ent-kaurane diterpenoids from Chinese liverworts and their antitumor activities through Michael addition as detected in situ by a fluorescence probe

Article abstract of DOI:10.1021/acs.jmedchem.5b00208

It is generally accepted that the origin of the cytotoxicity of ent-kaurane diterpenoids is due to the formation of reactive oxygen species (ROS) and that the α,β-unsaturated carbonyl is a pivotal moiety. Herein we demonstrate the isolation of 32 new and 12 known ent-kaurane diterpenoids from two Chinese liverworts. These compounds and three semisynthesized derivatives were screened against human cancer cell lines. The results revealed that their anticancer activities are caused by ROS formation through Michael modification of the protein thiols and depletion of glutathione unselectively. We also found that N-acetylcysteine reverses the cytotoxicity of these diterpenoids by forming Michael adducts, not through a well-recognized ROS scavenging pathway as previously reported. In situ intracellular thiol detection helped us visualize the intracellular distribution of the diterpenoids and determine the potency of their cytotoxicity. An alkaline analogue was found to be more selective because of the altered subcellular distribution.

Full text of DOI:10.1021/acs.jmedchem.5b00208

Article
pubs.acs.org/jmc
ent-Kaurane Diterpenoids from Chinese Liverworts and Their
Antitumor Activities through Michael Addition As Detected in Situ
by a Fluorescence Probe
Zhaomin Lin,†,§ Yanxia Guo,‡,§ Yanhui Gao,† Shuqi Wang,† Xiaoning Wang,† Zhiyu Xie,† Huanmin Niu,‡
Wenqiang Chang,† Lei Liu,† Huiqing Yuan,‡ and Hongxiang Lou*,†
†Department of Natural Products Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical
Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of China
‡Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan
250012, People’s Republic of China
S
* Supporting Information
ABSTRACT: It is generally accepted that the origin of the cytotoxicity of ent-
kaurane diterpenoids is due to the formation of reactive oxygen species (ROS)
and that the α,β-unsaturated carbonyl is a pivotal moiety. Herein we demonstrate
the isolation of 32 new and 12 known ent-kaurane diterpenoids from two
Chinese liverworts. These compounds and three semisynthesized derivatives
were screened against human cancer cell lines. The results revealed that their
anticancer activities are caused by ROS formation through Michael modification
of the protein thiols and depletion of glutathione unselectively. We also found
that N-acetylcysteine reverses the cytotoxicity of these diterpenoids by forming
Michael adducts, not through a well-recognized ROS scavenging pathway as
previously reported. In situ intracellular thiol detection helped us visualize the
intracellular distribution of the diterpenoids and determine the potency of their
cytotoxicity. An alkaline analogue was found to be more selective because of the
altered subcellular distribution.
In this work, a small library of ent-kaurane diterpenoids was
built by ongoing chemical investigations on the Chinese
liverworts Jungermannia fauriana and Jungermannia hyalina in
our laboratory.19−22 Twenty new (1−20) and four known
(21−24) ent-kaurane diterpenoids from the liverwort J. fauriana
and 12 new (25−36) and 11 known (22−24 and 37−44)
structures from J. hyalina (Figure 1) were isolated separately,
and three ent-kaurane derivatives (45−47) were semisynthe-
sized from stevioside (Scheme 1).23 Herein we report the
discovery of new ent-kaurane diterpenoids and their cytotoxic
effects on human cancer cell lines. The relationship between
selectivity and intracellular distribution as detected by our
developed in situ imaging method was investigated. A possible
way to improve the selectivity to kill cancer cells by altering the
drug subcellular distribution was also preliminarily explored.
INTRODUCTION
■
Natural products have a profound impact upon both chemical
biology and drug discovery, and the great structural diversity of
natural products with various interesting biological character-
istics has always provided medicinal chemists an important
source of inspiration in their search for new molecular entities
with pharmacological activities.1−3 Among them, ent-kaurane
diterpenoids are remarkable chemical markers in both liver-
worts4−8 and some other higher plants.9−11 They exhibit
considerable pharmacological activities, including antitumor,
antituberculosis, antibacterial, and anti-inflammatory ef-
fects.11−13 Some of them have recently been developed into
cancer therapeutic agents.14,15 The widely accepted mechanism
for these anticancer agents is the Michael addition of soft
nucleophiles, such as thiols and protein sulfhydryl groups, onto
the α,β-unsaturated ketone moiety, leading to the deactivation
of SH enzymes or SH coenzymes.16−18 Despite some progress,
the poor selectivity has restricted the development of these
diterpenoids into drugs, which may be ascribed to the poor
selected intracellular distribution and multitargeting.11 The
diversity of the diterpenoids provides us a good opportunity to
explore the structure−activity relationship (SAR), the intra-
cellular distribution, and the target molecules to improve the
state of art of the area.
RESULTS AND DISCUSSION
■
Structure Elucidation. The EtOH extract of J. fauriana was
repeatedly chromatographed over MCI gel, silica gel, Sephadex
LH-20, and further semipreparative HPLC to yield 20 new (1−
20) and four known (21−24) ent-kaurane diterpenoids. By a
Received: February 4, 2015
© XXXX American Chemical Society
A
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Figure 1. Structures of compounds 1−24 isolated from the liverwort J. fauriana and compounds 22−44 from the liverwort J. hyalina.
similar method, 12 new (25−36) and 11 known (22−24 and
37−44) diterpenoids were isolated from J. hyalina.
(δC 38.7) and of the olefinic proton H-11 (δH 5.55) with C-10
(δC 38.7), C-12 (δC 43.0), and C-13 (δC 74.4) confirmed the
double bond between C-9 and C-11. The HMBC correlations
of H2-17 (δH 6.03 and 5.64) with C-12, C-13, and C-15 (δC
201.8) revealed the presence of the oxygenated quaternary
carbon at C-13. Both the oxygenated methylene (δH 3.50 and
3.04) and the methyl at δH 0.84 showed HMBC correlations to
C-3 (δC 35.2), C-4 (δC 38.2), and C-5, which assigned the
location of the hydroxyl group at C-18 or C-19. The nuclear
Overhauser effect spectroscopy (NOESY) correlations of the
oxygenated methylene to H-5 (δH 1.73) and H-6β (δH 1.98−
2.08) and of the methyl at δH 0.84 to CH3-20 (δH 1.13) (Figure
S1) suggested that the hydroxyl group is at C-18 and the α-CH3
at C-4. To determine the absolute configuration, compound 1
was subjected to single-crystal X-ray diffraction analysis with Cu
Kα radiation, which unambiguously confirmed the structure
and the depicted absolute configuration to be that of an ent-
kaurane diterpenoid (Figure S2). Accordingly, 1 was elucidated
as 13,18-dihydroxy-ent-9(11),16-kauradien-15-one.
Compound 1, isolated as colorless crystals (MeOH), was
assigned the molecular formula C20H28O3 on the basis of the
NMR data and an [M + NH4]+ ion at m/z 334.2375 (calcd
334.2377) found by high-resolution electrospray ionization
mass spectrometry (HR-ESI-MS), which indicated seven
indices of hydrogen deficiency. The IR spectrum showed
absorption bands of hydroxyl (3390 cm−1) and carbonyl (1722
1
cm−1) groups. The H NMR spectrum of 1 (Table S1 in the
Supporting Information) displayed signals for two tertiary
methyl groups at δH 0.84 (s) and 1.13 (s), a pair of oxygenated
methylene protons at δH 3.50 (d, J = 11.4 Hz) and 3.04 (d, J =
11.4 Hz), an olefinic proton at δH 5.55 (t, J = 3.0 Hz), and an
exocyclic methylene at δH 6.03 (s) and 5.64 (s). The 13C NMR
data (Table S5), analyzed with the help of heteronuclear single-
quantum correlation (HSQC) data, indicated that 20 carbons
of 1 are two methyls, nine methylenes, two methines, and seven
quaternary carbons. The aforementioned spectral data allowed
the assignment of an ent-kaurane diterpenoid skeleton.24−26 In
the heteronuclear multiple-bond correlation (HMBC) spec-
trum (Figure S1 in the Supporting Information), the long-range
correlations of CH3-20 (δH 1.13) with C-9 (δC 150.2) and C-10
The same strategy was used to determine the structures of
the new compounds 2−20 and 25−36 as shown in Figure 1
(for the detailed elucidation, see the Supporting Information).
The known compounds were identified by comparison of their
B
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a
Scheme 1. Synthesis of Stevioside Derivatives 45−47
a
Reagents and conditions: (a) NaIO4, H2O, room temperature, 16 h; KOH, H2O, reflux, 1 h. (b) SeO2, t-BuOOH, THF, room temperature, 20 h.
(c) PDC, DMF, room temperature, 12 h. (d) CH3I, DMF, room temperature, 8 h. (e) BrCH2CH2Br, K2CO3, DMF, room temperature, 4 h. (f) N-
methylpiperazine, DMF, reflux, 10 h.
NMR and MS data with reported data (see the Supporting
Information for details).
kaurane diterpenoid skeleton has no significant effect on the
efficacy.
