Design, synthesis and biological evaluation of valepotriate derivatives as novel antitumor agents

Article abstract of DOI:10.1039/c6ra27478a

Natural products remain the largest resources of lead compounds that can be used to develop novel anticancer drug candidates. Based on deacetylisovaltratum, a natural product with promising anticancer activity, herein we designed and synthesized of a series of valepotriate derivatives with a novel skeleton from commercially available genipin. In addition, a structure-activity relationship study demonstrated the importance of an epoxy group on the C1-position and the preferable size of the sidechain ((5-methylhexanoyl)oxy) on the C-7 position of valepotriates for their cytotoxic activities. The most potent compound 1e showed moderate to good IC₅₀ values against various cancer cells, ranging from 10.7 to 50.2 μM, which are comparable to that of deacetylisovaltratum. Additionally, we demonstrate that mitochondrion-mediated apoptosis would be its mechanism of action, thus enlightening the further development of novel valepotriate derivatives.

Full text of DOI:10.1039/c6ra27478a

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Design, synthesis and biological evaluation of
valepotriate derivatives as novel antitumor agents†
Cite this: RSC Adv., 2017, 7, 31899
c
Bo Zhang,‡ab Ruiying Guo,‡c Yongzhou Hu,c Xiaowu Dong,
Nengming Lin,b
a
a
b
a
*
*
Xiaoyang Dai, Honghai Wu, Shenglin Ma and Bo Yang
Natural products remain the largest resources of lead compounds that can be used to develop novel
anticancer drug candidates. Based on deacetylisovaltratum, a natural product with promising anticancer
activity, herein we designed and synthesized of a series of valepotriate derivatives with a novel skeleton
from commercially available genipin. In addition, a structure–activity relationship study demonstrated the
importance of an epoxy group on the C1-position and the preferable size of the sidechain ((5-
methylhexanoyl)oxy) on the C-7 position of valepotriates for their cytotoxic activities. The most potent
compound 1e showed moderate to good IC50 values against various cancer cells, ranging from 10.7 to
50.2 mM, which are comparable to that of deacetylisovaltratum. Additionally, we demonstrate that
mitochondrion-mediated apoptosis would be its mechanism of action, thus enlightening the further
development of novel valepotriate derivatives.
Received 29th November 2016
Accepted 12th June 2017
DOI: 10.1039/c6ra27478a
rsc.li/rsc-advances
metrocarcinoma and cervical cancer in ancient China, whereas
the biological components responsible for its anticancer activity
are not fully recognized.10,11 Indeed, a variety of pharmacologi-
1 Introduction
Natural product research continues to explore a variety of lead
compounds, which are potential templates for the development
of new anticancer drug candidates.1–3 However, challenges
remain unsolved in either the isolation or purication of
bioactive natural products; thus semisynthesis becomes a pref-
erable method to obtain bioactive compounds.4 The semi-
synthetic strategy is widely used when the precursor molecules
are structurally complex, or difficult produce by total synthesis,
such as anticancer drugs (i.e. paclitaxel, etoposide, and irino-
tecan), antimalarial drugs (i.e. roxithromycin) and antibiotics
(i.e. cexime) (Fig. 1).
Valerian is a very important genus of plants used as
a medicinal herb in many areas of the world, the roots and
rhizomes of which have been used for the treatment of epilepsy,
hysteria, nervous disorders, neurasthenia and emotional
stress.5–9 In particular, Patrinia heterophylla Bunge, belonging to
the genus Valerian, showed anticancer activity against
cally active components (e.g. monoterpenes, valepotriates, and
sesquiterpenes) have been isolated and characterized.12,13
Among them, valepotriates, belonging to the family of iridoids
with a 10-carbon basic skeleton, showed their signicant
activity against cancer cells.1–3,14 Recently, we found that
deacetylisovaltratum, a compound belonging to the class of
valepotriates, could effectively cause G2/M-phase arrest in
gastric cancer cells by disrupting tubulin polymerization, and
inducing mitochondrion-dependent apoptosis.15,16 Considering
the unique structure of epoxy group in deacetylisovaltratum, the
alkylating properties could be responsible for its potent anti-
aZhejiang Province Key Laboratory of Anti-Cancer Drug Research, Institute of
Pharmacology and Toxicology, School of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, Zhejiang 310058, P. R. China. E-mail: yang924@zju.edu.cn;
Tel: +86-57188208400
bTranslational Medicine Research Center, Nanjing Medical University, Affiliated
Hangzhou Hospital, Hangzhou First People's Hospital, Hangzhou, Zhejiang 310006,
P. R. China. E-mail: mashenglin@medmail.com.cn; Tel: +86-57156007908
cZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory
of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310058, P. R. China
† Electronic supplementary information (ESI) available: NMR spectra of the
synthesized compounds. See DOI: 10.1039/c6ra27478a
‡ These authors contributed equally to this work.
Fig. 1 Examples of semisynthetic drugs.
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length of aliphatic side chain on cytotoxicity against a variety of
cancer cells. More signicantly, some compounds showed
superior cytotoxic activities than that of deacetylisovaltratum.
In addition, mitochondrion-mediated apoptosis was signi-
cantly observed, which would be the exact action mechanism of
its cytotoxic activities, thus enlightening the further develop-
ment of novel valepotriate derivatives.
2 Results and discussion
2.1 The synthesis of valepotriate derivatives
Fig. 2 Murakami's synthetic strategy.
