Synthesis and Evaluation of Cleistanthin A Derivatives as Potent Vacuolar H+-ATPase Inhibitors

Article abstract of DOI:10.1111/cbdd.12538

Twelve new glycosides and alkane derivatives of cleistanthin A were designed and synthesized. Their in vitro antiproliferative activity was investigated against HCT-116, HepG2, A549, Hela tumor cell lines and HEK293 cell by MTT assay. Most of these compounds displayed antiproliferative effects on four cancer cells at submicromolar concentration, but they were less potent than cleistanthin A Moreover, they showed no antiproliferative effects on HEK293 cell at 200 nm. The most potent compounds, 3e and 4a, have been shown to inhibit the activity of vacuolar H⁺-ATPase (V-ATPase) and neutralize the pH of lysosomes at submicromolar concentrations.

Full text of DOI:10.1111/cbdd.12538

Chem Biol Drug Des 2015; 86: 691–696
Research Article
Synthesis and Evaluation of Cleistanthin A Derivatives
as Potent Vacuolar H -ATPase Inhibitors
+
+
Yu Zhao, Yapeng Lu, Jinlong Ma and Li Zhu*
raacetate, as potent vacuolar H -ATPase (V-ATPase)
inhibitors (10).
Institute of Nautical Medicine, Nantong University,
Nantong 226001, China
These results prompted us to design and synthesize a
library of cleistanthin A derivatives to explore novel V-AT-
Pase inhibitors with the hope of optimizing the anticancer
activity of cleistanthin A. Herein, we report the syntheses,
antiproliferative activity, V-ATPase inhibitory effects, and
lysosomal pH analysis of these glycosides and alkyl ethers
derivatives of cleistanthin A (Figure 1). Some of these deriva-
tives showed strong potent V-ATPase inhibitory activity.
*
Corresponding author: Li Zhu, zhulili65@163.com
Twelve new glycosides and alkane derivatives of cleis-
tanthin A were designed and synthesized. Their in vitro
antiproliferative activity was investigated against HCT-
1
16, HepG2, A549, Hela tumor cell lines and HEK293
cell by MTT assay. Most of these compounds dis-
played antiproliferative effects on four cancer cells at
submicromolar concentration, but they were less
potent than cleistanthin A Moreover, they showed no
antiproliferative effects on HEK293 cell at 200 nM. The
most potent compounds, 3e and 4a, have been shown
Methods and Materials
+
to inhibit the activity of vacuolar H -ATPase (V-ATPase)
Chemistry
and neutralize the pH of lysosomes at submicromolar
concentrations.
Materials and General Methods’ Commercial grade
reagents and solvents were used without further purifica-
tion, except where noted. Flash chromatography was per-
formed on silica gel. The purity of all compounds was
judged by TLC analysis (single-spot/two-solvent systems)
+
Key words: Cleistanthin A, inhibitor, synthesis, vacuolar H -
ATPase
1
13
using a UV lamp. H NMR and C NMR spectra were
taken on 400 MHz spectrometer with tetramethylsilane
Received 12 October 2014, revised 6 January 2015 and
accepted for publication 2 February 2015
(TMS) as an internal standard, and chemical shifts were
recorded in ppm values.
The regulation of intracellular pH and modulation of
intra-organellar acidity has been implicated in various physi-
ological functions. The maintenance of intracellular pH is
Typical synthetic procedure for synthesis of 3a,
3
c, and 3e
+
A mixture of the cleistanthin A (0.10 mmol), glycosyl tri-
controlled by the vacuolar H -ATPases (V-ATPases), which
ꢀ
chloroacetimidate (0.13 mmol), and powered 4 A molecu-
are a class of ATP-driven multisubunit proton pumps found
in all eukaryotic cells (1). Significantly, scientific evidence
suggests that the acidic microenvironment is critical to
managing cancer development, progression and metasta-
sis. Thus, V-ATPases play a vital role in tumor invasion and
metastasis development, which are responsible for micro-
environment 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.
lar sieves in anhydrous CH
20 °C under N2 for 10 min (Scheme 1). A solution of
BF O (0.13 mmol) in CH Cl was added dropwise.