In Vitro Antiproliferation Activity. Cytotoxicity screening
of the diterpenoid library against 13 human cancer cell lines
indicated that compounds 1−5, 14−16, 23, and 45−47, which
contain an exo-methylene cyclopentanone system, possess
strong activity, while all of the other compounds are inactive
(IC50 > 50 μM) (Table 1). The screening results also suggested
that the active compounds showed comparable cytotoxic
activities against cancer and normal cell lines, revealing poor
selectivity with a low selectivity index (SI). An SAR
investigation (Figure 2) showed that the exo-methylene
cyclopentanone system in the D ring is pivotal for the
cytotoxicity. Any destruction of this system, e.g., by saturation
of the methylene group (compounds 6−13, 17−22, and 24) or
the ketone group (compounds 25−44) led to complete loss of
cytotoxicity. Comparison of the IC50 values of 1 and 15 with
those of 4 and 16 indicates that the C13-OH should be
beneficial to the cytotoxicity. Moreover, compounds with a
hydroxyl group at C-11 (e.g., 23) also showed increased
activity. However, compounds 1 and 3, analogues of 5 bearing a
hydroxyl group on the methyl group at C-4, proved to be less
potent (5.5 μM for 1 and 6.6 μM for 3) than compound 5 (3.2
μM) against the PC3 cell line. The activity further decreased
upon oxidation of the hydroxymethyl group to a formyl group
(2, 11.4 μM) or a carboxylic acid (45, 30.1 μM), although the
loss of activity could be partially reversed by esterification of the
carboxylic acid (46, 6.1 μM). Comparison of the activities of 5
and 14 suggests that rearrangement of the D ring in the ent-
ROS-Induced Cell Death by Diterpenoids. Reactive
oxygen species (ROS) can modulate the activities and
expressions of many transcription factors and signaling proteins
that are involved in stress response and cell survival through
multiple mechanisms.27−29 Compounds that promote ROS
generation in cancer cells show promising anticancer
activity.30−33 The ent-kaurane-type diterpenoids oridonin and
adenanthin were reported to interact with thioredoxin/
thioredoxin reductase34 in acute myeloid leukemia or
peroxiredoxin I and II18 in acute promyelocytic leukemia
through increased intracellular ROS, which in turn induces
apoptotic cell death or cell differentiation. However, in their
research, the role of induced ROS was unintentionally wrongly
designed using N-acetylcysteine (NAC) as an antioxidant to
reverse the ROS-initiated apoptosis.18,34
To investigate whether ROS are involved in cell death
induced by our compounds, we measured cellular changes in
the ROS levels after treatment with the test diterpenoid 46
using the ROS-sensitive fluorometric probe 2′,7′-dichlorofluor-
escein diacetate (DCFH-DA). As expected, 46 elevated the
ROS levels of treated cells in a time-dependent manner (Figure
3A,B). NAC, a ROS scavenger, dramatically diminished the 46-
induced ROS level and significantly attenuated the inhibitory
effect of 46 on cell viability (Figure 3A,C), which was
consistent with literature reports.18,34 However, NAC possesses
a mercapto moiety, which has the potential to react with the
α,β-unsaturated carbonyl moiety of diterpenoids. To verify this
C
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hypothesis, we first obtained the conjugated compound 46−
NAC by incubating 46 with NAC. Then we detected the
probable reaction after addition of 46 to medium containing
NAC by HPLC−MS/MS. As shown in Figure 3D, blank cell
culture medium, medium with 5 mM NAC, and medium with
10 μM 46 were used as controls, and the conjugated product
46−NAC was easily detected in the medium when NAC was
added to the test medium containing 46. Meanwhile, the
prototype compound 46 was hardly detected. These results
indicate that NAC reverses the cytotoxicity of diterpenoids not
through ROS scavenging but rather through reaction with the
compounds that results in loss of efficacy. The end-conjugated
product is not toxic to cancer cells (IC50 > 50 μM). Therefore,
it is not correct to use NAC as an antioxidant to investigate the
role of ROS in cell death of such diterpenoids.18,34−37
Glutathione (GSH), a thiol-based intracellular antioxidant, is
a major ROS scavenging agent in cells.38 Depletion of GSH
would result in ROS accumulation and cell death.39,40 Again
using compound 46, we detected its effect to decrease the level
of cellular GSH. The results showed that the GSH content in
the PC3 cells was reduced upon treatment with 46 in a time-
dependent manner (Figure 4A). Meanwhile, the levels of GSSG
(the oxidized form GSH) decreased after a slight increase, and
the total cellular glutathione (GSH + GSSG) decreased in a
time-dependent manner. It is noteworthy that GSH is the most
abundant intracellular non-protein sulfhydryl-containing com-
pound. As we have demonstrated that 46 can react with the
sulfhydryl group of NAC, we supposed that the compound
might also react with GSH. As expected, conjugated 46−GSH
was distinctly detected in the treated cells by HPLC−MS/MS,
as shown in Figure 4B. The antioxidant function of GSH is
accomplished largely by the redox cycle of GSH and GSSG.40
Because of the formation of the carbon−sulfur bond, the
activity of GSH cannot be restored, resulting in a decrease of
the content of both GSH and GSSG. Collectively, these results
clearly demonstrate that 46 depletes intracellular GSH through
a conjugation reaction.
In Situ Detection of the Diterpenoids with a
Fluorescence Probe. The coumarin derived azide 51 is a
fluorescence probe,41 as it reacts with thiol to form fluorescent
aminocoumarin 52, enabling simple and sensitive quantification
of hydrogen sulfide (Scheme S1 in the Supporting
Information). By modifying this method, we found that it is
also valid for the detection of intracellular thiol-based
ingredients. As illustrated above, the ent-kaurane diterpenoids
exert their biofunctions by interacting with intracellular thiols.
The unreacted thiols can be detected by 51. On the basis of this
mechanism, the intracellular distribution of the diterpenoid can
be monitored by observing the fluorescence using the probe.
Here, with PC3 cells and the sulfhydryl group scavenger N-
ethylmaleimide (NEM)42−44 as a control, the performance of
46 was investigated by confocal microscopy.
As anticipated, incubation of PC3 cells with 51 resulted in a
robust intracellular fluorescence signal, while preincubation
with NEM reduced the level of fluorescence markedly (Figure
5A). With similar results, the level of fluorescence after
preincubation of 46 decreased in a concentration- and time-
dependent manner (Figure 5A,B). The intracellular fluores-
cence intensity, the statistical result by confocal fluorescence
image analysis (Figure 5A,B), in turn demonstrated the potency
of the cytotoxic activity. As shown in Table 1, 46 exhibited
good cytotoxicity toward all of the tested cell lines. Its
nonselective characteristic can be partly explained by the probe
D
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Figure 2. SAR summary of requirements for cytotoxicity. All of the IC50 values were measured against PC3 cells.
Figure 3. (A) Effect of 46 on the generation of ROS. PC3 cells exposed to 6 μM 46 for 4 h were collected, stained with DCFH-DA, and detected by
flow cytometry. The antioxidant NAC (5.0 mM) was added for 1 h prior to 46. (B) Statistical analysis of the relative ratio of ROS formation, **, P <
0.01 and ***, P < 0.001 compared with the control group or NAC-untreated group. (C) Effect of the antioxidant on 46-induced cell death. PC3 cells
were pretreated with 5 mM NAC for 1 h and then incubated with 6 μM 46 in the presence or absence of antioxidant for 24 h. Cell viability was then
assessed by MTT assay. Data are shown as mean SD. ***, P < 0.001. (D) Detection of compounds 46 and 46−NAC by HPLC−MS/MS: (a)
standard substances; (b) cell culture medium; (c) cell culture medium with 5 mM NAC; (d) cell culture medium with 10 μM 46; (e) cell culture
medium with 5 mM NAC and 10 μM 46.
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fluorescence intensities were inversely proportional to the
cytotoxicities of the compounds, suggesting that 51 can be
applied as a probe to indicate the potency of these diterpenoids
to kill the cancer cells.
The distribution behavior of a drug within a cell is an
important variable in both activity and differential selectivity.
Altered intracellular distribution of a drug is a way to improve
drug selectivity.45 From stevioside as a starting material,
compound 47 was synthesized by introducing an N-
methylpiperazine group. Again, by means of the above-
established in situ detection method, the lysosomotropic ability
and cytotoxic selectivity of 47 were tested. It is known that the
lysosomes in cancer cells are more numerous and larger and
that they have greater cathepsin activity than those in normal
cells, and the release of cathepsins into the cytosol may result in
apoptosis or necrosis.46−48 A cytotoxicity test found that 47
showed higher activity toward PC3 cells (IC50 = 8.2 μM) while
showing lower toxicity toward normal RWPE1 cells (IC50
=
29.3 μM), with improved selectivity (SI = 3.6). We also
observed that lysosomes swelled or fused after treatment with
47 (Figure 5D). In situ detection with 51 still found a high
value of the fluorescence intensity within the cells, indicating
that there were more free thiols in the cytosol (Figure 5E).
Meanwhile, the lower fluorescence in organelles suggests a high
accumulation of 47. The altered subcellular distribution of 47
led to cell death through the lysosome pathway by cathepsin B
release, as shown in Figure 5F.
CONCLUSION
■
We have constructed a small library of ent-kaurane diterpenoids
by isolation of natural products from liverworts and semisyn-
thesis. Cytotoxicity screening showed that having an exo-
methylene cyclopentanone moiety in these ent-kauranes is vital
to their biological activity. The presence of a hydroxyl group on
the C ring increased the activity, while the methyl oxidation at
C-4 decreased the toxicity. Investigation of the mechanism
revealed that these compounds kill the cancer cells through
ROS-mediated apoptosis by the Michael reaction with the
thiol-based proteins and depletion of intracellular GSH. A
coumarin-based probe has been shown to be useful for
detecting the cellular distribution of the diterpenoids by its
reaction with free thiols. An alkaline derivative showed greatly
improved selectivity to kill cancer cells as a result of its
lysomotropic property. This provides a clue to develop this
type of active diterpenoid into druggable candidates.
EXPERIMENTAL SECTION
■
General Materials and Methods. Melting points were
determined on an X-6 melting point apparatus (Beijing TECH
Instrument Co. Ltd.) and are uncorrected. Optical rotations were
measured on a PerkinElmer 241 MC polarimeter. UV spectra were
obtained with an Agilent 8453E UV−vis spectroscopy system.
Electronic circular dichroism (CD) spectra were obtained on a
Chirascan spectropolarimeter. IR spectra were recorded on a Nicolet
iN 10 Micro FTIR spectrometer. NMR spectra were obtained using a
Bruker Avance DRX-600 spectrometer operating at 600 (1H) or 150
(13C) MHz with tetramethylsilane as an internal standard. HR-ESI-MS
was carried out on an LTQ Orbitrap XL instrument. HPLC was
performed on an Agilent 1200 system equipped with a G1311A
isopump, a G1322A degasser, and a G1315D DAD detector using a
ZORBAX SB-C18 column (9.4 mm × 250 mm, 5 μm). All of the
solvents used were of analytical grade. MCI gel (CHP20P, 75−150
μm, Mitsubishi Chemical Industries Ltd.), C18 reversed-phase silica gel
(150−200 mesh, Merck), and Sephadex LH-20 (25−100 μm;
Figure 4. (A) Effect of 46 on the cellular GSH and GSSG levels. PC3
cells were exposed to 6 μM 46 for the indicated times. The results are
presented as relative content of the treated cells compared with the
control group. (B) Detection of 46−GSH by HPLC−MS/MS: (a)
standard substances; (b) control group; (c) cells treated with 6 μM 46
for 8 h.
staining results. In Figure 5A, compared with that of the control
cells, the fluorescence of the drug-treated cells decreased
homogeneously, suggesting the poor selectivity distribution.
PC3 cells treated with different compounds at the same
concentration, followed by incubation with 51, were examined
by confocal microscopy. As shown in Figure 5C, the
F
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Figure 5. (A) Response of 51 with NEM and increasing concentrations of 46. PC3 cells exposed to 5 μM NEM and different concentrations of 46
for 4 h were incubated with 30 μM 51 for 1 h in the dark and then examined by confocal microscopy (magnification 63× oil). The fluorescence
intensities of different cell groups were quantitatively analyzed by confocal microscopy. (B) Quantitative analysis of the fluorescence intensities of
different cell groups incubated with 30 μM 51 after treatment with 46 (6 μM) for different times. (C) Quantitative analysis of the fluorescence
intensities of different cell groups incubated with 30 μM 51 after treatment with 5 μM 14, 15, and 45 for 4 h as measured by confocal microscopy.
