with EtOAc/ petroleum
ether (1:4 v/v) gave the product 4 as a colorless oil (89%). 1H NMR (400 Hz, CDCl3) (only S
on the basis of literature [19]) δ 5.53 (q, J=4.8, 9.6 Hz, 1H), 5. 41 (d, J=4.4 Hz, 1H), 4. 11(t,
J=4.4 Hz, 1H), 3. 81 (dd, J=4.4,12 Hz, 1H), 3.65-3. 61 (m, 1H), 3. 54, 3. 46 (2s, 6H),
3.52-3.48 (m, 2H), 1. 38 (d, J=4.8 Hz, 3H), LRMS (ESI-TOF) (m/z) [M+Na]+ calcd for
C9H16O5Na 227.1, found 227.1, Spectral data for 4 matched those previously reported [19] .
4.1.3 1, 2-Di-O-acetyl-3, 4-di-O-methyl-xylopyranose (6)
To a stirring solution of compound 4 (2.673 g, 13.1 mmol) in dioxane was added 1 M
H2SO4 (4 mL). The reaction mixture was heated under reflux for 2 h. Afterward, the solution
was cooled, neutralized with saturated Na2CO3 and evaporated under reduced pressure.
Purification by column chromatography on silica eluting with EtOAc/ petroleum ether (1:1
v/v) gave the product 5 as colorless oil (80%).1H NMR (400Hz, CDCl3) (α/β: 1.7/1): α: δ 4.92
(d, J=7.2 Hz, 1H), 4.08 (dd, J=2.8, 12.4 Hz, 1H), 3.97 (dd, J=4, 12.4 Hz, 1H), 3.79-3.61 (m,
3H), 3.51, 3.46 (s, 6H); β: δ 4.28 (d, J=9.2 Hz, 1H), 4.08 (dd, J=2.8, 12.4 Hz, 1H), 3.97 (dd,
J=4, 12.4 Hz, 1H), 4.40-3.28 (m, 3H), 3.58, 3.47 (s, 6H).
To a stirring solution of compound 5 (1.7 g, 9.5 mmol) in pyridine was added Ac2O (3.5
mL) and DMAP (58 mg, 0.48 mmol). The reaction mixture was stirred for 2 h at room
temperature. The solution was partitioned between EtOAc (100 mL) and H2O (50 mL). The
organic layer was separated, washed with 2 M HCl (3×100 mL) and water (1×100 mL), brine
(2×40 mL), dried over NaSO4 and evaporated under reduced pressure. Purification by column
chromatography on silica eluting with EtOAc/ petroleum ether (1:4 v/v) gave the product 6 as
a colorless oil (88%). 1H NMR (400Hz, CDCl3) (α/β: 1.3/1): α: δ 6.17 (d, J=3.6 Hz, 1H), 4.88
6

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(dd, J=3.6, 6.0 Hz, 1H), 3.90 (dd, J=5.6, 11.6 Hz, 1H), 3.59, 3.56 (s, 2H), 3.57, 3.51 (2s, 6H),
3.43-3.41 (m, 1H), 2.15, 2.08 (2s, 6H); β: δ 5.63 (d, J=7.2 Hz, 1H), 4.97 (t, J=8.0 Hz, 1H),
4.10-4.04 (m, 1H), 3.53,3.48 (2s, 6H), 3.40-3.34 (m, 3H), 2.10,2.09 (2s, 6H); LRMS
(ESI-TOF) (m/z) [M+Na]+ calcd for C11H18O7Na 285.1, found 285.0, Spectral data for 6
matched those previously reported [19] .
4.1.4 4-O-[2′′-O-acetyl-3′′, 4′′-di-O-methyl-β-D-xylopyranosyl]-diphyllin (8)
To a stirring solution of compound 6 (1 g, 3.8 mmol) in CH2Cl2 was added 35%
HBr-HOAc (1 mL) at 0 °C. The reaction mixture was stirred for 2 h at room temperature. The
reaction mixture was diluted with CH2Cl2 (3×100 mL), washed with water (1×100 mL) and
saturated NaHCO3 (1×100 mL), brine (2×50 mL) and the organic layer was dried on NaSO4.
The mixture was purified by flash chromatography (EtOAc/ petroleum ether, 1/4, v/v) to give
7 as a yellowish oil (72%). This compound was easily decomposed and used without further
purification.