The synthesis of compounds 1a–h started with genipin is
illustrated in Scheme 1. At rst, genipin 2 was treated by
Amberlyst-15 resins in methanol to afford methyl acetal 3,
which was submitted to phenylsuldation using diphenyldi-
sulde and tributylphosphane in toluene to give phenylsulde
4. Aer acidic hydrolysis of the methyl acetal with 10% HCl–
THF, the resulting hemiacetal 5 was coupled with isovaleric acid
in the presence of isovaleric acid, 1,10-carbonyldiimidazole
(CDI) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to yield
isovaleryl acetal 6. Aer oxidation of 6 with oxone in methanol,
the resulting sulfoxide was subjected to Mislow–Evans rear-
rangement with trimethyl phosphite in MeOH to afford allyl
alcohol 7. In this rearrangement procedure, the reaction time
was quite crucial for the conversion, which should be carefully
monitored by TLC analysis. Then, stereoselective epoxidation
associated with the adjacent hydroxyl group using tert-butyl
hydroperoxide (TBHP) and vanadium oxyacetylacetonate
[VO(acac)2]17,18,21,22 afforded epoxyalcohols 8.18,22 Finally, desired
compounds 1a–h was obtained by introduction of the appro-
priate aliphatic acid group to C-7 of compound 7 by Mitsunobu
inversion using diethyl azodicarboxylate (DEAD) and triphe-
nylphosphine (Fig. 3).
cancer activity. Moreover, the structural diversity might change
its physicochemical properties and also its intracellular activi-
ties. However, due to the lack of structural diversity in natural
valepotriates, as well as the difficulty in synthetic procedures,
the structural–activity relationship (SAR) studies of valepotriate
analogues is very rare, which hampers the development of
valepotriate as novel anti-cancer agents. Therefore, the explo-
ration of the crucial structural requirement of valepotriates for
anticancer activities is particularly needed. In this study, we
intend to acquire series of valepotriate derivatives via semi-
synthesis and to study the biological activity of these
compounds.
Recently, Murakami et al. reported the concise synthesis of
5,6-dihydrovaltrate from commercially available iridoid geni-
pin, providing a novel semisynthetic route in structurally opti-
mization of valepotriates (Fig. 2).17,18 As part of our continued
interest in the area of chemical modication of natural prod-
ucts,19,20 herein, we designed, synthesized a series of valepo-
triate derivatives with novel skeleton from genipin using slightly
modied Murakami's method. Noteworthy, some crucial SAR
clues were found aer investigating the effect of epoxy and
Scheme 1 Synthesis of valepotriate derivatives. Reagents and conditions: (a) Amberlyst-15, MeOH; (b) PhSSPh, Tol; (c) 10%HCl, THF; (d) isovaleric
acid, DBU, CDI; (e) oxone, MeOH; (f) (MeO)3P, MeOH; (g) TPHP, VO(acac)2, Tol; (h) appropriate acid, DEAD, Ph3P, Tol.
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replacement of (5-methylhexanoyl)oxy group with aromatic (1d)
and cyclohexyl (1c) substituents reduced its cytotoxic activity.
Based on our efforts in structural optimization of valepotriate
analogues, we successfully discovered a potent semisynthetic
compound 1e bearing unique skeleton distinguished from
deacetylisovaltratum which was previously identied as potent
natural anti-cancer agent.
Fig. 3 The structures of valepotriate derivatives 1a–1h in this study.
2.3 Cytotoxicity of 1e in human lung cancer H1975 cells
Human lung cancer H1975 cell harbours both L858R and
T790M mutation in epidermal growth factor receptor (EGFR),
thus it is resistant to rst-generation EGFR tyrosine inhibitor
such as getinib or erlotinib. As recently reported, several
plants-derived compounds possessed anti-proliferation
activity against H1975, and the IC50 value varied from 3 to
15 mM.24–26 In our study, the cytotoxicity of 1e was determined
by CCK-8 assay in human lung cancer H1975 cells (Fig. 4).
1e exhibited both time- and concentration-dependent cyto-
toxicity against H1975 cells. The IC50 values of 48 and 72 h
treatment with 1e were 27.4 and 13.1 mM respectively.
2.2 Cytotoxicity of compounds 1a–h against various cancer
cells
To investigate whether these compounds could suppress the
growth of cancer cells, anti-proliferative effect was tested
against various cancer cell lines, including pancreatic cancer
cells (SW1990, BXPC3, CAPAN2, CFPAC, PANC1), breast cancer
cells (BT474, MCF7, MDA-MB-231), gastric cancer cells (AGC,
HGC-27, KATOIII) and lung cancer cells (H1795) (Table 1). As
expected, some of these compounds (i.e. 1c, 1d, 1e) showed
comparable cytotoxic activities as the parental compound
deacetylisovaltratum, suggesting that the novel skeleton with
methoxycarbonyl on C4-position of valepotriate remained the
anticancer activities. Moreover, we found epoxy group on C1-
position of valepotriate was very crucial by comparing with
the anticancer activities of 7a and deacetylisovaltratum, which
was in consistent with the work of Murakami et al.22 In another
study, R. Bos et al. reported the cytotoxicity of valepotriates
analogues against GL4 and COLO 320 cells with IC50 values
ranging from 1 to 6 mM.12 Additionally, three valepotriate
isomers isolated by S. Lin et al. displayed moderate cytotoxicity
against various cancer cell lines with IC50 values varied from 2.8
to 8.3 mM.23 In this study, 1e showed comparable cytotoxic
activity as previously reported valepotriate analogues. In further
SAR analysis, either shortening or prolonging the sidechain (i.e.