(D) PC3 cells exposed to 46 (6 μM) and 47 (8 μM) for 2 h were stained with LysoTracker Red and Hoechst 33342 and then measured by confocal
microscopy. (E) Quantitative analysis of the fluorescence intensities of different cell groups incubated with 30 μM 51 after exposure to different
compounds at the indicated concentrations for 4 h. All values are expressed as mean SD. *, P < 0.05 and **, P < 0.01. (F) Western blotting
analysis of cathepsin B. PC3 cells were treated with 47 (8 μM) and 46 (6 μM) for the indicated times. GAPDH served as the loading control.
Pharmacia Biotek, Denmark) were used for column chromatography
(CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical
Co., Ltd.) were used for thin-layer chromatography. Spots were
visualized with UV light or by spraying with H2SO4/EtOH (1:9 v/v)
followed by heating. The purities of all biologically evaluated
compounds were established as ≥95% by HPLC analysis using two
different gradients with UV detection (210, 254, and 280 nm).
Plant Material. The liverworts J. fauriana and J. hyalina were
collected from Maoer Mountain, Guangxi Zhuang Autonomous
Region, P. R. China, in April 2011 and authenticated by Prof. Yuan-
Xin Xiong, College of Life Sciences, Guizhou University, P. R. China.
Voucher specimens (no. 20110417-16 and no. 20110409-02) were
deposited at the Department of Natural Products Chemistry, School of
Pharmaceutical Sciences, Shandong University, P. R. China.
Extraction and Isolation. The air-dried powders of the plant
material of J. fauriana (100.0 g) were extracted with 95% EtOH at
room temperature (3 × 1.5 L, each for 1 week). Evaporation of the
solvent in vacuo provided a dark residue (8.0 g), which was suspended
in H2O (150 mL) and partitioned with Et2O (3 × 150 mL)
successively. The Et2O extract (5.1 g) was separated by MCI gel
column chromatography (MeOH/H2O, 3:7 to 9:1) to give six
fractions (1−6). Fraction 2 (2.4 g) was chromatographed on a silica
gel column [petroleum ether (60−90 °C)/acetone, 200:1 to 1:1] to
give five subfractions (2.1−2.5). Fraction 2.2 (848.0 mg) was
chromatographed on Sephadex LH-20 (MeOH) to obtain two
fractions (2.2.1 and 2.2.2). Fraction 2.2.2 was separated by reversed-
phase C18 HPLC (MeOH/H2O, 7:3) and then purified by
semipreparative HPLC (MeOH/H2O, 80:20) to give 4 (3.4 mg, tR
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= 27.0 min), 21 (37.0 mg, tR = 36.0 min), 22 (116.0 mg, tR = 40.2
min), 23 (45.1 mg, tR = 30.9 min), 16 (2.3 mg, tR = 19.7 min), a (18.0
mg, tR = 33.2 min), and b (12.2 mg, tR = 28.8 min). a and b were
further purified by semipreparative HPLC (acetonitrile/H2O, 70:30)
to give two pairs of compounds: 5 (10.0 mg, tR = 31.3 min) and 6 (3.2
mg, tR = 30.0 min) as well as 14 (9.0 mg, tR = 26.7 min) and 18 (1.9
mg, tR = 25.3 min). Fraction 2.3 (129.0 mg) was chromatographed on
Sephadex LH-20 (MeOH) to obtain three fractions (2.3.1−2.3.3).
Further separation of fraction 2.3.2 (89.0 mg) by reversed-phase C18
HPLC (MeOH/H2O, 3:2) and then by semipreparative HPLC
(MeOH/H2O, 62:38) yielded 8 (5.3 mg, tR = 20.9 min), 19 (6.5 mg,
tR = 16.5 min), and c (24.0 mg, tR = 23.1 min), which was purified by
semipreparative HPLC (acetonitrile/H2O, 50:50) to give 2 (8.6 mg, tR
= 25.2 min) and 9 (12.0 mg, tR = 23.1 min). Fraction 2.4 (651.0 mg)
was chromatographed on Sephadex LH-20 (MeOH) and then
separated by reversed-phase C18 HPLC (MeOH/H2O, 7:3) to obtain
three fractions (2.4.1−2.4.3). Fraction 2.4.1 was purified by semi-
preparative HPLC to give 20 (5.9 mg, tR = 9.6 min), d (173.0 mg, tR =
15.3 min), e (54.0 mg, tR = 10.6 min), and f (18.0 mg, tR = 12.4 min),
which were further purified by semipreparative HPLC to afford 1 and
7 (72.0 mg/71.0 mg, 28.6 min/26.1 min; acetonitrile/H2O, 40:60), 15
and 17 (3.5 mg/45.0 mg, 14.6 min/13.7 min; acetonitrile/H2O,
45:55), and 3 and 10 (2.6 mg/13.0 mg, 14.9 min/13.7 min;
acetonitrile/H2O, 40:60), respectively. Fraction 2.5 (215.0 mg) was
purified by Sephadex LH-20 column chromatography (MeOH)
followed by semipreparative HPLC using MeOH/H2O (68:32) as
the mobile phase to yield 11 (1.2 mg, tR = 21.0 min), 12 (3.2 mg, tR =
24.5 min), and 13 (9.0 mg, tR = 25.7 min). Fraction 3 (1.0 g) was also
subjected to silica gel CC (petroleum ether/acetone, 200:1 to 5:1),
which gave six fractions (3.1−3.6). Compound 24 (25.0 mg) was
obtained from fraction 3.3 (92.0 mg) by Sephadex LH-20 column
chromatography (CH2Cl2/MeOH, 1:1).
separated on Sephadex LH-20 (MeOH) and then separated using
reversed-phase C18 HPLC (MeOH/H2O, 6:4 to 9:1) to yield four
subfractions (3.2.1−3.2.4). Fraction 3.2.3 (28.0 mg) was purified by
semipreparative HPLC (acetonitrile/H2O, 85:15) to yield 31 (2.4 mg,
tR = 20.4 min). Fraction 3.2.4 (33.4 mg) was purified by the same
method (acetonitrile/H2O, 95:5) to give 44 (9.0 mg, tR = 31.6 min),
40 (8.0 mg, tR = 33.1 min), and 43 (5.0 mg, tR = 35.7 min).
13,18-Dihydroxy-ent-9(11),16-kauradien-15-one (1). Colorless
needle crystals (MeOH); mp 164−166 °C; [α]2D5 = −21.90 (c 0.137,
MeOH); UV (MeOH) λmax (log ε) 350 (2.22), 227 (3.66), 208 (3.81)
nm; CD (MeOH) λmax (Δε) 355 (+1.06), 278 (−0.36) nm; IR νmax
1
3390, 2945, 1722, 1651 cm−1; for H and 13C NMR data, see Tables
S1 and S5; HR-ESI-MS (positive mode) m/z 334.2375 [M + NH4]+
(calcd for C20H32O3N, 334.2377).
13-Hydroxy-ent-9(11),16-kauradien-18-al-15-one (2). Colorless
oil; [α]2D5 = +18.52 (c 0.108, MeOH); UV (MeOH) λmax (log ε)
206 (3.78) nm; CD (MeOH) λmax (Δε) 354 (+0.72), 311 (+0.60),
1
269 (−0.14) nm; IR νmax 3425, 2931, 1718, 1647, 1456 cm−1; for H
and 13C NMR data, see Tables S1 and S5; HR-ESI-MS (positive
mode) m/z 332.2220 [M + NH4]+ (calcd for C20H30O3N, 332.2220).
13,19-Dihydroxy-ent-9(11),16-kauradien-15-one (3). Colorless
needle crystals (MeOH); mp 169−170 °C; [α]2D5 = +112.5 (c 0.080,
MeOH); UV (MeOH) λmax (log ε) 206 (3.72) nm; CD (MeOH) λmax
(Δε) 354 (+0.74), 278 (−0.22), 208 (+0.40) nm; IR νmax 3328, 2956,
1708, 1641, 1462 cm−1; for 1H and 13C NMR data, see Tables S1 and
S5; HR-ESI-MS (positive mode) m/z 334.2393 [M + NH4]+ (calcd for
C20H32O3N, 334.2377).
18-Hydroxy-ent-9(11),16-kauradien-15-one (4). Colorless oil;
[α]2D5 = −27.40 (c 0.073, MeOH); UV (MeOH) λmax (log ε) 243
(3.38), 202 (3.61) nm; CD (MeOH) λmax (Δε) 321 (+0.35), 245
1
(−0.15) nm; IR νmax 3417, 2926, 1712, 1647 cm−1; for H and 13C
NMR data, see Tables S1 and S5; HR-ESI-MS (positive mode) m/z
301.2161 [M + H]+ (calcd for C20H29O2, 301.2162).
The air-dried powders of the plant material of J. hyalina (290.0 g)
were extracted with 95% EtOH at room temperature (3 × 1.5 L, each
for 1 week). Evaporation of the solvent in vacuo provided a dark
residue (14.1 g), which was separated by MCI gel CC (MeOH/H2O,
3:7 to 9:1) to yield three fractions (1−3). Fraction 2 (2.6 g) was
chromatographed on a silica gel column [petroleum ether (60−90
°C)/acetone, 200:1 to 1:1] to give six subfractions (2.1−2.6). Fraction
2.2 (420.0 mg) was separated on Sephadex LH-20 (MeOH) to yield
24 (52.0 mg) and three new fractions (2.2.1−2.2.3). Fraction 2.2.2
(820.0 mg) was separated using reversed-phase C18 HPLC (MeOH/
H2O, 6:4 to 9:1) to yield five subfractions (2.2.2.1−2.2.2.5). Fraction
2.2.2.1 (18.0 mg) was purified by semipreparative HPLC (MeOH/
H2O, 78:22) to yield 33 (2.4 mg, tR = 12.2 min), 35 (1.8 mg, tR = 14.6
min), and 30 (3.2 mg, tR = 17.9 min). Fraction 2.2.2.3 (23.5 mg) was
separated using semipreparative HPLC (MeOH/H2O, 80:20) to
obtain 28 (15.0 mg, tR = 15.0 min). Fraction 2.2.2.4 (27.0 mg) was
purified by semipreparative HPLC (MeOH/H2O, 84:76) to yield 42
(2.0 mg, tR = 21.5 min), 34 (3.5 mg, tR = 23.4 min), and 22 (7.0 mg, tR
= 28.4 min). Fraction 2.2.2.5 (25.2 mg) was separated using
semipreparative HPLC (MeOH/H2O, 90:10) to obtain 38 (11.0
mg, tR = 24.5 min). Fraction 2.3 (346.0 mg) was chromatographed on
Sephadex LH-20 (MeOH) and separated using reversed-phase C18
HPLC (MeOH/H2O, 7:3 to 9:1) to yield five subfractions (2.3.1−
2.3.5). Fraction 2.3.2 (75.4 mg) was separated by semipreparative
HPLC (MeOH/H2O, 72:28) to give a (41.0 mg, tR = 24.0 min) and b
(5.8 mg, tR = 26.7 min), which were further purified by semi-
preparative HPLC (acetonitrile/H2O, 50:50) to give 26 (1.6 mg, tR =
7.3 min), 25 (34.0 mg, tR = 11.4 min), and 27 (3.4 mg, tR = 8.8 min).