To a stirring solution of diphyllin (584.4 mg, 1.53 mmol) and TBAB (489.6 mg, 1.53
mmol) in CHCl3 (20 mL) was added aqueous 0.1 mol•L-1 NaOH (30 mL). After stirring for 10
min at 40 °C, compound 7 (864 mg, 3.06 mmol) was added, and the two-phase reaction
mixture was stirred for 6 h at 40 °C. Then CHCl3 (20 mL) was added and the resulting
organic phase was washed with water (2×40 mL), brine (2×50 mL), dried on NaSO4, and the
solvent was evaporated under reduced pressure. The residue was purified by column
chromatography on silica eluting with CH2Cl2/MeOH (200:1 v/v) to afford 8 as a white solid
1
(76%). H NMR (400 Hz, CDCl3): δ: 7.61 (d, J=1.6 Hz, 1H), 7.05 (s, 1H), 6.97 (d, J=8 Hz,
1H), 6.82 (t, J=6.4 Hz, 2H), 6.10 (d, J=1.2 Hz, 1H), 6.05 (d, J=1.2 Hz, 1H), 5.46-5.43 (m, 2H),
5.36-5.30 (m, 1H), 5.13 (d, J=6.4 Hz, 1H), 4.19 (dd, J=4.0,11.6 Hz, 1H), 4.08 (s, 3H), 3.81 (s,
3H), 3.62 (s, 3H), 3.52 (s, 3H), 3.50 (m, 1H), 3.46 (t, J=7.6 Hz, 1H), 3.40-3.35 (m, 1H), 2.17
(s, 3H); LRMS (ESI-TOF) (m/z) [MH]+ calcd for C30H31O12 583.2, found 583.0, Spectral data
for 8 matched those previously reported[15] .
4.1.5 4-O-[3′′, 4′′-Di-O-methyl-β-D-xylopyranosyl]-diphyllin (9, Cleistanthin-A)
The solid 8 (220 mg, 0.378 mmol) was dissolved in MeOH (5 mL), and then K2CO3 (104
mg, 0.756 mmol) was added. After stirring at room temperature for 2 h, the solution was
neutralized with 1 mol•L-1 HCl. The mixture was diluted with CH2Cl2 (3×100 mL), washed
7

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with water (1×100 mL), brine (2×50 mL) and the organic layer was dried on NaSO4. The
residue was purified by column chromatography on silica eluting with CH2Cl2/MeOH (200:1
v/v) to afford Cleistanthin A as a white solid (81%). m.p. 139-142 °C; [α]58927: -65° (CHCl3, c
1.0); 1H NMR (400 Hz, CDCl3): δ 7.92 (s, 1H, 5-ArH-), 7.07 (s, 1H, 8-ArH-), 6.97 (d, J=8 Hz,
1H, 6′-ArH-), 6.82 (t, J=7.6 Hz, 2H, 2′, 5′-ArH-), 6.10 (d, J=1.2 Hz, 1H, 7′-CH2-), 6.05 (s, 1H,
7′-CH2-), 5.53-5.41 (m, 2H, 3a-CH2-), 5.05 (d, J=7.6 Hz, 1H, 1′′-CH-), 4.13 (dd, J=3.6,12.0
Hz, 1H, 5′′-CH-), 4.05 (s, 3H, 6-OMe), 3.95-3.91 (m, 1H, 2′′-CH-), 3.81 (s, 3H, 7-OMe), 3.69
(s, 3H, 3′′-OMe), 3.50 (s, 3H, 4′′-OMe), 3.47 (q, J=4.0,7.2 Hz, 1H, 4′′-CH-), 3.40-3.33 (m, 2H,
3′′, 5′′-CH-), 3.27 (s, 1H, 2′′-OH); 13C NMR (CDCl3): δ 151.8, 150.2, 147.5, 147.5, 144.1,
130.7, 128.4, 123.6, 1119.2, 110.7, 108.2, 106.1, 101.2, 82.0, 81.9, 78.2, 71.0, 70.9, 67.3, 61.6,
60.1, 58.0, 56.2, 55.8, 29.7; HRMS (ESI-TOF) (m/z) [M+Na]+ calcd for C28H28O11Na
563.15293, found 563.15072, Spectral data for 9 matched those previously reported[15] .