1a, 1b, 1f, 1g, 1h) undermined its biological activities, revealing
that (5-methylhexanoyl)oxy group on C-7 position would be
preferable lipophilicity towards cancer cells. Meanwhile, the
2.4 Compound 1e triggered apoptosis in H1975 cells
Apoptosis was usually involved in the proliferation inhibitory
effect caused by natural compound.27 Annexin V/PI staining was
used to characterize the early and late apoptotic cells aer cells
were treated with indicated concentrations of 1e for 48 h.28 In
H1975 cells, treatment with 20 mM of 1e yielded a 35% apoptosis
rate, which was in consistent with the IC50 value of 48 h treat-
ment (Fig. 5A). By comparing the apoptosis induction activity of
1e with other natural derived agents in H1975 cells, 1e exhibited
superior anti-cancer activity than cordycepin, but slightly less
effective than shikonin.24,25 Meanwhile, depolarized mitochon-
dria membrane potential was detected aer 24 h treatment with
20 mM of 1e (Fig. 5B). In addition, DAPI stain was used to verify
the occurrence of apoptosis. Aer 48 h treatment with 20 mM of
1e, nucleus with shrunk size and intensied uorescence were
seen under uorescence microscopy. Moreover, apoptotic
Table 1 Cytotoxic activities of valepotriate derivatives 1a–h against various cancer cellsa
Cytotoxic activities, IC50 (mM)
Cell lines
Cancer type
DI
7a
1a
1b
1c
1d
1e
1f
1g
1h
SW1990
BXPC3
CAPAN2
CFPAC
PANC1
BT474
MCF7
MDA-MB-231
AGS
Pancreatic cancer
34.9
14.4
17.5
13.0
14.8
24.3
27.5
23.1
7.7
85.2
24.6
63.7
87.7
>100
N.T.
51.2
51.0
53.2
25.7
N.T.
21.0
59.1
48.2
37.4
47.4
86.9
97.8
77.4
42.8
26.3
47.5
N.T.
25.8
>100
>100
53.8
52.0
N.T.
>100
>100
>100
>100
>100
N.T.
93.0
40.2
28.1
33.9
29.5
48.0
21.4
18.1
12.7
28.9
20.9
N.T.
28.2
54.9
37.1
68.0
60.9
66.9
54.0
19.8
27.3
36.9
28.2
N.T.
15.9
26.1
25.1
31.6
27.9
20.6
50.2
10.7
13.2
22.9
21.5
N.T.
11.3
N.T.
N.T.
40.7
54.3
N.T.
N.T.
N.T.
N.T.
55.7
>100
48.8
N.T.
N.T.
N.T.
>100
32.0
N.T.
N.T.
N.T.
N.T.
59.5
54.0
51.2
N.T.
N.T.
N.T.
>100
53.9
N.T.
N.T.
N.T.
N.T.
>100
26.6
92.5
N.T.
Breast cancer
Gastric cancer
Lung cancer
HGC-27
KATOIII
H1975
14.8
32.2
14.3
a
N.T. indicates not detectable.
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Fig. 7 1e triggered apoptosis in H1975 cells. (A) After treated with 1e
for 48 h, cells were lysed and proteins were analysed by western blot.
(B) After treated with 1e for 48 h, cells were lysed and caspase-3
activity was analysed using Caspase-3 Colorimetric Assay Kit (Bio-
vision, Milpitas, CA, US).
Fig. 4 The inhibitory effect of 1e on human lung cancer cell line
H1975. H1975 cells were seeded in 96-well plates and then treated
with 1e as indicated. Treatment was washed off after 24, 48, 72 or 96 h
and cell viability was calculated using CCK-8 assay.
2, activation of caspase-3 and cleavage of poly-(ADP-ribose)-
polymerase (PARP) was closely associated with the apoptosis
induced by isoliquiritigenin.29 In this study, to verify the role of
mitochondrion in 1e triggered apoptosis, we performed western
blots to analyze apoptosis-related proteins. As shown in Fig. 7A,
20 mM of 1e induced an elevated level of Bax, and meanwhile the
expression of Bcl-2 was decreased. In addition, decreased
protein level of procaspase-9 and cleaved PARP were found aer
treatment with 20 mM of 1e, indicating that 1e could trigger
mitochondrion-dependent apoptosis. In addition, 20 mM of 1e
signicantly induced an increase in caspase-3 activity (Fig. 7B),
which was not found in 10 mM of 1e treatment. Meanwhile, the
protein expression of XIAP was found unchanged aer treat-
ment with 1e, suggesting that XIAP was not involved in 1e
activated caspase-3. The anti-cancer activity of 1e was compa-
rable with other plants derived compounds. For instance, Wang
and et al. reported mitochondrion-dependent apoptosis
induced by cordycepin at the concentration of 15 mM in Hꢀ11975
cells.25 Lu and et al. found that treatment with 150 mg mL of
sulfated galactoglucan could activate caspase-3 in H1975 cells.30
These results indicated that 1e as a derivative of deacetyliso-
valtratum obtained from semi-synthesis, achieved superior
anticancer activity than the parental compound. Moreover, the
anti-proliferation ability was at a similar level of other plants
derived compounds.
Fig. 5 1e triggered mitochondrion-dependent apoptosis in H1975
cells. (A) H1975 cells were seeded in 6-well plate and cultured for 24 h.