Fraction 2.3.3 (15.2 mg) was purified by semipreparative HPLC
(acetonitrile/H2O, 50:50) to give 39 (0.7 mg, tR = 32.0 min). Fraction
2.3.4 (53.7 mg) was purified by the same method (acetonitrile/H2O,
60:40) to give 29 (2.8 mg, tR = 18.9 min), 37 (9.0 mg, tR = 22.3 min),
32 (2.0 mg, tR = 23.7 min), and 23 (23.0 mg, tR = 26.9 min).
Furthermore, separation of fraction 2.3.5 (13.0 mg) by semipreparative
HPLC (MeOH/H2O, 80:20) yielded 41 (1.9 mg, tR = 25.6 min) and
36 (2.7 mg, tR = 28.3 min). Fraction 3 (1.6 g) was chromatographed
on a silica gel column [petroleum ether (60−90 °C)/acetone, 200:1 to
5:1] to give five subfractions (3.1−3.5). Fraction 3.2 (350.0 mg) was
13-Hydroxy-ent-9(11),16-kauradien-15-one (5). Colorless needle
crystals (MeOH); mp 141−143 °C; [α]2D5 = −86.54 (c 0.104, MeOH);
UV (MeOH) λmax (log ε) 205 (3.77) nm; CD (MeOH) λmax (Δε) 221
1
(+0.61) nm; IR νmax 3376, 2937, 1709, 1644 cm−1; for H and 13C
NMR data, see Tables S1 and S5; HR-ESI-MS (positive mode) m/z
301.2159 [M + H]+ (calcd for C20H29O2, 301.2162).
(16R)-13-Hydroxy-ent-9(11)-kauren-15-one (6). Colorless needle
crystals (MeOH); mp 148−150 °C; [α]2D5 = +83.33 (c 0.144, MeOH);
UV (MeOH) λmax (log ε) 230 (3.08), 264 (1.96) nm; CD (MeOH)
λmax (Δε) 303 (+3.81), 237 (−0.89) nm; IR νmax 3364, 2927, 1733,
1456 cm−1; for 1H and 13C NMR data, see Tables S1 and S5; HR-ESI-
MS (positive mode) m/z 303.2320 [M + H]+ (calcd for C20H31O2,
303.2319).
(16R)-13,18-Dihydroxy-ent-9(11)-kauren-15-one (7). Colorless
needle crystals (MeOH); mp 172−174 °C; [α]2D5 = +25.21 (c 0.119,
MeOH); UV (MeOH) λmax (log ε) 300 (2.32), 201 (3.61) nm; CD
(MeOH) λmax (Δε) 303 (+3.57), 234 (−1.16), 213 (+0.42) nm; IR
1
νmax 3408, 2949, 1731, 1460 cm−1; for H and 13C NMR data, see
Tables S1 and S5; HR-ESI-MS (positive mode) m/z 336.2532 [M +
NH4]+ (calcd for C20H34O3N, 336.2533).
(16S)-13,18-Dihydroxy-ent-9(11)-kauren-15-one (8). Colorless
needle crystals (MeOH); mp 182−184 °C; [α]2D5 = +111.11 (c
0.081, MeOH); UV (MeOH) λmax (log ε) 300 (2.16), 224 (3.04), 201
(3.52) nm; CD (MeOH) λmax (Δε) 306 (+3.08), 238 (−0.41), 210
1
(+0.56) nm; IR νmax 3417, 2931, 1733, 1459 cm−1; for H and 13C
NMR data, see Tables S1 and S5; HR-ESI-MS (positive mode) m/z
336.2533 [M + NH4]+ (calcd for C20H34O3N, 336.2533).
(16R)-13-Hydroxy-ent-9(11)-kauren-18-al-15-one (9). Colorless
oil; [α]2D5 = +47.06 (c 0.085, MeOH); UV (MeOH) λmax (log ε)
279 (2.37), 204 (3.55) nm; CD (MeOH) λmax (Δε) 304 (+2.48), 235
(−0.68), 210 (+0.58) nm; IR νmax 3418, 2937, 1726, 1456 cm−1; for
1H and 13C NMR data, see Tables S2 and S5; HR-ESI-MS (positive
mode) m/z 334.2372 [M + NH4]+ (calcd for C20H32O3N, 334.2377).
(16R)-13,19-Dihydroxy-ent-9(11)-kauren-15-one (10). Colorless
needle crystals (MeOH); mp 164−166 °C; [α]2D5 = +195.88 (c
0.097, MeOH); UV (MeOH) λmax (log ε) 203 (3.42) nm; CD
(MeOH) λmax (Δε) 303 (+3.41), 233 (−1.17), 212 (+0.68) nm; IR
H
DOI: 10.1021/acs.jmedchem.5b00208
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Journal of Medicinal Chemistry
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1
νmax 3389, 2956, 1722, 1661, 1461 cm−1; for H and 13C NMR data,
see Tables S2 and S5; HR-ESI-MS (positive mode) m/z 336.2533 [M
+ NH4]+ (calcd for C20H34O3N, 336.2533).
Tables S3 and S6; HR-ESI-MS (positive mode) m/z 336.2548 [M +
NH4]+ (calcd for C20H34O3N, 336.2533).
6α,15β-Dihydroxy-ent-16-kauren-11-one (25). Colorless needle
crystals (MeOH); mp 167−169 °C; [α]2D5 = +207.92 (c 0.101,
MeOH); UV (MeOH) λmax (log ε) 208 (2.79) nm; CD (MeOH) λmax
(Δε) 310 (+0.78), 221 (+1.05) nm; IR νmax 3522, 2919, 1687, 1659,
1415 cm−1; for 1H and 13C NMR data, see Tables S3 and S6; HR-ESI-
MS (positive mode) m/z 336.2535 [M + NH4]+ (calcd for
C20H34O3N, 336.2533).
(16R)-6α,13,18-Trihydroxy-ent-9(11)-kauren-15-one (11). White
amorphous solid (MeOH); [α]2D5 = +100.00 (c 0.070, MeOH); UV
(MeOH) λmax (log ε) 295 (2.29), 202 (3.60) nm; CD (MeOH) λmax
(Δε) 304 (+3.81), 233 (−1.22), 209 (+1.00) nm; IR νmax 3424, 2990,
1724, 1466 cm−1; for H and 13C NMR data, see Tables S2 and S5;
1
HR-ESI-MS (positive mode) m/z 352.2482 [M + NH4]+ (calcd for
C20H34O4N, 352.2482).
6α,7α,15β-Trihydroxy-ent-16-kauren-11-one (26). Colorless nee-
dle crystals (MeOH); mp 173−175 °C; [α]2D5 = +175.93 (c 0.108,
MeOH); UV (MeOH) λmax (log ε) 204 (2.75) nm; CD (MeOH) λmax
(Δε) 310 (+0.40), 222 (+1.18) nm; IR νmax 3415, 2948, 1688, 1408
(16R)-3α,11β-Dihydroxy-ent-kauran-15-one (12). Colorless nee-
dle crystals (MeOH); mp 196−197 °C; [α]2D5 = −108.33 (c 0.120,
MeOH); UV (MeOH) λmax (log ε) 201 (3.32) nm; CD (MeOH) λmax
(Δε) 288 (+0.29), 235 (−0.09) nm; IR νmax 3457, 2934, 1724, 1464
1
cm−1; for H and 13C NMR data, see Tables S3 and S6; HR-ESI-MS
(positive mode) m/z 352.2483 [M + NH4]+ (calcd for C20H34O4N,
352.2482).
cm−1; for H and 13C NMR data, see Tables S2 and S5; HR-ESI-MS
1
(positive mode) m/z 338.2692 [M + NH4]+ (calcd for C20H36O3N,
338.2690).
7α,15β-Dihydroxy-ent-16-kauren-11-one (27). Colorless needle
crystals (MeOH); mp 209−211 °C; [α]2D5 = +183.67 (c 0.098,
MeOH); UV (MeOH) λmax (log ε) 305 (1.55), 206 (2.79) nm; CD
(MeOH) λmax (Δε) 310 (+0.61), 222 (+1.42) nm; IR νmax 3518, 2950,
(16S)-3α,11β-Dihydroxy-ent-kauran-15-one (13). Colorless nee-
dle crystals (MeOH); mp 209−210 °C; [α]2D5 = −62.50 (c 0.080,
MeOH); UV (MeOH) λmax (log ε) 305 (2.51), 203 (2.95) nm; CD
(MeOH) λmax (Δε) 330 (−0.05), 249 (+0.08) nm; IR νmax 3425, 2931,
1
1701, 1058 cm−1; for H and 13C NMR data, see Tables S3 and S6;
HR-ESI-MS (positive mode) m/z 336.2533 [M + NH4]+ (calcd for
C20H34O3N, 336.2533).
1725, 1444 cm−1; for H and 13C NMR data, see Tables S2 and S5;
1
HR-ESI-MS (positive mode) m/z 338.2689 [M + NH4]+ (calcd for
C20H36O3N, 338.2690).
15β-Hydroxy-ent-16-kauren-11-one (28). Colorless needle crystals
(MeOH); mp 140−142 °C; [α]2D5 = +186.29 (c 0.102, MeOH); UV
(MeOH) λmax (log ε) 306 (1.58), 207 (2.82) nm; CD (MeOH) λmax
(Δε) 311 (+0.96), 222 (+1.61) nm; IR νmax 3389, 2921, 1692, 1655
13-Hydroxyjungermannenone B (14). Colorless needle crystals
(MeOH); mp 171−173 °C; [α]2D5 = −267.44 (c 0.086, MeOH); UV
(MeOH) λmax (log ε) 350 (2.51), 269 (2.97), 207 (3.88) nm; CD
(MeOH) λmax (Δε) 357 (−1.47), 281 (+0.66), 245 (−0.29) nm; IR
1
cm−1; for H and 13C NMR data, see Tables S3 and S6; HR-ESI-MS
(positive mode) m/z 320.2582 [M + NH4]+ (calcd for C20H34O2N,
320.2584).