4.1.6
4-O-[2′′′, 3′′′, 4′′′, 6′′′-O-tetra-O-acetyl-β-D-glucopyranosyl (1→2)-3′′,
4′′-di-O-methyl-β-D-xylopyranosyl]-diphyllin (10, Cleistanthoside-A tetraacetate).
A mixture of the Cleistanthin A (54 mg, 0.1 mmol), glucopyranosyl trichloroacetimidate
(64 mg, 0.13 mmol), and powered 4 Å molecular sieves in anhydrous CH2Cl2 (3 mL) was
stirred at -20 °C under N2 for 10 min. A solution of BF3·Et2O (21 mg, 0.15 mmol) in CH2Cl2
was added drop-wise. After being stirred at room temperature for 3 h, the mixture was diluted
with CH2Cl2 (3×10 mL), washed with saturated NaHCO3 (1×10 mL), brine (2×20 mL) and
the organic layer was dried on NaSO4. The residue was purified by column chromatography
on silica eluting with EtOAc/petroleum ether (1:2 v/v) to afford Cleistanthoside A tetraacetate
1
as a white solid (64%). m.p. 135-137 °C; [α]58927: -38° (CHCl3, c 1.0); H NMR (400 Hz,
CDCl3): δ 7.83 (s, 1H, 5-ArH), 7.08 (s, 1H, 8-ArH), 6.97 (d, J=8 Hz, 1H, 6′-ArH), 6.85-6.79
(m, 2H, 2′, 5′-ArH), 6.10 (d, J=19.2 Hz, 2H, 7′-CH2-), 5.55-5.41 (m, 2H, 3a-CH2-), 5.28 (t,
J=9.2 Hz, 1H, 3′′′-CH-), 5.14-5.05 (m, 3H, 1′′′, 2′′′, 4′′′-CH-), 4.92 (d, J=7.2 Hz, 1H, 1′′-CH-),
4.18 (d, J=2 Hz, 1H, 6′′′-CH-), 4.14 (s, 3H, 6-OMe), 4.04 (dd, J=5.2, 12 Hz, 1H, 5′′–CH2-),
3.94 (dd, J=12, 20 Hz, 2H, 2′′, 6′′′-CH-), 3.81 (s, 3H, 7-OMe), 3.73 (d, J=10 Hz, 1H, 5′′′-CH-),
3.62 (s, 3H, 3′′-OMe), 3.47 (s, 3H, 4′′-OMe), 3.42-3.39 (q, J=4.8, 8.8 Hz, 1H, 4′′-CH-), 3.29 (t,
J=8.4 Hz, 1H, 3′′-CH-), 3.12 (t, J=12 Hz, 1H, 5′′-CH-), 2.12 (s, 3H, 2′′′-OCOMe), 2.04 (s, 3H,
3′′′-OCOMe), 2.01 (s, 3H, 4′′′-OCOMe), 1.81 (s, 3H, 6′′′-OCOMe); 13C NMR (CDCl3): δ
8

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170.4, 170.4, 169.9, 169.4, 167.2, 151.8, 150.2, 147.5, 136.3, 130.8, 128.3, 123.6, 119.2,
110.7, 108.2, 106.7, 103.2, 101.9, 101.3, 101.2, 84.5, 79.9, 77.4, 77.3, 77.1, 76.8, 73.0, 72.0,
71.8, 68.0, 60.8, 58.5, 29.7, 20.9, 20.7, 20.6, 20.3; HRMS (ESI-TOF) (m/z) [M+Na]+ calcd for
C42H46O20Na 893.24801, found 893.24757, Spectral data for 10 matched those previously
reported[15] .