Cells were treated with 1e for 48 h before staining and followed by
flow cytometric analysis. (B) H1975 cells were seeded in 6-well plate
and cultured for 24 h. Cells were treated with 1e for 12 h before JC-1
stain and flow cytometric analysis. Three independent experiments
were quantitatively analyzed. Each bar represented the mean ꢁ SD.
***P < 0.001.
bodies could be observed at 20 mM of 1e, suggesting that H1975
cells were undergoing apoptosis (Fig. 6).
2.5 Compound 1e induced caspase-dependent apoptosis
3 Conclusion
Mitochondrion plays a critical role in the apoptosis caused by
natural compounds. In H1975 cells, reduced expression of Bcl-
In this study, we designed and synthesized of a series of vale-
potriate derivatives from genipin using slightly modied Mur-
akami's method. Among these derivatives, 1e which contained
a novel skeleton with methoxycarbonyl on C4-position and (5-
methylhexanoyl)oxy group on C-7 position possessed stronger
anticancer activity than its parental compound deacetylisoval-
tratum. Noteworthy, specic SAR has been found: (1) natural
derived group at 1st position is better than others; (2) different
substituents at the 7th position can be critical for its bioactivity;
(3) the length of aliphatic side chain is crucial to its anticancer
activity. Finally, 1e has been found with superior potency than
lead compound deacetylisovaltratum. In addition, 1e induced
mitochondrion-mediated apoptosis was signicantly observed,
Fig. 6 H1975 cells were seeded in 6-well plate and cultured for 24 h.
Cells were treated with 1e for 48 h before DAPI stain and observed
under fluorescence microscopy. Yellow arrows indicates apoptotic
bodies. Scale bar ¼ 20 mm.
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which might be the action mechanism of its cytotoxic activities, 2.79 (dd, J ¼ 16.5, 8.6 Hz, 1H), 2.69 (t, J ¼ 7.5 Hz, 1H), 2.03–1.97
thus enlightening the further development of novel valepotriate (m, 1H), 1.87 (dd, J ¼ 14.8, 6.8 Hz, 1H), 1.76–1.68 (m, 1H). LC-
derivatives.
MS: 319 (M + H).
4.1.4 Conversion from hemiacetal (5) to hemiester (6). A
solution of CDI (14.4 g, 88 mmol) in CH2Cl2 (70 mL) was treated
with isovaleric acid (14.0 mL, 88 mmol) and DBU (264 mL, 1.76
mmol) at 0 ꢂC for 10 min. The mixture was added to a solution
4 Experimental section
4.1 Chemistry
Proton NMR spectra were obtained on a Bruker AVII 500 or 400 of the 5 (2.8 g, 8.8 mmol) in CH2Cl2 (10 mL), then the whole was
NMR spectrometer (500 or 400 MHz) by use of CDCl3, CD3OD, or stirred at rt overnight. Aer the reaction mixture was poured
DMSO-d6 as solvent. Carbon-13 NMR spectra were obtained into aqueous saturated NaCl, the whole was extracted with
a Bruker spectrometer (125 MHz) by use of CDCl3 CD3OD or EtOAc. The organic layer was successively washed with 5%
DMSO-d6 as solvent. Carbon-13 chemical shis are referenced aqueous HCl, aqueous saturated NaHCO3, and aqueous satu-
to the central peak of CDCl3 (d 77.0 ppm) or DMSO-d6 (d 39.5 rated NaCl and dried over Na2SO4. Concentrate the organic layer
ppm). Multiplicities are recorded by the following under reduced pressure to give a residue, which was puried by
abbreviations: s, singlet; d, double; t, triplet; q, quartet; m, silica gel column chromatography (PE/EtOAc ¼ 10 : 1) to afford
multiplet; J, coupling constant (Hz). ESI-MS spectra were ob- 6a (2.8 g, 80%). 1H NMR (500 MHz, CDCl3) d 7.44 (d, J ¼ 1.1 Hz,
tained from Shimadzu LCMS-2020 mass spectrometer. Unless 1H), 7.33–7.26 (m, 4H), 7.20 (ddd, J ¼ 7.2, 3.9, 1.3 Hz, 1H), 5.95
otherwise noted, reagents and solvent were obtained from (d, J ¼ 6.9 Hz, 1H), 5.67 (s, 1H), 3.76 (d, J ¼ 14.2 Hz, 1H), 3.71 (s,
commercial suppliers and without further purication.
3H), 3.49–3.44 (m, 1H), 3.19 (dd, J ¼ 11.4, 4.5 Hz, 1H), 2.97 (t, J ¼
4.1.1 Conversion from genipin (2) to methyl acetal (3). To 7.3 Hz, 1H), 2.76 (dd, J ¼ 16.7, 8.2 Hz, 1H), 2.31–2.07 (m, 4H),
a solution of genipin (3.57 g, 16.14 mmol) in MeOH (30 mL) was 0.96 (dd, J ¼ 6.6, 4.2 Hz, 6H). LC-MS: 425 (M + Na+).