1
νmax 3478, 2926, 1717, 1651, 1473 cm−1; for H and 13C NMR data,
see Tables S2 and S5; HR-ESI-MS (positive mode) m/z 301.2149 [M
+ H]+ (calcd for C20H29O2, 301.2162).
1α,15β-Dihydroxy-ent-16-kaurene (29). Colorless needle crystals
(MeOH); mp 182−185 °C; [α]2D5 = −86.54 (c 0.104, MeOH); UV
(MeOH) λmax (log ε) 208 (2.77) nm; CD (MeOH) λmax (Δε) 355
(+0.76), 276 (−0.44), 221 (+0.61) nm; IR νmax 3376, 2937, 1709,
1644 cm−1; for 1H and 13C NMR data, see Tables S4 and S6; HR-ESI-
MS (positive mode) m/z 322.2739 [M + NH4]+ (calcd for
C20H36O2N, 322.2741).
1α,15α-Dihydroxy-ent-16-kaurene (30). Colorless needle crystals
(MeOH); mp 166−168 °C; [α]2D5 = +218.18 (c 0.055, MeOH); UV
(MeOH) λmax (log ε) 205 (2.96) nm; CD (MeOH) λmax (Δε) 311
(+0.11), 211 (−1.05) nm; IR νmax 3328, 2927, 1445, 1057 cm−1; for
1H and 13C NMR data, see Tables S4 and S6; HR-ESI-MS (positive
mode) m/z 322.2740 [M + NH4]+ (calcd for C20H36O2N, 322.2741).
9β,15β-Dihydroxy-ent-16-kaurene (31). Colorless needle crystals
(MeOH); mp 165−166 °C; [α]2D5 = +100.00 (c 0.070, MeOH); UV
(MeOH) λmax (log ε) 209 (2.84) nm; CD (MeOH) λma1x (Δε) 210
(+0.24) nm; IR νmax 3409, 2929, 1713, 1459 cm−1; for H and 13C
NMR data, see Tables S4 and S6; HR-ESI-MS (positive mode) m/z
322.2743 [M + NH4]+ (calcd for C20H36O2N, 322.2741).
11α,15β-Dihydroxy-ent-16-kaurene (32). Colorless needle crystals
(MeOH); mp 179−181 °C; [α]2D5 = +127.91 (c 0.086, MeOH); UV
(MeOH) λmax (log ε) 209 (2.83) nm; CD (MeOH) λmax (Δε) 308
(−0.07), 213 (+0.53) nm; IR νmax 3365, 2929, 1449, 1052 cm−1; for
1H and 13C NMR data, see Tables S4 and S6; HR-ESI-MS (positive
mode) m/z 322.2739 [M + NH4]+ (calcd for C20H36O2N, 322.2741).
1α,7α-Dihydroxy-ent-16-kaurene (33). Colorless needle crystals
(MeOH); mp 174−176 °C; [α]2D5 = +56.19 (c 0.133, MeOH); UV
(MeOH) λmax (log ε) 206 (2.77) nm; CD (MeOH) λma1x (Δε) 219
(+1.02) nm; IR νmax 3385, 2928, 1457, 1028 cm−1; for H and 13C
NMR data, see Tables S4 and S6; HR-ESI-MS (positive mode) m/z
322.2738 [M + NH4]+ (calcd for C20H36O2N, 322.2741).
7α-Acetoxy-1α-hydroxy-ent-16-kaurene (34). Colorless needle
crystals (MeOH); mp 172−174 °C; [α]2D5 = +38.46 (c 0.130,
MeOH); UV (MeOH) λmax (log ε) 209 (2.73) nm; CD (MeOH) λmax
(Δε) 218 (+0.28) nm; IR νmax 3445, 2925, 1728, 1704, 1242 cm−1; for
1H and 13C NMR data, see Tables S4 and S6; HR-ESI-MS (positive
mode) m/z 364.2843 [M + NH4]+ (calcd for C22H38O3N, 364.2846).
1α,7α-Dihydroxy-ent-15-kaurene (35). Colorless needle crystals
(MeOH); mp 159−161 °C; [α]2D5 = +66.17 (c 0.136, MeOH); UV
(MeOH) λmax (log ε) 209 (2.70) nm; CD (MeOH) λmax (Δε) 218
13,18-Dihydroxyjungermannenone B (15). Colorless needle
crystals (MeOH); mp 183−185 °C; [α]2D5 = −110.00 (c 0.100,
MeOH); UV (MeOH) λmax (log ε) 350 (2.54), 205 (3.89) nm; CD
(MeOH) λmax (Δε) 357 (−1.27), 282 (+0.59), 247 (−0.20) nm; IR
1
νmax 3416, 2931, 1720, 1649, 1436 cm−1; for H and 13C NMR data,
see Tables S2 and S5; HR-ESI-MS (positive mode) m/z 334.2376 [M
+ NH4]+ (calcd for C20H32O3N, 334.2377).
18-Hydroxyjungermannenone B (16). Colorless oil; [α]D25
=
−150.00 (c 0.080, MeOH); UV (MeOH) λmax (log ε) 227 (3.34),
207 (3.46) nm; CD (MeOH) λmax (Δε) 319 (−0.75), 268 (+0.25),
206 (−0.38) nm; IR νmax 3478, 2950, 1718, 1656, 1435 cm−1; for H
1
and 13C NMR data, see Tables S2 and S5; HR-ESI-MS (positive
mode) m/z 301.2167 [M + H]+ (calcd for C20H29O2, 301.2162).
16α,17-Dihydro-13,18-dihydroxyjungermannenone B (17). Col-
orless needle crystals (MeOH); mp 202−204 °C; [α]2D5 = −68.63 (c
0.102, MeOH); UV (MeOH) λmax (log ε) 300 (2.42), 229 (3.07), 203
(3.58) nm; CD (MeOH) λmax (Δε) 300 (−3.75), 240 (+1.41), 206
(−0.20) nm; IR νmax 3481, 2938, 1720, 1681, 1390 cm−1; for H and
1
13C NMR data, see Tables S2 and S6; HR-ESI-MS (positive mode) m/
z 336.2535 [M + NH4]+ (calcd for C20H34O3N, 336.2533).
16α,17-Dihydro-13-hydroxyjungermannenone B (18). White
amorphous solid (MeOH); [α]2D5 = −100.92 (c 0.109, MeOH); UV
(MeOH) λmax (log ε) 300 (2.40), 229 (3.08), 204 (3.52) nm; CD
(MeOH) λmax (Δε) 300 (−3.09), 236 (+1.15), 211 (−0.33) nm; IR
νmax 3472, 2935, 1724, 1457 cm−1; for H and 13C NMR data, see
1
Tables S3 and S6; HR-ESI-MS (positive mode) m/z 320.2578 [M +
NH4]+ (calcd for C20H34O2N, 320.2584).
18-Formyl-16α,17-dihydro-13-hydroxyjungermannenone B (19).
Colorless oil; [α]2D5 = −155.84 (c 0.077, MeOH); UV (MeOH) λmax
(log ε) 300 (2.57), 244 (3.26), 205 (3.50) nm; CD (MeOH) λmax
(Δε) 306 (−1.74), 257 (+0.34), 238 (+0.29), 211 (−0.25) nm; IR
1
νmax 3416, 2938, 1729, 1664, 1454 cm−1; for H and 13C NMR data,
see Tables S3 and S6; HR-ESI-MS (positive mode) m/z 334.2374 [M
+ NH4]+ (calcd for C20H32O3N, 334.2377).
16α,17-Dihydro-13,19-dihydroxyjungermannenone B (20). Col-
orless needle crystals (MeOH); mp 198−199 °C; [α]2D5 = −134.62 (c
0.052, MeOH); UV (MeOH) λmax (log ε) 300 (2.63), 202 (3.77) nm;
CD (MeOH) λmax (Δε) 300 (−3.94), 235 (+1.81), 209 (−0.69) nm;
IR νmax 3428, 2932, 1721, 1453 cm−1; for H and 13C NMR data, see
1
I
DOI: 10.1021/acs.jmedchem.5b00208
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(+0.31) nm; IR νmax 3371, 2928, 1461, 1018 cm−1; for H and 13C
NMR data, see Tables S4 and S6; HR-ESI-MS (positive mode) m/z
322.2740 [M + NH4]+ (calcd for C20H36O2N, 322.2741).
g, 0.79 mmol) for 10 h under reflux. Then 10 mL of water was added
to the resulting solution, which was extracted with EtOAc (15 mL ×
2). The combined extracts were washed with brine, dried over MgSO4,
and evaporated under vacuum to obtain compound 50 (75.0 mg),
which was then dissolved in 2 mL of DMF and reacted with PDC
(75.0 mg, 0.20 mmol) for 12 h at room temperature. Then 10 mL of
water was added to the resulting solution, which was extracted with
EtOAc (15 mL × 2). The combined extracts were washed with brine,
dried over MgSO4, and evaporated under vacuum to obtain the crude
product, which was then purified by flash chromatography to give
compound 47 (40.0 mg, 29%). Colorless oil; HR-ESI-MS (positive
mode) m/z 459.3216 [M + H]+ (calcd for C27H43O4N2, 459.3217);
1H NMR (CDCl3, 600 MHz) δH 6.04 (1H, s, H-17a), 5.44 (1H, s, H-
17b), 2.59 (3H, s, −NCH3), 1.20 (3H, s, H3-18), 0.93 (3H, s, H3-20);
13C NMR (CDCl3, 150 MHz) δC 39.8 (C-1), 18.8 (C-2), 37.9 (C-3),
15β-Acetoxy-ent-kaur-16-en-18-oic Acid (36). Colorless needle
crystals (MeOH); mp 186−188 °C; [α]2D5 = +11.36 (c 0.088, MeOH);
UV (MeOH) λmax (log ε) 206 (2.91) nm; CD (MeOH) λmax (Δε) 221
1
(−0.15) nm; IR νmax 3362, 2930, 1730, 1708 cm−1; for H and 13C
NMR data, see Tables S4 and S6; HR-ESI-MS (positive mode) m/z
378.2639 [M + NH4]+ (calcd for C22H38O4N, 378.2639).
ent-13-Hydroxy-15-kaurene-19-acid (45). To a solution of
stevioside (2.0 g, 2.48 mmol) in 150 mL of deionized water was
added NaIO4 (3.0 g, 14.02 mmol). The mixture was stirred for 16 h
with hydrogen at room temperature and then at 0 °C. Then KOH
(15.0 g, 267.86 mmol) was added slowly to the resulting solution,
which was subsequently refluxed for 1 h. The resulting solution was
cooled, acidified with acetate to a pH of 3−4, and extracted with ethyl
ether (3 × 100 mL). The combined extracts were washed with distilled
water and brine, dried over MgSO4, and evaporated under vacuum to
obtain the crude product, which was then purified by flash
chromatography to give 500.0 mg of steviol. To a solution of steviol
(400.0 mg, 1.26 mmol) in 12 mL of THF were added SeO2 (100.0 mg,
0.90 mmol) and t-BuOOH (680 μL, 7.08 mmol) in 12 mL of THF.