4.1.7 4-O-[β-D-glucopyranosyl (1→2)-3′′, 4′′-di-O-methyl-β-D-xylopyranosyl]-diphyllin
(11, Cleistanthoside-A)
The solid Cleistanthoside A tetraacetate (87 mg, 0.1 mmol) was dissolved in MeOH (5
mL), and then K2CO3 (34.5 mg, 0.25 mmol) was added. After stirring for 60 min at room
temperature the solution was neutralized with 1 mol•L-1 HCl and concentrated under reduced
pressure. The residue was purified by column chromatography on silica eluting with
CH2Cl2/MeOH (30:1 v/v) to afford Cleistanthoside A as a white solid (83%). m.p. 237-242 °C;
1
[α]58927: -5° (MeOH, c 1.0); H NMR (400 Hz, CDCl3): δ 7.82 (s, 1H, 5-ArH), 6.96 (s, 1H,
8-ArH), 6.89 (t, J=12, Hz, 1H, 6’ -ArH), 6.68 (s, 1H, 2′-ArH), 6.64 (s, 1H, 5′-ArH), 6.04 (t,
J=14.4 Hz, 2H, 7′-CH2-), 5.52-5.40 (m, 2H, 3a-CH2-), 5.24-4.97 (m, 4H, 6′′′, 5′′-CH2-, 1′′′,
2′′′-CH-), 4.92 (t, J=4.4 Hz, 1H, 1′′-CH-), 4.15 (dd, J=7.2, 14 Hz, 1H, 5′′′-CH-), 4.03 (s, 3H,
6-OMe), 3.97 (d, J=9.6 Hz, 1H, 2′′′-CH-), 3.80 (s, 2H, 5′′-CH2-, 3′′-CH-), 3.73 (d, J=12 Hz,
3H, 7-OMe), 3.64 (d, J=2.8 Hz, 4H, 3′′-OMe), 3.52-3.49 (m, 3H, 6′′′-CH2-, 3′′′, 4′′′-CH-), 3.39
(d, J=3.6 Hz, 3H, 4′′-OMe), 3.14 (obsc, 1H, 4′′-CH-), 2.05 and 1.28-1.21 and 0 (obsc, 4H, 2′′′,
3′′′, 4′′′, 6′′′-OH); 13C NMR (CDCl3): δ 170.1, 151.8, 150.2, 147.4, 144.0, 143.7, 135.7, 130.6,
128.34, 126.9, 123.6, 119.2, 110.7, 108.1, 106.0, 104.4, 101.5, 101.2, 76.2, 74.4, 69.8, 67.6,
61.7, 61.1, 60.4, 60.2, 58.0, 55.4, 55.7; HRMS (ESI-TOF) (m/z) [M+Na]+ calcd for
C34H38O16Na 725.20575, found 725.20275, Spectral data for 11 matched those previously
reported[15] .
4.2 In Vitro Antiproliferative Assays
Antiproliferative cellular assays were conducted as described using MCF-7, HeLa,
HepG2, HCT-116, and U251. All cell lines were cultured in a 37 °C incubator with a 5% CO2
environment. Compounds were dissolved in DMSO with a concentration of 10 mM. Cells
were seeded into 96-well plates at a concentration of 2×103 per well, cultured for 24 h,
exposed to the compounds at various concentrations for cancer cell viability experiments,
cells were cultured for 72 h and viability was determined through the use of the MTT assay.
MTT assay data are shown of three independent experiments.
9

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4.3 Activity of V-ATPase Assays
The human liver hepatocellular carcinoma cell line (HepG2) was used for V-ATPase
activity assay. Briefly, cells were grown in DMEM supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100
mg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. HepG2 cells were
exposed to the compounds at various concentrations, cultured for 72 h, and collected. To
evaluate V-ATPase activity, the release of phosphate was measured in spectrophotometer by
colorimetric assay according to the manufacturer’s instruction. Absorption (340 nm) of
samples at 0 min, the 5th minute, and the 10th minute after beginning of the reaction was
measured in reaction medium of malachite green to determine the V-ATPase activity. The
experiment was repeated three times.
4.4 Lysosomal pH Analysis
HepG2 cells were grown on coverslips in six-well plates and loaded with 100 nM
LysoTracker Red and treated with 100nM 9 or 11 for 3 h. Coverslips were washed with PBS,
fixed for 10 min with 1% formaldehyde, washed again with PBS, and mounted on slides for
fluorescence or DIC imaging using a Leica DMRXA2 confocal microscope and Simple PCI
software.
Acknowledgements
The studies in the laboratory were supported by the natural science foundation of Jiangsu
Province (BK2011390), Priority Academic Program Development of Jiangsu Higher
Education Instititutions (PAPD), National Natural Science Foundation of China (Grant No.
31171143 & 21201105).