added Amberlyst-15 (10.51 g). The mixture was stirred rt over-
4.1.5 Conversion from 6 to alcohol (7). To a solution of 6
night, and the resulting mixture was ltered and washed by (750 mg, 1.87 mmol) in MeOH (30 mL) was added oxone
EtOAc, and the combined ltrates were concentrated under (687 mg, 1.11 mmol) at 0 ꢂC for 1 h. Then the reaction was
reduced pressure to give the light yellow oil. Aer being puried quenched by aqueous saturated Na2SO3 for 10 min, and the
by silica gel column chromatography (PE/EtOAc ¼ 2 : 1) to mixture was extracted with EtOAc. The organic layer was washed
afford 3 (3.2 g, 84%).1H NMR (500 MHz, CDCl3) d 7.49 (s, 1H), by aqueous saturated NaCl and dried over Na2SO4. Concentra-
5.81 (s, 1H), 4.46 (d, J ¼ 8.1 Hz, 1H), 4.22 (s, 2H), 3.70 (s, 3H), tion of the organic layer under reduced pressure gave the crude
3.56 (s, 3H), 3.17 (q, J ¼ 8.4 Hz, 1H), 2.85 (ddd, J ¼ 16.5, 8.5, sulfoxide. To a solution of crude sulfoxide in MeOH (20 mL) was
1.3 Hz, 1H), 2.58 (t, J ¼ 8.0 Hz, 1H), 2.09–2.02 (m, 1H). LC-MS: added (MeO)3P (331 mL, 2.8 mmol) and the mixture was heated
241 (M + H).
under reux for 5 h. Aer it's fully reacted, the mixture was
4.1.2 Conversion from methyl acetal (3) to phenylsulde cooled to rt and poured into aqueous saturated NaCl, then the
(4). To a solution of 2 (10.6 g, 44.1 mmol) in Tol (80 mL) was mixture was extracted by EtOAc, and the organic layer was
added PhSSPh (8.47 g, 55.2 mmol) and tributylphosphine (13.2 washed by aqueous saturated NaCl and dried over Na2SO4.
mL, 53.1 mmol). The mixture was stirred at rt for 3 h, the Concentrate the organic layer under reduced pressure to give
mixture was concentrated under reduced pressure to give a residue, which was puried by silica gel column chromatog-
a residue, which was puried by silica gel column chromatog- raphy (PE/EtOAc ¼ 2 : 1) to afford 7 (430 mg, 80%). 1H NMR (500
raphy (from PE to PE/EtOAc ¼ 10 : 1) to afford 4 (13.88 g, 94%). MHz, CDCl3) d 7.56–7.53 (m, 1H), 7.42 (d, J ¼ 1.2 Hz, 1H), 7.35
1H NMR (500 MHz, CDCl3) d 7.51 (d, J ¼ 0.9 Hz, 1H), 7.33–7.30 (dd, J ¼ 4.3, 3.1 Hz, 1H), 5.89 (d, J ¼ 6.7 Hz, 1H), 5.43–5.40 (m,
(m, 2H), 7.29–7.26 (m, 2H), 7.19 (d, J ¼ 7.3 Hz, 1H), 5.68 (s, 1H), 1H), 5.31 (t, J ¼ 1.8 Hz, 1H), 4.48 (t, J ¼ 4.2 Hz, 1H), 4.10 (q, J ¼
4.51 (d, J ¼ 7.7 Hz, 1H), 3.71 (s, 3H), 3.62–3.58 (m, 1H), 3.56 (s, 7.1 Hz, 1H), 3.81 (d, J ¼ 12.7 Hz, 3H), 3.72 (s, 3H), 3.25 (d, J ¼
3H), 3.12 (td, J ¼ 8.2, 0.9 Hz, 1H), 2.77 (dd, J ¼ 16.4, 8.5 Hz, 2H), 7.4 Hz, 1H), 2.95–2.89 (m, 1H), 2.31–2.21 (m, 2H), 2.18 (ddd, J ¼
2.05 (dd, J ¼ 8.2, 1.4 Hz, 1H). LC-MS: 333 (M + H).
13.5, 6.7, 4.4 Hz, 1H), 2.12 (dt, J ¼ 13.7, 6.8 Hz, 1H), 2.03 (s, 2H),
4.1.3 Conversion from phenylsulde (4) to hemiacetal (5). 1.89 (ddd, J ¼ 13.7, 8.0, 6.0 Hz, 1H), 1.24 (t, J ¼ 7.1 Hz, 2H), 0.96
To a solution of 4 (5.0 g, 15.1 mmol) in THF (80 mL) was added (dd, J ¼ 6.6, 2.6 Hz, 7H), 0.90–0.78 (m, 1H). LC-MS: 311 (M + H).
aqueous 10% HCl (39 mL). The mixture was stirred at 60 ꢂC for
4.1.6 Conversion from alcohol (7a) to target compound 1a
16 h. The mixture was cooled to rt and poured into aqueous to 1h. To a solution of 7 (46.5 mg, 0.15 mmol) in toluene (10 mL)
saturated NaHCO3, then the whole was extract with EtOAc (50 was added VO(acac)2 (3.9 mg, 0.015 mmol) and 70% TBHP (62
mL ꢃ 3). The combined organic layer was washed by aqueous mL, 0.45 mmol) until 7 was fully consumed. Then, the reaction
saturated NaCl and dried over Na2SO4. Then the organic layer was quenched by aqueous saturated NaHCO3 for 15 min, and the
was concentrated under reduced pressure to give a residue, mixture was extracted with EtOAc. The organic layer was washed
which was puried by silica gel column chromatography (from by aqueous saturated NaCl and dried over Na2SO4. The crude
PE/EtOAc ¼ 10 : 1 to PE/EtOAc ¼ 2 : 1) to afford 5 (1.98 g, 41.3%, epoxide was given by the concentration of organic layer under
1
40% recovery of 4). H NMR (500 MHz, CDCl3) d 7.49 (s, 1H), reduced pressure. Under the protection of N2, the solution of
7.32 (d, J ¼ 7.5 Hz, 2H), 7.29–7.24 (m, 2H), 7.18 (t, J ¼ 7.3 Hz, crude epoxide in toluene was added DEAD (56 mL, 0.15 mmol) at
ꢂ
1H), 5.71 (s, 1H), 4.85 (d, J ¼ 8.3 Hz, 1H), 3.87 (d, J ¼ 14.4 Hz, the presence of Ph3P and acid at 0 C. Then, the mixture was
1H), 3.71 (s, 3H), 3.58 (t, J ¼ 6.6 Hz, 1H), 3.12 (q, J ¼ 8.2 Hz, 1H), stirred at rt for 2 h. The whole was concentrated under reduced
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pressure and directly puried by silica gel column chromatog- 1H), 3.05 (d, J ¼ 4.6 Hz, 1H), 2.95 (dd, J ¼ 9.8, 5.2 Hz, 2H), 2.85–
raphy (PE/EtOAc ¼ 10 : 1) to afford the target compounds.