After 20 of stirring at room temperature, saturated brine with a slight
amount of NaHSO3 was added to the resulting solution, which was
then extracted twice with EtOAc. The combined extracts were washed
with brine, dried over MgSO4, and evaporated under vacuum to obtain
the crude product, which was then purified by flash chromatography to
give compound 48 (210.0 mg, powder, 51%). Compound 48 (70.0
mg, 0.21 mmol) and PDC (73.0 mg, 0.19 mmol) were reacted in 1.5
mL of DMF for 12 h at room temperature. Then saturated brine was
poured into the resulting solution, which was extracted with EtOAc.
The combined extracts were washed with brine, dried over MgSO4,
and evaporated under vacuum to obtain the crude product, which was
then purified by flash chromatography to give compound 45 (50.0 mg,
72%). Colorless needles (CHCl3); HR-ESI-MS (positive mode) m/z
43.8 (C-4), 56.0 (C-5), 20.1 (C-6), 32.9 (C-7), 55.1 (C-8), 50.1 (C-9),
40.1 (C-10), 20.7 (C-11), 39.2 (C-12), 77.0 (C-13), 44.8 (C-14),
208.4 (C-15), 151.3 (C-16), 115.3 (C-17), 28.8 (C-18), 177.2 (C-19),
15.6 (C-20), 61.1 (C-1′), 56.1 (C-2′), 51.1 (C-3′, C-5′), 54.3 (C-4′, C-
6′), 44.8 (−NCH3).
X-ray Crystal Structure Analysis. A colorless crystal of 1 was
obtained from MeOH. Intensity data were collected on a Bruker
APEX DUO diffractometer equipped with an APEX II CCD using Cu
Kα radiation. Cell refinement and data reduction were performed with
Bruker SAINT. The structures were solved by direct methods using
SHELXS-97.49 Refinements were performed with SHELXL-97 using
full-matrix least-squares with anisotropic displacement parameters for
all of the non-hydrogen atoms. The H atoms were placed in calculated
positions and refined using a riding model. Molecular graphics were
computed with SHELXS-97. Crystallographic data for the structure of
1 (excluding structure factor tables) have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication
CCDC 1046802. Copies of the data can be obtained free of charge on
application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K.
[fax: Int. +44(0) (1223) 336 033; e-mail: deposit@ccdc.cam.ac.uk].
Crystal Data for 13,18-Dihydroxy-ent-9(11),16-kauradien-15-one
(1). C20H28O3, MW = 316.43, orthorhombic, a = 13.0132(11) Å, b =
21.6913(16) Å, c = 6.3473(6) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V
= 1791.7(3) Å3, T = 293(2) K, space group P2, Z = 4, μ(Cu Kα) =
0.643 mm−1, 3845 reflections measured, 2634 independent reflections
(Rint = 0.0321). The final R1 values were 0.0512 (I > 2σ(I)) and 0.0676
(all data). The final wR(F2) values were 0.1169 (I > 2σ(I)) and 0.1312
(all data). The goodness of fit on F2 was 1.017, and the Flack
parameter was −0.1(4).
Synthesis of Compound 46−NAC. To a solution of compound
46 (10.0 mg) in 1.5 mL of MeOH was added 5.0 mg of NAC in 0.5
mL of water. The pH of the mixture was adjusted to 9 with aqueous
ammonia. Then the mixture was stirred for 0.5 h at room temperature
to give the target compound. HR-ESI-MS (positive mode) m/z
510.2517 [M + H]+ (calcd for C26H40O7NS, 510.2520).
Synthesis of Compound 46−GSH. To a solution of compound
46 (10.0 mg) in 1.5 mL of MeOH was added 5.0 mg of GSH in 0.5
mL of water. The pH of the mixture was adjusted to 9 with aqueous
ammonia. Then the mixture was stirred for 0.5 h at room temperature
to give the target compound. HR-ESI-MS (positive mode) m/z
654.3039 [M + H]+ (calcd for C31H48O10N3S, 654.3055).
350.2327 [M + NH4]+ (calcd for C20H32O4N, 350.2326); H NMR
1
(CDCl3, 600 MHz) δH 6.05 (1H, s, H-17a), 5.44 (1H, s, H-17b), 1.27
(3H, s, H3-18), 1.03 (3H, s, H3-20); 13C NMR (CDCl3, 150 MHz) δC
39.6 (C-1), 18.7 (C-2), 37.5 (C-3), 43.5 (C-4), 55.9 (C-5), 20.0 (C-6),
32.7 (C-7), 55.1 (C-8), 50.2 (C-9), 40.2 (C-10), 20.7 (C-11), 39.1 (C-
12), 77.1 (C-13), 44.8 (C-14), 208.3 (C-15), 151.1 (C-16), 115.1 (C-
17), 28.7 (C-18), 182.6 (C-19), 15.4 (C-20).
ent-13-Hydroxy-15-kaurene-19-acid Methyl Ester (46). To a
solution of compound 45 (30.0 mg, 0.09 mmol) in 2 mL of DMF
were added anhydrous K2CO3 (32.0 mg, 0.23 mmol) and MeI (28.0
μL, 0.45 mmol). The mixture was stirred for 8 h at room temperature.
Then 3 mL of water were added to the resulting solution, which was
extracted with EtOAc (10 mL × 2). The combined extracts were
washed with brine, dried over MgSO4, and evaporated under vacuum
to obtain the crude product, which was then purified by flash
chromatography to give compound 46 (21.0 mg, 68%). Colorless
needles (CHCl3); HR-ESI-MS (positive mode) m/z 364.2482 [M +
NH4]+ (calcd for C21H34O4N, 364.2482); 1H NMR (CDCl3, 600
MHz) δH 6.04 (1H, s, H-17a), 5.43 (1H, s, H-17b), 3.66 (3H, s,
−OCH3), 1.20 (3H, s, H3-18), 0.89 (3H, s, H3-20). 13C NMR (CDCl3,
150 MHz) δC 39.8 (C-1), 18.8 (C-2), 37.8 (C-3), 43.7 (C-4), 56.0 (C-
5), 20.0 (C-6), 32.8 (C-7), 55.1 (C-8), 50.2 (C-9), 40.0 (C-10), 20.7
(C-11), 39.1 (C-12), 77.0 (C-13), 44.7 (C-14), 208.5 (C-15), 151.4
(C-16), 115.4 (C-17), 28.7 (C-18), 177.8 (C-19), 15.3 (C-20), 51.3
(−OCH3).
ent-13-Hydroxy-15-kaurene-19-acid N-Methylpiperazine Ethyl
Ester (47). To a solution of compound 48 (100.0 mg, 0.30 mmol)
in 5 mL of DMF were added 1,2-dibromoethane (0.24 mL, 2.78
mmol) and anhydrous K2CO3 (190.0 mg, 1.37 mmol). The mixture
was stirred for 4 h at room temperature. Then 10 mL of water was
added to the resulting solution, which was extracted with EtOAc (15
mL × 2). The combined extracts were washed with brine, dried over
MgSO4, and evaporated under vacuum to obtain compound 49 (120.0
mg), which was then dissolved in 10 mL of water and reacted with N-
methylpiperazine (0.10 mL, 0.90 mmol) and anhydrous K2CO3 (110.0
HPLC−MS/MS Analysis. The HPLC−MS/MS system consisted
of an Agilent 1260 series binary pump (Agilent Technologies, Palo
Alto, CA, USA) and a Shiseido 5100 autosampler connected to an API
4000 mass spectrometer (Applied Biosystems Sciex, Ontario, Canada)
using a turbospray ionization source (ESI). The instrument was
interfaced to a computer running Applied Biosystems Analyst version
1.4.2 software.
Compounds were separated on a ZORBAX SB-C18 column (5 μm,
150 mm × 2.1 mm I.D., Agilent Science Inc., USA) through a 4 mm ×
3 mm precolumn (Security Guard C18 cartridge, Phenomenex, Inc.)
maintained at room temperature. The mobile phase consisted of
acetonitrile−0.5% formic acid/water (70:30 v/v) (pH 2.5) and was set
at a flow rate of 0.25 mL/min. The detector was operated at unit
resolution in the positive multiple-reaction monitoring (+MRM)
J
DOI: 10.1021/acs.jmedchem.5b00208
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mode using the transitions of the protonated molecular ions of 46 (m/
z 347.3; m/z 269.2) and 46−NAC (m/z 510.3; m/z 347.3). The
optimized parameters were as follows: the curtain gas, gas1, and gas2
(nitrogen) pressures were 25, 50, and 40 psi, respectively; the dwell
time was 200 ms; the source temperature was 500 °C; the ion spray
voltage was 4800 V. The declustering potential and collision energy
were 60 and 20 V for 46 and 50 and 25 V for 46−NAC, respectively.
Compounds were separated on a ZORBAX SB-C18 column (5 μm,
150 mm × 2.1 mm I.D., Agilent Science Inc., USA) through a 4 mm ×
3 mm precolumn (Security Guard C18 cartridge, Phenomenex, Inc.)
maintained at room temperature. The mobile phase consisted of
acetonitrile−0.5% formic acid/water (80:20 v/v) (pH 2.5) and was set
at a flow rate of 0.30 mL/min. The detector was operated at unit
resolution in the negative multiple-reaction monitoring (−MRM)
mode using the transitions of the protonated molecular ions of 46−
GSH (652.3 m/z; 306.2 m/z). The optimized parameters were as
follows: the curtain gas, gas1, and gas2 (nitrogen) pressures were 15,
30, and 50 psi, respectively; the dwell time was 200 ms; the source
temperature was 500 °C; the ion spray voltage was −4200 V. The
declustering potential and collision energy were −65 and −27 V,
respectively, for46−GSH.