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Eur. J. Pharmacol. 667(2011) 330-338
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Figure Caption
Fig.1. Diphyllin and its glycosides
Scheme 1. Synthesis of xylopyranosyl bromide 7
Scheme 2. Synthesis of Cleistanthin-A, Cleistanthoside A and Cleistanthoside A tetraacetate
Fig.2. Inhibition of V-ATPase activity by CA and CleT
Fig.3. Lysosomal pH analysis of HepG2 treated with CA, CT and left untreated (con) for 48 h.
Acidity of lysosomes was visualized using a pH-dependent LysoTracker and confocal
microscopy. One representative picture of three independent sets is shown
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Table1. Optimization of the reaction conditions for the second glycosylation.
Entry
1
Solvent
MeCN
T (°C)
rt
Reagent
NBS
Donor
12
Yielda (%)
no product
12
2
3
MeCN
-20
-20
NIS, TMSOTf
TMSOTf
<5%
<5%
13
CH2Cl2
13
14
14
4
5
6
CH2Cl2
CH2Cl2
-20
-20
40
BF3·Et2O
AgOTf
64
no product
no product
CHCl3/H2O
TBAB, NaOH
aThe yields after column chromatography are given in brackets.

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Table 2. In vitro anti-proliferative activity of CA and CT
Compd
IC50 (nM)
MCF-7
190
HeLa
67
HepG2
36
HCT-116
31
U251
97
CA
CleA
>200
92
>200
74
>200
39
>200
82
>200
101
CleT
Paclitaxel
45
96
84
69
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Highlights
• Three diphyllin glycosides CA, CleA and CleT have been synthesized.
• CA and CleT showed anti-proliferative activity at nanomolar concentrations.
• CA and CleT showed potent inhibitory effect on V-ATPase.
• CA and CleT were identified as novel V-ATPase inhibitors.

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Supporting Information
Synthesis and Identification of Cytotoxic Diphyllin
Glycosides as Vacuolar H+-ATPase Inhibitors
Zhitao Zhang, Jinlong Ma, Jie Hui, Li Zhu, Yu Zhao*
Institute of Nautical Medicine, Nantong University, Nantong 226001, China
 Corresponding author. Tel.: +86 513 85051797; fax: +86 513 85051796.
E‐mail addresses: zhaoyoo@163.com

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Table of Contents
1. General Experimental Procedures
S3
S4-S11
S12-S13
S14-S20
S21
2. NMR Spectra of Compounds
3. HSQC Spectra of Compounds
4. MS/MS Accurate of Compounds
5. The Fitting Curve of CA and CleT
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General Experimental Procedures
All air sensitive and moisture reactions were conducted under nitrogen in
vacuum dried flask. Tetrahydrofuran were freshly distilled from
sodium/benzophenone while CH2Cl2 was freshly distilled from phosphorus pentoxide.
Other solvents and chemicals obtained from commercial sources were used without
further purification. TLC was performed on precoated silica plates. UV light was used
to detect the spots. Flash chromatography was performed on silica gel (100-200 mesh).
1H NMR and 13C NMR spectra were taken on 400 MHz spectrometer with
tetramethylsilane (TMS) as an internal standard, and chemical shifts were recorded in
ppm values. Ion electrospray ionization (ESI) mass spectra were carried out on the
instrument equipped with a turbo ionspray source.
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3, 4-Di-O-acetyl-1, 2-O-[(R, S)-ethylidenyl]-D-xylopyranose (2) 1H NMR (400 Hz,
CDCl3)
1, 2-O-[(R, S)-ethylidenyl]-D-xylopyranose (3) 1H NMR (400 Hz, CDCl3)
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1
3, 4-Di-O-methyl-1, 2-O-[(S)-ethylidenyl]-D-xylopyranose (4) H NMR (400 Hz,
CDCl3)
3, 4-Di-O-methyl-xylopyranose (5) 1H NMR (400 Hz, CDCl3)
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1, 2-Di-O-acetyl-3, 4-di-O-methyl-xylopyranose (6) 1H NMR (400 Hz, CDCl3)
1
4-O-[2′′-O-acetyl-3′′, 4′′-di-O-methyl-β-D-xylopyranosyl]-diphyllin (8) H NMR
(400 Hz, CDCl3)
S6