ꢁEt
After being stirred at room temperature for 4 h, the mixture
was diluted with CH Cl (3 9 10 mL), washed with satu-
rated NaHCO (1 9 10 mL), brine (2 9 20 mL), and the
organic layer was dried on Na SO . The residue was puri-
2 2
Cl (3 mL) was stirred at
ꢀ
3
2
2
2
2
2
3
2
4
fied by column chromatography on silica gel eluting with
EtOAc/petroleum ether (1:2 v/v) to afford a solid.
000 000 000 000
-O-[2 , 3 , 4 , 6 -O-tetra-O-acetyl-b-D-
galactopyranosyl (1?2)-3 , 4 -di-O-methyl- b-D-
4
0
0
00
The natural lignan, diphyllin, has been proved to possess
potent V-ATPase inhibitory activity with the IC50 value
xylopyranosyl]-diphyllin (3a)
27
white solid, yield 55%, m.p. 143–145 °C; [a]589 : ꢀ29.7°
17 nM (9). Recently, we have indentified two cytotoxic di-
1
(
CHCl , c 1.0); H NMR (400 MHz, CDCl ): d 7.85 (s, 1H,
3
3
phyllin glycosides, cleistanthin A and cleistanthoside A tet-
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12538
691

Zhao et al.
Figure 1: Cleistanthin A and its derivatives.
0
5
-ArH), 7.08 (s, 1H, 8-ArH), 6.98(d, J = 8.4 Hz, 1H, 6 -
concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel eluting
with CH Cl /MeOH (30:1 v/v) to afford a solid.
0
0
ArH), 6.86–6.79 (m, 2H, 2 , 5 -ArH), 6.10 (d, J = 20.8 Hz,
H, 7 -CH -), 5.58–5.50 (m, 2H, 3a-CH -), 5.46 (d,
J = 2.4 Hz, 1H, 3 -CH-), 5.31 (t, J = 8.4 Hz, 1H, 4 -CH-
0
2
2
2
2
2
0
00
000
000 000
)
1
, 5.10–5.05 (m, 2H, 1 ,2 -CH-), 4.86 (d, J = 7.2 Hz, 1H,
0
0
000
00
00
-CH-), 4.16 (s, 3H, 6-OMe), 4.10–4.01 (m, 3H, 6 –
4-O-[b-D-galactopyranosyl (1?2)-3 , 4 -di-O-
methyl-b-D-xylopyranosyl]-diphyllin (3b)
00 00 000
CH
s, 3H, 7-OMe), 3.63 (s, 3H, 3 -OMe), 3.48 (s, 3H, 4 -
2 2
- 9 2, 5 -CH -), 3.97–3.89 (m, 2H, 2 , 5 -CH-), 3.82
0
0
00
27
(
white solid, yield 90%, m.p. 239–242 °C; [a]589 : ꢀ45°
0
0
1
OMe), 3.44-3.41 (q, J = 4.4, 8.4 Hz, 1H, 4 -CH-), 3.29 (t,
J = 8.4 Hz, 1H, 3 -CH-), 3.14 (t, J = 9.6 Hz, 1H, 5 -CH-),
.15 (s, 3H, 2 -OCOMe), 2.12 (s, 3H, 3 -OCOMe), 2.02
s, 3H, 4 -OCOMe), 1.87(s, 3H, 6 -OCOMe); C NMR
CDCl ): d 170.2, 169.4, 151.7, 150.1, 147.5, 147.4,
36.3, 131.1, 130.7, 128.4, 127.2, 123.6, 119.2, 110.7,
08.2, 106.0, 103.5, 102.0, 101.3, 85.0, 80.0, 79.7, 71.0,
0.7, 69.1, 67.4, 67.0, 62.9, 60.9, 60.6, 58.3, 56.5, 55.8,
(MeOH, c 1.0); H NMR (400 MHz, CDCl
3
): d 7.86 (s,
0
0
00
1H, 5-ArH), 7.05 (d, J = 8 Hz, 1H, 8-ArH), 6.99 (s, 1H,
0
00
000
0
0
2
(
(
1
1
7
2
6 -ArH), 6.93 (d, J = 5.6 Hz, 1H, 2 -ArH), 6.81 (d,
0
00
000
13
0 0
J = 8 Hz, 1H, 5 -ArH), 6.13 (s, 2H, 7 -CH -), 5.58 (dd,
2
3
J = 15.2, 31.6 Hz, 2H, 3a-CH
2
-), 5.20 (t, J = 6.4 Hz, 1H,
000 00 000
1 -CH-), 4.80–4.76 (m, 2H, 5 ,6 -CH -), 4.64 (d,
2
00 00 000
J = 7.6 Hz, 1H, 1 -CH-), 4.48 (t, J = 4.8 Hz, 2H, 2 , 5 -
0
0
000
CH-), 4.07–4.01 (m, 2H, 5 -CH -, 2 -CH-), 3.98 (s, 3H,
2
+
000 00
6-OMe), 4.10–4.01 (m, 3H, 6 -CH - 9 2, 5 -CH -),
2 2
0.9, 20.7, 20.4; HRMS (ESI-TOF) (m/z) [M + Na] calcd
00 000
for C42
H
46
O
20Na 893.24801, found 893.24920.