Cell Culture and Treatments. The human prostate carcinoma
cell lines PC3, DU145, and LNCaP, the lung cancer cell line NCI-
H1299, the breast carcinoma cell line MDA-MB231, the colon cancer
cell line LOVO, the urinary bladder carcinoma cell line T24, and the
myelogenous leukemia cell lines K562 and HL-60 were cultured in
RPMI 1640 medium (Hyclone, Logan, UT) supplemented with 10%
fetal bovine serum (FBS) (Hyclone), 100 units/mL penicillin, and 100
μg/mL streptomycin. The human astrocytoma cell lines A172 and SH-
SY5Y and the osteosarcoma cell line Saos-2 were cultured in DMEM/
HIGH GLUCOSE medium supplemented with 10% FBS, 5% mare
serum (Hyclone), 100 units/mL penicillin, and 100 μg/mL
streptomycin. The human ovarian cancer cell line SKOV3 was
cultured in McCoy’s 5A medium (Hyclone, Logan, UT) supplemented
with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.
The human prostate epithelial cell line RWPE-1 was maintained in
Keratinocyte1 medium (K-SFM) supplemented with 50 mg/L bovine
pituitary extract and 5 μg/L epidermal growth factor (Gibco, Grand
Island, NY). The cells were maintained in 5% CO2 at 37 °C until they
reached approximately 50−70% confluence and were then treated with
various concentrations of compounds. Dimethyl sulfoxide (DMSO)
was used as the control vehicle.
Cell Proliferation Assay. An MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide, Sigma, St. Louis, MO] assay
was used to measure the proliferation of cells treated with different
compounds in 96-well plates. After treatment with vehicle, the
compounds alone, or cisplatin as a positive control for 24 h, the cells
were incubated with 10 μL of MTT (5 mg/mL) for 4 h. The formazan
product was then solubilized with 100 μL of DMSO. The absorbance
was measured at 570 nm using a microplate reader (Bio-Rad, USA).
The IC50 value was calculated based on percent cell viability using
GraphPad Prism 5.0 (La Jolla, CA). All of the experiments were
conducted at least three times. In some experiments, cells were
exposed to 5 mM NAC (Sigma-Aldrich, USA) for 1 h prior to
treatment with compound 46. The cell proliferations in response to
the compounds were analyzed using the MTT assay.
Microscopy. PC3 cells were seeded on 6 mm round glass
coverslips and placed at the bottom of 24-well plates. After different
treatments, cells on glass coverslips were fixed with cold methanol/
acetone (1:1) for 5 min. After washing, cells were incubated with 30
μM probe 51 for 1 h at room temperature in the dark. Fluorescence
images were captured using a confocal microscope (Carl Zeiss). The
mean fluorescence intensity of cells was measured using ZEN 2010
software. Cells were defined using a free-form selection tool.
aggregated LysoTracker in acidic compartments using a confocal
microscope.
ROS Measurement. Intracellular ROS accumulation was moni-
tored using the fluorescent probe DCFH-DA. After treatment with 46
for the indicated times, PC3 cells were incubated with 10 μM DCFH-
DA at 37 °C for 30 min. Cells were washed, collected, resuspended in
PBS, and analyzed immediately using flow cytometry (Becton
Dickinson, USA). In some experiments, cells were pretreated with 5
mM NAC for 1 h prior to exposure with 46 and analyzed for ROS
generation.
GSH and GSSG Analyses. Cellular GSH and GSSG levels were
measured by the method of Griffith.50 PC3 cells exposed to 6 μM 46
for the indicated times were collected and resuspended in a protein
removal agent to collect supernatant by centrifugation after repeated
freezing and thawing. Total GSH and GSSG were measured after the
addition of dithiobis(2-nitrobenzoic acid) (DNTB) at an absorbance
of 412 nm. GSSG was selectively measured after assaying samples in
which GSH was masked by pretreatment with 2-vinylpyridine. The
difference between the two values gave the GSH level in the cells.
Western Blotting. Cells were collected and lysed with RIPA buffer
containing a fresh protease inhibitor mixture (50 μg/mL aprotinin, 0.5
mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium
orthovanadate, 10 mM NaF, and 10 mM glycerol phosphate). Protein
concentrations were quantified using BCA (bicinchoninic acid) assay.
Equal amounts of proteins were separated by SDS-PAGE (12%) and
electrotransferred onto nitrocellulose membranes. The membranes
were blocked with 5% nonfat milk in TBST buffer (20 mM Tris-
buffered saline and 0.5% Tween 20) for 1 h at room temperature
followed by incubation with the corresponding primary antibodies
overnight at 4 °C. After washing with TBST buffer, secondary
antibodies were used at 1:2000 dilutions for 45 min at room
temperature. Immunoblot proteins were visualized using an enhanced
chemiluminescence detection system (Millipore, Germany) followed
by exposure to X-ray film. The primary antibodies cathepsin B and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used.
Statistical Analysis. Data are presented as mean
SD for
triplicate experiments. The statistical significance of the differences
between treated groups and controls was calculated using Student’s t
test. P < 0.05 was considered statistically significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
Structure elucidation of compounds 2−20 and 25−36; 1H and
13C NMR data for compounds 1−20 and 25−36; X-ray
crystallographic structure of 1; key 1H−1H COSY, HMBC, and
NOESY correlations and 1D and 2D NMR, HR-ESI-MS, CD,
and IR spectra of compounds 1−20 and 25−36; H and 13C
1
NMR spectra of compounds 45−47; and a spreadsheet
containing SMILES data (CSV). This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +86-531-8838-2012. Fax: +86-531-8838-2019. E-mail:
louhongxiang@sdu.edu.cn.
Author Contributions
§Z.L. and Y.G. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
To measure the volume of lysosomes, PC3 cells in various stages
after treatment were incubated at 37 °C for 30 min with 100 ng/mL
LysoTracker (Molecular Probes) and 10 μg/mL Hoechst 33342
(Sigma-Aldrich). Cells were then washed with phosphate-buffered
saline (PBS) for measurement of the fluorescence derived from the
This work was financially supported by the National Natural
Science Foundation of China (81273383 and 81473107). We
acknowledge Prof. M. Li (Shandong University) for providing
the fluorescence probe. We are grateful to Dr. Paul E.
K
DOI: 10.1021/acs.jmedchem.5b00208
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Journal of Medicinal Chemistry
Article
(16) Santagata, S.; Xu, Y.; Wijeratne, E. M. K.; Kontnik, R.; Rooney,
C.; Perley, C. C.; Kwon, H.; Clardy, J.; Kesari, S.; Whitesell, L.;
Lindquist, S.; Gunatilaka, A. A. L. Using the heat-shock response to
discover anticancer compounds that target protein homeostasis. ACS
Chem. Biol. 2012, 7, 340−349.
(17) Wijeratne, E. M. K.; Bashyal, B. P.; Liu, M. X.; Rocha, D. D.;
Gunaherath, G. M. K. B.; U’Ren, J. M.; Gunatilaka, M. K.; Arnold, A.
E.; Whitesell, L.; Gunatilaka, A. A. L. Geopyxins A−E, ent-Kaurane
Diterpenoids from Endolichenic Fungal Strains Geopyxis aff. majalis
and Geopyxis sp. AZ0066: Structure−Activity Relationships of
Geopyxins and Their Analogues. J. Nat. Prod. 2012, 75, 361−369.
(18) Liu, C. X.; Yin, Q. Q.; Zhou, H. C.; Wu, Y. L.; Pu, J. X.; Xia, L.;
Liu, W.; Huang, X.; Jiang, T.; Wu, M. X.; He, L. C.; Zhao, Y. X.; Wang,
X. L.; Xiao, W. L.; Chen, H. Z.; Zhao, Q.; Zhou, A. W.; Wang, L. S.;
Sun, H. D.; Chen, G. Q. Adenanthin targets peroxiredoxin I and II to
induce differentiation of leukemic cells. Nat. Chem. Biol. 2012, 8, 486−
493.
(19) Guo, D. X.; Zhu, R. X.; Wang, X. N.; Wang, L. N.; Wang, S. Q.;
Lin, Z. M.; Lou, H. X. Scaparvin A, a novel caged cis-clerodane with an
unprecedented C-6/C-11 bond, and related diterpenoids from the
liverwort Scapania parva. Org. Lett. 2010, 12, 4404−4407.
(20) Wang, L. N.; Zhang, J. Z.; Li, X.; Wang, X. N.; Xie, C. F.; Zhou,
J. C.; Lou, H. X. Pallambins A and B, unprecedented hexacyclic 19-
nor-secolabdane diterpenoids from the Chinese liverwort Pallavicinia
ambigua. Org. Lett. 2012, 14, 1102−1105.
(21) Liu, N.; Li, R. J.; Wang, X. N.; Zhu, R. X.; Wang, L.; Lin, Z. M.;
Zhao, Y.; Lou, H. X. Highly oxygenated ent-pimarane-type
diterpenoids from the Chinese liverwort Pedinophyllum interruptum
and their allelopathic activities. J. Nat. Prod. 2013, 76, 1647−1653.
(22) Lin, Z. M.; Guo, Y. X.; Wang, S. Q.; Wang, X. N.; Chang, W. Q.;
Zhou, J. C.; Yuan, H.; Lou, H. Diterpenoids from the Chinese
liverwort Heteroscyphus tener and their antiproliferative effects. J. Nat.
Prod. 2014, 77, 1336−1344.
(23) Zhang, D. Y.; Tang, Y.; Wang, K.; Wu, X. M.; Hua, W. Y.
Synthesis and antitumor activity of ent-kaurane diterpenoids. J. China
Pharm. Univ. 2010, 41, 20−25.
(24) Nagashima, F.; Kasai, W.; Kondoh, M.; Fujii, M.; Watanabe, Y.;
Braggins, J. E.; Asakawa, Y. New ent-kaurene-type diterpenoids
possessing cytotoxicity from the New Zealand liverwort Jungermannia
species. Chem. Pharm. Bull. 2003, 51, 1189−1192.
Floreancig (University of Pittsburgh) for helpful suggestion on
the preparation of the manuscript.
ABBREVIATIONS USED
■
SAR, structure−activity relationship; ROS, reactive oxygen
species; NAC, N-acetylcysteine; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide; IC50, half-maximal
inhibitory concentration; SI, selectivity index; DCFH-DA,
2′,7′-dichlorofluorescein diacetate; GSH, glutathione; GSSG,
oxidized form of GSH; PBS, phosphate-buffered saline; DMSO,
dimethyl sulfoxide; BCA, bicinchoninic acid; BSA, bovine
serum albumin; TBST, Tris-buffered saline and Tween 20;
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresis
REFERENCES
■
(1) Newman, D. J. Natural products as leads to potential drugs: an
old process or the new hope for drug discovery? J. Med. Chem. 2008,
51, 2589−2599.
(2) Lee, K. H. Discovery and Development of Natural Product-
Derived Chemotherapeutic Agents Based on a Medicinal Chemistry
Approach. J. Nat. Prod. 2010, 73, 500−516.
(3) Newman, D. J.; Cragg, G. M. Natural products as sources of new
drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 2012, 75,
311−335.