2.75 (m, 1H), 2.36 (dd, J ¼ 7.7, 5.7 Hz, 1H), 2.26–2.19 (m, 4H),
1
Compound 1a (26.6 mg, 43.2%): colorless oil H NMR (500 2.14–2.06 (m, 1H), 1.87 (ddd, J ¼ 14.8, 5.9, 4.3 Hz, 1H), 1.68 (s,
MHz, CDCl3) d 7.42 (d, J ¼ 1.0 Hz, 1H), 6.11 (d, J ¼ 5.6 Hz, 1H), 1H), 1.57–1.52 (m, 2H), 1.34 (dd, J ¼ 7.4, 3.6 Hz, 2H), 0.96 (dd, J
5.03 (dd, J ¼ 7.7, 4.1 Hz, 1H), 3.73 (d, J ¼ 3.1 Hz, 3H), 3.25 (d, J ¼ ¼ 6.6, 2.0 Hz, 6H), 0.89–0.86 (m, 3H). LC-MS: 425 (M + H).
7.4 Hz, 1H), 3.06 (d, J ¼ 4.6 Hz, 1H), 2.95 (d, J ¼ 4.6 Hz, 1H),
Compound 1g (15 mg, 22.1%): colorless oil 1H NMR (500
2.86–2.75 (m, 1H), 2.37 (dd, J ¼ 7.7, 5.6 Hz, 1H), 2.24 (t, J ¼ MHz, CDCl3) d 7.42 (d, J ¼ 1.1 Hz, 1H), 6.11 (d, J ¼ 5.6 Hz, 1H),
6.8 Hz, 2H), 2.10 (t, J ¼ 6.1 Hz, 3H), 2.04–1.98 (m, 1H), 1.89 (dd, J 5.01 (dd, J ¼ 7.7, 4.2 Hz, 1H), 3.74 (s, 3H), 3.25 (d, J ¼ 7.5 Hz, 1H),
¼ 14.8, 1.4 Hz, 1H), 0.96 (dd, J ¼ 6.6, 1.8 Hz, 6H), 0.91 (d, J ¼ 3.05 (d, J ¼ 4.6 Hz, 1H), 2.95 (d, J ¼ 4.7 Hz, 2H), 2.85–2.76 (m,
6.6 Hz, 6H). 13C NMR (126 MHz, CDCl3) d 170.92, 170.04, 165.64, 1H), 2.36 (dd, J ¼ 7.6, 5.8 Hz, 1H), 2.28–2.18 (m, 4H), 2.14–2.07
150.33, 109.57, 87.30, 76.21, 75.26, 64.40, 50.41, 47.01, 42.25, (m, 1H), 1.87 (ddd, J ¼ 14.8, 5.8, 4.4 Hz, 1H), 1.67 (td, J ¼ 14.5,
42.04, 41.74, 36.07, 29.85, 24.61, 24.59, 21.30, 21.24. LC-MS: 411 7.0 Hz, 1H), 1.58–1.50 (m, 2H), 1.25 (d, J ¼ 4.0 Hz, 7H), 0.96 (dd, J
(M + H).
Compound 1b (28.5 mg, 51.6%): colorless oil H NMR (500
¼ 6.6, 2.0 Hz, 6H), 0.87 (t, J ¼ 6.9 Hz, 3H). LC-MS: 453 (M + H).
1
Compound 1h (18 mg, 25.0%): colorless oil 1H NMR (500
MHz, CDCl3) d 7.41 (d, J ¼ 1.3 Hz, 1H), 6.12 (d, J ¼ 5.8 Hz, 1H), MHz, CDCl3) d 7.41 (d, J ¼ 1.1 Hz, 1H), 6.11 (d, J ¼ 5.7 Hz, 1H),
5.00 (dd, J ¼ 7.7, 4.4 Hz, 1H), 3.74 (s, 3H), 3.25 (d, J ¼ 7.7 Hz, 5.01 (dd, J ¼ 7.7, 4.2 Hz, 1H), 3.73 (s, 3H), 3.29–3.19 (m, 1H), 3.05
1H), 3.05 (d, J ¼ 4.6 Hz, 1H), 2.97 (d, J ¼ 4.6 Hz, 1H), 2.82 (dd, J ¼ (d, J ¼ 4.6 Hz, 1H), 2.95 (d, J ¼ 4.6 Hz, 1H), 2.81 (dt, J ¼ 15.4,
7.8, 7.0 Hz, 1H), 2.59 (s, 3H), 2.34 (dd, J ¼ 7.6, 5.9 Hz, 1H), 2.24 7.9 Hz, 1H), 2.36 (dd, J ¼ 7.6, 5.8 Hz, 1H), 2.26–2.19 (m, 4H), 2.10
(dd, J ¼ 7.1, 5.9 Hz, 2H), 2.15–2.06 (m, 1H), 1.90–1.83 (m, 1H), (dt, J ¼ 13.6, 6.8 Hz, 1H), 1.87 (ddd, J ¼ 14.8, 5.8, 4.4 Hz, 1H),
0.96 (dd, J ¼ 6.6, 1.9 Hz, 6H). LC-MS: 369 (M + H).