3.97–3.89 (m, 2H, 2 , 5 -CH-), 3.57 (s, 3H, 7-OMe),
00 00 000 000 000
.53–3.40 (m, 5H, 3 , 4 , 3 , 4 -CH-, 6 -CH -), 3.38
2
s, 3H, 3 -OMe), 3.33 (s, 3H, 4 -OMe);
3
(
0
0
00
13
C
NMR
Typical synthetic procedure for synthesis of 3b,
d, and 3f
The solid of peracetyl glycoside (3a, 3c, 3e) (0.1 mmol)
(CDCl
3
): d 169.4, 151.9, 150.4, 147.4, 144.4, 135.0,
3
130.1, 129.1, 128.9, 128.7, 126.9, 124.0,119.2, 111.3,
108.4, 105.9, 104.5, 102.6, 101.8, 101.5, 83.5, 79.6,
was dissolved in MeOH (5 mL), and then, K2CO3
78.9, 77.7, 75.8, 73.8, 71.5, 68.3, 67.5, 60.6, 57.7;
+
(0.25 mmol) was added. After stirring at room temperature
HRMS (ESI-TOF) (m/z) [M + Na] calcd for C34
H
38
O
16Na
for 60 min, the solution was neutralized with 1 M HCl and
725.20575, found 725.20724.
6
92
Chem Biol Drug Des 2015; 86: 691–696

+
Cleistanthin A Derivatives as Vacuolar H -ATPase Inhibitors
Scheme 1: Synthesis of cleistanthin A derivatives.
Typical synthetic procedure for synthesis of 4a–4e
To a stirring solution of cleistanthin A (0.22 mmol) in
DMSO (5 mL) was added NaOH (1.76 mmol) (Scheme 1).
After stirring at room temperature for 10 min, bromoe-
thane (4.4 mmol) was added. The reaction mixture was
stirred for 5 h at room temperature and then was diluted
Sciences (Shanghai, China). All cell lines were cultured in
a 37 °C incubator with a 5% CO2 environment. Com-
pounds were dissolved in DMSO with a concentration of
10 mM. Cells were seeded into 96-well plates at a con-
3
centration of 2 9 10 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.
2 2
with CH Cl (50 mL), washed with water (2 9 10 mL),
brine (2 9 20 mL). The organic layer was dried on
Na SO and concentrated under reduced pressure. The
2
4
residue was purified by column chromatography on silica
gel eluting with EtOAc/petroleum ether (1:2 v/v) to afford
a solid.
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-inacti-
vated fetal bovine serum, 2 mM L-glutamine, 100 units/mL
penicillin, and 100 mg/mL streptomycin at 37 °C in a
0
0
00
00
4
-O-[2 -O-ethyl-3 , 4 -Di-O-methyl-b-D-
xylopyranosyl]-diphyllin (4a)
2
7
white solid, yield 83%, m.p. 137–140 °C; [a]589 : ꢀ59.7°
1
(
5
CHCl , c 1.0); H NMR (400 MHz, CDCl ): d 7.81 (s, 1H,
humidified atmosphere with 5% CO . HepG2 cells were
3
3
2
0
-ArH-), 7.09 (s, 1H, 8-ArH-), 6.97 (d, J = 9.2 Hz, 1H, 6 -
ArH-), 6.85–6.79 (m, 2H, 2 , 5 -ArH-), 6.10 (s, 1H, 7 -CH
, 6.05 (s, 1H, 7 -CH
exposed to the compounds at various concentrations, cul-
tured for 72 h, and collected. To evaluate V-ATPase activ-
ity, the release of phosphate was measured in
spectrophotometer by colorimetric assay according to the
manufacturer’s instruction. Absorption (340 nm) of sam-
ples at 0 min, the 5th min, and the 10th min after begin-
ning of the reaction was measured in reaction medium of
malachite green to determine the V-ATPase activity. The
experiment was repeated three times.