(4) Asakawa, Y. Chemosystematics of the Hepaticae. Phytochemistry
2004, 65, 623−669.
(5) Asakawa, Y. Liverwortspotential source of medicinal
compounds. Curr. Pharm. Des. 2009, 14, 3067−3088.
(6) Asakawa, Y.; Ludwiczuk, A.; Nagashima, F. Phytochemical and
biological studies of bryophytes. Phytochemistry 2013, 91, 52−80.
(7) Asakawa, Y.; Ludwiczuk, A.; Nagashima, F. Chemical Constituents
of Bryophytes: Bio- and Chemical Diversity, Biological Activity, and
Chemosystematics; Progress in the Chemistry of Organic Natural
Products, Vol. 95; Springer: Berlin, 2013.
(8) Nagashima, F.; Kondoh, M.; Uematsu, T.; Nishiyama, A.; Saito,
S.; Sato, M.; Asakawa, Y. Cytotoxic and apoptosis-inducing ent-
kaurane-type diterpenoids from the Japanese liverwort Jungermannia
truncata NEES. Chem. Pharm. Bull. 2002, 50, 808−813.
(9) Zheng, H.; Chen, Q.; Zhang, M.; Lai, Y.; Lei, L.; Shu, P.; Zhang,
J.; Xue, Y.; Luo, Z.; Li, Y.; Yao, G.; Zhang, Y. Cytotoxic ent-Kaurane
Diterpenoids from Salvia cavaleriei. J. Nat. Prod. 2013, 76, 2253−2262.
(10) Zhan, R.; Li, X. N.; Du, X.; Wang, W. G.; Dong, K.; Su, J.; Li, Y.;
Pu, J. X.; Sun, H. D. Bioactive ent-Kaurane diterpenoids from Isodon
rosthornii. J. Nat. Prod. 2013, 76, 1267−1277.
(11) Sun, H. D.; Huang, S. X.; Han, Q. B. Diterpenoids from Isodon
species and their biological activities. Nat. Prod. Rep. 2006, 23, 673−
698.
(12) Luo, X.; Pu, J. X.; Xiao, W. L.; Zhao, Y.; Gao, X. M.; Li, X. N.;
Zhang, H. B.; Wang, Y. Y.; Li, Y.; Sun, H. D. Cytotoxic ent-Kaurane
diterpenoids from Isodon rubescens var. lushiensis. J. Nat. Prod. 2010, 73,
1112−1116.
(25) Nagashima, F.; Kondoh, M.; Fujii, M.; Takaoka, S.; Watanabe,
Y.; Asakawa, Y. Novel cytotoxic kaurane-type diterpenoids from the
New Zealand liverwort Jungermannia species. Tetrahedron 2005, 61,
4531−4544.
(26) Qu, J. B.; Zhu, R. L.; Zhang, Y. L.; Guo, H. F.; Wang, X. N.; Xie,
C. F.; Yu, W. T.; Ji, M.; Lou, H. X. ent-Kaurane diterpenoids from the
liverwort Jungermannia atrobrunnea. J. Nat. Prod. 2008, 71, 1418−
1422.
(27) Trachootham, D.; Lu, W.; Ogasawara, M. A.; Nilsa, R. D.;
Huang, P. Redox regulation of cell survival. Antioxid. Redox Signaling
2008, 10, 1343−1374.
(28) Rhee, S. G. H2O2, a necessary evil for cell signaling. Science
2006, 312, 1882−1883.
(29) Dickinson, B. C.; Chang, C. J. Chemistry and biology of reactive
oxygen species in signaling or stress responses. Nat. Chem. Biol. 2011,
7, 504−511.
(13) Aquila, S.; Weng, Z. Y.; Zeng, Y. Q.; Sun, H. D.; Ríos, J. L.
Inhibition of NF-κB activation and iNOS induction by ent-kaurane
diterpenoids in LPS-stimulated RAW264.7 murine macrophages. J.
Nat. Prod. 2009, 72, 1269−1272.
́
(30) Alexandre, J.; Batteux, F.; Nicco, C.; Chereau, C.; Laurent, A.;
Guillevin, L.; Weill, B.; Goldwasser, F. Accumulation of hydrogen
peroxide is an early and crucial step for paclitaxel-induced cancer cell
death both in vitro and in vivo. Int. J. Cancer 2006, 119, 41−48.
(31) Trachootham, D.; Zhou, Y.; Zhang, H.; Demizu, Y.; Chen, Z.;
Pelicano, H.; Chiao, P. J.; Achanta, G.; Arlinghaus, R. B.; Liu, J.;
Huang, P. Selective killing of oncogenically transformed cells through a
ROS-mediated mechanism by β-phenylethyl isothiocyanate. Cancer
Cell 2006, 10, 241−252.
(14) Zhou, G. B.; Kang, H.; Wang, L.; Gao, L.; Liu, P.; Xie, J.; Zhang,
F. X.; Weng, X. Q.; Shen, Z. X.; Chen, J.; Gu, L. J.; Yan, M.; Zhang, D.
E.; Chen, S. J.; Wang, Z. Y.; Chen, Z. Oridonin, a diterpenoid extracted
from medicinal herbs, targets AML1-ETO fusion protein and shows
potent antitumor activity with low adverse effects on t(8;21) leukemia
in vitro and in vivo. Blood 2007, 109, 3441−3456.
(15) Gu, Z. M.; Wu, Y. L.; Zhou, M. Y.; Liu, C. X.; Xu, H. Z.; Yan,
H.; Zhao, Y.; Huang, Y.; Sun, H. D.; Chen, G. Q. Pharicin B stabilizes
retinoic acid receptor-α and presents synergistic differentiation
induction with ATRA in myeloid leukemic cells. Blood 2010, 116,
5289−5297.
(32) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer
cells by ROS-mediated mechanisms: a radical therapeutic approach?
Nat. Rev. Drug Discovery 2009, 8, 579−591.
L
DOI: 10.1021/acs.jmedchem.5b00208
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(33) Gorrini, C.; Harris, I. S.; Mak, T. W. Modulation of oxidative
stress as an anticancer strategy. Nat. Rev. Drug Discovery 2013, 12,
931−947.
(34) Zhen, T.; Wu, C. F.; Liu, P.; Wu, H. Y.; Zhou, G. B.; Lu, Y.; Liu,
J. X.; Liang, Y.; Li, K. K.; Wang, Y. Y.; Xie, Y. Y.; He, M. M.; Cao, H.
M.; Zhang, W. N.; Chen, L. M.; Petrie, K.; Chen, S. J.; Chen, Z.
Targeting of AML1-ETO in t(8;21) leukemia by oridonin generates a
tumor suppressor-like protein. Sci. Transl. Med. 2012, 4, No. 127ra38.
(35) Huang, J.; Wu, L.; Tashiro, S.; Onodera, S.; Ikejima, T. Reactive
oxygen species mediate oridonin-induced HepG2 apoptosis through
p53, MAPK, and mitochondrial signaling pathways. J. Pharmacol. Sci.
2008, 107, 370−379.
(36) Yu, Z. Y.; Liang, Y. G.; Xiao, H.; Shan, Y. J.; Dong, B.; Huang,
R.; Fu, Y. L.; Zhao, Z. H.; Liu, Z. Y.; Zhao, Q. S.; Wang, S. Q.; Chen, J.
P.; Mao, B. Z.; Cong, Y. W. Melissoidesin G, a diterpenoid purified
from Isodon melissoides, induces leukemic-cell apoptosis through
induction of redox imbalance and exhibits synergy with other
anticancer agents. Int. J. Cancer 2007, 121, 2084−2094.
(37) Liao, Y. J.; Bai, H. Y.; Li, Z. H.; Zou, J.; Chen, J. W.; Zheng, F.;
Zhang, J. X.; Mai, S. J.; Zeng, M. S.; Sun, H. D.; Pu, J. X.; Xie, D.
Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via
downregulation of Skp2 and apoptosis induction through ROS/JNK/
c-Jun pathway in hepatocellular carcinoma cells. Cell Death Dis. 2014,
5, No. e1137.
(38) Dickinson, D. A.; Forman, H. J. Cellular glutathione and thiols
metabolism. Biochem. Pharmacol. 2002, 64, 1019−1026.
(39) Chatterjee, A. Reduced glutathione: a radioprotector or a
modulator of DNA-repair activity? Nutrients 2013, 5, 525−542.
(40) Lu, S. C. Regulation of glutathione synthesis. Mol. Aspects Med.
2009, 30, 42−59.
(41) Li, W.; Sun, W.; Yu, X.; Du, L.; Li, M. Coumarin-based
fluorescent probes for H2S detection. J. Fluoresc. 2013, 23, 181−186.
(42) Yuan, L.; Jian, W.; Zhang, D.; Aubry, A. F.; Arnold, M. E.
Application of a stabilizer cocktail of N-ethylmaleimide and phenyl-
methanesulfonyl fluoride to concurrently stabilize the disulfide and
ester containing compounds in a plasma LC−MS/MS assay. J. Pharm.
Biomed. Anal. 2014, 88, 552−561.
(43) Kim, J. A.; Kang, Y. S.; Park, S. H.; Kim, H. W.; Cho, S. Y.; Lee,
Y. S. Role of reactive oxygen species in apoptosis induced by N-
ethylmaleimide in HepG2 human hepatoblastoma cells. Eur. J.
Pharmacol. 2001, 433, 1−6.
(44) Yellaturu, C. R.; Bhanoori, M.; Neeli, I.; Rao, G. N. N-
Ethylmaleimide Inhibits Platelet-Derived Growth Factor BB-Stimu-
lated Akt Phosphorylation via Activation of Protein Phosphatase 2A. J.
Biol. Chem. 2002, 277, 40148−40155.
(45) Rajendran, L.; Knolker, H. J.; Simons, K. Subcellular targeting
̈
strategies for drug design and delivery. Nat. Rev. Drug Discovery 2010,
9, 29−42.
(46) Mohamed, M. M.; Sloane, B. F. Cysteine cathepsins:
multifunctional enzymes in cancer. Nat. Rev. Cancer 2006, 6, 764−775.
(47) Mizushima, N.; Levine, B.; Cuervo, A. M.; Klionsky, D. J.
Autophagy fights disease through cellular self-digestion. Nature 2008,
451, 1069−1075.
(48) Boya, P.; Kroemer, G. Lysosomal membrane permeabilization in
cell death. Oncogene 2008, 27, 6434−6451.
(49) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277,
319−343.
(50) Griffith, O. W. Determination of glutathione and glutathione
disulfide using glutathione reductase and 2-vinylpyridine. Anal.
Biochem. 1980, 106, 207−212.
M
DOI: 10.1021/acs.jmedchem.5b00208
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