1.57–1.50 (m, 2H), 1.25 (s, 12H), 0.96 (dd, J ¼ 6.6, 2.0 Hz, 6H), 0.87
1
Compound 1c (27 mg, 41.2%): H NMR (500 MHz, CDCl3) (t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 172.67, 171.06,
d 7.42 (d, J ¼ 1.2 Hz, 1H), 6.10 (d, J ¼ 5.3 Hz, 1H), 5.01 (dd, J ¼ 166.68, 151.33, 110.64, 88.36, 77.23, 76.28, 65.38, 51.45, 47.97,
7.6, 3.8 Hz, 1H), 3.73 (s, 3H), 3.25 (dd, J ¼ 13.5, 6.6 Hz, 1H), 3.06 43.08, 42.74, 37.10, 34.25, 31.85, 30.82, 29.40, 29.25, 29.22, 29.09,
(d, J ¼ 4.6 Hz, 1H), 2.93 (d, J ¼ 4.6 Hz, 1H), 2.80–2.73 (m, 1H), 25.62, 24.79, 22.66, 22.27, 22.25, 14.10. LC-MS: 481 (M + H).
2.39 (dd, J ¼ 7.6, 5.3 Hz, 1H), 2.23 (dd, J ¼ 15.4, 8.6 Hz, 3H), 2.10
(m, 1H), 1.91 (ddd, J ¼ 14.9, 5.3, 3.9 Hz, 1H), 1.79 (s, 2H), 1.70 (s,
4.2 Pharmacological assay
2H), 1.62 (d, J ¼ 9.5 Hz, 1H), 1.57 (d, J ¼ 4.1 Hz, 3H), 1.40–1.31
(m, 2H), 0.96 (dd, J ¼ 6.6, 1.9 Hz, 6H). 13C NMR (126 MHz, RPMI-1640 medium and fetal bovine serum (FBS) were
CDCl3) d 174.83, 171.08, 166.65, 151.35, 110.59, 88.27, 77.23, purchased from Gibco, BRL (Grand Island, NY, USA). Human
76.24, 65.55, 51.41, 48.01, 43.06, 42.85, 37.02, 30.93, 28.79, pancreatic cancer cell lines (SW1990, BXPC3, CAPAN2, CFPAC,
28.75, 25.66, 25.62, 25.35, 25.31, 22.26. LC-MS: 437 (M + H).
PANC1), human breast cancer cell lines (BT474, MCF7, MDA-
Compound 1d (24 mg, 37.2%): colorless oil 1H NMR (500 MB-231), human lung cancer cell line (H1975) and human
MHz, CDCl3) d 7.94 (dd, J ¼ 8.3, 1.2 Hz, 2H), 7.56 (s, 1H), 7.49 (d, gastric cancer cell lines (AGC, HGC-27, KATOIII) were
J ¼ 1.3 Hz, 1H), 7.43 (t, J ¼ 7.8 Hz, 2H), 6.20 (d, J ¼ 5.1 Hz, 1H), purchased from Shanghai Institutes for Biological Sciences,
5.30 (s, 1H), 3.67 (s, 3H), 3.33 (dt, J ¼ 7.7, 6.4 Hz, 1H), 3.12 (d, J ¼ CAS (Shanghai, China). DAPI was purchased from Sigma-
4.5 Hz, 1H), 3.04 (d, J ¼ 4.5 Hz, 1H), 2.90–2.81 (m, 1H), 2.48 (dd, Aldrich (St Louis, MO, US). The Annexin V-FITC Apoptosis Kit
J ¼ 7.6, 5.2 Hz, 1H), 2.25 (t, J ¼ 6.9 Hz, 2H), 2.18–2.07 (m, 2H), was purchased from BD (Franklin Lakes, NJ, US). The primary
0.96 (dd, J ¼ 6.6, 3.5 Hz, 6H). 13C NMR (126 MHz, CDCl3) antibodies against Bax, Bcl-2, Mcl-1, procaspase-9, poly-ADP-
d 171.14, 166.60, 165.41, 151.32, 133.31, 129.67, 128.43, 110.70, ribose polymerase (PARP), XIAP and b-actin were purchased
88.25, 77.43, 77.23, 65.72, 51.40, 48.20, 43.07, 37.02, 31.17, from Abcam Inc. (Cambridge, MA, US). Caspase-3 Colorimetric
25.65, 22.26. LC-MS: 431 (M + H).
Assay Kit was purchased from Biovision (Milpitas, CA, US).