0
0
0
2
-
0
)
(
6
7
2
-), 5.51–5.41 (m, 2H, 3a-CH
00 00
2
-), 4.83
d, J = 7.6 Hz, 1H, 1 -CH-), 4.14–4.06 (m, 4H, 5 -CH-,
00 00
-OMe), 4.03–3.96 (m, 2H, 2 , 5 -CH-), 3.81 (s, 3H,
-OMe), 3.69 (s, 3H, 3 -OMe), 3.50 (s, 4H, 3 -CH, 4 -
OMe), 3.41–3.52 (m, 1H, -CH -), 3.25 (t, J = 8.8 Hz, 1H,
-), 3.08 (t, J = 11.6 Hz, 1H, 4 -CH-), 1.36 (t,
J = 6.8 Hz, 3H, -CH
0
0
00
00
2
00
-
CH
2
13
3
); C NMR (CDCl
3
): d 169.8, 151.8,
1
1
8
1
50.9, 147.5, 144.2, 136.2, 130.8, 130.7, 128.3, 126.9,
23.6, 119.2, 110.7, 108.2, 106.2, 104.7, 101.2, 100.8,
5.6, 81.5, 79.3, 69.0, 67.3, 63.5, 61.0, 58.8, 56.3, 55.8,
Lysosomal pH analysis
+
5.8; HRMS (ESI-TOF) (m/z) [M + Na]
calcd for
HepG2 cells were grown on coverslips in six-well plates
and loaded with 100 nM LysoTracker Red and treated with
different concentration of compounds diphyllin, 3e and 4a
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.
C H O11Na 591.1842, found 591.1853.
3
0 32
Spectroscopic data of 3c, 3e, 3d, 3f, 4b, 4c, 4e can be
found in Supporting information.
In vitro antiproliferative assays
Human hepatocellular carcinoma (HepG2), human non-
small-cell lung tumor (A549), human colon carcinoma
(
(
HCT-116), human embryonic kidney 293 cell line
HEK293), and human cervical carcinoma (HeLa) were
Statistical analysis
Data were presented as means ꢂ SD and analyzed by
SPSS software (SPSS 19.0; SPSS Inc., Chicago, IL, USA).
obtained from the Cell Bank of the Chinese Academy of
Chem Biol Drug Des 2015; 86: 691–696
693

Zhao et al.
Pictures were processed with Photoshop software. Mean
values were obtained from at least three independent
experiments.
A549, Hela, and a normal cell HEK293, were used to
determine their antiproliferation activity employing a MTT
assay. The average inhibitory concentrations of 50% were
shown in Table 1.
Results and Discussion
Most of these compounds displayed antiproliferative
effects on four cancer cells at submicromolar concentra-
tion, but they were less potent than cleistanthin A. These
derivatives showed antiproliferative selectively for HCT-116
cell line, with a potency of over several times that of other
three cell lines. Moreover, they showed no antiproliferative
effects on normal HEK293 cell at 200 nM. In the glyco-
sides derivatives, 3b, 3d, and 3f showed weak antiprolifer-
ative activity, while peracetyl glycosides 3a, 3c, and 3e
possessed significant effects, which suggested the hydro-
phobic glycosyl moieties were critical to their antiprolifera-
tive activity. In particular, the glycoside 3e having an
acetylated a-L-arabinose exhibited the best inhibitory effect
on these four cancer cells. This result prompted us to fur-
ther design a series of compounds with hydrophobic ter-
minals by introducing hydrophobic C2-C6 alkane chains.
We found all these five alkane derivatives 4a–4e pos-
sessed strong antiproliferation activity similar to the control
drug paclitaxel. Moreover, these derivatives showed com-
paratively better activity against HCT-116 cell line than
other three cell lines.
Chemistry
Previously, we have reported the synthesis of cleistanthin A
from diphyllin. Therefore, we could use the synthesized
cleistanthin A as staring material in this study. We also
found that the glycosylation of cleistanthin A could be
achieved effectively by BF ꢁEt O catalyzed Schmidt glyco-
3
2
sylation (10). In the first part of this study, six diglycosides
3
a–3f bearing different glycosyl moities were synthesized
employing Schmidt glycosylation from cleistanthin A. Briefly,
the glycosylation of cleistanthin A with glycosyl trichloroace-
timidate was performed under the catalysis of BF ꢁEt O (1.5
3
2
ꢁ
ꢀ
equiv) in the presence of 4 A MS in CH
2
Cl
2
at ꢀ20 °C to
give per-acetylated glycosides (3a, 3c, 3e) in 55–75% yield
11,12). The following deacetylation with K CO in methanol
(
2
3
was performed smoothly to produce glycosides (3b, 3d, 3f)
in 90–98% yields (Scheme 1). The assignments of H NMR
signals were made on the basis of the comparison of natu-
rally occurring diphyllin glycosides and synthesized diphyllin
glycosides in our laboratory.
1
Secondly, the etherification reaction of cleistanthin A and
alkyl bromide in the presence of NaOH afforded the alkyl-
ation products 4a–4e in 77–83% yields.
V-ATPase inhibitory activity
Based on the above MTT results, 3e and 4a were used
for V-ATPase activity assay (Figure 2). The human liver
hepatocellular carcinoma (HepG2) was treated with 3e and
4a in a range of concentrations (0, 25, 50, 100 nM,
respectively), and then, the activity of V-ATPase was
examined by V-ATPase activity assay kit (10). Previously,
Antiproliferation activity
With twelve cleistanthin A derivatives in hand, four cancer
cell lines from various solid tumors, HCT-116, HepG2,
Table 1: In vitro antiproliferative activity of these compounds
IC50 ꢂ SD (nM)
Compounds
HepG2
HCT-116
A549
Hela
HEK293
1
7.9 ꢂ 1.2
77.5 ꢂ 2.3
>200
0.9 ꢂ 0.5
95.0 ꢂ 1.2
>200
4.4 ꢂ 0.7
19.2 ꢂ 2.3
56.1 ꢂ 0.1
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
5.5 ꢂ 1.0
2
2
3
3
3
3
3
3
4
4
4
4
4
a
128.1 ꢂ 0.2
>200
a
b
c
d
e
f
30.3 ꢂ 2.6
10.9 ꢂ 2.4
15.4 ꢂ 0.5
60.2 ꢂ 0.6
>200
>200
>200
>200
25.2 ꢂ 1.6
>200
7.7 ꢂ 1.2
>200
18.9 ꢂ 0.2
>200
78.3 ꢂ 2.2
>200
13.9 ꢂ 0.6
0.8 ꢂ 0.5
5.2 ꢂ 0.8
25.4 ꢂ 4.2
>200
>200
>200
>200
a
b
c
d
e
26.3 ꢂ 2.1
23.5 ꢂ 0.7
57.5 ꢂ 2.2
30.0 ꢂ 1.2
27.7 ꢂ 2.4
13.6 ꢂ 0.7
3.5 ꢂ 1.6
0.8 ꢂ 0.3
6.0 ꢂ 0.9
6.2 ꢂ 0.4
7.8 ꢂ 0.6
1.1 ꢂ 1.3
5.8 ꢂ 1.5
4.2 ꢂ 0.5
23.9 ꢂ 4.8
18.6 ꢂ 0.6
12.4 ꢂ 0.2
4.3 ꢂ 1.6
46.2 ꢂ 2.5
36.3 ꢂ 0.2
80.7 ꢂ 4.6
67.6 ꢂ 3.2
44.5 ꢂ 2.5
32.2 ꢂ 3.2
Paclitaxel
NT, not detect.
All experiments were independently performed at least three times.
6
94
Chem Biol Drug Des 2015; 86: 691–696

+
Cleistanthin A Derivatives as Vacuolar H -ATPase Inhibitors
TopoII and tubulin. Therefore, we believe that diphyllin gly-
cosides are multitargeted antitumoral natural compounds.
The lysosome acidity was examined in HepG2 cells at vari-
ous concentrations of diphyllin, 3e and 4a (Figure 3).