Compound 1e (30 mg, 44.2%): colorless oil 1H NMR (500 Human lung cancer cell line H1975 was purchased from
MHz, CDCl3) d 7.42 (s, 1H), 6.10 (d, J ¼ 5.3 Hz, 1H), 5.05 (dt, J ¼ Shanghai Institutes for Biological Sciences, CAS (Shanghai,
7.5, 3.8 Hz, 1H), 3.73 (d, J ¼ 2.4 Hz, 3H), 3.26 (dd, J ¼ 13.7, China). Cells were cultured with RPMI-1640 medium containing
7.3 Hz, 1H), 3.06 (dd, J ¼ 4.5, 2.2 Hz, 1H), 2.91 (d, J ¼ 4.6 Hz, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at
1H), 2.78 (dtd, J ¼ 10.6, 7.8, 2.9 Hz, 1H), 2.41–2.36 (m, 1H), 2.28– 37 ꢂC, 5% CO2 humidied atmosphere. 1e was dissolved in
2.20 (m, 2H), 2.20–2.14 (m, 1H), 2.10 (dt, J ¼ 13.6, 6.8 Hz, 1H), DMSO at the concentration of 100 mM.
1.93–1.86 (m, 1H), 1.56–1.36 (m, 4H), 1.30–1.25 (m, 2H), 1.21–
4.2.1 Cell viability assay. H1975 cells were counted in log-
1.12 (m, 2H), 0.99–0.94 (m, 6H), 0.89–0.78 (m, 6H). 13C NMR arithmic phase and 5000 cells were placed in 96-well plates.
(126 MHz, CDCl3) d 175.15, 175.07, 171.07, 166.64, 151.38, Aer treatment with 1e (10, 20 and 30 mM), cells were incubated
110.60, 88.29, 88.28, 77.25, 76.28, 76.22, 65.62, 51.42, 48.08, for an additional 2 h with CCK-8 reagent (100 mL per mL
48.05, 47.35, 47.19, 43.06, 42.88, 37.17, 31.58, 31.51, 31.05, medium) and the absorbance was read at 450 nm using
29.53, 29.52, 25.61, 25.32, 25.27, 22.57, 22.51, 22.27. LC-MS: 453 a microplate reader (Sunnyvale, CA, USA). Cell proliferation
(M + H).
inhibition rates were calculated according to the following
Compound 1f (30 mg, 47.2%): colorless oil 1H NMR (500 formula: the proliferation inhibition ratio (%) ¼ 1 ꢀ [(A1 ꢀ A3)/
MHz, CDCl3) d 7.42 (d, J ¼ 1.3 Hz, 1H), 6.11 (d, J ¼ 5.6 Hz, 1H), (A2 ꢀ A3)] ꢃ 100, where, A1 is the OD value of drug experimental
5.02 (dd, J ¼ 7.7, 4.2 Hz, 1H), 3.74 (s, 3H), 3.25 (d, J ¼ 7.6 Hz, group, A2 is the OD value of blank control group, A3 is the OD
31904 | RSC Adv., 2017, 7, 31899–31906
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value of the RPMI1640 medium without cells. The IC50 (50%
inhibitory concentration) value, which represents the concen-
tration of the drug that demonstrates 50% of cell growth inhi-
bition, was calculated by nonlinear regression analysis using
GraphPad Prism soware (San Diego, CA, USA). Assays were
performed on three independent experiments.
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4.2.2 DAPI stain. H1975 cells (2ꢃ104 cells per well) were
cultured in 24-well plates. Aer treatment with 1e for 48 h, cells
were xed with 4% paraformaldehyde for 20 min, and stained
with DAPI for 20 min at 37 ꢂC. Aer washing with PBS, cells were
observed under a uorescence microscope (Nikon, Ti-E, Japan).
4.2.3 Apoptosis assay by ow cytometry. Exponentially
growing cells were seeded in 6-well plates (5ꢃ104 per well) and
cultured overnight in a 5% CO2 atmosphere at 37 ꢂC. Aer
treatment with 1e for 24 h, cells were harvested and washed with
PBS. Then cells were stained with Annexin V-FITC Apoptosis Kit
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4.2.4 Caspase-3 activity assay. Caspase-3 Colorimetric
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Acknowledgements
This work was supported by Hangzhou Major Science and Tech-
nology Project (20112312A01, Shenglin Ma; 20172016A01, Nengm- 22 S. Tamura, K. Fujiwara, N. Shimizu, S. Todo and
ing Lin), National Natural Science Foundation of China (81603144, N. Murakami, Bioorg. Med. Chem., 2010, 18, 5975–5980.
Bo Zhang), Natural Science Foundation of Zhejiang Province 23 S. Lin, P. Fu, T. Chen, J. Ye, X. W. Yang and W. D. Zhang, J.
(LQ15H310001, Bo Zhang), Scientic Research Foundation of Asian Nat. Prod. Res., 2017, 19, 15–21.
Zhejiang Health and Family Planning Commission (2015KYA177, 24 X. Li, X. X. Fan, Z. B. Jiang, W. T. Loo, X. J. Yao, E. L. Leung,
Bo Zhang), Fundamental Research Funds for the Central Univer-
L. W. Chow and L. Liu, Pharmacol. Res., 2017, 115, 45–55.
sities (2016QNA7030, Xiaoyang Dai), Science and Technology 25 Z. Wang, X. Wu, Y. N. Liang, L. Wang, Z. X. Song, J. L. Liu and
Project of Zhejiang Province (2016C33067, Xiaowu Dong).
Z. S. Tang, Molecules, 2016, 21(10), 1267.
26 L. H. Li, P. Wu, J. Y. Lee, P. R. Li, W. Y. Hsieh, C. C. Ho,
C. L. Ho, W. J. Chen, C. C. Wang, M. Y. Yen, S. M. Yang
and H. W. Chen, PLoS One, 2014, 9, e104203.
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