HepG2 cells were stained with a pH-sensitive fluorescence
dye (LysoTracker Red) and sequential loaded with diphyl-
lin, 3e and 4a. As shown in Figure 3, the amount of red
fluorescence decreased in cells treated with diphyllin, 3e
and 4a compared with untreated cells. The V-ATPase was
completely suppressed at the concentrations of 60 nM 3e
or 4a and 3 lM diphyllin, which indicated they were more
potent than diphyllin. The results also implied that the
modulation of lysosomes acidity by 3e and 4a was consis-
tent with their inhibitory effect on V-ATPase activity.
Figure 2: Inhibition of V-ATPase activity by 3e and 4a.
we measured the inhibitory of V-ATPase activity of cleis-
tanthin A Treatment of HepG2, it decreased <10% the
percentage of activity of V-ATPase at 25 nM (10). While
treatment of HepG2 with 3e and 4a at 25 nM significantly
decreased the percentage of activity of V-ATPase to 34%
and 18%, respectively, which proved they were more
potent than cleistanthin A. But we found their V-ATPase
inhibitory activity did not matched well with their antiprolif-
eration activity. We deduced that the inhibition of activity
of V-ATPase was not the only mode of action of their
antiproliferation activity, and they could also act on both
Conclusion
In summary, we have designed and synthesized a new
series of cleistanthin A derivatives bearing glycosyl moieties
or chain alkanes as potential V-ATPase inhibitors. Most of
these compounds displayed antiproliferative effects on four
cancer cells at submicromolar concentration, but they were
less potent than cleistanthin A. The acetylated a-L-arabino-
side 3e and C2 alkane 4a possessed the best antiprolifera-
Figure 3: Lysosomal pH analysis of HepG2
treated with diphyllin, 3e and 4a 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.
Chem Biol Drug Des 2015; 86: 691–696
695

Zhao et al.
tive activity in vitro against four cancer cell lines. Moreover,
these derivatives showed no antiproliferative effects on nor-
mal HEK293 cell at 200 nM. They also displayed potent
inhibitory activity against V-ATPase. Lysosomal pH analysis
illuminated that they inhibited the HepG2 lysosomal acidifi-
cation at submicromolar concentrations. Compounds 3e
and 4a possessed promising V-ATPase inhibitory activity,
which need to be studied further.
as novel inhibitors of the sarco/endoplasmic reticulum
calcium ATPase. Bioorg Med Chem;17:6613–6619.
6. Lebreton S., Jaunbergs J., Roth M.G., Ferguson D.A.,
Brabander J.K. (2008) Evaluating the potential of vacu-
olar ATPase inhibitors as anticancer agents and multi-
gram synthesis of the potent salicylihalamide analog
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7
. Petrangolini G., Supino R., Pratesi G. (2006) Effect of a
novel vacuolar-H -ATPase inhibitor on cell and tumor
+
Acknowledgments
response to camptothecins.
J
Pharmacol Exp
Ther;318:939–946.
The studies in the laboratory were supported by the Natu-
ral Science Foundation of Nantong City (BK2014069), Pri-
ority Academic Program Development of Jiangsu Higher
Education Instititutions (PAPD), National Natural Science
Foundation of China (Grant No. 31471141).
8. Sugimoto Y., Konoki K., Murata M. (2009) Design, syn-
thesis, and biological evaluation of fluorinated ana-
logues of salicylihalamide. J Med Chem;52:798–806.
9. Sorensen M.G., Henriksen K., Wulff A.V., Dziegiel
M.H., Karsdal M.A. (2007) Diphyllin, a novel and natu-
rally potent V-ATPase inhibitor, abrogates acidification
of the osteoclastic resorption lacunae and bone resorp-
tion. J Bone Miner Res;22:1640–1648.
Conflict of interest
1
0. Zhang Z.T., Ma J.L., Zhu L., Zhao Y. (2014) Synthesis
and identification of cytotoxic diphyllin glycosides as
All authors declare that they have no conflict of interest.
vacuolar
Ht-ATPase
inhibitors.
Eur
J
Med
Chem;82:466–471.
1
1
1. Song G.P., Yang S., Zhang W. (2009) Discovery of the
first series of small molecule H5N1 entry inhibitors.
J Med Chem;52:7368–7371.
2. Utille J.P., Jeacomine I. (2007) Synthesis of a library
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Additional Supporting Information may be found in the
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Appendix S1. Characterization for all the synthesized
compounds.
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